hymecromone and 4-methylumbelliferylguanidinobenzoate

Human trypsin 4 is an unconventional serine protease that possesses an arginine at position 193 in place of the highly conserved glycine. Although this single amino acid substitution does not affect steady-state activity on small synthetic substrates, it has dramatic effects on zymogen activation, interaction with canonical inhibitors, and substrate specificity toward macromolecular substrates. To study the effect of a non-glycine residue at position 193 on the mechanism of the individual enzymatic reaction steps, we expressed wild type human trypsin 4 and its R193G mutant. 4-Methylumbelliferyl 4-guanidinobenzoate has been chosen as a substrate analogue, where deacylation is rate-limiting, and transient kinetic methods were used to monitor the reactions. This experimental system allows for the separation of the individual reaction steps during substrate hydrolysis and the determination of their rate constants dependably. We suggest a refined model for the reaction mechanism, in which acylation is preceded by the reversible formation of the first tetrahedral intermediate. Furthermore, the thermodynamics of these steps were also investigated. The formation of the first tetrahedral intermediate is highly exothermic and accompanied by a large entropy decrease for the wild type enzyme, whereas the signs of the enthalpy and entropy changes are opposite and smaller for the R193G mutant. This difference in the energetic profiles indicates much more extended structural and/or dynamic rearrangements in the equilibrium step of the first tetrahedral intermediate formation in wild type human trypsin 4 than in the R193G mutant enzyme, which may contribute to the biological function of this protease.

Topics: Acylation; Brain; Entropy; Glycine; Hot Temperature; Humans; Hydrolysis; Hymecromone; Kinetics; Molecular Conformation; Mutation; Thermodynamics; Time Factors; Trypsin

Human tryptase-beta (HTbeta) is a unique serine protease exhibiting a frame-like tetramer structure with four active sites directed toward a central pore. Potent inhibition of HTbeta has been attained using CRA-2059. This compound has two phenylguanidinium head groups connected via a linker capable of spanning between two active sites. The properties of the CRA-2059:HTbeta interaction were defined in this study. Tight-binding reversible inhibition was observed with an inhibition constant (Ki) of 620 pM, an association rate constant of 7x10(7) M(-1) s(-1) and a relatively slow dissociation rate constant of 0.04 s(-1). Bivalent inhibition was demonstrated by displacement of p-aminobenzamidine from the primary specificity pocket with a stoichiometry, [CRA-2059]0/[HTbeta]0, of 0.5. The potency of the bivalent interaction was illustrated by CRA-2059 inhibition of HTbeta, 24% or 53% inhibited by pre-incubation with an irreversible inhibitor. Two interactions were observed consistent with mono- and bi-valent binding; the Ki value for bivalent inhibition was at least 10(4)-fold lower than that for monovalent inhibition. Comparison of the affinities of CRA-2059 and phenylguanidine for HTbeta finds an approximate doubling of the free energy change upon bivalent binding. This doubling suggests that the linker portion minimally hinders the binding of CRA-2059 to HTbeta. The potency of CRA-2059 is thus attributable to effective bivalent binding.

Topics: Benzamidines; Binding Sites; Binding, Competitive; Cross-Linking Reagents; Dioxoles; Fluorescent Dyes; Guanidines; Humans; Hymecromone; Kinetics; Osmolar Concentration; Protein Binding; Recombinant Proteins; Serine Endopeptidases; Serine Proteinase Inhibitors; Spectrometry, Fluorescence; Sulfones; Thermodynamics; Tryptases

The mechanism by which the protein cofactor, tissue factor, enhances the activity of its cognate serine protease, coagulation factor VIIa (FVIIa), has been studied using the fluorogenic ester substrate 4-methylumbelliferyl p'-guanidinobenzoate (MUGB). Kinetic data were collected at pH 8.4 and pH 7.6 in the presence and absence of soluble tissue factor (sTF; recombinant human tissue factor containing only the extracellular domain). Pre-steady-state techniques allowed the determination of the individual rate constants for acylation (k2) and deacylation (k3) of the sTF.FVIIa complex as well as the dissociation constant for the noncovalent Michaelis complex with MUGB. Alternative methods were required for determination of these parameters for free FVIIa due to extremely slow hydrolysis of MUGB in the absence of sTF. Under all experimental conditions, deacylation was found to be rate-limiting. The major effect of sTF was to raise the affinity of FVIIa for MUGB (31-fold at pH 8.4 and 36-fold at pH 7.6); only minor changes in k2 and k3 were observed. Thus, we conclude that for the ester substrate MUGB, sTF exerts greater allosteric effects on substrate binding than on the later steps involved in the catalytic pathway.

Topics: Allosteric Regulation; Binding Sites; Factor VIIa; Humans; Hydrolysis; Hymecromone; Kinetics; Models, Chemical; Protein Binding; Recombinant Proteins; Solubility; Spectrometry, Fluorescence; Thromboplastin

Dansyl fluoride (Dan-F), an active site directed fluorescent inhibitor of guanidinobenzoatase (GB), has been used for the location of tumour cells in frozen sections of human squamous cell carcinoma and colonic carcinoma tissues. The tumour cell surfaces having active GB bind Dan-F and fluoresce blue. The surrounding normal epithelial lung cell surfaces fail to bind Dan-F and hence lack fluorescence, whilst the normal colon cell surfaces have another isoenzymic form of GB, bind Dan-F and fluoresce blue. Kinetic studies have shown that Dan-F is an irreversible inhibitor of GB, and Dan-GB complexes are not dissociated with SDS and high salt concentration. However hydroxylamine (1 M) can dissociate Dan-GB complexes in the presence of 0.1% SDS, both on membrane-bound and in free solution. These studies suggest that Dan-F is a potent inhibitor of GB, and in very low concentration (3 x 10(-8) M) can be used as a novel fluorescent probe for the location of tumour cells in histological sections of human tissues.

Topics: Aminacrine; Binding Sites; Carboxylic Ester Hydrolases; Colonic Neoplasms; Coloring Agents; Dansyl Compounds; Endopeptidases; Enzyme Activation; Histocytochemistry; Humans; Hydroxylamine; Hydroxylamines; Hymecromone; Isoenzymes; Lung Neoplasms; Microscopy, Fluorescence; Serine Proteinase Inhibitors; Sodium Dodecyl Sulfate; Spectrometry, Fluorescence

Pentosan polysulphate (PPS), a highly negatively charged polysaccharide, is a significant inhibitor of an isoenzymic form of a cell surface protease referred to as guanidinobenzoatase GB, associated with colonic carcinoma tissues in frozen sections and free GB in solution, in a concentration-dependent manner. However PPS failed to recognise and bind to the isoenzymic form of GB associated with normal colon epithelial cell surfaces. Texas red labelled PPS (TR-PPS) binds to the tumour cell surfaces of colonic carcinoma and colonic polyps and these cells fluoresce red, whilst the normal colon cell surfaces failed to bind the TR-PPS, and hence lacked red fluorescence. Polysulphonated suramin also selectively recognised and inhibited the colonic carcinoma GB isoenzyme. The kinetic data indicated that this inhibition was not caused by a mere polyanionic effect, since highly sulphated heparin failed to show a significant inhibition of colonic carcinoma GB, however trypan blue did show 50% inhibition. Kinetic studies have also shown that PPS is a non-competitive, reversible inhibitor of colonic carcinoma GB, with an apparent Km 6.8 x 10(-7) M. Gel analysis has shown that PPS binds to another site, distinct from the active centre, and after binding PPS changed the conformation of GB. These studies suggest that TR-PPS is a potent inhibitor of colonic carcinoma GB, and can be used as a novel fluorescent probe for the location of tumour cells in frozen sections of human colon tissues. PSS could also have potential as a vehicle for the transport of cytotoxic compounds to carcinoma cells of the colon.

Topics: Carboxylic Ester Hydrolases; Cell Membrane; Chromatography, Affinity; Colonic Neoplasms; Dansyl Compounds; Electrophoresis, Polyacrylamide Gel; Endopeptidases; Fluorescent Dyes; Heparin; Humans; Hymecromone; Isoenzymes; Kinetics; Microscopy, Fluorescence; Pentosan Sulfuric Polyester; Protease Inhibitors; Protein Conformation; Suramin; Trypan Blue; Xanthenes

The relationship between serum and tumour cell surface proteolytic enzymes and the development of muscle breakdown in cancer cachexia has been studied in a murine model of the condition (MAC16). The surface of the MAC16 tumour cells carried a proteolytic enzyme referred to as guanidinobenzoatase (GB). Serum from mice also contained an enzyme (referred to as MSE) which cleaved the trypsin inhibitor 4-methylumbelliferyl-p-guanidinobenzoate as a true substrate, but there was no relationship with weight loss or the presence or absence of tumour and the level of this serum enzyme. Polyunsaturated fatty acids (PUFAs) were shown to be inhibitors of MSE at microM concentrations and one PUFA, eicosapentaenoic acid (EPA) was found to be a non-competitive inhibitor of both MSE and GB. The effect of EPA was specific since other proteolytic enzymes, trypsin, esterase and tissue plasminogen activator were unaffected by concentrations inhibiting GB and MSE. MSE and GB are two different enzymes which possess some common properties. However, GB is likely to be significant for tumour development since MSE is also found in normal mouse serum.

Topics: Animals; Cachexia; Carboxylic Ester Hydrolases; Eicosapentaenoic Acid; Endopeptidases; Hymecromone; Male; Membrane Proteins; Mice; Mice, Inbred Strains; Neoplasms, Experimental; Rhodamines; Tissue Plasminogen Activator; Weight Loss

Three guinea pig testicular, low-molecular-weight, acid-stable inhibitors specific for trypsin-like proteinases were isolated, purified, and characterized. The procedure comprised acid extraction of testicular acetone powder, pH precipitation of the extract, gel filtration of the supernatant on Sephadex G-100 and G-50, ion-exchange chromatography on SP-Sephadex, followed by QAE-Sephadex. Final purification was by rechromatography on Sephadex G-50 superfine gel. The three proteinase inhibitors were labeled A, B, and Cnb, the latter to denote nonbinding of Cnb to the QAE-Sephadex. Components A and Cnb showed competitive, whereas B showed noncompetitive, inhibition against trypsin. All three inhibitors were active against trypsin but were ineffective against chymotrypsin. The inhibition constants, Ki, were obtained using trypsin-catalyzed hydrolysis of the N-benzyloxycarbonyl-L-arginyl amide of 7-amino-4-trifluoro-methylcoumarin (CBZ-Arg-AFC) at pH 8.0. The values were calculated to be, for A, 1.5 x 10(-8) M; for B, 1.5 x 10(-8) M; and, for Cnb, 2.2 x 10(-7) M. The Ki values calculated from inhibition of trypsin-catalyzed hydrolysis of the active site titrant 4-methylumbelliferyl-p-guanidinobenzoate (MUGB) using Easson-Stedman plots were, for A, 7.7 x 10(-9) M; for B, 6.7 x 10(-9) M; and, for Cnb, 1.4 x 10(-7) M. The Mrs as determined by active site titration with MUGB were A, 11.2 kDa; B, 10.5 kDa; Cnb, 17.0 kDa. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis gave Mr values for A of 11 kDa, for B of 4 kDa, and for Cnb of 19 kDa. The discrepancy in Mr values for B indicates that it may function as a dimer or trimer in the active state.

Topics: Animals; Chromatography, Ion Exchange; Chymotrypsin; Coumarins; Electrophoresis, Polyacrylamide Gel; Guinea Pigs; Hymecromone; Kinetics; Male; Protease Inhibitors; Spectrophotometry, Ultraviolet; Sperm Maturation; Substrate Specificity; Testis; Trypsin; Trypsin Inhibitors

Two acrosin inhibitors, 4'-methylumbelliferyl 4-guanidinobenzoate and 2'-carbomethoxyphenyl 4-guanidinobenzoate, were tested for mutagenicity in the transplacental micronucleus assay and the mouse bone marrow micronucleus assay. The compounds were administered intraperitoneally at doses of 125 mg/kg and 250 mg/kg to pregnant mice. Fetal peripheral blood and maternal bone marrow cells were examined at 36 h for the frequency of micronucleated polychromatic erythrocytes. Neither compound induced micronuclei in maternal or fetal tissues. The ratio of polychromatic erythrocytes to normochromatic erythrocytes was not affected by the drug treatments indicating that the compounds had no effect on the cell cycle or mitosis in these tissues and that they were not cytotoxic. Both compounds, which show promise as vaginal contraceptives, were not mutagenic in this study.

Topics: Animals; Bone Marrow; Bone Marrow Cells; Erythrocytes; Female; Fetus; Guanidines; Hematopoietic System; Hymecromone; Mice; Micronucleus Tests; Mitosis; Mutagens; Placenta; Pregnancy; Staining and Labeling

Topics: Benzoylarginine-2-Naphthylamide; Carboxylic Ester Hydrolases; Cell Membrane; Endopeptidases; Fluorescent Dyes; Hymecromone; Rhodamines; Substrate Specificity; Tissue Plasminogen Activator

It has recently been reported that the fluorogenic serine proteolytic active site titrant, 4-methyl-umbelliferyl-p-guanidinobenzoate (MUGB), cannot be employed in this capacity for tissue-type plasminogen activator (TPA) [Geiger, M., and Binder, B.R. (1987) Biochim. Biophys. Acta 912, 34-40]. Since this observation has such important ramifications in this area of research, we have studied the reaction of MUGB with recombinant (rec)TPA under a variety of experimental conditions and find that MUGB is indeed an effective titrant of rec-two chain TPA (recTCTPA) at 4 degrees, a condition under which the deacylation rate constant is diminished to the point that acylation can be readily observed. The KS for the interaction of MUGB with recTCTPA is 43 microM-46 microM, the acylation rate constant, k2, is approximately 3.6 min-1-4.2 min-1, and the rate constant for deacylation of p-guanidinobenzoyl-recTCTPA is 0.084 min-1-0.110 min-1. This same recTCTPA, after treatment with diisopropylfluorophosphate, does not react with MUGB. Single-chain TPA (recSCTPA) has been found to acylate more slowly than its two-chain counterpart and to exhibit a higher degree of turnover of the acyl-enzyme with this reagent. These results demonstrate that the active site concentration of TCTPA can be accurately determined by titration with MUGB, a consideration which is essential to the proper kinetic evaluation of this agent and its genetic variants. On the other hand, the presteady state kinetic characteristics for MUGB toward SCTPA are not favorable for its use as a titrant with this form of the enzyme.

Topics: Acylation; Binding Sites; Hymecromone; Isoflurophate; Kinetics; Recombinant Proteins; Tissue Plasminogen Activator; Umbelliferones

There are at least two binding sites for the mouse egg zona pellucida on the surface of mouse sperm: a site with galactosyltransferase (GT) activity inhibitable by uridine-5'-diphosphate-dialdehyde (UDPd) and alpha-lactalbumin, and a trypsin inhibitor-sensitive (TI) site that hydrolyzes guanidinobenzoate (GB) esters. Characterization of GT activity gave the Km for UDP galactose as 37 microM with N-acetylglucosamine as galactose acceptor, and Vmax as 0.37 pmol/min/10(6) sperm. UDP galactose from 12.5-100 microM inhibited sperm binding to zona-intact eggs in a concentration-dependent manner with close correlation to GT activity (r = 0.95). To assess the independence and spatial relationship of the two types of site, cross-perturbation studies were performed. p-Nitrophenyl-GB, a low molecular mass inhibitor specific for the TI site, had no effect on the enzyme activity of the GT site. Conversely, UDPd, a specific inhibitor of GT, had no effect on GB hydrolysis. Weak inhibitions were found when soybean trypsin inhibitor (SBTI) was included with the GT assay and when GB hydrolysis was assayed in the presence of alpha-lactalbumin or asialo-agalacto-(alpha 1-acid glycoprotein). Acid-solubilized zona protein (ASZP) weakly inhibited the GT reaction, while stronger inhibition was seen with chymotrypsin-solubilized zona protein (CSZP). ASZP inhibited sperm binding to zonae with the same concentration dependence associated with inhibition of GB hydrolysis, but the inhibition of GT enzyme activity was on the same order as that found with SBTI, indicating that ASZP was only binding to the TI site under enzyme assay conditions. The results support the hypothesis that the two types of site are independent in binding their specific zona ligands, but are close enough for steric perturbation of the enzyme activity of one site by macromolecules bound to the other. The different interactions of solubilized zona preparations with the GT site under enzyme assay conditions are an indication that conditions which favor the enzyme activity of the site may interfere with the physiological binding functions of the site.

Topics: Animals; Binding Sites; Female; Fluorometry; Galactosyltransferases; Hydrolysis; Hymecromone; Male; Mice; Mice, Inbred Strains; Ovum; Solubility; Sperm-Ovum Interactions; Spermatozoa; Trypsin Inhibitors; Zona Pellucida

Topics: Humans; Hydrolysis; Hymecromone; Kinetics; Protein Binding; Serum Albumin; Umbelliferones

The applicability of 4-methylumbelliferyl-p-guanidinobenzoate hydrochloride (MUGB) as active-site titrant for tissue-type plasminogen activator (t-PA) was studied in comparison to urokinase. Although t-PA was capable of cleaving MUGB, active-site titration of t-PA (one-chain form as well as two-chain form) with MUGB was not possible, whereas MUGB titration of urokinase could be performed. We therefore studied the kinetics of the interaction of these two plasminogen activators with MUGB. The equilibrium dissociation constant, KS, for the interaction between MUGB and urokinase was 2.9 X 10(-6) M, and for the interaction with t-PA 3.13 X 10(-5) M. However, one main requirement for active-site titration, namely a stable acyl enzyme intermediate (ES'), was only fulfilled for MUGB urokinase but not for MUGB t-PA. Whereas for the reaction of MUGB and urokinase the first-order acylation rate constant k2 was found to be about 10(6)-times higher than the first-order deacylation rate constant k3 (k2 = 3.76 X 10(-1) s-1, k3 = 3.7 X 10(-7) s-1), the k2/k3 ratio for the reaction of MUGB and t-PA (one- and two-chain form) was 0.77 to 3.85. Therefore, urokinase and t-PA differ in their reaction with this fluorogenic substrate and MUGB cannot be used for active-site titration of tPA.

Topics: Binding Sites; Humans; Hymecromone; Isoflurophate; Kinetics; Spectrometry, Fluorescence; Tissue Plasminogen Activator; Umbelliferones; Urokinase-Type Plasminogen Activator

Certain aryl 4-guanidinobenzoates (AGs; inhibitors of proteinases, including the sperm enzyme acrosin) have been shown to be more potent vaginal contraceptives in rabbits and less toxic than nonoxynol-9, the active ingredient of most marketed vaginal contraceptive formulations. To determine if these AGs can contact sperm and inhibit acrosin when mixed with the entire human ejaculate for a short period of time (roughly imitating clinical conditions), the inhibitors were added to semen at various concentrations for 2 min, after which the seminal plasma and unbound inhibitor were removed from the sperm by Ficoll centrifugation. Subsequently, the total arginine amidolytic activity of the spermatozoa was determined spectrophotometrically after a combined treatment that resulted in extraction, proacrosin activation, and reaction with substrate. Dose-response curves were prepared. All AGs studied were effective inhibitors of the amidolytic activity under these conditions, with ED50 values (the dose levels at which half of the acrosin associated with 10(6) sperm is inhibited) ranging from 10(-5) to 10(-7) M. To determine the effect on the proteolytic activity of individual spermatozoa, the experiment was repeated with 4'-acetamidophenyl 4-guanidinobenzoate (AGB), and the protease released from the sperm was measured by the gelatin-plate assay. The inhibition results were similar to those obtained by extraction of the spermatozoa and measurement of amidolytic activity. Thus, when mixed with the human ejaculate, AGs interact rapidly with spermatozoa to inhibit both their arginine amidolytic and proteolytic activity (probably due primarily or only to inhibition of acrosin) and remain bound even after removal of the seminal plasma. These data encourage further study of the compounds for contraceptive purposes.

Topics: Acrosin; Benzoates; Contraceptive Agents, Female; Endopeptidases; Gelatin; Guanidines; Humans; Hymecromone; In Vitro Techniques; Male; Protease Inhibitors; Spectrophotometry; Spermatozoa

The first contact of mammalian gametes is the binding of the spermatozoon to the zona pellucida of the egg. Previous work has shown that binding of the spermatozoon to the zona in the mouse occurs prior to the acrosome reaction and that trypsin inhibitors block this initial binding. This suggests that the sperm surface contains a trypsinlike binding site that functions by an active site mechanism to effect initial zona binding. When suspensions of twice-washed spermatozoa were incubated with the serine protease active site titrant, 4-methylumbelliferyl p-guanidinobenzoate (MUGB), the titrant was hydrolyzed at a rate of 8 pmoles/min-10(6) cells. MUGB was found to inhibit the binding of spermatozoa to the zona pellucida. The degree of inhibition and the rate of hydrolysis of MUGB by washed spermatozoa depend on the concentration of titrant, with half maximal effects at 13 microM and a linear correlation with r = 0.99. The analogous lysyl and arginyl trypsin substrates containing 7-amino-4-methylcoumarin as the fluorogenic leaving group were not hydrolyzed under the same conditions and did not inhibit zona binding. Both binding of sperm to zona-intact eggs and the hydrolysis of MUGB by sperm are inhibited by p-nitrophenyl guanidinobenzoate, soybean trypsin inhibitor, and acid-solubilized zonae. The linear correlation coefficients of the inhibition of sperm binding and MUGB hydrolysis by these three substances are greater than 0.92. This "trypsinlike" sperm site is essential for sperm binding to the zona: its stereospecificity is unique in that it reacts with trypsin inhibitors but not with trypsin substrates.

Topics: Animals; Binding Sites; Cell Membrane; Female; Hydrolysis; Hymecromone; Male; Mice; Sperm-Ovum Interactions; Spermatozoa; Trypsin; Trypsin Inhibitors; Zona Pellucida

A pre-steady state kinetic analysis of the stimulation by monovalent cations of the activity of bovine activated protein C (APC) and a proteolytic fragment of APC, des-1-41-light chain activated protein C (GDAPC), toward the substrate, 4-methylumbelliferyl p-guanidinobenzoate, has been undertaken. With the cations Na+ and Cs+, at least two cation sites, or classes of sites, on APC were found to be important to the kinetic effects observed. For GDAPC, with both monovalent cations investigated, a single cation-binding site, or class of sites, of kinetic importance was discovered. The most general mechanism that fits all kinetic data was a rapid equilibrium type, with the cation(s) (A) and substrate (S) binding to the enzyme in a random fashion. Cations were found to be essential activators, and only formation of the EAS or EA2S complex led to product generation. For each enzyme, stimulation of the reaction rates was found to be chiefly due to a dramatic enhancement by monovalent cations of the rate constant (k2) for acylation of the enzyme since the dissociation constant (Ks) for enzyme-substrate interactions was increased in the presence of cations, and the deacylation rate constant (k3) was not affected by these activators.

Topics: Animals; Binding Sites; Cations, Monovalent; Cattle; Cesium; Enzyme Activation; Hymecromone; Kinetics; Peptide Fragments; Protein C; Sodium

We evaluated four methods purported to distinguish between individuals homozygous or heterozygous for cystic fibrosis (CF) and normal controls: (1) detection of a protein in the serum by isoelectric focusing at pH 8.5, (2) detection of a lectinlike factor in the serum by hemagglutination, (3) isolation of CF-lectin from the serum by affinity chromatography, and (4) measurement of MUGB-reactive proteases in the plasma. The results were disappointing. The detection of CF protein by isoelectric focusing was unreliable; it could be identified in only 46% of heterozygotes and 66% of homozygotes, with a false positive rate of 17%. Detection of a lectinlike factor by hemagglutination was also found to be unreliable and irreproducible. The lectin isolated by affinity chromatography was not specific for the CF gene. No significant differences were found in the MUGB titers of the three populations tested. However, low titers (MU less than 200 nmol/ml) were found in 33% of homozygotes and heterozygotes and in 17% of normal controls. We conclude that none of these methods is suitable for carrier detection in cystic fibrosis.

Topics: Adult; Blood Proteins; Calgranulin A; Cystic Fibrosis; Heterozygote; Homozygote; Humans; Hymecromone; Lectins; Middle Aged

Amniotic fluids were obtained from 19 mothers who had previously given birth to a child with cystic fibrosis. Measurement of methylumbelliferylguanidinobenzoate (MUGB) reactive proteases suggested that all 19 would have unaffected babies. Amongst the first 10 cases to come to term there were 5 infants with cystic fibrosis. It is concluded that MUGB protease titration is not suitable for the early prenatal diagnosis of cystic fibrosis.

Topics: Amniotic Fluid; Cystic Fibrosis; Female; Gestational Age; Humans; Hymecromone; Peptide Hydrolases; Pregnancy; Prenatal Diagnosis; Umbelliferones

Measurement of 4-methylumbelliferyl p-guanidinobenzoate (MUGB)-hydrolyzing activity in the plasma of normal controls, cystic fibrosis (CF) heterozygotes, and CF homozygotes did not support previously reported (35) differences in MUGB-hydrolyzing activity. We identified human plasma albumin as the major source of MUGB-hydrolyzing activity by comparison of our plasma results to those obtained with physiologic concentrations of commercial albumin samples. Substantiating evidence was obtained from gel filtration experiments and correlation of albumin levels in CF plasma with MUGB titers. We found essentially no proteolytic activity towards dinitrophenylprotamine sulfate associated with commercial albumin samples. It appears that the reaction between human albumin and MUGB represents a weak esterase activity, perhaps involving the acylation of a specific site(s) on the protein. Hypoalbuminemia has been documented (8) in some CF patients. Low albumin concentrations, indicated by MUGB titers less than 190 nmole methylumbelliferone/ml plasma, were found in 42% of CF homozygotes, 6% of heterozygotes, and 4% of controls. Gel filtration studies of a normal amniotic fluid supernatant indicated that albumin was the major MUGB-hydrolyzing substance in this fluid. We conclude that MUGB abnormalities are not associated with the basic gene defect in CF and cannot be used as the basis of a test for intrauterine or heterozygote detection.

Topics: Adolescent; Adult; Amniotic Fluid; Child; Child, Preschool; Chromatography, Gel; Cystic Fibrosis; Genetic Carrier Screening; Humans; Hydrolysis; Hymecromone; Middle Aged; Prenatal Diagnosis; Serum Albumin; Umbelliferones

Topics: Clinical Enzyme Tests; Cystic Fibrosis; Heterozygote; Humans; Hymecromone; Peptide Hydrolases; Serum Albumin; Umbelliferones

4-methylumbelliferylguanidinobenzoate (MUGB) reactivity in plasma from patients with cystic fibrosis and in amniotic fluid from pregnancies leading to children with cystic fibrosis, has been reported to be significantly decreased. We have so far been unable to confirm these findings and have therefore reexamined this reactivity using diisopropylfluorophosphate (DEF), another active site titrant of serine proteases. We have shown that MUGB and DFP are reacting with the same molecules in plasma and amniotic fluid. Using crossed immunoelectrophoresis and SDS-polyacrylamide gel electrophoresis of 3H-DFP labelled plasma and amniotic fluid we have obtained strong evidence that the main contribution of MUGB and DFP reactive protein in plasma and amniotic fluid is identical to serum albumin. The use of MUGB reactivity in amniotic fluid in pregnancies at risk for cystic fibrosis must therefore be reconsidered.

Topics: Amniotic Fluid; Cystic Fibrosis; Endopeptidases; Female; Humans; Hymecromone; Isoflurophate; Pregnancy; Prenatal Diagnosis; Serine Endopeptidases; Serum Albumin; Substrate Specificity

In contrast to the original reports by others, the hydrolysis of 4-methylumbelliferylguanidinobenzoate ((MUGB), and active-site titrant of serine proteases, was found not to be significantly different in plasma samples from patients with cystic fibrosis (CF), obligate heterozygotes, and normal controls. Since the steady-state turnover of substrate is the dominant term of MUGB hydrolysis, the assay in its present form does not meet the requirements of an active-site titration. The reproducibility and reliability of the assay are hampered by (1) high blank values due to non-specific hydrolysis, (2) the absence of a defined endpoint of the incubation, (3) the non-linearity of the assay with respect to plasma concentration, (4) the temperature-dependent evolution of esterase activity during incubation, and (5) most importantly the loss of activity during storage of plasma samples. Cleavage of MUGB catalysed by the imidazole ring of histidine accounts for a significant portion of the activity in plasma.

Topics: Adolescent; Adult; Child; Child, Preschool; Cystic Fibrosis; Drug Stability; Enzyme Activation; Female; Histidine; Humans; Hydrolysis; Hymecromone; Kinetics; Male; Methods; Peptide Hydrolases; Umbelliferones

Low molecular weight, synthetic proteinase inhibitors that inhibit sperm-associated acrosin, were released systemically in female mice at a constant rate from minipumps. The release was timed so that, after mating, the minipumps were depleted of inhibitor before blastocyst implantation took place. Three of the inhibitors: 4-aminobenzamidine (ABD), 4-nitrophenyl-4-guanidino-benzoate (NPGB) and 4-methylumbelliferone-4-guanidinobenzoate (MUGB) caused a 50% decrease in fertility, the last two at very low concentrations. The fourth inhibitor, benzamidine (BD), which is also the weakest inhibitor of mouse acrosin and in vitro fertilization, had no effect. These results show that at least one of the processes leading to fertilization or early blastogenesis, is dependent on proteolytic activity and that the systemic application of proteinase inhibitors inhibits conception. MUGB possessed low toxicity and a high margin of safety, encouraging the development of phenol derivatives of guanidinobenzoic acid as contraceptive agents.

Topics: Acrosin; Amidines; Animals; Benzamidines; Benzoates; Contraceptive Agents, Female; Drug Implants; Female; Hymecromone; Mice; Protease Inhibitors; Umbelliferones

Topics: Amniotic Fluid; Cystic Fibrosis; False Negative Reactions; Female; Humans; Hymecromone; Isoelectric Focusing; Molecular Weight; Peptide Hydrolases; Pregnancy; Prenatal Diagnosis; Reference Values

Kinetic studies were performed to evaluate the interaction of benzamidine (BD), 4-aminobenzamidine (ABD). 4'-nitrophenyl 4-guanidinobenzoate (NPGB), and 4'-methylumbelliferyl 4-guanidinobenzoate (MUGB) with mouse acrosin. The Michaelis constant of mouse acrosin towards alpha-N-benzoyl-L-arginine ethyl ester and the sensitivity of mouse acrosin to inhibitors differed from those reported for other species. NPGB and MUGB were much more active inhibitors of acrosin than BD and ABD. Plots of percentage fertilization versus acrosin inhibitor concentration were generated for all 4 compounds. Linear dose-response curves were obtained and gave ED50 values (50% inhibition of fertilization) of 230 microM for BD, 27 microM for ABD, 35 nM for MUGB, and 13 nM for NPGB. The relative antifertility activity of the compounds paralleled their inhibitory activity towards mouse acrosin, strongly indicating that the inhibition of fertilization is obtained through the inhibition of acrosin. Since the dose-response curves were linear, the mouse in-vitro fertilization system may be useful to screen acrosin inhibitors for their antifertility potency. MUGB should have low toxicity and may have potential as a contraceptive agent.

Topics: Acrosin; Animals; Benzamidines; Benzoates; Dose-Response Relationship, Drug; Fertilization in Vitro; Hymecromone; Kinetics; Mice; Protease Inhibitors; Trypsin Inhibitors

Topics: Amniocentesis; Amniotic Fluid; Clinical Enzyme Tests; Cystic Fibrosis; False Negative Reactions; Female; Humans; Hymecromone; Peptide Hydrolases; Pregnancy; Pregnancy Complications; Prenatal Diagnosis; Umbelliferones

Topics: Amniocentesis; Amniotic Fluid; Clinical Enzyme Tests; Cystic Fibrosis; False Positive Reactions; Female; Humans; Hymecromone; Peptide Hydrolases; Pregnancy

Ehrlich ascites tumour cells grown in mice have a cell-surface trypsin-like neutral protease (TLNP) which is not inhibited by high-mol.-wt inhibitors of trypsin. This enzyme is inhibited by low concentrations of zinc, which may be removed by chelating agents, with the consequent return of enzymic activity. Gold, provided as the drugs aurothiomalate or auranofin, also inhibits TLNP. The gold can be removed from the enzyme by incremental addition of thiols. The mechanisms of gold transfer to the active site to cause inhibition and subsequent removal of gold with reactivation of TLNP, have been shown to be controlled by reversible thiol-exchange reactions.

Topics: Aminopeptidases; Animals; Auranofin; Aurothioglucose; Carcinoma, Ehrlich Tumor; Cathepsin H; Cathepsins; Cell Membrane; Cells, Cultured; Cysteine Endopeptidases; Dithiothreitol; Dose-Response Relationship, Drug; Edetic Acid; Gold; Gold Sodium Thiomalate; Hymecromone; Mice; Zinc

Topics: Amino Acid Sequence; Animals; Benzoates; Binding Sites; Carboxypeptidases; Carboxypeptidases A; Cattle; Chymotrypsin; Chymotrypsinogen; Guanidines; Hymecromone; Protein Conformation; Trypsin; Trypsinogen

Previous studies in our laboratory have suggested that measurement of methylumbelliferylguanidinobenzoate (MUGB) reactive proteases in midtrimester amniotic fluid is potentially of value for the intrauterine detection of cystic fibrosis. Thirty-nine pregnancies of obligate heterozygotes of cystic fibrosis and 12 cases where one parent has a sib with cystic fibrosis have been previously monitored by means of quantitative and qualitative measurements of MUGB in amniotic fluid. In all but one case the diagnosis was accurately ascertained in the midtrimester and confirmed after delivery. The measurement of MUGB reactivity in midtrimester amniotic fluid by three different procedures--quantitative analysis, polyacrylamide isoelectric focusing, and column filtration--appears to provide a practical and reliable approach for the intrauterine detection of cystic fibrosis.

Topics: Amniotic Fluid; Cystic Fibrosis; Female; Heterozygote; Humans; Hymecromone; Pregnancy; Pregnancy Trimester, Second; Prenatal Diagnosis

Topics: Amniotic Fluid; Cystic Fibrosis; Electrophoresis, Polyacrylamide Gel; Female; Humans; Hymecromone; Pregnancy; Pregnancy Trimester, Second; Umbelliferones

An arginine esterase activity similar to that observed in plasma has been demonstrated in second trimester and term human amniotic fluid. Like plasma, the protease(s) hydrolyzed esters of arginine, were reactive towards 4-methylumbelliferylguanidinobenzoate (MUGB), a sensitive active site titrant of trypsin-like enzymes, and had a pI of 5.1--5.4. The pH optimum for proteolytic activity was 8.0. This protease activity was inhibited by soybean trypsin inhibitor (STI), benzamidine and (p-nitrophenyl)-p'-guanidinobenzoate (NPGB), and was insensitive to 1-chloro-3-tosylamido-7-amino-2-heptanone (TLCK) and p-hydroxymercuribenzoic acid (HMB). Upon gel filtration, two MUGB-reactive fractions were observed, one with an apparent molecular weight of 200,000 and the other, 100,000. Both fractions had arginine esterase activity and appeared to be sensitive to inhibition by STI and benzamidine. The mean MUGB titre value (nmoles of 4-methylumbelliferone released per ml amniotic fluid) for 300 mid-trimester amniotic fluids was 11.40 +/- 2.40 nmoles MU/ml. The mean specific activity was 2.36 +/- 0.41 nmoles MU/mg protein. Two amniotic fluids from pregnancies which delivered children with cystic fibrosis (CF) were analyzed in blind samples sent from other laboratories. The MU titre values obtained were 4.73 and 4.32 with specific activities of 1.24 and 1.30 respectively. A third was identified in our screening program of amniotic fluids obtained from amniocenteses done for the intrauterine detection of genetic abnormalities. The MU titre value was 5.52 nmoles/ml with a specific activity of 1.34. The specific activities of these fluids when compared to the controls were significantly different (p less than 0.001). The mean titre value for 23 term amniotic fluids samples was 8.14 +/- 1.69 nmoles MU/ml. The mean specific activity was 3.37 +/- 0.76 nmoles MU/mg protein. A term amniotic fluid obtained from a woman who delivered a baby with CF showed a markedly reduced level of MUGB reactivity (3.01 nmole/ml). The specific activity was 1.06 which was significantly different from the control term fluids. The MU titre values and specific activities of amniotic fluids obtained from abnormal pregnancies (such as those with neural tube defects, chromosomal abnormalities and polymorphisms, abortions and stillbirths) and fluids with elevated alphafetoprotein and maternal blood contaminants did not significantly vary from the mean control values (Table 3).

Topics: Amniotic Fluid; Arginine; Chloroform; Cystic Fibrosis; Ellagic Acid; Female; Guanidines; Humans; Hydrolysis; Hymecromone; Peptide Hydrolases; Pregnancy; Pregnancy Trimester, Second; Pregnancy Trimester, Third; Prenatal Diagnosis; Umbelliferones

Over 1,000 specimens of midtrimester human amniotic fluid have been analyzed for 4-methylumbelliferylguanidinobenzoate (MUGB) reactivity. The mean specific activity value (nanomoles of methylumbelliferone (MU) formed per milligram of protein) for the control population (1,037) was 2.37 +/- 0.41. The mean specific activity values from a series of specimens of amniotic fluid derived from abnormal pregnancies (chromosomal and biochemical abnormalities, confirmed neural tube defects, abortions/stillbirths) were found not to vary significantly from value in the control population. The mean specific activity value of blood-tinged samples centrifuged to remove the red blood cell contaminants prior to freezing at -20 degrees C did not vary from the value for the control group, in contrast to the value for samples containing hemolyzed red blood cells (1.76 +/- 0.30). The mean specific activity values for all four groups (control, abnormal, spun and unspun) were found to be significantly different (p less than 0.001) from the mean specific activity value of 1.24 +/- 0.12 for the four cystic fibrosis (CF) amniotic fluids. In addition, upon isoelectric focusing of the control and CF fluids on polyacrylamide gels, pH 5-7, subsequently stained for alpha-N-benzoyl-L-arginine ethyl ester activity, the controls gave a pattern of five activity bands with a pl of 5.1 to 5.4, whereas the CF fluids consistently gave only four. These data suggest the potential value of the MUGB-reactive protease assay for the intrauterine detection of cystic fibrosis.

Topics: Amniocentesis; Amniotic Fluid; Cystic Fibrosis; Female; Humans; Hymecromone; Peptide Hydrolases; Pregnancy; Umbelliferones

Thirteen pregnancies of obligate heterozygotes of cystic fibrosis have been prospectively monitored utilizing quantitative and qualitative measurements of methylumbelliferylguanidinobenzoate (MUGB) reactive proteases in amniotic fluid. In each case the diagnosis was accurately ascertained in midtrimester and confirmed after delivery. The measurement of MUGB reactivity in midtrimester amniotic fluid by three different procedures, quantitative analysis, polyacrylamide isoelectric focusing, and column filtration appears to provide a practical and reliable approach for the intrauterine detection of cystic fibrosis.

Topics: Amniotic Fluid; Chromatography, Gel; Cystic Fibrosis; Female; Humans; Hymecromone; Isoelectric Focusing; Peptide Hydrolases; Pregnancy; Prenatal Diagnosis; Umbelliferones

Protease activity in plasma is assayed using 4-methylumbelliferylguanidinobenzoate. The assay is modified by carrying out the reaction in the presence and absence of benzamidine, a competitive inhibitor of trypsin-like proteases. The parameters of the assay are described in detail. Using this assay, our earlier demonstration of a deficiency of protease activity in plasma of patients with cystic fibrosis is confirmed. The activity, corrected for the nonspecific hydrolysis of 4-methylumbelliferylguanidinobenzoate by benzamidine, is expressed as nanomoles of 4-methylumbelliferone released per milliliter plasma. Under standard conditions, the activity in plasma activated with chloroform-ellagic acid was 127.2 +/- 23.1 in 7 controls, 70.4 +/- 11.7 in 11 obligate heterozygotes, and 48.7 +/- 16.6 in 12 patients with cystic fibrosis. Identical results were obtained when unactivated plasma was used. These data demonstrate that the judicious use of specific inhibitors such as benzamidine might be useful in assaying low levels of protease activity in crude systems.

Topics: Benzamidines; Child; Cystic Fibrosis; Guanidines; Humans; Hymecromone; Peptide Hydrolases; Trypsin Inhibitors; Umbelliferones

Protease activity, assayed using 4-methylumbelliferylguanidinobenzoate, an active site titrant of certain proteases, is significantly deficient in plasma of patients with cystic fibrosis. The deficiency can be demonstrated with both chloroform-ellagic acid activated plasma in which the proteases can hydrolyze esters of arginine and unactivated plasma in which the proteases have negligible activity towards these esters. The deficiency can also be demonstrated by separation of the proteases by isoelectric focusing on polyacrylamide gels or by chromatography on agarose columsn. Since protease deficiency can be demonstrated with unactivated plasma, the deficiency in cystic fibrosis is probably due to a reduced number of protease molecules rather than their decreased catalytic efficiency.

Topics: Adolescent; Arginine; Binding Sites; Child; Child, Preschool; Cystic Fibrosis; Guanidines; Humans; Hymecromone; In Vitro Techniques; Isoelectric Focusing; Molecular Weight; Peptide Hydrolases; Umbelliferones