Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound   Target Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound

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Query: EC:3.4.21.4 (trypsin)
42,187 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

An acrosin inhibitor was isolated from bull seminal plasma by gel filtration on Sephadex G-50 fine and ion-exchange chromatography on CM-Sephadex. The inhibitor is a basic polypeptide (pl greater than or equal to 10.5) of molecular weight 6 200 (calculated from amino acid composition). Its N-terminal amino group is blocked. The inhibitor is not strictly specific in its effect since it also inhibits trypsin and to a lesser degree chymotrypsin, in addition to bull and boar acrosin.
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PMID:Isolation of basic acrosin inhibitor from bull seminal plasma (BUSI II). 39 4

Acidic extracts of washed, ejaculated human spermatozoa contain, besides acrosin, two proteinase inhibitors, a trypsin-chymotrypsin (elastase) inhibitor (HUSI-I) and a trypsin-acrosin inhibitor (HUSI-II). Using the indirect immunofluorescence technique these inhibitors could be localized in the spermatozoa. Ejaculated spermatozoa were treated with monospecific antibodies raised in rabbits against HUSI-I and HUSI-II, respectively, and with fluorescein-labeled IgG from goat directed against the rabbit IgG. If acetone-fixed spermatozoa were used, fluorescence appeared only in a small ring near or at the equatorial segment of the spermatozoa. After prefixation of washed spermatozoa with 0.36% formaldehyde, however, distribution of both inhibitors in the region of the acrosomal caps could clearly be demonstrated. Present results suggest that they are attached at the plasma membrane. Obviously, in the case of human spermatozoa the inhibitors are relatively easily detached together with the membrane so that prefixation is necessary to achieve proper localization.
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PMID:Localization of seminal plasma proteinase inhibitors in human spermatozoa as revealed by the indirect immunofluorescence technique. 79 87

Proteolytic activity in the acrosomes of ejaculated bull spermatozoa was demonstrated using an autoradiographic film as a gelatin substrate. Incubation of the spermgelatin adducts at +37 degrees C and 94% humidity, which was kept constant by ventilating an incubator with water-saturated compressed air, yielded reproducible results. Gelatin depolymerisation started adjacent to the posterior segment of the acrosome within 30 to 60 s after application of individual spermatozoa to the substrate membrane and, finally, increased to a white circular digestion area enveloping the entire sperm head. The observed gelatinolysis seems to be mainly caused by acrosin, the trypsin-like acrosomal proteinase. This conclusion is supported by the positive correlation (r = +0.83, P is less than or equal to 0.01) found between the mean values of the lysis areas of individual spermatozoa on gelatin films and the acrosin activity of the sperm population measured with Bz-Arg-OEt as substrate after acidic extraction of the spermatozoa. In addition, prior saturation of the substrate layers with acrosin inhibitor (SSPI-I, II) from boar seminal plasma prevented the lysis reaction. Extraction of acrosin from the spermatozoa before application to the gelatin membranes resulted in a complete loss of any proteolytic activity. If spermatozoa were stored for 4 to 6 days at +4 degrees C or -20 degrees C in Tris buffer and afterwards applied to the substrate layer, lysis areas of individual spermatozoa differed markedly. Spermatozoa from undiluted ejaculated frozen at -20 degrees C showed no proteolytic effect on gelatin films. In general, there was a high correlation (r = +0.83, P is less than or equal 0.01) between the number of "living cells" characterized by live-dead staining and the percentage of spermatozoa active on the substrate membranes.
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PMID:The lysis effect of bull spermatozoa on gelatin substrate film methodical investigations. 118 Dec 57

1. A simple method is given for isolating from ram spermatozoa a water-soluble form of acrosin (a trypsin-like enzyme) which is about 25% pure. It is free from an acrosin inhibitor which is located in the spermatozoa. 2. In the hydrolysis of N-alpha-benzoyl-l-arginine ethyl ester the degree of activation of acrosin by Ca(2+), and by some other cations, is dependent on the extent of contamination by the inhibitor. In 50mm-Tris-HCl buffer (pH8.2) activation by Ca(2+) did not exceed 40%, but acrosin that is partially inhibited may be activated by up to 300%: this is due to cation-mediated protection of acrosin against the inhibitor. 3. Increasing concentrations of buffers (e.g. Tris) also activate acrosin but at above certain buffer concentrations Ca(2+) no longer exerts an activating effect and may become inhibitory. Ca(2+) is also inhibitory when added to assay systems involving anionic buffers with chelating properties. This is due to a fall in pH. 4. The above results suggest reasons for conflicting conclusions in papers dealing with the effects of Ca(2+) on acrosin activity. 5. Inhibition of acrosin by the Kunitz pancreatic trypsin inhibitor is increased on addition of Ca(2+). Inhibitions of trypsin by the acrosin inhibitor and by the Kunitz inhibitor are insensitive to Ca(2+). 6. Like trypsin, acrosin is activated, up to 60%, by 2-methyl-propan-2-ol, dimethyl sulphoxide, and some other water-miscible solvents. Effects of cations and solvents tend to be additive and a common maximum acrosin activity can be achieved with various concentrations of solvent, salts and buffer in the assay system. Activation by solvents is increased when low concentrations of the acrosin inhibitor are present. 7. Activations of acrosin by salts and by solvents are more pronounced when the substrate is N-alpha-benzoyl-dl-arginine 2-naphthylamide. 8. K(m) values for ram acrosin (about 0.2mm) are much higher than those for trypsin, and k(cat.) values are slightly higher than those for trypsin. Considerations of the influences of ions and dimethyl sulphoxide on the activities and kinetic constants of acrosin and trypsin suggest that conformational changes are the factors mainly responsible for the reported activations of acrosin. 9. The following conclusions are reached. (a) Acrosin plays a role in the penetration of the sperm cell into the egg without becoming detached from the acrosomal membrane. (b) The enzyme is a peripheral membrane protein which may be classed as a cathepsin. (c) The susceptibility of the activity of soluble acrosin to cations and solvents points to a flexible molecule, i.e. one lacking conformational restraints imposed by association (presumably ionic) with the acrosomal membrane.
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PMID:Studies on ram acrosin. Isolation from spermatozoa, activation by cations and organic solvents, and influence of cations on its reaction with inhibitors. 119 Dec 54

1. Titration in sodium barbiturate buffer of acrosin, a serine proteinase from sperm acrosomes, with the ester substrate 4-methylumbelliferyl p-guanidinobenzoate gave rise to an incomplete 'burst' of 4-methylumbelliferone. Studies of the effects on the reaction of activators of acrosin (Ca2+, water-miscible solvents) showed that titrations carried out in barbiturate buffer containing 1M-CaCl2 and diluted with 0.2 vol. of dimethylsulphoxide produced a rapid quantitative burst within 4 min at 20 degrees C. 2. The net post-burst production of 4-methylumbelliferone was neglibible because (a) the acyl-enzyme was very stable, and (b) the slow post-burst formation of 4-methylumbelliferone (turnover of acyl-enzyme) was virtually equal to the slow photolytic destruction of 4-methylumbelliferone that was liberated during the burst. 3. The standard procedure permits titrations of 20-100pmol of acrosin, i.e. amounts normally taken for conventional rate assays, and with these amounts the impurities present in crude enzme fractions did not interfere. The burst was judged to be quantitative on the basis of comparisons with titrations of acrosin with p-nitrophenyl p'-quanidinobenzoate. 4. The burst reaction of trypsin with the 4-methylumbelliferyl ester was inhibited by high Ca2+ concentrations and by dimethyl sulphoxide. 5. The association and dissociation of complexes of both acrosin and trypsin with protein-type inhibitors (Kunitz pancreatic trypsin inhibitor and a spermatozoal acrosin inhibitor) are rather slow. It is thus possible, in certain cases, to use the ester to titrate both total enzyme in an inhibitor-enzyme mixture and net enzyme, i.e. the stoicheiometric excess of enzyme over inhibitor.
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PMID:Studies on ram acrosin. Fluorimetric titratiion of operational molarity with 4-methylumbelliferyl p-guanidinobenzoate. 119 Dec 55

A new acrosin inhibitor with a relative molecular mass of about 8000 was isolated to apparent homogeneity from ejaculated boar spermatozoa. The inhibitor is effective against boar acrosin and bovine trypsin. It interacts with polyvalent antibodies against the acrosin inhibitor from boar seminal plasma, but differs from all known acrosin inhibitors in its amino acid composition and N-terminal sequence.
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PMID:Variability of acrosin inhibitors in boar reproductive tract. 180 45

The kinetic properties and inhibitor sensitivity of human sperm phospholipase A2 (PLA2; EC 3.1.1.4) were studied. Phospholipase activity was isolated from human spermatozoa by acid extraction. Hydrolysis of dipalmitoyl phosphatidylcholine was specific to the sn-2 position. Activity was sensitive to product inhibition (60% inhibition by 0.1 mM lysophosphatidylcholine). The effects of Ca2+ and sodium deoxycholate on enzyme activity were biphasic; maximal activities were observed at 0.5 mM concentration of each agent. PLA2 was stimulated (135%) by 3% dimethylsulfoxide and was inhibited by elevated ionic strength (approximately 70% inhibition with either 0.2 M NaCl or 0.2 M KCl). Two molecular forms of PLA2 were kinetically distinguishable, one with an apparent Michaelis constant and maximal reaction velocity of 3.0 microM and 0.64 mlU/mg protein and the other with respective constants of 630 microM and 32.0 mlU/mg protein. Both forms of the enzyme were Ca2+ dependent and heat stable; however, the low-Km activity was less resistant to 60 degrees C preincubation at pH 7.5 (28% inactivation of low-Km activity after 45 min, as compared to no effect on high-Km activity). Quinacrine was a noncompetitive PLA2 inhibitor with Kis for low- and high-Km activities of 0.42 mM and 0.49 mM, respectively. Trifluoperazine (calmodulin antagonist) inhibited the high-Km activity noncompetitively (Ki = 87 microM) and the low-Km activity by a mechanism consistent with the removal of a nonessential activator. Dissociation and rate constants for inactivation of low- and high-Km activities by p-bromophenacyl bromide were 0.28 mM and 0.032 min-1, and 0.73 mM and 0.066 min-1, respectively. PLA2 was inhibited by p-nitrophenyl-p'-guanidinobenzoate, at higher concentrations (10(-4)-10(-3) M) than required to inhibit trypsinlike proteinases; p-aminobenzamidine, another potent trypsin/acrosin inhibitor, stimulated (approximately 40%) PLA2 at concentrations from 2-5 mM but inhibited PLA2 (40-50%) at a concentration of 10 mM. MnCl2 (5mM) inhibited low- and high-Km PLA2 activities by 77% and 76%, respectively. Quinacrine (0.4 mM), trifluoperazine (20 microM), p-bromophenacyl bromide (20 microM), and MnCl2 (5 mM) were tested as inhibitors of the ionophore A23187-induced human acrosome reaction. Inhibition was noted only with quinacrine (32%) and MnCl2 (93%). The effect of MnCl2 was restricted to an interaction with A23187, rather than with PLA2; p-Bromophenacyl bromide inhibited (P less than 0.05) PLA2 (29%) when added to intact spermatozoa but had no effect on the acrosome reaction. PLA2 inhibition was poorly correlated with the acrosome reaction.(ABSTRACT TRUNCATED AT 400 WORDS)
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PMID:Characterization and inhibitor sensitivity of human sperm phospholipase A2: evidence against pivotal involvement of phospholipase A2 in the acrosome reaction. 226 93

Using immunoaffinity chromatography on a Sepharose 4B column with adsorbed antibodies to the basic inhibitor in bovine organs (Kunitz-type), a proteinase inhibitor was isolated from boar seminal vesicle fluid. The isolated protein inhibited acrosin, trypsin, plasmin and chymotrypsin, but not kallikrein. Its molecular weight determined by gel filtration on Sephadex G-50 was 9,500 (+/- 500) and by SDS electrophoresis in polyacrylamide gel 12,000 (+/- 500) daltons. The protein was demonstrated by immunoprecipitation only in boar seminal vesicle fluid and seminal plasma, and by indirect immunofluorescence on ejaculated spermatozoa and in the epithelium of boar seminal vesicles. This inhibitor is the first acrosin inhibitor specific for the genital organs, which evidently belongs to the group of Kunitz type inhibitors, to be described.
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PMID:A Kunitz type of proteinase inhibitor isolated from boar seminal vesicle fluid. 293 36

A new acrosin inhibitor was isolated to apparent homogeneity from the fluid of boar seminal vesicles. The inhibitor is immunologically related to the polyvalent trypsin-kallikrein inhibitor from bovine lung known as aprotinin. A crude preparation of the acrosin inhibitor was prepared by immunoaffinity chromatography on anti-aprotinin antibodies bound to Sepharose 4B column. The inhibitor was further purified by affinity chromatography on trypsin immobilized on a Sepharose 4B column, by ion-exchange chromatography on CM-Sephadex C-25, and by reversed-phase high-performance liquid chromatography on a C18 column. The relative molecular mass (Mr) of the inhibitor is about 7,000 as estimated from dodecyl sulfate-polyacrylamide gel electrophoresis. Its amino-acid composition was determined, the sequence of the first 8 amino-acid residues from the N-terminus is Thr-Arg-Asp-Phe-Pro-Pro-Asp-Gly-...
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PMID:An acrosin inhibitor from boar seminal vesicle fluid immunologically related to the trypsin-kallikrein inhibitor (Kunitz). 320 71

Acrosin (acrosomal proteinase; EC 3.4.21.10) is a sperm-specific serine proteinase implicated in sperm penetration of the mammalian oocyte. Previously, we had shown that human acrosin, unlike human trypsin (EC 3.4.21.4), was inhibited by beta-D-fructose and related carbohydrates. The present study was undertaken to more fully elucidate the mechanism of action of fructose as an acrosin inhibitor, and to further differentiate the kinetic properties of acrosin from those of trypsin. Fructose produced a complex pattern of inhibition. At relatively low concentrations (10-60 mM), fructose acted as a competitive inhibitor with an apparent inhibition constant of 13 mM. In contrast, at high concentrations (80-320 mM), fructose behaved as a noncompetitive inhibitor, with an apparent inhibition constant of 205 mM. A Hill plot of enzyme activity as a function of fructose concentration suggested only a single binding site for fructose (slope = -0.90). The pattern of inhibition is not consistent with an enzyme containing only a single catalytic site, based either upon steady-state or rapid equilibrium assumptions; however, good agreement between observed and simulated data were obtained based upon the assumption of two catalytic sites with equal or similar binding and catalytic constants. The data suggested that fructose interacts with a single binding site (Ki = 8 mM) which alters both catalytic sites to produce an enzyme species having a higher apparent Michaelis constant and lower kcat as compared to the uninhibited enzyme. Fructose had no effect upon the rate of acrosin inactivation by either diisopropylfluorophosphate or tosyl-lysine-chloromethylketone, suggesting that neither substrate binding nor acylation were altered by this agent. The above data indicate substantial differences between the catalytic properties of human acrosin and those of trypsin.
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PMID:Evidence for multiple catalytic sites of human acrosin from kinetic evaluation of fructose-induced acrosin inhibition. 389 12


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