sepharose and 2-diethylaminoethanol

Topics: Adsorption; Anions; Cations; Chromatography, Ion Exchange; Dextrans; Electrochemistry; Ethanolamines; Isoelectric Point; Peptides; Proteins; Sepharose

A lysosomal glycosidase, β-glucuronidase, has been purified to homogeneity, from the soluble extracts of a freshwater mussel, L. corrianus, by a series of chromatography techniques involving phenyl-Sepharose, ion exchange, affinity and gel filtration chromatography. In native PAGE, β-glucuronidase resolved into a single band and the molecular mass determined by gel filtration chromatography was found to be 250 kDa. Zymogram analysis with 4-methyl umbelliferyl β-glucuronide substrate validated the purified enzyme as β-glucuronidase. In SDS-PAGE, the purified enzyme was resolved into four sub-units with molecular weights around 90, 75, 65, and 50 kDa, respectively, and two of the subunits (90 and 50 kDa) cross-reacted with human β-glucuronidase antiserum. The optimum pH and temperature of the purified glycosidase were 5.0 and 70 °C, respectively. The enzyme kinetics parameters, substrate affinity (K

Topics: Animals; Bivalvia; Chromatography, Affinity; Chromatography, Gel; Circular Dichroism; Ethanolamines; Glucuronidase; Humans; Hydrogen-Ion Concentration; Ions; Kinetics; Lysosomes; Metals; Molecular Dynamics Simulation; Molecular Weight; Sepharose; Spectrophotometry, Ultraviolet; Temperature

The purpose of this study was to study polysaccharides isolated from Polygonatum sibiricum to establish the structure-activity relationships of the active substances and to discover the optimal fraction for further development and application. Four polysaccharides fractions (PSP1, PSP2, PSP3 and PSP4) from P. sibiricum were obtained by DEAE-Sepharose Fast Flow ion-exchange chromatography. Acid hydrolysis and FT-IR spectral and NMR spectral analyses were employed for structural analysis. Our results illustrated that PSP with different chemical structure and monosaccharide composition showed different abilities to activate phagocytic activity in vitro. According to the preliminary screening results in vitro, the newly identified water-soluble polysaccharides of PSP3 were selected for further evaluation in vivo. The results demonstrated that PSP3 possessed an immunomodulatory function and could be regarded as a promising candidate as an immunomodulator.

Topics: Ethanolamines; Immunologic Factors; Magnetic Resonance Spectroscopy; Polygonatum; Polysaccharides; Sepharose; Spectroscopy, Fourier Transform Infrared

Helicobacter pylori neutrophil-activating protein (HP-NAP) is involved in H. pylori-associated gastric inflammation. HP-NAP is also a vaccine candidate, a possible drug target, and a potential diagnostic marker for H. pylori-associated diseases. Previously, we purified recombinant HP-NAP by one-step diethylaminoethyl (DEAE) negative mode chromatography by collecting the unbound fraction at pH 8.0 at 4°C. It remains unclear why HP-NAP does not bind to DEAE resins at the pH above its isoelectric point during the purification. To investigate how pH affects the surface net charge of HP-NAP and its binding to DEAE resins during the purification, recombinant HP-NAP expressed in Escherichia coli was subjected to DEAE negative mode chromatography at pH ranging from 7.0 to 9.0 at 25°C and the surface charge of purified HP-NAP was determined by capillary electrophoresis. A minimal amount of HP-NAP was detected in the elution fraction of DEAE Sepharose resin at pH 8.5, whereas recombinant HP-NAP was detected in the elution fraction of DEAE Sephadex resin only at pH 7.0 and 8.0. The purified recombinant HP-NAP obtained from the unbound fractions was not able to bind to DEAE resins at pH 7.0 to 9.0. In addition, the surface charge of the purified HP-NAP was neutral at pH 7.0 to 8.0 and was either neutral or slightly negative at pH 8.5 and 9.0. However, recombinant HP-NAP purified from gel-filtration chromatography was able to bind to DEAE Sepharose resin at pH 7.0 to 9.0 and DEAE Sephadex resin at pH 7.0. At pH 8.5 and 9.0, only the negatively charged species of HP-NAP were found. Thus, recombinant HP-NAP with different charge status can be differentially purified by DEAE negative mode chromatography and gel-filtration chromatography. Furthermore, the charge distribution on the surface of HP-NAP, the presence of impure proteins, and the overall net charge of the resins all affect the binding of HP-NAP to DEAE resins during the negative purification.

Topics: Bacterial Proteins; Chromatography, Gel; Chromatography, Ion Exchange; DEAE-Dextran; Electrochemistry; Electrophoresis, Capillary; Ethanolamines; Helicobacter Infections; Helicobacter pylori; Humans; Hydrogen-Ion Concentration; Ion Exchange Resins; Neutrophils; Recombinant Proteins; Sepharose

This study aims to characterise biochemically urease from an atypical Campylobacter lari, namely urease-positive thermophilic Campylobacter (UPTC). Urease was purified from cells of a Japanese UPTC isolate (CF89-12) using phenyl-Sepharose chromatography. Two protein components (estimates molecular masses 24 kDa and 61 kDa) were obtained that appeared to be structural proteins of urease (subunits A and B), and these were fractionated by sodium dodecyl sulphate-polyacrylamide gel electrophoresis (PAGE). The native molecular weight for the final purified UPTC urease was estimated to be approximately 186,000 Da which is close to the calculated molecular weight (182,738 Da) based on all six open reading frames of UPTC CF89-12 urease genes (ureA, B, E, F, G and H), as described previously. Moreover, an active band was observed on phenol red staining after a nondenaturing native PAGE of the crude extract from the UPTC cells. In addition, the purified urease of UPTC CF8912 showed enzyme activity over a broad pH range (pH 6-10), with maximal activity at pH 8.0. The urease was also stable against heat treatment, with almost no loss of enzyme activity seen following 60-min incubation at temperatures of 20-60 degrees C. Urease subunits A and B were identified immunologically by Western blot analysis with rabbit anti-urease alpha (A) and beta (B) raised against Helicobacter pylori.

Topics: Blotting, Western; Campylobacter lari; Catalysis; Chromatography, Agarose; Dose-Response Relationship, Drug; Electrophoresis, Polyacrylamide Gel; Enzyme Inhibitors; Ethanolamines; Ethylmaleimide; Hydroxyurea; Sepharose; Temperature; Thiourea; Urease

Using agarose coated gigaporous polystyrene microspheres as a base support, a novel anion exchanger (DEAE-AP) has been developed after functionalization with diethylaminoethyl chloride. The gigaporous structure, static adsorption behavior, and chromatographic properties of DEAE-AP medium were characterized and compared with those of commercially available resin DEAE Sepharose Fast Flow (DEAE-FF). The results implied that there existed some through pores in DEAE-AP microspheres, which effectively reduced resistance to stagnant mobile phase mass transfer by inducing convective flow of mobile phase in the gigapores of medium. As a consequence, the column packed with DEAE-AP exhibited low column backpressure, high column efficiency, high dynamic binding capacity and high protein resolution at high flow velocity up to 2600cm/h. In conclusion, all the results suggested that the gigaporous absorbent is promising for high-speed protein chromatography.

Topics: Chromatography, High Pressure Liquid; Chromatography, Ion Exchange; Ethanolamines; Microspheres; Polystyrenes; Proteins; Sepharose; Technology, Pharmaceutical

This study reports the purification and characterization of a novel raw starch digesting alpha-amylase from a newly isolated Bacillus sp. YX-1. Maximum alpha-amylase activity (53 U mL(-1)) was obtained at 45 degrees C after 44 h of incubation. The enzyme was purified using ammonium sulfate precipitation, ion exchange and gel filtration chromatography, and showed a molecular weight of 56 kDa by SDS-PAGE. This enzyme exhibited maximum activity at pH 5.0, performed stability over a broad range of pH 4.5-11.0, and was optimally active at 40-50 degrees C. The enzyme preparation had a strong digesting ability towards various raw starches and efficiently hydrolyzed raw corn starch at a concentration of 20% and pH 5.0, which were normally used in the starch industries, in a period of 12h. By analyzing its partial amino acid sequences, the enzyme was proposed to be a novel alpha-amylase.

Topics: alpha-Amylases; Ammonium Sulfate; Bacillus; Biotechnology; Chromatography; Chromatography, Gel; Chromatography, Ion Exchange; Dextrans; DNA, Ribosomal; Ethanolamines; Hydrogen-Ion Concentration; Hydrolysis; Sepharose; Temperature; Time Factors

A convection interaction media (trade name CIM, BIA Separation, Ljubljana, Slovenia) isobutyl monolithic disc was prepared by incubating a CIM epoxy monolithic disc with isobutylamine, and it was then applied to the purification of secondary alcohol dehydrogenase (S-ADH) and primary alcohol oxidase (P-AOD). Both enzymes were adsorbed on this column and eluted with high purity. Thus, S-ADH was purified to an electrophoretically homogeneous state by four column chromatographies using CIM DEAE-8 and CIM C4-8 tube monolithic columns, blue-Sepharose column and CIM isobutyl disc monolithic column. P-AOD was also purified to an electrophoretically homogeneous state by three column chromatographies of CIM DEAE-8 tube, CIM C4-8 tube and CIM isobutyl disc columns.

Topics: Alcohol Dehydrogenase; Alcohol Oxidoreductases; Butylamines; Chromatography, Liquid; Electrophoresis, Polyacrylamide Gel; Ethanolamines; Reproducibility of Results; Sepharose

Arginine methylation is a post-translational modification found in many RNA-binding proteins. Heterogeneous nuclear ribonucleoprotein K (hnRNP K) from HeLa cells was shown, by mass spectrometry and Edman degradation, to contain asymmetric N(G),N(G)-dimethylarginine at five positions in its amino acid sequence (Arg256, Arg258, Arg268, Arg296, and Arg299). Whereas these five residues were quantitatively modified, Arg303 was asymmetrically dimethylated in <33% of hnRNP K and Arg287 was monomethylated in <10% of the protein. All other arginine residues were unmethylated. Protein-arginine methyltransferase 1 was identified as the only enzyme methylating hnRNP K in vitro and in vivo. An hnRNP K variant in which the five quantitatively modified arginine residues had been substituted was not methylated. Methylation of arginine residues by protein-arginine methyltransferase 1 did not influence the RNA-binding activity, the translation inhibitory function, or the cellular localization of hnRNP K but reduced the interaction of hnRNP K with the tyrosine kinase c-Src. This led to an inhibition of c-Src activation and hnRNP K phosphorylation. These findings support the role of arginine methylation in the regulation of protein-protein interactions.

Topics: Amino Acid Sequence; Arginine; CSK Tyrosine-Protein Kinase; DNA Methylation; Dose-Response Relationship, Drug; Embryo, Mammalian; Ethanolamines; HeLa Cells; Heterogeneous-Nuclear Ribonucleoprotein K; Humans; Mass Spectrometry; Methylation; Microscopy, Fluorescence; Molecular Sequence Data; Phosphorylation; Plasmids; Protein Binding; Protein Biosynthesis; Protein Interaction Mapping; Protein Processing, Post-Translational; Protein Structure, Tertiary; Protein-Arginine N-Methyltransferases; Protein-Tyrosine Kinases; Recombinant Proteins; Repressor Proteins; RNA; Sepharose; Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; src-Family Kinases; Stem Cells; Transcription, Genetic; Transfection

Plasma membranes of the yeast, Candida utilis, were solubilized with octyl-beta-D-glucopyranoside and a fraction enriched in the lactate carrier was obtained with DEAE-Sepharose anion-exchange chromatography, after elution with 0.4 M NaCl. The uptake of lactic acid into proteoliposomes, containing the purified protein fraction and cytochrome c oxidase, was dependent on a proton-motive force and the transport specificity was consistent with the one of C. utilis intact cells. Overall, we have obtained a plasma membrane fraction enriched in the lactate carrier of C. utilis in which the transport properties were preserved. Given the similarities between the lactate transport of C. utilis and the one of mammalian cells, this purified system could be further explored to screen for specific lactate inhibitors, with potential therapeutic applications.

Topics: Biochemistry; Biological Transport; Biotechnology; Candida; Cell Membrane; Chromatography; Chromatography, Ion Exchange; Dose-Response Relationship, Drug; Electron Transport Complex IV; Ethanolamines; Lactates; Lactic Acid; Monocarboxylic Acid Transporters; Protons; Sepharose

A thermostable superoxide dismutase (SOD) from a Thermomyces lanuginosus strain (P134) was purified to homogeneity by fractional ammonium sulfate precipitation, ion-exchange chromatography on DEAE-Sepharose, Phenyl-Sepharose hydrophobic interaction chromatography, and gel filtration on Sephacryl S-100. The molecular mass of a single band of the enzyme was estimated to be 22.4 kDa, using sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Using gel filtration on Sephacryl S-100, the molecular mass was estimated to be 89.1 kDa, indicating that this enzyme was composed of four identical subunits of 22.4 kDa each. The SOD was found to be inhibited by NaN3, but not by KCN or H2O2, suggesting that the SOD in T. lanuginosus was of the manganese superoxide dismutase type. The SOD exhibited maximal activity at pH 7.5. The optimum temperature for the activity was 55 degrees C. It was thermostable at 50 and 60 degrees C and retained 55% activity after 60 min at 70 degrees C. The half-life of the SOD at 80 degrees C was approximately 28 min and even retained 20% activity after 20 min at 90 degrees C.

Topics: Ammonium Sulfate; Ascomycota; Chromatography, Gel; Chromatography, Ion Exchange; Electrophoresis, Polyacrylamide Gel; Ethanolamines; Hot Temperature; Hydrogen Peroxide; Hydrogen-Ion Concentration; Kinetics; Manganese; Sepharose; Signal Transduction; Superoxide Dismutase; Temperature; Time Factors

The feasibility of applying expanded bed adsorption technology to recombinant protein recovery from extracts of transgenic canola (rapeseed) was assessed. The extraction step results in a suspension of high solids content that is difficult to clarify. The coarse portion of the solids can be removed easily, and our aim was to operate the expanded bed in the presence of the recalcitrant particulates. Recombinant beta-glucuronidase (rGUS) produced in transgenic canola seed was the model system. Diethylaminoethyl (DEAE) and Streamline DEAE resin exhibited similar binding and elution properties for both rGUS and native canola proteins. More than 95% of native canola proteins did not bind to DEAE resins at pH 7.5, whereas the bound proteins were fractionated by two-step salt elution into two groups with the first peak, containing 70% of total bound proteins, at 20 mS/cm, followed by elution of rGUS at 50 mS/cm. The adsorption isotherm was only slightly influenced by the presence of up to 14 mg solids/mL extract; C(m) and K(d) changed by -1% and +39%, respectively. Bed expansion was semiquantitatively predictable from physical properties of the fluid together with Stokes's law and the Richardson-Zaki correlation for both clarified and partially clarified extracts. The presence of 1.4% solids did not change rGUS breakthrough behavior of the expanded bed; however, a small difference between expanded bed and packed bed was observed early in the sample loading stage, during which bed expansion adjusts. Canola solids moved through the column in approximately plug flow with no detriment to bed stability. Seventy-two percent recovery of 34-fold purified rGUS was obtained after initial loading of 1.4% (w/w) solids extract to 25% breakthrough.

Topics: Adsorption; Brassica rapa; Chromatography, Ion Exchange; Ethanolamines; Feasibility Studies; Glucuronidase; Ion Exchange Resins; Plant Extracts; Plants, Genetically Modified; Recombinant Proteins; Seeds; Sepharose

Abundant flavin binding sites have been found in membranes of plants and fungi. With flavin mononucleotide-agarose affinity columns, riboflavin-binding activity from microsomes of Cucurbita pepoL. hypocotyls was purified and identified as a specific PIP1-homologous protein of the aquaporin family. Sequences such as gi|2149955 in Phaseolus vulgaris, PIP1b of Arabidopsis thaliana, and NtAQP1 of tobacco are closely related. The identification as a riboflavin-binding protein was confirmed by binding tests with an extract of Escherichia coli cells expressing the tobacco NtAQP1 as well as leaves of transgenic tobacco plants that overexpress NtAQP1 or were inhibited in PIP1 expression by antisense constructs. When binding was assayed in the presence of dithionite, the reduced flavin formed a relatively stable association with the protein. Upon dilution under oxidizing conditions, the adduct was resolved, and free flavin reappeared with a half time of about 30 min. Such an association can also be induced photochemically, with oxidized flavin by blue light at 450 nm, in the presence of an electron donor. Several criteria, localization in the plasma membrane, high abundance, affinity to roseoflavin, and photochemistry, argue for a role of the riboflavin-binding protein PIP1 as a photoreceptor.

Topics: Amino Acid Sequence; Aquaporins; Arabidopsis Proteins; Butanols; Cell Membrane; Chromatography, Affinity; Coloring Agents; Cucurbita; Dithionite; Ethanolamines; Flavin Mononucleotide; Membrane Proteins; Molecular Sequence Data; Photochemistry; Plant Proteins; Protein Binding; Riboflavin; Sepharose; Sequence Analysis, Protein; Solubility; Tritium; Trypsin

Viruses were characterized by their adsorption to DEAE-Sepharose or by their elution from octyl-Sepharose by using buffered solutions of sodium chloride with different ionic strengths. Viruses whose adsorption to DEAE-Sepharose was reduced most rapidly by an increase in the sodium chloride concentration were considered to have the weakest electrostatic interactions with the solids; these viruses included MS2, E1, and phiX174. Viruses whose adsorption to DEAE-Sepharose was reduced least rapidly were considered to have the strongest electrostatic interactions with the column; these viruses included P1, T4, T2, and E5. All of the viruses studied adsorbed to octyl-Sepharose in the presence of 4 M NaCl. Viruses that were eluted most rapidly following a decrease in the concentration of NaCl were considered to have the weakest hydrophobic interactions with the column; these viruses included phiX174, CB4, and E1. Viruses that were eluted least rapidly from the columns after the NaCl concentration was decreased were considered to have the strongest hydrophobic interactions with the column; these viruses included f2, MS2, and E5.

Topics: Adsorption; Animals; Ethanolamines; Osmolar Concentration; Sepharose; Sodium Chloride; Viruses

The alternative pathway of complement shares its terminal components (C3 and C5 through 9) with the classical pathway, but has several unique components, including factors D, B, and P (properdin). This unit presents methods for assaying total alternative pathway activity and the activity of factors B and D. Radial immunodiffusion (RID) can also be used to measure factor D, B, and P concentrations.

Topics: Chromatography, Liquid; Complement C3; Complement C5; Complement Hemolytic Activity Assay; Erythrocytes; Ethanolamines; Humans; Lysine; Plasma; Sensitivity and Specificity; Sepharose

Chick kainate binding protein was solubilized from cerebellar membranes and purified (x19) by use of two chromatographic steps. Measurements of [3H]kainate binding and GTPase activity in the different fractions reveal a consistent decrease of GTPase activity as the purification proceeds so that no GTPase is detectable after the final purification step. This fact, in the context of the differential involvement in nucleotide recognition of some critical amino acid residues in the p-loop motif of GTPases and in the guanine nucleotide-binding sequence of ionotropic glutamate receptors, together with significant discrepancies concerning the activity of individual nucleotides, suggests that both guanine nucleotide-recognizing sequences are unlikely to be alternative expressions of the same functional domain.

Topics: Animals; Binding, Competitive; Cerebellum; Chickens; Concanavalin A; Cytidine Monophosphate; Ethanolamines; GTP Phosphohydrolases; Receptors, Kainic Acid; Sepharose; Tritium

Bovine milk xanthine oxidase (XO) was isolated and purified from milk fat globule membrane (MFGM). The method included the following steps: solubilization of XO from MFGM in 200 mM dithiothreitol (DTT) at pH 8.0, fractionation of solubilized proteins with ammonium sulfate, chromatography on DEAE-Sepharose with gradient elution, and rechromatography of the XO fraction for final purification. The method is highly reproducible, is comparatively simple, and provides highly pure enzyme. Purified XO, analyzed by (8%) SDS-PAGE, had only one band of 140-150 kDa. XO showed a high specific activity of 2.5 units/mg of protein and an A280: A450 ratio of 4.8.

Topics: Animals; Cattle; Chemical Precipitation; Chromatography, Ion Exchange; Ethanolamines; Membranes; Milk; Sepharose; Solubility; Xanthine Oxidase

A previous lectin binding study demonstrated the presence of high molecular-mass mucin-like glycoproteins (HMGP) on the surface of hamster tracheal surface epithelial (HTSE) secretory cells (Proc. Natl. Acad. Sci. USA 1987;84:9304). In the present study, we intended to isolate and characterize these HMGP from the plasma membrane of the primary HTSE cells and then to determine whether or not these membrane HMGP are Muc-1 mucins, a type of mucins originally discovered on the surface of some carcinomas. A subcellular fraction enriched with the plasma membrane was obtained using a sucrose density gradient centrifugation. This fraction contained high molecular-mass glycoconjugates which were excluded from Sepharose CL-4B gel. Biochemical characterization of these glycoconjugates revealed the following characteristics: (1) susceptibility to both pronase and mild alkaline treatments, but totally resistant to proteoglycan-digesting enzymes; (2) partitioning in the detergent phase of Triton X-114 and resistance to digestion by phosphatidylinositol phospholipase C or D; (3) a buoyant density of 1.5 g/ml based on CsCl density gradient centrifugation; (4) polydispersity in terms of both size and charge density; and (5) lack of immunoreactivity with an anti-Muc-1 mucin antibody. We conclude that the plasma membrane of HTSE cells at confluence contains HMGP, which seem to be the integral membrane proteins but different from Muc-1 mucins, and that these membrane HMGP appear to share some similarities with secreted mucins in terms of size and charge.

Topics: Animals; Antibodies; Cell Fractionation; Cell Membrane; Centrifugation; Cesium; Chlorides; Chromatography; Cricetinae; Detergents; Electrophoresis, Polyacrylamide Gel; Epithelial Cells; Ethanolamines; Glycoproteins; Male; Mesocricetus; Molecular Weight; Mucins; N-Acetylneuraminic Acid; Octoxynol; Phosphatidylinositol Diacylglycerol-Lyase; Phospholipase D; Polyethylene Glycols; Precipitin Tests; Rabbits; Sepharose; Trachea; Tritium; Type C Phospholipases

A method for selective extraction of photosystem 2 (PS 2) pigment-protein complexes (PPC) from pea thylakoids and their purification from Sepharose DEAE 6B was developed. It was shown that extraction of thylakoids (3 mg chlorophyll/ml) with Triton X-100 (Triton X-100:chlorophyll = 15-20:1 w/w) in the presence of 1 M sucrose, 1 M NaCl, 50 mM MES in the solubilization medium (pH 5.0), tenfold dilution with 50 mM MES buffer (pH 5.0), and centrifugation at 16,000 g for 10 min provided selective extraction of PS 2 PPC. Spectral, electrophoretic and functional properties of the isolated PPC were determined. This method allowed us to isolate native pigment-protein oxygen-evolving complexes (core complexes) and D1-D2-cytochrome b559 complexes of the reaction centre (RC). The core complex immobilized on a Sepharose DEAE 6B column was treated with 2 mM hydroxylamine or 1 M CaCl2. In the former case, modification by hydroxylamine led to the removal of 4 atoms of Mn, but not of the 33-kDa protein; in the latter case, modification by CaCl2 led to the removal of the 33 kDa protein. The modified core complexes were unstable upon native electrophoresis in the presence of n-dodecyl-beta-d-maltoside and deriphat-160. We suggest that the structural organization of the Mn cluster and 33 kDa protein stabilized the core complex and increased its stability upon the action of the detergents.

Topics: Chloroplasts; Chromatography, Ion Exchange; Ethanolamines; Hydrogen-Ion Concentration; Indicators and Reagents; Intracellular Membranes; Light-Harvesting Protein Complexes; Photosynthetic Reaction Center Complex Proteins; Pisum sativum; Sepharose; Sodium Chloride; Viscosity

We have explored various chromatographic procedures with the intention of establishing an isolation procedure that would allow us to isolate a large quantity of PSA-ACT (prostate specific antigen-alpha 1-antichymotrypsin) complex either from patients' sera or from incubation mixtures of free PSA and protease inhibitors. We found that at pH 7.2, both free PSA and PSA-ACT molecules are negatively charged and bind to the DEAE-Sepharose column. However, they could be separated from each other using a linear gradient of NaCl at pH 7.2. Both free PSA and PSA-ACT molecules were also found to be retained by the Con A Sepharose column because of the carbohydrate moiety of the PSA molecule. These two molecules were not separable by Con A chromatography. These two molecules apparently differ in their isoelectric points and were well separated by chromatofocusing using a pH gradient from pH 9 to 6. It appears that chromatofocusing can also be used to identify the isoforms of free PSA because of its high resolving power. The large difference in molecular size between free PSA and PSA-ACT complex allowed their separation by gel filtration chromatography on a column containing either S-100, S-200, or S-300 gel. S-200 gel appeared to be the best for the separation of free PSA from PSA-ACT and for the removal of other contaminating serum proteins.(ABSTRACT TRUNCATED AT 250 WORDS)

Topics: alpha 1-Antichymotrypsin; Chromatography; Chromatography, Gel; Concanavalin A; Dextrans; Ethanolamines; Humans; Prostate-Specific Antigen; Sepharose

We have recently diagnosed aspartylglucosaminuria (AGU) in four members of a Canadian family. AGU is a lysosomal storage disease in which asparagine-linked glycopeptides accumulate to particularly high concentrations in liver, spleen and thyroid of affected individuals. A lesser accumulation of these glycopeptides is seen in the kidney and brain, and they are also excreted in the urine. The altered metabolism in AGU results from a deficiency of the enzyme aspartylglucosaminidase (1-aspartamido-beta-N-acetylglucosamine amidohydrolase), which hydrolyses the asparagine to N-acetylglucosamine linkages of glycoproteins and glycopeptides. We have used human liver as a source of material for the purification of aspartylglucosaminidase. The enzyme has been purified to homogeneity by using heat treatment, (NH4)2SO4 fractionation, and chromatography on concanavalin A-Sepharose, DEAE-Sepharose, sulphopropyl-Sephadex, hydroxyapatite, DEAE-cellulose and Sephadex G-100. Enzyme activity was followed by measuring colorimetrically the N-acetylglucosamine released from aspartylglucosamine at 56 degrees C. The purified enzyme protein ran at a 'native' molecular mass of 56 kDa in SDS/12.5%-PAGE gels, and the enzyme activity could be quantitatively recovered at this molecular mass by using gel slices as enzyme source in the assay. After denaturation by boiling in SDS the 56 kDa protein was lost with the corresponding appearance of polypeptides alpha,beta and beta 1, lacking enzyme activity, at 24.6, 18.4 and 17.4 kDa respectively. Treatment of heat-denatured enzyme with N-glycosidase F resulted in the following decreases in molecular mass; 24.6 to 23 kDa and 18.4 and 17.4 to 15.8 kDa. These studies indicate that human liver aspartylglucosaminidase is composed of two non-identical polypeptides, each of which is glycosylated. The N-termini of alpha,beta and beta 1 were directly accessible for sequencing, and the first 21, 26 and 22 amino acids respectively were identified.

Topics: Amino Acid Sequence; Aspartylglucosylaminase; Dextrans; Electrophoresis, Polyacrylamide Gel; Ethanolamines; Glycoside Hydrolases; Humans; Hydrolases; Liver; Macromolecular Substances; Molecular Sequence Data; Sepharose; Sodium Dodecyl Sulfate

A new method for the isolation of glutathione reductase which successively utilizes chromatography on 2'-5'-ADP-Sepharose 4B and DEAE-Sepharose CL 6B, is described. With these two steps, it was possible to purify to homogeneity the glutathione reductase from gerbil liver. Some molecular properties of the purified enzyme are reported.

Topics: Animals; Chromatography, Affinity; Chromatography, Ion Exchange; Ethanolamines; Gerbillinae; Glutathione Reductase; Isoelectric Focusing; Liver; Sepharose