hymecromone and 4-methylumbelliferyl-glucoside

Pompe disease, a rare, autosomal, recessive, inherited, lysosomal storage disorder, is caused by mutations in the acid α-glucosidase (GAA) gene leading to a deficiency of the lysosomal GAA enzyme. Some GAA mutations eliminate all enzymatic activities, causing severe infantile Pompe disease; others allow residual GAA activity and lead to middle adulthood forms. Here, we report a cohort of 12 patients, belonging to 11 unrelated families, with infantile Pompe disease. The mutational analysis of GAA gene revealed a novel c.1494G > A (p.Trp498X) mutation in one patient and three known mutatio,ns including the c.1497G > A (p.Trp499X) mutation, in two patients, the c.1927G > A (p.Gly643Arg) mutation in one patient and the common c.236_246del (p.Pro79ArgfsX13) mutation in eight patients. The high prevalence of c.236_246del mutation in our cohort (58%) was supported by the existence of a common founder ancestor that was confirmed by its segregation of similar SNPs haplotype, including four intragenic SNPs of GAA gene. In addition, a 3D structure model and a docking were generated for the mutant p.Gly643Arg using the crystal structure of human GAA as template and the 4-methylumbelliferyl-α-D-glucopyranoside as substrate. The results showed that the arginine at position 643 caused electrostatic changes in neighboring regions, leading to the repulsion between the amino acids located in the catalytic cavity of the GAA enzyme, thus restricting access to its substrate. These structural defects could cause the impairment of the transport and maturation previously reported for p.Gly643Arg mutation.

Topics: alpha-Glucosidases; Catalytic Domain; Female; Glucosides; Glycogen Storage Disease Type II; Humans; Hymecromone; Infant; Male; Molecular Docking Simulation; Mutation; Protein Binding

Epidermal β-glucocerebrosidase (GBA1), an acid β-glucosidase normally located in lysosomes, converts (glucosyl)ceramides into ceramides, which is crucial to generate an optimal barrier function of the outermost skin layer, the stratum corneum (SC). Here we report on two developed in situ methods to localize active GBA in human epidermis:

Topics: Benzoxazines; Boron Compounds; Cyclohexanols; Enzyme Assays; Epoxy Compounds; Fluorescent Dyes; Gene Expression; Glucosides; Glucosylceramidase; Humans; Hymecromone; Skin; Staining and Labeling; Tissue Culture Techniques

Thermostable enzymes derived from Thermotoga maritima have attracted worldwide interest for their potential industrial applications. Structural analysis and docking studies were preformed on T. maritima β-glucosidase enzyme with cellobiose and pNP-linked substrates. The 3D structure of the thermostable β-glucosidase was downloaded from the Protein Data Bank database. Substrates were downloaded from the PubCehm database and were minimized using MOE software. Docking of BglA and substrates was carried out using MOE software. After analyzing docked enzyme/substrate complexes, it was found that Glu residues were mainly involved in the reaction, and other important residues such as Asn, Ser, Tyr, Trp, and His were involved in hydrogen bonding with pNP-linked substrates. By determining the substrate recognition pattern, a more suitable β-glucosidase enzyme could be developed, enhancing its industrial potential.

Topics: Amino Acid Sequence; Benzyl Alcohols; beta-Glucosidase; Cellobiose; Glucosides; Hydrogen Bonding; Hymecromone; Kinetics; Molecular Conformation; Molecular Docking Simulation; Nitrophenylgalactosides; Software; Substrate Specificity; Thermotoga maritima

Three tetrahydropyridoimidazole-type glycosidase inhibitors have been synthesized with the 3-deoxy ribo- and arabino-, and 3-deoxy-3-fluoro gluco-configurations and two of them screened for activity against α- and β-gluco- and mannosidase enzymes. Only one substance, the 3-deoxy-3-fluoro-derivative of the gluco-configured tetrahydropyridoimidazole was found to have any activity against a single enzyme, sweet almond β-glucosidase, and even then at a level 100-fold lower than that of the corresponding simple gluco-configured tetrahydropyridoimidazole thereby underlining the importance of the 3-hydroxy group in the key substrate-enzyme interactions.

Topics: Enzyme Assays; Enzyme Inhibitors; Glucosidases; Glucosides; Hymecromone; Imidazoles; Plant Proteins; Saccharomyces cerevisiae Proteins; Substrate Specificity

Pompe disease is caused by a deficiency in acid α-glucosidase (GAA) and results in progressive, debilitating, and often life-threatening symptoms. Newborn screening has led to the early diagnosis of Pompe disease, but the best algorithm for screening has not yet been established.. GAA and neutral α-glucosidase (NAG) activities in dried blood spots (DBSs) were assayed using 4-methylumbelliferyl-β-d-glucopyranoside as the substrate. We also measure α-galactosidase A (GLA) activity in DBSs for comparison. A total of 473,738 newborns were screened for Pompe disease, and the data were analyzed retrospectively to determine the best screening algorithm.. The fluorescence assay used in the screening possessed good reproducibility, but the NAG/GAA ratio was superior in separating the true-positive from the false-positive cases. An NAG/GAA cutoff ratio≥60 produces a positive predictive value (PPV) of 63.4%, and in our sample, only two cases of later-onset Pompe disease would have been missed. The GLA/GAA ratio is not as effective as the NAG/GAA ratio.. A suitable control enzyme can improve the performance of newborn screening. Newborn screening for Pompe disease can be performed using the NAG/GAA ratio as a cutoff even in the presence of GAA partial deficiency.

Topics: Algorithms; alpha-Galactosidase; alpha-Glucosidases; Base Sequence; Glucosides; Glycogen Storage Disease Type II; Humans; Hymecromone; Infant, Newborn; Molecular Sequence Data; Neonatal Screening; Reproducibility of Results; Taiwan

A high yield of beta-glucosidase (EC 3.2.1.21) of 159.1 U/g-solid activity on 4-nitrophenyl beta-d-glucopyranoside (pNPG) was achieved by rice bran-based solid-state fermentation (SSF) of the recently characterized fungus Penicillium citrinum YS40-5. The enzyme was both thermophilic and acidophilic at the optimized temperature and pH of 70 degrees C and 5.0, respectively. Over 95% of the original beta-glucosidase activity was maintained after a prolonged storage at ambient temperature for 4 weeks. The kinetic parameters V(max), K(m) and K(I) were 85.93 U/mg, 1.2 mM and 17.59 mM with pNPG, and 72.49 U/mg, 32.17 mM and 8.29 mM with cellobiose, respectively. The protein band with beta-glucosidase activity was characterized by native PAGE followed by MUG-zymogram analysis, and its identity confirmed by nanoLC-MS/MS. A 3.43-fold synergistic effect by combining this beta-glucosidase with Trichoderma reesei cellulases was observed, indicating this enzyme could potentially be used for improving the efficiency of cellulosic bioconversion.

Topics: Amino Acid Sequence; beta-Glucosidase; Biotechnology; Carbon; Cellulose; DNA, Ribosomal; Enzyme Stability; Fermentation; Glucosides; Hydrogen-Ion Concentration; Hymecromone; Ions; Kinetics; Mass Spectrometry; Metals; Molecular Sequence Data; Nitrogen; Oryza; Penicillium; Phylogeny; Temperature; Trichoderma

Three beta-glucosidases (At1g66270-BGLU21, At1g66280-BGLU22, and At3g09260-BGLU23) were purified from the roots of Arabidopsis and their cDNAs were expressed in insect cells. In addition, two beta-glucosidase binding protein cDNAs (At3g16420; PBPI and At3g16430; PBPII) were expressed in Escherichia coli and their protein products purified. These binding proteins interact with beta-glucosidases and activate them. BGLU21, 22 and 23 hydrolyzed the natural substrate scopolin specifically and also hydrolyzed to some extent substrates whose aglycone moiety is similar to scopolin (e.g. esculin and 4-MU-glucoside). In contrast, they hydrolyzed poorly DIMBOA-glucoside and did not hydrolyze pNP- and oNP-glucosides. We determined the physicochemical properties of native and recombinant BGLUs, and found no differences between them. They were stable in a narrow pH range (5-7.5) and had temperature and pH optima for activity at 35 degrees C and pH 5.5, respectively. As for thermostability, >95% of their activity was retained at 40 degrees C but dramatically decreased at >50 degrees C. The apparent K(m) of native and recombinant enzymes for scopolin was 0.73 and 0.81 mM, respectively, and it was 5.8 and 9.7 mM, respectively, for esculin. Western blot analysis showed that all three enzymes were exclusively expressed in roots of seedlings but not in any other plant part or organ under normal conditions. Furthermore, spatial expression patterns of all eight genes belonging to subfamily 3 were investigated at the transcription level by RT-PCR.

Topics: Amino Acid Sequence; Arabidopsis; Arabidopsis Proteins; Carrier Proteins; Cellulases; Coumarins; Enzyme Activation; Esculin; Gene Expression Regulation, Enzymologic; Gene Expression Regulation, Plant; Glucosides; Hydrogen-Ion Concentration; Hydrolysis; Hymecromone; Molecular Sequence Data; Plant Roots; Protein Binding; Protein Stability; Temperature

It has been recently reported that tea flavanols, including epigallocatechin gallate (EGCG), efficiently inhibit glucosidase II in liver microsomes. Since glucosidase II plays a central role in glycoprotein processing and quality control in the endoplasmic reticulum we investigated the possible contribution of endoplasmic reticulum stress and unfolded protein response (UPR) to the pro-apoptotic activity of EGCG in mouse hepatoma cells. The enzyme activity measurements using 4-methylumbelliferyl-alpha-d-glucopyranoside substrate confirmed the inhibition of glucosidase II in intact and alamethicin-permeabilized cells. EGCG treatment caused a progressive elevation of apoptotic activity as assessed by annexin staining. The induction of CHOP/GADD153, the cleavage of procaspase-12 and the increasing phosphorylation of eIF2alpha were revealed in these cells by Western blot analysis while the induction of endoplasmic reticulum chaperones and foldases was not observed. Time- and concentration-dependent depletion of the endoplasmic reticulum calcium stores was also demonstrated in the EGCG-treated cells by single-cell fluorescent detection. The massive alterations in the endoplasmic reticulum morphology revealed by fluorescent microscopy further supported the development of UPR. Collectively, our results indicate that EGCG interferes with protein processing in the endoplasmic reticulum presumably due to inhibition of glucosidase II and that the stress induces an incomplete unfolded protein response with dominantly pro-apoptotic components.

Topics: alpha-Glucosidases; Animals; Antineoplastic Agents; Apoptosis; Calcium; Catechin; Dose-Response Relationship, Drug; Endoplasmic Reticulum; Eukaryotic Initiation Factor-2; Fluorescent Antibody Technique; Glucosides; Glycoside Hydrolase Inhibitors; Hymecromone; Liver Neoplasms, Experimental; Mice; Phosphorylation; Protein Folding; Stress, Physiological; Time Factors; Transcription Factor CHOP

Sulfated sialic acid (SiaS) is a unique sialic acid (Sia) derivative in which an additional anionic group is attached to a carboxylated monosaccharide. Very little is known about the occurrence and biologic function of SiaS, due to the limitations of analytical methods to detect it in minute amounts. In this study, to develop methods and probes for detecting and pursuing the functions of SiaS, we developed sensitive chemical and immunochemical detection methods. First, we synthesized as model compounds 4-methylumbelliferyl glycosides of 8-O- and 9-O-sulfated Sia consisting of N-acetylneuraminic acid (Neu5Ac), N-glycolylneuraminic acid (Neu5Gc), and deaminoneuraminic acid (Kdn). Second, we applied fluorometric high performance liquid chromatography (HPLC) analysis to these synthetic glycosides. After acid hydrolysis of the samples, the liberated SiaS were labeled with a fluorescent reagent, 1,2-diamino-4,5-methylenedioxybenzene, and analyzed on fluorometric HPLC. We established an optimal elution condition for successful separation of 8-O- and 9-O-sulfated Neu5Ac, Neu5Gc, and Kdn on HPLC. Third, we generated a monoclonal antibody (mAb) 2C4 against SiaS using sea urchin egg components as the immunogen. mAb.2C4 recognizes both 8-O-sulfated Neu5Ac (Neu5Ac8S) and Neu5Gc8S, whereas the previously prepared mAb.3G9 only recognizes Neu5Ac8S. Finally, using the fluorometric HPLC and monoclonal antibodies, we demonstrated that glycoconjugates from sea urchin sperm exclusively contained Neu5Ac8S, whereas those from eggs contained Neu5Gc8S. Furthermore, we clarified the quantitative differences in the SiaS content in eggs and sperm from two different species of sea urchins. Immunostaining using mAb.2C4 showed that Neu5Gc8S is localized in the cortical granules in unfertilized eggs, whereas it is localized in the outer surface of the fertilization layer as well as in the inner surface of fertilized eggs. Thus, 8-O-sulfation is dependent on the species, gametic cell-type, site-localization of the eggs, and glycoconjugates.

Topics: Acids; Animals; Antibodies, Monoclonal; Antibody Specificity; Blotting, Western; Chromatography, High Pressure Liquid; Enzyme-Linked Immunosorbent Assay; Epitopes; Female; Fluorometry; Glucosides; Glycoconjugates; Hemicentrotus; Hydrogen-Ion Concentration; Hydrolysis; Hymecromone; Male; Ovum; Sensitivity and Specificity; Sialic Acids; Spermatozoa; Strongylocentrotus purpuratus

Abracris flavolineata midgut contains a processive exo-beta-glucanase (ALAM) with lytic activity against Saccharomyces cerevisiae, which was purified (yield, 18%; enrichment, 37 fold; specific activity, 1.89 U/mg). ALAM hydrolyses fungal cells or callose from the diet. ALAM (45 kDa; pI 5.5; pH optimum 6) major products with 0.6 mM laminarin as substrate are beta-glucose (61%) and laminaribiose (39%). Kinetic data obtained with laminaridextrins and methylumbelliferyl glucoside suggest that ALAM has an active site with at least six subsites. The best fitting of kinetic data to theoretical curves is obtained using a model where one laminarin molecule binds first to a high-affinity accessory site, causing active site exposure, followed by the transference of the substrate to the active site. The two-binding-site model is supported by results from chemical modifications of amino acid residues and by ALAM action in MUbetaGlu plus laminarin. Low laminarin concentrations increase the modification of His, Tyr and Asp or Glu residues and MUbetaGlu hydrolysis, whereas high concentrations abolish modification and inhibit MUbetaGlu hydrolysis. Our data indicate that processivity results from consecutive transferences of substrate between accessory and active site and that substrate inhibition arises when both sites are occupied by substrate molecules abolishing processivity.

Topics: Animals; Binding Sites; Ethyldimethylaminopropyl Carbodiimide; Glucan 1,4-beta-Glucosidase; Glucans; Glucosides; Grasshoppers; Hydrogen-Ion Concentration; Hydroxymercuribenzoates; Hymecromone; Kinetics; Male; Models, Chemical; Polysaccharides; Saccharomyces cerevisiae

Two brothers with the childhood variant of type II glycogenosis (GSD-IIb) treated with nutrition and exercise therapy (NET) from a young age showed an unusually benign course. Muscle biopsy from the older brother, which showed characteristic vacuolar glycogen accumulation at age 2, had reverted to normal by age 16. A muscle biopsy from the younger brother was normal at 5 years. It is uncertain whether this anomalous evolution was spontaneous (nature) or due to the symptomatic therapy (nurture), but NET should be considered in patients with GSD-IIb until enzyme replacement or gene therapy become generally available.

Topics: DNA; Exercise Therapy; Glucan 1,4-alpha-Glucosidase; Glucosides; Glycogen; Glycogen Storage Disease Type IIb; Humans; Hymecromone; Infant; Male; Muscle, Skeletal; Nutritional Support; Treatment Outcome

The beta-glucosidase activity is involved in the hydrolysis of several important compounds for the development of varietal wine flavour. The aim of the present study was to investigate the production of beta-glucosidase in a number of wine-related yeast strains and to measure and identify this activity over the course of grape juice fermentation.. beta-glucosidase activity was measured as the amount of 4-methylumbelliferone released from 4-methylumbelliferyl-beta-d-glucopyranoside substrate. Intact cells of some grape and wine-spoilage yeasts showed beta-glucosidase activity much higher than those observed in wine yeasts "sensu stricto". During fermentation, three Saccharomyces cerevisiae strains, one Hanseniaspora valbyensis strain and one Brettanomyces anomalus strain showed beta-glucosidase activity both intra- and extracellularly.. In the studied strains, beta-glucosidase activity was at its maximum when the cells were in the active growth phase. However, a lowering of medium pH to values around 3 during fermentation led to total loss of activity.. During the course of this study, a new, rapid and reproducible method to assay beta-glucosidase activity was developed. The fact that Saccharomyces and non-Saccharomyces yeast strains are able to express beta-glucosidase activity during the alcoholic fermentation sheds new light on the contribution of these yeasts in the aroma expression of wines.

Topics: beta-Glucosidase; Colony Count, Microbial; Culture Media; Ethanol; Fermentation; Fluorometry; Food Microbiology; Glucosides; Hydrogen-Ion Concentration; Hymecromone; Saccharomyces cerevisiae; Time Factors; Vitis; Wine; Yeasts

To determine the mechanism of the hydrolysis of 4-methylumbelliferyl-beta-D-glucopyranoside (beta-MUG) by germinating and outgrowing spores of Bacillus species.. Spores of B. atrophaeus (formerly B. subtilis var. niger, Fritze and Pukall 2001) are used as biological indicators of the efficacy of ethylene oxide sterilization by measurement of beta-MUG hydrolysis during spore germination and outgrowth. It was previously shown that beta-MUG is hydrolysed to 4-methylumbelliferone (MU) during the germination and outgrowth of B. atrophaeus spores (Chandrapati and Woodson 2003), and this was also the case with spores of B. subtilis 168. Germination of spores of either B. atrophaeus or B. subtilis with chloramphenicol reduced beta-MUG hydrolysis by almost 99%, indicating that proteins needed for rapid beta-MUG hydrolysis are synthesized during spore outgrowth. However, the residual beta-MUG hydrolysis during spore germination with chloramphenicol indicated that dormant spores contain low levels of proteins needed for beta-MUG uptake and hydrolysis. With B. subtilis 168 spores that lacked several general proteins of the phosphotransferase system (PTS) for sugar uptake, beta-MUG hydrolysis during spore germination and outgrowth was decreased >99.9%. This indicated that beta-MUG is taken up by the PTS, resulting in the intracellular accumulation of the phosphorylated form of beta-MUG, beta-MUG-6-phosphate (beta-MUG-P). This was further demonstrated by the lack of detectable glucosidase activity on beta-MUG in dormant, germinated and outgrowing spore extracts, while phosphoglucosidase active on beta-MUG-P was readily detected. Dormant B. subtilis 168 spores had low levels of at least four phosphoglucosidases active on beta-MUG-P: BglA, BglH, BglC (originally called YckE) and BglD (originally called YdhP). These enzymes were also detected in spores germinating and outgrowing with beta-MUG, but levels of BglH were the highest, as this enzyme's synthesis was induced ca 100-fold during spore outgrowth in the presence of beta-MUG. Deletion of the genes coding for BglA, BglH, BglC and BglD reduced beta-MUG hydrolysis by germinating and outgrowing spores of B. subtilis 168 at least 99.7%. Assay of glucosidases active on beta-MUG or beta-MUG-P in extracts of dormant and outgrowing spores of B. atrophaeus revealed no enzyme active on beta-MUG and one enzyme that comprised > or =90% of the phosphoglucosidase active on beta-MUG-P. Partial purification and amino-terminal sequence analysis of this phosphoglucosidase identified this enzyme as BglH.. Generation of MU from beta-MUG by germinating and outgrowing spores of B. atrophaeus and B. subtilis is mediated by the PTS-driven uptake and phosphorylation of beta-MUG, followed by phosphoglucosidase action on the intracellular beta-MUG-P. The major phosphoglucosidase catalyzing MU generation from beta-MUG-P in spores of both species is probably BglH.. This work provides new insight into the mechanism of uptake and hydrolysis of beta-MUG by germinating and outgrowing spores of Bacillus species, in particular B. atrophaeus. The research reported here provides a biological basis for a Rapid Readout Biological Indicator that is used to monitor the efficacy of ethylene oxide sterilization.

Topics: Amino Acid Sequence; Bacillus subtilis; Electrophoresis, Polyacrylamide Gel; Glucosides; Hydrolysis; Hymecromone; Molecular Sequence Data; Spores, Bacterial

Pyrococcus furiosus laminarinase (LamA, PF0076) is an endo-glycosidase that hydrolyzes beta-1,3-glucooligosaccharides, but not beta-1,4-gluco-oligosaccharides. We studied the specificity of LamA towards small saccharides by using 4-methylumbelliferyl beta-glucosides with different linkages. Besides endo-activity, wild-type LamA has some exo-activity, and catalyzes the hydrolysis of mixed-linked oligosaccharides (Glcbeta4Glcbeta3Glcbeta-MU (Glc = glucosyl, MU = 4-methylumbelliferyl)) with both beta-1,4 and beta-1,3 specificities. The LamA mutant E170A had severely reduced hydrolytic activity, which is consistent with Glu170 being the catalytic nucleophile. The E170A mutant was active as a glycosynthase, catalyzing the condensation of alpha-laminaribiosyl fluoride to different acceptors. The best condensation yields were found at pH 6.5 and 50 degrees C, but did not exceed 30%. Depending on the acceptor, the synthase generated either a beta-1,3 or a beta-1,4 linkage.

Topics: Amino Acid Substitution; Catalytic Domain; Enzyme Stability; Glucan Endo-1,3-beta-D-Glucosidase; Glucosides; Glycosylation; Hydrogen-Ion Concentration; Hydrolases; Hymecromone; Mutation, Missense; Oligosaccharides; Pyrococcus furiosus; Substrate Specificity; Temperature

Two alpha-glucosidase encoding genes (aglA and aglB) from Bifidobacterium adolescentis DSM 20083 were isolated and characterized. Both alpha-glucosidases belong to family 13 of the glycosyl hydrolases. Recombinant AglA (EC 3.2.1.10) and AglB (EC 3.2.1.20), expressed in Escherichia coli, showed high hydrolytic activity towards isomaltose and pnp-alpha-glucoside. The K(m) for pnp-alpha-glucoside was 1.05 and 0.47 mM and the V(max) was 228 and 113 U mg(-1) for AglA and AglB, respectively. Using pnp-alpha-glucoside as substrate, the pH optimum for AglA was 6.6 and the temperature optimum was 37 degrees C. For AglB, values of pH 6.8 and 47 degrees C were found. AglA also showed high hydrolytic activity towards isomaltotriose and, to a lesser extent, towards trehalose. AglB has a high preference for maltose and less activity towards sucrose; minor activity was observed towards melizitose, low molecular weight dextrin, maltitol, and maltotriose. The recombinant alpha-glucosidases were tested for their transglucosylation activity. AglA was able to synthesize oligosaccharides from trehalose and sucrose. AglB formed oligosaccharides from sucrose, maltose, and melizitose.

Topics: alpha-Amylases; alpha-Glucosidases; Amino Acid Sequence; Bifidobacterium; Cloning, Molecular; Glucosides; Glycosylation; Hymecromone; Molecular Sequence Data; Recombinant Proteins; Sequence Alignment; Substrate Specificity

An 85-kDa beta-glucosidase/xylosidase (BGX1) was purified from the axenically grown phytopathogenic oomycete, Phytophthora infestans. The bgx1 gene encodes a predicted 61-kDa protein product which, upon removal of a 21 amino acid leader peptide, accumulates in the apoplastic space. Extensive N-mannosylation accounts for part of the observed molecular mass difference. BGX1 belongs to family 30 of the glycoside hydrolases and is the first such oomycete enzyme deposited in public databases. The bgx1 gene was found in various Phytophthora species, but is apparently absent in species of the related genus, Pythium. Despite significant sequence similarity to human and murine lysosomal glucosylceramidases, BGX1 demonstrated neither glucocerebroside nor galactocerebroside-hydrolyzing activity. The native enzyme exhibited glucohydrolytic activity towards 4-methylumbelliferyl (4-MU) beta-D-glucopyranoside and, to lesser extent, towards 4-MU-D-xylopyranoside, but not towards 4-MU-beta-D-glucopyranoside. BGX1 did not hydrolyze carboxymethyl cellulose, cellotetraose, chitosan or xylan, suggesting high substrate specificity and/or specific cofactor requirements for enzymatic activity.

Topics: Amino Acid Sequence; Animals; Bacterial Proteins; Base Sequence; beta-Glucosidase; DNA, Fungal; Glucosides; Glucosylceramidase; Glycoside Hydrolases; Humans; Hymecromone; Mice; Molecular Sequence Data; Oomycetes; Species Specificity; Substrate Specificity; Xylosidases

The pore of the translocon complex in the endoplasmic reticulum (ER) is large enough to be permeated by small molecules, but it is generally believed that permeation is prevented by a barrier at the luminal end of the pore. We tested the hypothesis that 4-methylumbelliferyl alpha-d-glucopyranoside (4MalphaG), a small, neutral dye molecule, cannot permeate an empty translocon pore by measuring its activation by an ER resident alpha-glucosidase, which is dependent on entry into the ER. The basal entry of dye into the ER of broken Chinese hamster ovary-S cells was remarkably high, and it was increased by the addition of puromycin, which purges translocon pores of nascent polypeptides, creating additional empty pores. The basal and puromycin-dependent entries of 4MalphaG were mediated by a common, salt-sensitive pathway that was partially blocked by spermine. A similar activation of 4MalphaG was observed in nystatin-perforated cells, indicating that the entry of 4MalphaG into the ER did not result simply from the loss of cytosolic factors in broken cells. We reject the hypothesis and conclude that a small, neutral molecule can permeate the empty pore of a translocon complex, and we propose that translationally inactive, ribosome-bound translocons could provide a pathway for small molecules to cross the ER membrane.

Topics: Animals; Biological Transport; CHO Cells; Cricetinae; Detergents; Endoplasmic Reticulum; Glucosides; Hymecromone; Osmolar Concentration; Salts; Spermine

Activities of the extracellular enzymes beta-glucosidase and phosphatase and bacterial densities were investigated during the filtration process at several sites in a groundwater recharge plant at the Ruhr river (Hengsen recharge plant in Schwerte. Germany). Low numbers of microorganisms and low levels of activity in this type of habitat, compared to most surface waters, caused methodological problems when determining microbial activity. In this study, fluorigenic model substrates, which enable hydrolytic rates as low as 1 nmol (L x h)(-1) to be measured, were used to determine extracellular enzyme activities. Highest activities were determined in surface water (107 nmol (L x h)(-1) for beta-glucosidase and 252 nmol (L x h)(-1) for phosphatase). which decreased during the filtration process in the gravel prefilter and the main sand filter until the end of subsurface flow (1.6 nmol (L x h)(-1) and 6.8 nmol (L x h)(-1), respectively). Similarly, bacterial numbers decreased from 3.4 x 10(6) to 0.29 x 10(6) cells mL(-1). These data showed that microbial activity within the prefilter and the shallow layers of the sand filter had the greatest impact on water quality. In addition to its involvement in the continuous purification of surface water, the microbial community in the sand filter probably acts as a biological buffer against ephemeral increases in the loads of organic matter and nutrients in the recharge plant.

Topics: beta-Glucosidase; Colony Count, Microbial; Extracellular Space; Filtration; Fresh Water; Germany; Glucosides; Hymecromone; Models, Biological; Phosphoric Monoester Hydrolases; Silicon Dioxide; Water Supply

Topics: alpha-Glucosidases; Animals; Disaccharides; Entamoeba histolytica; Glucosides; Hydrogen-Ion Concentration; Hydrolysis; Hymecromone; Substrate Specificity

The determination of the high-resolution structure of the Thermomonospora fusca endocellulase E2 catalytic domain makes it ideal for exploring cellulase structure-function relationships. Here we present binding parameters (Kd, DeltaH degrees, and DeltaS degrees) describing the interaction of E2 with 4-methylumbelliferyl glycosides, determined by titrating the quenching of ligand fluorescence in equilibrium binding experiments. Quenched MU(Glc)2/E2 complexes were used as indicators in displacement titrations to measure the binding of natural glycosides and also of a nonhydrolyzable cellotetraose analogue. Binding of MU(Glc)2 and cellotriose were also determined by titration calorimetry. The results show that E2 binds glycosides exclusively in its active-site cleft, with high affinity and specificity. The observed patterns of ligand hydrolysis and the results with MU(Glc)2 as a substrate indicated that ligands bound to E2 with their nonreducing ends in position -2, consistent with the position of cellobiose in the E2cd structure. Polymerase chain reaction (PCR) mutagenesis of the conserved residue Tyr 73 (in E2 binding subsite -1) to Phe and Ser produced enzymes with lower activity but higher binding affinities, indicating that the volume of the subsite -1 binding pocket is crucial for enzyme function. Similarly, MUXylGlc (with its xylosyl unit located in position -1) bound with 100-fold higher affinity than MU(Glc)2. These results are similar to those for the related Trichoderma reesei exocellulase CBH II. The binding data were compared with that previously reported for CBH II and interpreted in terms of the functional differences between endo- and exocellulases.

Topics: Actinomycetales; Bacterial Proteins; Binding Sites; Calorimetry; Cellobiose; Cellulase; Glucose; Glucosides; Glycosides; Hydrogen-Ion Concentration; Hymecromone; Kinetics; Ligands; Mutagenesis, Site-Directed; Phenylalanine; Serine; Spectrometry, Fluorescence; Thermodynamics; Titrimetry; Tyrosine

The carbohydrate binding site of Bacillus licheniformis 1,3-1,4-beta-D-glucan 4-glucanohydrolase was probed with a series of synthetic 4-methylumbelliferyl beta-D-glucan oligosaccharides (1a-e). The title enzyme is a retaining endo-glycosidase that has an extended carbohydrate binding site composed of four glucopyranosyl binding subunits on the non-reducing end from the scissile glycosidic bond, plus two or three subsites on the reducing end. Subsites -II to -IV have a stabilizing effect on the enzyme-substrate transition state complex in the rate-determining step leading to a glycosyl-enzyme intermediate, with subsite -III having a larger effect (-3.5 kcal mol-1). Since KM values decrease from the mono- to the tetrasaccharide, part of the effect is due to ground stabilization of the Michaelis complex. On the other hand, the chromophoric trisaccharide 1c and the homologous nonchromogenic tetrasaccharide 2b, which locates a glucopyranosyl unit in subsite +I, have almost identical KM values, the difference in reactivity being a consequence of an 18-fold increase of kcat for 2b. Therefore, interactions between subsite +I and the substrate appear to be mainly used to lower the energy of the transition state in the glycosylation step, rather than in the stabilization of the Michaelis complex. Finally, the pH dependence of the kinetic parameters for the hydrolysis of 1c, and the pH-dependent enzyme inactivation by a water-soluble carbodiimide (EAC) suggest two essential groups with pKa values of 5.5 and 7.0 in the free enzyme. The latter value is shifted up to 1.5 pH units upon binding of substrate in the non-covalent enzyme-substrate complex.

Topics: Bacillus; beta-Glucans; Binding Sites; Glucans; Glucosides; Glycoside Hydrolases; Hydrogen-Ion Concentration; Hydrolysis; Hymecromone; Kinetics; Oligosaccharides; Stereoisomerism; Substrate Specificity

The degradation of chitin involves a diverse array of enzymes, some with overlapping substrate specificities. In order to distinguish between different types of enzymes, specific substrates are needed. Toward this end, two new fluorogenic substrates containing thio-glycosidic linkages, 4-methylumbelliferyl N,N'-diacetyl-4-thio-beta-chitobioside (Mu-TCB) and N,N',N"-triacetyl-4,4'-dithio-beta-chitotrioside (Mu-TCT) are described. The substitution of the glycosidic oxygens (except the one that links oligosaccharide with the fluorogenic aglycon) with a sulfur atom resulted in resistance of these compounds to N-acetyl-beta-hexosaminidases while they were specific substrates for the newly discovered chitodextrinase from Vibrio furnissii (Keyhani,N.O. and Roseman,S. (1996) J. Biol. Chem., 271, 33414-33424) and some bacterial chitinases. The enzyme kinetics of these 4-S-linked substrates, Mu-TCB and Mu-TCT, as well as the O-linked 4-methylumbelliferyl N,N'-diacetyl-beta-chitobioside (Mu-CB) and N,N',N"-triacetyl-beta-chitotrioside (Mu-CT) with the chitodextrinase were studied and compared. The usefulness of the substrates for screening for chitodextrinase and/or chitinase activity was demonstrated.

Topics: Carbohydrate Sequence; Chitinases; Fluorescent Dyes; Glucosides; Hymecromone; Kinetics; Molecular Sequence Data; Oligosaccharides; Substrate Specificity; Vibrio

Fluorogenic 6-acylamino-4-methylumbelliferyl-beta-D-glucosides were found to be poor substrates for the three known human beta-glucosidases, i.e., lysosomal and non-lysosomal glucocerebrosidases and cytosolic broad-specificity beta-glucosidase. However, homogenates of human tissues and human cell types showed significant enzymatic hydrolysis of 6-ethanoylamino-4-methylumbelliferyl-beta-D-glucoside (EMGlc) due to the activity of a hitherto undescribed beta-glucosidase, called here EMGlc-ase. It was shown that the isozyme is hardly active towards 4-methylumbelliferyl-beta-D-glucoside or glucosylceramide. EMGlc-ase exhibits maximal activity at pH 4.5 and 5.0 in the absence and presence of sodium taurocholate respectively. It is a soluble lysosomal enzyme with a discrete isoelectric point of about 5.0. EMGlc-ase is not inhibited by conduritol B-epoxide, is activated by sodium taurocholate and binds strongly to Concanavalin A. This enzyme is not deficient in relation to Gaucher disease.

Topics: Animals; beta-Glucosidase; Chromatography, Agarose; Chromatography, Gel; Fibroblasts; Fluorescence; Gaucher Disease; Glucosides; Glucosylceramidase; Humans; Hydrogen-Ion Concentration; Hydrolysis; Hymecromone; Isoelectric Point; Kidney; Liver; Lysosomes; Rats; Spleen; Subcellular Fractions; Substrate Specificity

Topics: beta-Glucans; Carbohydrate Sequence; Glucans; Glucosides; Glycoside Hydrolases; Hymecromone; Magnetic Resonance Spectroscopy; Molecular Sequence Data; Molecular Structure; Oligosaccharides

Glycogenosis type II (GSD II, Pompe disease) is an autosomal recessive lysosomal storage disease that results from a deficiency of acid alpha-glucosidase (GAA). Patients with this disorder are unable to break down lysosomal glycogen, which consequently accumulates in the lysosome. To evaluate enzyme replacement therapy for GSD II patients, we have expressed human GAA cDNA in Chinese hamster ovary-K1 cells utilising a vector that places the cDNA under the transcriptional control of the human polypeptide chain elongation factor 1 alpha gene promoter. A clonal cell line that secreted precursor recombinant GAA at approximately 18 mg.l-1.day-1 was identified. The precursor recombinant GAA was purified to homogeneity, had a molecular mass of 110 kDa as measured by SDS/PAGE, and was shown to have pH optima and kinetic parameters similar to those of GAA purified from human tissues. The partial N-terminal amino acid sequence of recombinant GAA conformed to that derived from the nucleotide sequence of the cloned cDNA. The recombinant enzyme was taken up by cultured fibroblasts and skeletal muscle cells from GSD II patients, and was shown to correct the storage phenotype. Endocytosed GAA was localised to the lysosome and showed evidence of intracellular processing to a more mature form. Activity levels increased up to twice the normal value and uptake was prevented if cells were cultured in the presence of mannose 6-phosphate.

Topics: alpha-Glucosidases; Amino Acid Sequence; Animals; Cells, Cultured; CHO Cells; Cricetinae; Endocytosis; Enzyme Precursors; Glucan 1,4-alpha-Glucosidase; Glucosides; Glycogen; Glycogen Storage Disease Type II; Humans; Hydrogen-Ion Concentration; Hymecromone; Lysosomes; Mannosephosphates; Molecular Sequence Data; Muscle, Skeletal; Peptide Elongation Factor 1; Peptide Elongation Factors; Protein Processing, Post-Translational; Recombinant Proteins; Sequence Analysis

Kinetic of hydrolysis, by lysosomal glycosidases, of their synthetic substrates were studied in systems of the Aerosol OT (AOT) reversed micelles in octane. Catalytic activity of all the tested enzymes, viz., GM1-galactosidase, beta-hexosaminidases A and B, neuraminidase, and galactocerebrosidase, in reversed micelles proved to be the same as or higher than in the water buffer. In the reversed micelles an effective inhibition of the enzymatic reactions by the resulting carbohydrates was however observed. The dependence of the enzymes' activity on the hydration degree was represented by curves with one or several maxima, corresponding to various oligomeric forms of the enzymes. Dependencies of effective Km on the hydration degree in reversed micelles are similar dependencies of the enzyme activities on the same parameter, which can be explained by corresponding changes of local substrate concentration near the enzyme active site. Dependencies of the enzymatic activity on the surfactant's concentration in the reversed micellar system were also studied. Catalytic activity of the soluble lysosomal glycosidases was found to be unaffected by the micelles concentration. Activity of the membrane lysosomal glycosidase, galactocerebrosidase, strongly increased when the surfactant's concentration decreased. Under optimal conditions the activity of galactocerebrosidase in reversed micelles was 10-fold as compared with its activity in the water buffer.

Topics: Catalysis; Dioctyl Sulfosuccinic Acid; Glucosides; Glycoside Hydrolases; Humans; Hydrolysis; Hymecromone; Kinetics; Micelles; Surface-Active Agents; Water

The intercellular lipids of the stratum corneum, which are highly enriched in ceramides, are critical for the mammalian epidermal permeability barrier. During the terminal stages of epidermal differentiation, the glucosylceramide content is dramatically reduced, while the content of free ceramides increases. To investigate whether beta-glucocerebrosidase (beta-GlcCer'ase) could be responsible for this change in lipid content, we characterized its activity in murine epidermis, compared enzyme activity to other murine tissues, and localized beta-GlcCer'ase activity within the epidermis. Epidermal extracts demonstrated linear 4-methylumbelliferyl-beta-D-glucose hydrolysis (to 3 h) with protein concentrations between 1 and 250 micrograms/ml. Whole epidermis contained comparable beta-glucosidase activity (9.1 +/- 0.4 nmol/min per mg DNA) to murine brain and liver, and 5-fold higher activity than spleen. Epidermal beta-glucosidase activity was stimulated greater than 15-fold by sodium taurocholate at pH 5.6, and inhibited at acidic pH (3.5-4.0). Bromoconduritol B epoxide (greater than or equal to 1.0 microM), inhibited epidermal enzyme activity by greater than 75%, while activity in brain, liver, and spleen was only inhibited by 6, 17, and 14%, respectively. Moreover, beta-GlcCer'ase mRNA expression in murine epidermis exceeded levels in liver, brain, and spleen. Finally, beta-GlcCer'ase activity was highest in the outer, more differentiated epidermal cell layers including the stratum corneum. In summary, mammalian epidermis contains an usually high percentage (approximately 75%) of beta-glucocerebrosidase activity, and the concentration of activity in the more differentiated cell layers may account for the replacement of glucosylceramide by ceramides in the outer epidermis.

Topics: Animals; beta-Glucosidase; Cell Differentiation; Cyclohexenes; Dose-Response Relationship, Drug; Epidermis; Glucosides; Glucosylceramidase; Hymecromone; Inositol; Male; Mice; Mice, Hairless; RNA, Messenger; Taurocholic Acid; Tissue Distribution

Research has shown that various percentages of fecal Escherichia coli isolates obtained from healthy subjects may be beta-glucuronidase negative. The ability to detect beta-glucuronidase activity among fecal E. coli isolates from healthy subjects may be affected by assay conditions. A study was conducted in which agar and broth media containing 4-methylumbelliferyl-beta-D-glucuronide (MUG) were used to examine beta-glucuronidase activities of fecal isolates from healthy swine. Rectal swabs were plated on MacConkey agar plus 100 mg of MUG per liter (MAC-MUG) and incubated at 35 degrees C for 24 h. Each of 986 isolates picked from MAC-MUG was inoculated into duplicate tubes of lauryl tryptose broth plus 50 mg of MUG per liter (LT-MUG). One set of tubes was incubated at 35 degrees C and the other set of tubes was incubated at 44.5 degrees C. Gas production and hydrolysis of MUG, indicated by fluorescence when observed with UV light with a wavelength of 360 nm, were determined after incubation for 24 and 48 h. A higher percentage (P less than 0.01) of isolates was MUG positive at 44.5 degrees C than at 35 degrees C after 24 h of incubation in LT-MUG. A higher percentage (P less than 0.01) of isolates was MUG positive after 48 h than after 24 h of incubation at both 35 and 44.5 degrees C. A lower percentage of isolates (P less than 0.05) was observed to be MUG positive on MAC-MUG agar compared with their MUG reactions in LT-MUG at 35 and 44.5 degrees C. Approximately 89% of the isolates identified were beta-glucuronidase-positive E. coli. The largest proportion of MUG-positive E. coli was detected with LT-MUG at 35 degrees C after 48 h of incubation.

Topics: Animals; Bacteriological Techniques; Escherichia coli; Evaluation Studies as Topic; Feces; Female; Fermentation; Glucosides; Glucuronidase; Hymecromone; Lactose; Male; Swine

Studies were conducted to investigate the mechanism by which acidic phospholipid-containing vesicles stimulate purified placental glucosylceramidase activity towards the water-soluble substrate 4-methylumbelliferyl-beta-D-glucopyranoside (MUGlc). Vesicles composed of pure phosphatidic acid (PA) or pure phosphatidylserine (PS) stimulated the activity of the enzyme about 20-fold. The inclusion of cholesterol and phosphatidylcholine (PC), beside PA, into the vesicles slightly improved their stimulatory effect. Further addition of oleic acid (OA) markedly increased the stimulation (50-fold). By ultracentrifugation and gel permeation procedures it was shown that, under optimal conditions for stimulation of the MUGlc hydrolysis by acidic phospholipid-containing vesicles, purified glucosylceramidase spontaneously binds to their surface. Interestingly, the molar fraction of the acidic phospholipid into the mixed vesicles, rather than its concentration in the assay, is the crucial parameter for activation and binding of the enzyme. The importance of glucosylceramidase association with appropriate vesicles for enzyme activation was indicated by observing that the presence of 0.2 M citrate-phosphate buffer (pH 5.5), that prevented the binding to PA-containing surfaces, also inhibited the enzyme activity. Our results indicate that the reconstitution of glucosylceramidase activity occurs through the spontaneous tight association of the enzymatic protein with preformed acidic phospholipid-containing vesicles.

Topics: Centrifugation, Density Gradient; Chromatography; Glucosides; Glucosylceramidase; Hydrogen-Ion Concentration; Hymecromone; Liposomes; Phospholipids

Using 4-methylumbelliferyl (MUF) beta-D-cellobioside as a substrate, the ability of cellobiohydrolase I from Trichoderma longibrachiatum to catalyze transglycosylation has been demonstrated. At substrate concentrations greater than 2 mM, the formation of MUF-tetrasaccharide was detected using HPLC. In the course of enzymatic reaction, a concentration of the transglycosylation product passed through a maximum, since at later stages of the reaction the product was further hydrolyzed. At MUF-beta-D-cellobioside concentrations of 2-10 mM, the maximum weight content of MUF-tetrasaccharide amounted to 1-4% of the total content of saccharides. In the reaction system, containing 2.5 mM MUF-beta-D-cellobioside and 10 mM MUF-beta-D-glucoside, MUF-trisaccharide was formed as the main transglycosylation product. In hydrolysis of natural substrates (cellulose and cellotriose) in the presence of MUF-beta-D-glucoside a formation of MUF-trisaccharide was also observed.

Topics: Cellobiose; Cellulose; Cellulose 1,4-beta-Cellobiosidase; Chromatography, High Pressure Liquid; Glucosides; Glycoside Hydrolases; Glycosylation; Hymecromone; Models, Chemical; Trichoderma; Trisaccharides

A 30-min fluorogenic test was developed for differentiation of members of the Candida parapsilosis group from other Candida species commonly encountered in clinical material. The fluorogenic substrate, 4-methylumbelliferyl-beta-D-glucoside, was utilized to assay beta-glucosidase activity. A total of 50 C. parapsilosis isolates and 135 isolates of four other Candida species were tested. Assay sensitivity and specificity were 100 and 99.3%, respectively. The procedure was adapted for use with a spectrofluorometer.

Topics: beta-Glucosidase; Candida; Glucosidases; Glucosides; Glycosides; Hymecromone; Predictive Value of Tests; Spectrometry, Fluorescence; Umbelliferones

Several studies have documented the association of blood and rectal-culture positivity for Streptococcus bovis with gastrointestinal neoplasia, especially colonic carcinoma. Conventional methods using bile-esculin hydrolysis, salt tolerance, and sugar fermentations to differentiate S. bovis from other streptococci are laborious, slow, and relatively expensive. Commercially available systems are costly and require at least 24 to 48 h of incubation. A rapid identification procedure for S. bovis and related bacteria was developed. The method uses a reagent containing two hydrolyzable substrates, p-nitrophenyl-alpha-D-galactopyranoside and 4-methylumbilliferyl-beta-D-glucoside, in the presence of 2.5% sodium deoxycholate. This combination test, performed with a rapid assay for L-pyrrolidonyl-aminopeptidase, could distinguish S. bovis, Streptococcus equinus, Enterococcus spp., Streptococcus pneumoniae, and the viridans group streptococci in culture within 30 min. Twelve species of the genera Streptococcus and Enterococcus were tested. The rapid method correlated well with conventional techniques. The reagents are readily available, inexpensive, and easy to make and can be stored in the refrigerator for at least 6 months.

Topics: Costs and Cost Analysis; Glucosides; Humans; Hydrolysis; Hymecromone; Nitrophenylgalactosides; Predictive Value of Tests; Pyroglutamyl-Peptidase I; Streptococcus; Time Factors

C16H17O8.1.5H2O, Mr = 364.33, monoclinic, C2, a = 14.314 (3), b = 6.851 (1), c = 18.178 (5) A, beta = 100.90 (2) degrees, V = 1750.46 A3, Z = 4, D chi = 1.382 Mg m-3, lambda(Mo K alpha) = 0.71069 A, mu = 0.933 mm(-1), F(000) = 768, T = 294 K, R = 0.078 for all 2160 reflections. The structure is characterized by the close stacking along the b axis of the planar 4-methylumbelliferyl ring system which is nearly perpendicular to b and the extensive hydrogen bonding scheme in which all hydroxyl groups are within 2.95 A of at least two other O atoms.

Topics: Computer Simulation; Glucosides; Glycosides; Hydrogen Bonding; Hymecromone; Models, Molecular; Molecular Conformation; Umbelliferones

Gaucher disease is an autosomal recessive sphingolipidosis associated with deficient glucocerebroside beta-glucosidase activity. It is a panethnic metabolic disorder, but the carrier frequency is particularly high among Ashkenazi Jews (estimated between 1:12-1:25). In order to establish a reliable and convenient biochemical assay method for differentiating asymptomatic Gaucher carriers from normal individuals, glucocerebroside beta-glucosidase activity was determined in peripheral blood lymphocytes and cultured skin fibroblasts of 11 Gaucher obligate heterozygotes using the authentic nonlabeled sphingolipid substrate N-palmitoyl dihydroglucocerebroside and the artificial fluorogenic substrate 4-methylumbelliferyl-beta-D-glucopyranoside (4MUGP). The level of lymphocyte beta-glucosidase activity on the glucocerebroside substrate was observed to range from 42-65% of that of the control mean, and there was no overlap of enzyme activity between the Gaucher heterozygotes and controls. However, when the artificial fluorogenic substrate 4MUGP was used, the level of beta-glucosidase activity in 2 of the Gaucher obligate heterozygotes was noted to overlap with that of the control individuals. Contrary to findings in the lymphocytes, cultured skin fibroblasts appear to be a reliable enzyme source for Gaucher carrier detection even when the artificial fluorogenic 4MUGP substrate was used, as the level of beta-glucosidase activity in all of the Gaucher obligate heterozygotes tested was intermediate and distinctly separated from that of the control persons. Using the lymphocyte glucocerebroside beta-glucosidase assay and fibroblast 4MUGP beta-glucosidase assay methods, we identified the carrier status in 3 other relatives and ruled it out in 4 others. These data suggest that nonlabeled glucocerebroside is a reliable and highly specific substrate for either lymphocyte or fibroblast beta-glucosidase activity assay in identifying asymptomatic Gaucher carriers. Use of the 4MUGP substrate for differentiating Gaucher heterozygotes from control persons, on the other hand, should be restricted to the fibroblast enzyme assay method, as considerable overlap of enzyme activity was noted in lymphocytes.

Topics: Adult; Cerebrosides; Clinical Enzyme Tests; Female; Fibroblasts; Gaucher Disease; Genetic Carrier Screening; Glucosides; Glucosylceramidase; Glucosylceramides; Glycosides; Humans; Hymecromone; Lymphocytes; Male; Pedigree; Quebec; Umbelliferones

Glucocerebrosidase is present in considerable amounts in human urine. The enzyme is stable in concentrated urine for several days when stored at 0 degrees C. Like tissue glucocerebrosidase, the urinary enzyme is inhibited by conduritol B-epoxide and hydrolyses not only glucocerebroside but also the synthetic substrate 4-methyl-umbelliferyl-beta-D-glucoside. The enzyme is deficient in urine from patients with Gaucher disease (type 1). It is possible to discriminate completely between patients with type 1 Gaucher disease and control subjects by measuring the ratio glucocerebrosidase/beta-hexosaminidase in urine. The value of this ratio (mean +/- SE) with the synthetic substrates 4-methylumbelliferyl-beta-glucoside and p-nitrophenyl-beta-N-acetylglucosaminide, respectively, was 34.2 +/- 3.7 (n = 24) in the controls and 2.1 +/- 0.9 (n = 21) in the patients.

Topics: Adolescent; Adult; beta-N-Acetylhexosaminidases; Child; Female; Gaucher Disease; Glucosidases; Glucosides; Glucosylceramidase; Hexosaminidases; Humans; Hymecromone; Inositol; Male; Middle Aged; Substrate Specificity

We have isolated from guinea-pig liver a broad-specificity beta-glucosidase of unknown function that utilizes as its substrate non-physiological aryl glycosides (e.g. 4-methylumbelliferyl beta-D-glucopyranoside, p-nitrophenyl beta-D-glucopyranoside). The present paper documents that this enzyme can be inhibited by various naturally occurring glycosides, including L-picein, dhurrin and glucocheirolin. In addition, L-picein, which acts as a competitive inhibitor of the broad-specificity beta-glucosidase (Ki 0.65 mM), is also a substrate for this enzyme (Km 0.63 mM; Vmax. 277,000 units/mg). Heat-denaturation, kinetic competition studies, chromatographic properties and pH optima all argue strongly that the broad-specificity beta-glucosidase is responsible for the hydrolysis of both the non-physiological aryl glycosides and L-picein. This paper demonstrates that beta-glucosidase can catalyse the hydrolysis of a natural glycoside, and may provide a key to understanding the function of this enigmatic enzyme. A possible role in the metabolism of xenobiotic compounds is discussed.

Topics: Animals; beta-Glucosidase; Glucosidases; Glucosides; Glycosides; Guinea Pigs; Hydrolysis; Hymecromone; Kinetics; Liver; Nitriles; Protein Denaturation; Substrate Specificity

We have defined one type of acid alpha-glucosidase and two types of neutral alpha-glucosidases from quail skeletal muscle on the basis of differences in the elution patterns on a DEAE-cellulose column. The appearance of the two neutral alpha-glucosidase isoenzymes was age-dependent. A decrease in acid alpha-glucosidase activity was demonstrated in Japanese quails with glycogenosis type II. The characteristics of these three alpha-glucosidase isoenzymes are described.

Topics: Age Factors; alpha-Glucosidases; Animals; Chromatography, Affinity; Chromatography, DEAE-Cellulose; Coturnix; Glucosidases; Glucosides; Glycogen Storage Disease Type II; Glycoside Hydrolase Inhibitors; Hydrogen-Ion Concentration; Hymecromone; Molecular Weight; Muscles; Substrate Specificity

We report on a 2-yr-old boy with progressive muscular weakness and respiratory failure. There was no clinical evidence of heart muscle involvement. Autopsy showed marked intralysosomal glycogen deposition in skeletal muscle and liver with no histological evidence of glycogen deposition in cardiac muscle. The activity of the lysosomal enzyme alpha-1,4-glucosidase was deficient in skin fibroblasts, skeletal muscle, cardiac muscle, and liver; however, the enzymatic activity in peripheral blood leukocytes was in the low normal range. The child's mother had normal enzymatic activity in leukocytes but heterozygote levels in skin fibroblasts.

Topics: alpha-Glucosidases; Child, Preschool; Fibroblasts; Genes, Recessive; Genetic Variation; Glucosides; Glycogen Storage Disease; Glycogen Storage Disease Type II; Humans; Hymecromone; Leukocytes; Male; Muscles; Muscular Diseases; Respiratory Insufficiency

'Acid' beta-glucosidase of human spleen, from either normal controls or patients with type 1 (adult) Gaucher disease, was incorporated into phosphatidylcholine liposomes. The non-incorporated (soluble) Gaucher-enzyme had a higher apparent molecular weight than had the corresponding control. Liposomal 'acid' beta-glucosidase prepared from Gaucher-spleen was more thermostable than was the corresponding normal enzyme; it was also stimulated by acidic lipids to a much lesser extent. The results suggest that the genetic mutation in type 1 (adult) Gaucher disease has multiple effects on the glycoprotein form of 'acid' beta-glucosidase.

Topics: beta-Glucosidase; Cell Membrane; Concanavalin A; Gaucher Disease; Glucosidases; Glucosides; Hot Temperature; Humans; Hydrogen-Ion Concentration; Hymecromone; Kinetics; Liposomes; Molecular Weight; Phosphatidylcholines; Solubility; Spleen

We show that sialosylgangliotetraosylceramide (GM1) is a potent activator of delipidated (sodium cholate- and 1-butanol-extracted) lysosomal rat liver glucocerebroside:beta-glucosidase. Stimulation of 4-methylumbelliferyl-beta-D-glucopyranoside hydrolysis by the beta-glucosidase was markedly dependent upon the concentration of GM1 in the assay medium. Estimations of critical micellar concentration (CMC) performed fluorometrically using the dye N-phenylnaphthylamine revealed two CMC values of GM1 above 18 degrees C; the CMC of the primary micelles (3.32 microM) was temperature-independent whereas that of the secondary micelles decreased with decreasing temperature (17.2 and 10.8 microM at 37 and 20 degrees C, respectively). In the temperature range of 18-39 degrees C, beta-glucosidase activity increased sharply when the GM1 concentration was above the CMC of the secondary micelles. Although a heat-stable factor, purified from the spleen of a patient with Gaucher's disease, had a profound effect on the activation of beta-glucosidase by GM1, it decreased the CMC only slightly (14.8 versus 17.2 microM at 37 degrees C). The heat-stable factor (8 micrograms/ml) changed the shape of the activation curve from sigmoidal to hyperbolic, suggesting that the heat-stable factor permits beta-glucosidase to be activated by primary micelles or monomers. The results of gel filtration chromatography and sucrose gradient centrifugation in H2O and D2O revealed that the activation of beta-glucosidase by GM1 was associated with an increase in the size of the enzyme from 45,800 to 178,500 daltons and an increase in the partial specific volume from 0.697 to 0.740 ml/g. The active, reconstituted beta-glucosidase appears to consist of 50% protein and 50% ganglioside (56 molecules/178,500 g). Concentrations of GM1 below the CMC of secondary micelles increased the rate of inactivation of the enzyme by the irreversible inhibitor conduritol B epoxide at 37 degrees C, indicating that GM1 monomers or primary micelles do interact with the enzyme, even though they do not increase the rate of hydrolysis of 4-methylumbelliferyl-beta-D-glucopyranoside by the enzyme.

Topics: Animals; beta-Glucosidase; Chemical Phenomena; Chemistry, Physical; Dose-Response Relationship, Drug; Enzyme Activation; G(M1) Ganglioside; Gangliosides; Gaucher Disease; Glucosidases; Glucosides; Hot Temperature; Humans; Hymecromone; Inositol; Kinetics; Liver; Lysosomes; Male; Micelles; Molecular Weight; Rats; Rats, Inbred Strains; Temperature

Studies were undertaken to characterize the beta-glucosidase activity in freshly homogenized liver from Sprague-Dawley rats. About 95% of the total beta-glucosidase activity was associated with the particulate fraction, whereas only about 3-7% was found in the cytosol. Storage of fresh liver at room temperature for several hours or repeated freezing and thawing of fresh rat liver prior to homogenization, solubilized 20-30% of the total hepatic beta-glucosidase activity. An additional 30% could be solubilized by extracting the particulate sediments with water or Triton X-100. The enzymatic activity in both the particulate and solubilized fractions optimally hydrolyzed 4-methylumbelliferyl-beta-D-glucoside as well as the glycolipid substrate, glucosylceramide, at an acidic pH. The rates of hydrolysis of either substrate by all subcellular fractions were stimulated by addition of sodium taurocholate or phosphatidylserine. The particulate, cytosolic and solubilized enzymes bound to concanavalin A, were inhibited by conduritol B epoxide and migrated more electronegatively on cellulose acetate than the cytosolic acid beta-glucosidase from human liver or spleen. These data indicated that the liver of Sprague-Dawley rats contained primarily the lysosomal acid beta-glucosidase ('glucocerebrosidase') and little, if any, 'nonspecific' beta-glucosidase. This, and the fact that about 60% of the rat hepatic beta-glucosidase could be solubilized by autolysis, freezing and rethawing or extraction with water, contrasts with the beta-glucosidases in human liver since about 80% of the total beta-glucosidase activity is cytosolic and does not hydrolyze glucosylceramide.(ABSTRACT TRUNCATED AT 250 WORDS)

Topics: Animals; beta-Glucosidase; Gaucher Disease; Glucosidases; Glucosides; Humans; Hymecromone; Liver; Lysosomes; Male; Rats; Rats, Inbred Strains; Solubility; Species Specificity; Subcellular Fractions

A procedure for the quantitation of the delivery of liposome contents into Catharanthus roseus protoplasts has been developed. The method is based on the uptake of liposome encapsulated methylumbelliferyl beta-D-glucoside and its enzymatic hydrolysis to yield fluorescent methylumbelliferone. Since the free glucoside is not taken up by the protoplasts to a significant extent, the delivery of material in the nanomole range can be measured with ease.

Topics: Biological Transport; Cell Membrane Permeability; Glucosidases; Glucosides; Hydrolysis; Hymecromone; Liposomes; Plants; Protoplasts; Spectrometry, Fluorescence; Substrate Specificity

Topics: alpha-Glucosidases; Animals; Glucosides; Glycosides; Guinea Pigs; Histocytochemistry; Hymecromone; Liposomes; Macrophages; Male; Microscopy, Fluorescence; Time Factors; Umbelliferones

This report describes a reliable and reproducible method for the identification of carriers of Type I Gaucher disease using blood platelets as the source of beta-glucosidase and 4-methylumbelliferyl-beta-D-glucoside as substrate. Platelet lysates have at least two identifiable beta-glucosidase activities with the synthetic substrate. One is maximally active at pH 5.0 in the absence of sodium taurocholate and the other at pH 5.6 in the presence of taurocholate. In platelets of Gaucher homozygotes and heterozygotes, the beta-glucosidase activity at pH 5.6 with the bile salt is reduced whereas the activity at pH 5.0 is the same in non-carriers, carriers and affected patients. In addition to differences in specific activity, the ratio of beta-hexosaminidase to beta-glucosidase activities is a useful parameter in the evaluation of the carrier state. Since carriers have normal activity of hexosaminidase and a reduced activity of beta-glucosidase, their mean activity ratio is about 70% higher than in non-carriers. Therefore we propose that the specific activity of beta-glucosidase at pH 5.6 in the presence of sodium taurocholate with the ratio of beta-hexosaminidase to beta-glucosidase serve as useful and reliable indices in the evaluation of the carrier state for Gaucher disease.

Topics: Adult; beta-Glucosidase; Blood Platelets; Gaucher Disease; Genetic Carrier Screening; Glucosides; Hexosaminidases; Homozygote; Humans; Hydrogen-Ion Concentration; Hymecromone; Kinetics; Leukocytes; Taurocholic Acid

The description in 1965 of glucocerebroside: beta-glucosidase as the enzymic defect in Gaucher's disease stimulated considerable research interest and effort toward establishing rapid, reliable, and inexpensive enzymic assays for diagnostic purposes and carrier detection. Here, we consider some of the methods currently in use in which the substrate is the synthetic glucoside, 4-methylumbelliferyl-beta-D-glucopyranoside, and leukocytes and fibroblasts are the sources of enzyme. We also consider the concepts of the "acid beta-glucosidase" and multiple forms of beta-glucosidase that have been proposed to explain the effectiveness of the fluorometric assays. Finally, we analyze the limitations of each method and discuss the difficulties involved in instituting heterozygote screening programs in the general population.

Topics: beta-Glucosidase; Clinical Enzyme Tests; Gaucher Disease; Glucosidases; Glucosides; Heterozygote; Humans; Hymecromone; Isoenzymes; Leukocytes

A procedure has been developed for the determination of glucocerebrosidase activity using the substrate analogue, 2-N-hexadecanoylamino-4-nitrophenyl-beta-D-glucopyranoside (HNGlu) with sodium taurocholate and oleic acid as activators. Cultured skin fibroblasts and amniotic fluid cells have been used as the enzyme source. It has been used successfully to confirm the diagnosis of two Type I and two Type II Gaucher patients. The procedure shows approximately a 15-fold increase in sensitivity over other procedures using HNGlu as substrate. Compared with 4-methylumbelliferyl-beta-D-glucoside, HNGlu proves to be a highly specific substrate for glucocerebrosidase with little or no hydrolysis by the beta-glucosidases present in fibroblast extracts. It is therefore the chromogenic substrate of choice for determining a glucocerebrosidase deficiency.

Topics: Cells, Cultured; Fibroblasts; Gaucher Disease; Glucosidases; Glucosides; Glucosylceramidase; Glycosides; Humans; Hymecromone; Methods; Skin; Substrate Specificity

The nature of the stimulatory action of the protein 'coglucosidase' on glucocerebrosidase was investigated with the use of highly purified cofactor from bovine spleen, radioactive glucosyl ceramide and methylumbelliferyl-beta-glucoside. A complex between glucosidase and either substrate could not be detected under equilibrium and non-equilibrium binding conditions. Complex formation between stimulating protein and the enzyme could be shown by the binding of the enzyme to an affinity column containing coglucosidase. This binding could be blocked by adding phosphatidylserine to the enzyme. The lipid also stimulated the enzyme. Additional evidence for binding of the enzyme to the two kinds of stimulators was the finding that they protected the enzyme against inactivation by N-ethylmaleimide and chloromercuriphenylsulfonate. A role for lipids in the stimulatory action of coglucosidase was shown by extracting lipids from the enzyme; this resulted in a loss of basal enzyme activity and of sensitivity to activation by the protein. Adding back to the lipids or phosphatidylserine increased the sensitivity of the delipidated enzyme to coglucosidase. Using the crude, unextracted enzyme we could show that low concentrations of phosphatidylserine augmented the effectiveness of coglucosidase but high concentrations of the lipid blocked the effect of the protein. It is proposed that lipids, particularly acidic ones, act on solubilized glucocerebrosidase to produce an enzyme conformation which allows binding and stimulation by coglucosidase. At higher lipid concentrations, the acidic lipids bind, in competition with coglucosidase, to the latter's binding site on the enzyme.

Topics: 4-Chloromercuribenzenesulfonate; Animals; Cattle; Enzyme Activation; Ethylmaleimide; Glucosidases; Glucosides; Glucosylceramidase; Glucosylceramides; Glycoproteins; Hymecromone; Kinetics; Phosphatidylserines; Protein Binding; Proteins; Saposins; Spleen

Topics: Adenocarcinoma; alpha-Glucosidases; Animals; Cell Line; Colonic Neoplasms; Disaccharides; Glucosidases; Glucosides; Glycogen; Humans; Hymecromone; Kinetics; Maltose; Mice; Mice, Nude

Both the common and a variant isozyme of acid alpha-glucosidase have been purified from a heterozygous placenta with CM-Sephadex, ammonium sulfate precipitation, dialysis, Amicon filtration, affinity chromatography by Sephadex G-100, and DEAE-cellulose chromatography. Three and two activity peaks, from the common and variant isozymes, respectively, were obtained by DEAE-cellulose chromatography using a linear NaCl gradient. The three peaks of activity of the common isozyme were eluted with 0.08, 0.12, and 0.17 M NaCl, whereas the two peaks of the variant, with 0.01 and 0.06 M NaCl. The pH optimum and thermal denaturation at 57 degrees C were the same in all enzyme peaks of both isozymes. Rabbit antiacid alpha-glucosidase antibodies produced against the common isozyme were found to cross-react with both peaks of the variant isozyme. The two isozymes shared antigenic identity and had similar Km's with maltose as substrate. Normal substrate saturation kinetics were observed with the common isozyme when glycogen was the substrate, but the variant produced an S-shaped saturation curve indicating a phase of negative and positive cooperativity at low and high glycogen concentrations, respectively. The activity of the variant was only 8.6% and 19.2% of the common isozyme when assayed with nonsaturating and saturating concentrations of glycogen, respectively. A similar rate of hydrolysis of isomaltose by both isozymes was found indicating that the reduced catalytic activity of the variant isozyme toward glycogen is not the result of a reduced ability of this enzyme to cleave the alpha-1,6 linkages of glycogen.

Topics: Chromatography; Electrophoresis; Female; Glucan 1,4-alpha-Glucosidase; Glucosidases; Glucosides; Glycogen; Humans; Hydrogen-Ion Concentration; Hymecromone; Immunologic Techniques; Isoenzymes; Isomaltose; Kinetics; Maltose; Placenta; Pregnancy; Temperature

Human leukocytes contain at least two isozymes of 4-methylumbelliferyl-beta-glucosidase acting optimally at pH 4.0 and 4.8; in Gaucher disease, only the former is deficient. Brief exposure of the leukocyte homogenate to pH 4.0 at room temperature results in irreversible inactivation of the pH 4.8 activity, while the activity at pH 4.0 remains unaffected. The more acidic isozyme is stimulated four- to fivefold by 0.2% sodium taurodeoxycholate (TDC) with a shift in the pH optimum to 5.0. The less acidic isozyme is completely suppressed in the presence of this detergent. Both leukocyte isozymes appear to be membrane-bound since gel filtration of Sephadex G-200 produces only one peak of activity located at the void volume, unlike in liver and kidney where a second peak also can be demonstrated. Heat inactivation analysis indicated that in controls, assayed in the absence of detergent, pH 4.0 activity is more thermostable than pH 4.8 activity. However, in Gaucher disease, the residual beta-glucosidase at pH 4.0 is just as thermolabile as the unaffected pH 4.8 activity. Heat inactivation of the enzyme in the presence of TDC resulted in rapid loss of activity, suggesting a direct effect of the bile salt on the configuration of the enzyme decreasing its thermal stability. In the absence of detergent, acid beta-glucosidase shows two K(m)'s, one at 3.2 mM and another at 0.9 mM. In the presence of detergent, only the higher K(m) at 3.3 mM is obtained. In patients with Gaucher disease and in obligate carriers, the K(m) remains essentially unaffected while the V(max) shows the expected deficiency.A reliable and reproducible selective assay technique has been developed for the diagnosis of Gaucher disease homozygotes and obligate heterozygotes and for the carrier screening of individuals at risk for this inherited disorder. The efficacy of this technique has been demonstrated by studying the activity in 42 controls, 26 patients, 32 obligate heterozygotes, and 23 healthy relatives of patients with Gaucher disease.

Topics: Adult; beta-Glucosidase; Child; Chromatography, Gel; Detergents; Female; Gaucher Disease; Genetic Carrier Screening; Glucosidases; Glucosides; Heterozygote; Homozygote; Humans; Hydrogen-Ion Concentration; Hymecromone; Isoenzymes; Kidney; Kinetics; Leukocytes; Liver; Male; Temperature

Leukocytes were isolated from 14 patients (7 males and 7 females ) with Gaucher disease of the Norrbottnian type (Type 3), 32 obligate heterozygotes (16 males and 16 females) for this disease and 20 controls (10 males and 10 females). After collection, the cells were transported in dry ice to the laboratory, where they were assayed. The assays were repeated after the cells had been stored for 12 months. beta-Glucosidase activity was assayed with D-[glucose-U-14C]glucosylceramide at pH 5.8 with Cutscum-Na-cholate as a detergent and 4-methylumbelliferyl-beta-glucoside at pH 4.1 with Triton-Na-taurocholate as a detergent. The activities of two marker enzymes, 4-methylumbelliferyl-beta-galactosidase and N-acetyl-beta-glucosaminidase, were assayed in aliquots of the same leukocyte samples. The activity of beta-galactosidase remained constant during storage, N-acetyl-beta-glucosaminidase increased, while beta-glucosidase decreased as assayed with the natural as well as with the artificial substrate. beta-Glucosidase activity was significantly lower in the female than in male controls and heterozygotes. When assayed with natural substrate beta-glucosidase activity in leukocytes from the male patients was 6--12% of the control mean value and 10--15% in those from the female patients. The corresponding figures found when the artificial substrate was used were 15--30% and 22--45%. The values for the heterozygotes were respectively 42--68% and 34--79% with the natural substrate, and 33--82% and 51--109% with the artificial substrate. No correlation was found between the age of the patient and the beta-glucosidase activity.

Topics: Acetylglucosamine; Acetylglucosaminidase; Adolescent; Adult; Age Factors; Aged; beta-Galactosidase; beta-Glucosidase; Child; Child, Preschool; Drug Stability; Female; Galactosides; Gaucher Disease; Glucosamine; Glucosidases; Glucosides; Glucosylceramidase; Heterozygote; Homozygote; Humans; Hymecromone; Leukocytes; Male; Middle Aged; Sex Factors

The rates of formation and dissociation of concanavalin A with some 4-methylumbelliferyl and p-nitrophenyl derivatives of alpha- and beta-D-mannopyranosides and glucopyranosides were measured by fluorescence and spectral stopped-flow methods. All processes examined were uniphasic. The second-order formation rate constants varied only from 6.8 x 10(4) to 12.8 x 10(4) M-1 x s-1, whereas the first-order dissociation rate constants ranged from 4.1 to 220 s-1, all at pH 5.0, I=0.3 M, and 25 degrees C. Dissociation rates thus controlled the value of the binding constant. The effect of temperature on these reactions was examined, from which enthalpies and entropies of activation and of reaction could be calculated. The effects of pH at 25 degrees C on the reaction rates of 4-methylumbelliferyl alpha-D-mannopyranoside and 4-methylumbelliferyl alpha-D-glucopyranoside with concanavalin A were examined. The value of the binding constant Kap (derived from the kinetics) at any pH could be related to the intrinsic binding constant K by the expression Kap = KaK(Ka + [H+])-1. The values of Ka, the ionization constant of the protein segment responsive to sugar binding, were 3 x 10(-4) M and 1 x 10(-4) M for 4-methylumbelliferyl alpha-D-mannopyranoside and 4-methylumbelliferyl alpha-D-glucopyranoside, respectively. The binding constant of p-nitrophenyl alpha-D-mannopyranoside is surprisingly much less sensitive to a pH change from 5.0 to 2.7. Ionic strength had little effect on the binding characteristics of 4-methylumbelliferyl alpha-D-mannopyranoside to concanavalin A at pH 5.2 and 25 degrees C.

Topics: Concanavalin A; Glucosides; Glycosides; Hydrogen-Ion Concentration; Hymecromone; Kinetics; Mannosides; Temperature; Thermodynamics

The 105 000g supernatant from human placental homogenates, prepared in the presence of sodium taurocholate and Cutscum, contained beta-glucosidase activity towards estrone glucoside as well as towards 4-methylumbelliferyl glucoside (4-MU-glucoside) and glucocerebroside. After partial purification, the estrone glucosidase was found to be active only after the addition of negatively charged phospholipid, whereas the other beta-glucosidases did not exhibit this requirement. The estrone glucosidase was separated from the 4-MU-glucosidase by chromatography on Sephadex G-200 with 0.1% sodium taurocholate in the eluting buffer. The estrone glucosidase was mainly contained in material with a pI of 4.7, while the 4-MU-glucosidase was distributed in fractions with pI values of 4.7 and 6.2 to 6.4. The partially purified estrone glucosidase had a pH optimum of 5.8, as distinct from that of 6.4 found for the 4-MU-glucosidase, and differed markedly from the 4-MU-glucosidase in its response to treatment with heat, sulfhydryl reagents, and detergents. Its sensitivity to changes in pH differed from those reported for glucocerebrosidase.

Topics: Detergents; Estrone; Female; Glucosidases; Glucosides; Hot Temperature; Humans; Hydrogen-Ion Concentration; Hymecromone; Phospholipids; Placenta; Pregnancy; Substrate Specificity; Sulfhydryl Reagents

The diagnosis of Pompe's disease by the assay of acid alpha-glucosidase in kidney and leucocytes was not previously possible because of the presence of another component which had activity at pH 4.0, but was not deficient in the disease. This problem was resolved either by the use of the inhibitors, turanose, maltose and citrate, or by isoelectric precipitation at pH 5.0, which enabled the estimation of acid alpha glucosidase in kidney and leucocytes.

Topics: alpha-Glucosidases; Glucosides; Glycogen Storage Disease; Glycogen Storage Disease Type II; Humans; Hymecromone; Kidney; Leukocytes; Umbelliferones

1. Cases of Gaucher's disease could not be distinguished from controls by the assay of beta-glucosidase activity in water homogenates of liver using 4-methylumbelliferyl-beta-D glucopyranoside. 2. Two peaks of beta-glucosidase activity were separated by Sephadex G-150 gel filtration in control and Gaucher livers. In the presence of the elution buffer pH profiles of peak I showed a deficiency at pH 3.5-4.5 in Gaucher's disease. Gaucher and control peak II had similar pH profiles with little or no activity at pH 3.0-4.0. 3. A clear distinction between homogenates of Gaucher and control liver was obtained by assay at pH 4.0 in the presence of elution buffer, or of sodium chloride, a component of the elution buffer.

Topics: Gaucher Disease; Glucosidases; Glucosides; Glucuronidase; Humans; Hydrogen-Ion Concentration; Hymecromone; Liver; Umbelliferones

The possible interference of neutral alpha-D-glucosidase in the diagnosis of Pompe's disease using 4-methylumbelliferyl-alpha-D-glucopyranoside as substrate for the assay of acid alpha-D-glucosidase was investigated. The pH profile of alpha-D-glucosidase in control skin fibroblasts and amniotic fluid cells showed two peaks of activity. The shape of the pH profile depended upon whether or not the extract was added to the buffer before the substrate. If extract was added to the buffer before the substrate, a greater separation was obtained between the two peaks of activity. The neutral alpha-D-glucosidase activity could be totally removed by preliminary precipitation at pH 5.0. Following acid region whilst Pompe's cells had no activity enabling a clear distinction to be made between carriers and the disease state.

Topics: Amniotic Fluid; Cells, Cultured; Drug Stability; Female; Fibroblasts; Glucosidases; Glucosides; Glycogen Storage Disease; Glycogen Storage Disease Type II; Humans; Hydrogen-Ion Concentration; Hymecromone; Kinetics; Pregnancy; Prenatal Diagnosis; Skin