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)

Further evidence is presented that the acrosomal proteinase acrosin exists as a zymogen precursor in freshly ejaculated boar spermatozoa. Autoactivation of proacrosin to acrosin takes place optimally at slightly alkaline pH and in the presence of calcium ions. Activation is considerably accelerated by catalytic amounts of trypsin or highly purified acrosin. A significant acceleration of the activation is also achieved by porcine pancreatic and urinary kallikrein, whereas chymotrypsin, plasmin, thrombin or urokinase showed no effect. Activation can be inhibited by p-amino-benzamidine and p-nitrophenyl p'-guanidino-benzoate. Electrophoretic analysis at different stages of activation revealed that during this process various molecular forms of acrosin are produced, apparently by limited proteolysis.
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PMID:Multiple forms of boar acrosin and their relationship to proenzyme activation. 0 66

The pH of the hamster sperm acrosome was estimated by a method based on the distribution of monoamines between membrane enclosed volumes maintaining pH gradients. A fluorescent amine, 9-aminoacridine, was used to permit both microscopic and fluorometric measurements of amine distribution. Cauda epididymal hamster sperm incubated with 9-aminoacridine accumulated the amine in the acrosomal volume. In the presence of NH4Cl or the ionophore Nigericin (compounds which discharge pH gradients) 9-aminoacridine fluorescence disappeared from the acrosome. Amine distribution between the acrosome and external volume was estimated by fluorometric measurement of sperm filtrates in the presence and absence of NH4Cl and Nigericin. These values, together with an estimated acrosomal volume of 0.4mu3 were used to calculate an acrosomal pH of less than 5. In addition, an acrosomal pH of 5 or less was obtained with 14C-methylamine. We suggest that such an acidic acrosomal pH of 5 or less could serve to inhibit the activation or autoactivation of the acrosomal zymogen proacrosin to acrosin, a trypsin-like enzyme involved in fertilization.
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PMID:The pH of the hamster sperm acrosome. 2 69

Two forms of proacrosin have been purified from ejacualted boar spermatozoa. The isolation method utilized benzamidine to inhibit the premeture activation of the zymogen and included pH precipitation, ammonium sulfate fractionation, and sodium chloride precipitation. Further purification was achieved by Sephadex G-200 FILTRATION OF THE PREPARATION AFTER IT WAS TREATED WITH 8 M urea. The overall proacrosin yield was 58% with a specific acitivity of 253 units/mg of protein. The molecular weights of the proacrosins determined by sodium dodecyl sulfate disc gel electrophoresis were 55,000 and 53,000. Proacrosin autoactivation followed the classical S-shaped activation curve and calcium was not required to obtain full activation. Time course activation studies in 0.1 M Tris/HCl, pH 8.4, at 0 degrees were monitored with sodium dodecyl sulfate-disc gel eletrophoresis and anlytical gel electrophoresis with staining techniques for protein and enzymatic activity. Under the conditions used, the zymogens were sequentially degraded to three different active specise of acrosin (alpha, beta, and gamma). The approximate molecualr weights of the acrosins were 49,000, 39,000, and 25,000 for the alpha, beta, and gamma forms, respectively. The autoactivation is concentration-dependent and can be proteolytically stimulated with either alpha- or beta-acrosin and trypsin, indicating the activation of proacrosin can via a bimolecular process.
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PMID:Boar proacrosin. Purification and preliminary activation studies of proacrosin isolated from ejaculated boar sperm. 84 51

Cock spermatozoa, like trypsin, induced a rapid fall in the viscosity of gelatin solutions but ram spermatozoa and inhibitor-free ram acrosin were ineffective. The gelatin-hydrolysing activity in cock spermatozoa was solubilized at pH 8 in the presence of calcium ions but comparable extracts of ram spermatozoa were inactive. Both extracts showed acrosin activity (assayed with benzoylarginine ethyl ester). The two catalytic activities of cock spermatozoa were each susceptible to the same trypsin inhibitors and during fractionations they were not separable. We deduce that cock acrosin, and probably some other avian acrosins, have the power to degrade dissolved gelatin while ram acrosin does not. The acrosin in cock spermatozoa, unlike that in ram spermatozoa, was inactivated at pH 2-7. Acid extracts of the former contain an inactive precursor of acrosin which undergoes spontaneous re-activation in buffers, pH 8, containing calcium ions. In this respect it resembles the proacrosin of rabbit testis.
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PMID:Comparison of neutral proteinase activities in cock and ram spermatozoa and observations on a proacrosin in cock spermatozoa. 127 37

Several studies suggest that acrosin, an acrosomal trypsin-like serine proteinase, plays a role in fertilization. The enzyme is present in an enzymatically inactive precursor form, called proacrosin and is believed to be converted to the enzymatically active form(s) through one/multiple physiological event(s) prior to the sperm penetration of the zona pellucida. Although, the proacrosin-acrosin system of several species has been well documented, the study of the enzyme system in bovine caput and cauda epididymis (where the maturation of spermatozoa occurs) has not been characterized. The present study demonstrates the quantification and partial characterization of the proacrosin-acrosin proteinase system in unpurified acrosomal extracts of bovine caput and cauda epididymal sperm. Proacrosin activation followed the sigmoidal type of activation curve. Activation experiments demonstrate that almost 80-90% of this protein exists in zymogen (proacrosin) form either in ejaculated or caput and cauda epididymal spermatozoa. Time-course activation studies showed that the zymogen in isolated spermatozoa was completely converted to active non-zymogen form in 3 and 5 h after removal from the cauda and caput regions, respectively, at pH 8.0 at 25 degrees C. This conversion was markedly inhibited by calcium in a dose dependent manner and the inhibition was reversible. On the other hand, calcium has a stimulatory effect on the hydrolytic activity of acrosin. These studies reveal that the proacrosin-acrosin system can be identified in crude extracts of bull epididymal and ejaculated sperm.
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PMID:Bovine epididymal sperm proacrosin-acrosin system: quantification and partial characterization. 150 54

Boar sperm acrosin is an acrosomal protease with trypsin-like specificity, and it functions in fertilization by assisting sperm passage through the zona pellucida by limited hydrolysis of this extracellular matrix. In addition to a proteolytic active site domain, acrosin binds the zona pellucida at a separate binding domain that is lost during proacrosin autolysis. In this study, we quantitate the binding of proacrosin to the physiological substrate for acrosin, the zona pellucida, and to a non-substrate, the polysulfated polysaccharide fucoidan. Binding was analogous to sea urchin sperm bindin that binds egg jelly fucan and the vitelline envelope of sea urchin eggs. Proacrosin was found to bind to fucoidan and to the zona pellucida with binding affinities similar to bindin interaction with egg jelly fucan. These interactions were competitively inhibited by similar relative molecular mass polysulfated polymers. Since bindin and proacrosin have distinctly different amino acid sequences, their interaction with acidic sulfate esters demonstrates an example of convergent evolution wherein different macromolecules localized in analogous sperm compartments have the same biological function. From cDNA sequence analysis of proacrosin, this binding may be mediated through a consensus sequence for binding sulfated glycoconjugates. Proacrosin binding to the zona pellucida may serve as both a recognition or primary sperm receptor, as well as maintaining the sperm on the zona pellucida once the acrosome reaction has occurred.
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PMID:The interaction of boar sperm proacrosin with its natural substrate, the zona pellucida, and with polysulfated polysaccharides. 190 70

Proteinase inhibitors have been shown to be capable of preventing various aspects of fertilization. Diisopropyl fluorophosphate (DFP) is an irreversible inhibitor of trypsin-like enzymes that is commercially available in a radiolabeled form. The experiments described herein were designed to determine if DFP would prevent sperm function in live, motile sperm and to identify the sperm proteins bound with DFP. DFP at 5 mM concentrations had no observable effect on sperm motility, but inhibited the penetration of zona-free hamster ova by human sperm (5.5%) compared to controls (33.5%). Acid extracts of motile sperm that had been incubated with radiolabeled DFP and collected by the swim-up procedure demonstrated the presence of radiolabeled DFP, and the autoradiography of the sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) gels of these extracts localized the uptake of radiolabeled DFP to proteins in the molecular weight region of the proacrosin-acrosin system. Acid-extracted proteinases from semen samples incubated with DFP demonstrated a concentration-dependent inhibition of both esterolytic hydrolysis of benzoyl-arginine ethyl ester on spectrophotometric analysis and proteolytic activity on gelatin SDS-PAGE zymography. DFP-labeled proteins were precipitated by highly specific antibodies to proacrosin. These results demonstrated that DFP is capable of inhibiting sperm function, and that it associates with the proacrosin-acrosin system in live motile sperm.
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PMID:Diisopropyl fluorophosphate labeling of sperm-associated proteinases. 211 Aug 39

Low-molecular-mass zymogen was extracted from boar spermatozoa together with proacrosin using 10% acetic acid supplemented with 10% glycerol, and was purified by the sequential use of gel filtration on Sephadex G-75 and (FPLC) reversed-phase chromatography. LMM zymogen represented approximately 5% of the latent trypsin-like activity present in the sperm extract. SDS-PAGE indicated a molecular mass of 33 kDa. The zymogen reacted with both mouse monoclonal and rabbit polyclonal antibodies to boar acrosin. Determination of the N-terminal sequence of 34 amino-acid residues revealed its identity with the known N-terminal sequence of boar proacrosin.
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PMID:Is sperminogen a modified proacrosin? Isolation, purification, and partial characterization of low-molecular-mass boar proacrosin. 211 Nov 46

Acrosin is a serine proteinase and located in a zymogen form, proacrosin, in the acrosome of the sperm. As deduced from the cDNA sequences for human and boar proacrosin, the enzyme is synthesized as a preproenzyme, preproacrosin, which contains a hydrophobic leader sequence. Using cDNA clones as probes, we have isolated the gene coding for human proacrosin from a human leucocyte genomic library and a human cosmid library, respectively. The gene contains four introns between 0.2 kb--4.5 kb in length. Similar to other serine proteinases, the coding sequence of the preproacrosin gene is spread over all the five exons of the gene and the three activesite residues His, Asp and Ser are encoded by three different exons. According to the exon-intron structure, preproacrosin is suggested to be closely related to the serine proteinase subfamily containing trypsin and kallikrein. However, the light chain of proacrosin seems to be similar to that of chymotrypsin. The coding of the serine active-site residue together with the proacrosin-specific proline-rich domain in one exon, namely exon E5, let us assume that the nucleotide sequence for the proline-rich domain was generated during evolution by intron-exon transfer from a foreign gene with subsequent intron excision. By primer extension analysis, the transcription initiation site of the preproacrosin mRNA could be assigned to the residue C at -74 nucleotides upstream from the translation initiation codon ATG. In contrast to most other eucaryotic genes, including the known testis-specific genes, typical TATA and CAAT box sequences in convential distances from the 5' end of the transcription start site could not be evaluated in the proacrosin gene.
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PMID:Nucleotide sequence and exon-intron organization of the human proacrosin gene. 211 85

In the present study, we identified cDNA clones of mouse acrosin from a testis lambda gt11 library. The deduced amino acid sequence indicates that mouse acrosin is initially synthesized as a single-chain polypeptide with a 16-residue signal peptide followed by a 23-residue light chain and then a 394-residue heavy chain; mouse acrosin zymogen contains 417 amino acid residues with a calculated molecular mass of 46,993 Da. The cDNA-derived sequence of mouse proacrosin shows a high degree of similarity with human and porcine proacrosins and major portions of bovine trypsin, including the active site residues, the recognition site for substrate, the location of 12 cysteine residues, and two potential N-glycosylation sites. The sequence homology suggests that mouse proacrosin is converted to a mature acrosin, which consists of the light and heavy chains with a combined molecular mass of 35,587 Da, by cleavage of the peptide bond between Arg23 and IIe24, and sequential removal of 23-, 26-, and 50-residue COOH-terminal segments. Using Northern blot analysis of RNAs from various mouse tissues, the acrosin gene transcript was present only in testis. The 1,800-base acrosin message was first detectable in 18-day-old testis. At the same time of testicular development, some of the acrosin mRNA was actually associated with polysomes. Also, in situ hybridization analysis suggests that the acrosin gene is expressed only in the round spermatid. Therefore, it is most likely that transcription of the mouse acrosin gene and subsequent translation of its mRNA first occur in the early stages of the round spermatid, and that the acrosin message is not under translational control.
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PMID:Primary structure of mouse proacrosin deduced from the cDNA sequence and its gene expression during spermatogenesis. 212 31


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