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)

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

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

Skin fibroblasts from patients with inherited adenomatosis of the large bowel (ACR-SF) possess alterations in actin microfilament (MF) organization which serve to distinguish "predisposed" cells from fibroblasts derived from normal individuals (NSF). MF bundle frequency and diameter were considerably reduced in ACR-SF compared to NSF. This deficit in MF density correlated with a 60% decline in cytoskeletal-associated actin half-life. Absence of a well-structured MF network in ACR-SF was reflected in relatively poor cell-to-substrate adhesion (as indicated by increased sensitivity to trypsin release) and extensive membrane ruffling. Unlike NSF, ACR-SF failed to develop well-defined vinculin-containing focal contacts although the cellular content of vinculin was approximately the same in both cell types. The relatively low substrate adhesivity and reduced incidence of adhesive structures (i.e., MF and associated focal contacts) which typify ACR-SF correlated with a sixfold increase in cellular plasminogen activator (PA) activity. This increased protease activity corresponded with a 50-70% reduction in the content of the PA inhibitor-like protein p50 in both the saponin-resistant undersurface matrix and the culture medium. Increased motility and reduced cell-to-substrate adhesion, involving several cellular structural elements, appear to be significant correlates of the "predisposed" phenotype in cultured fibroblasts.
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PMID:Analysis of actin microfilaments and cell-to-substrate adhesive structures in human fibroblasts from individuals genetically predisposed to colonic carcinoma. 207 Aug 20

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

In a series of experiments the influence of the trypsin inhibitors aprotinin (Trasylol) and TLCK (N-p-tosyl-L-lysin chloromethyl ketone) on the gelatinolytic activity of acrosin and motility of rabbit spermatozoa was tested. Ejaculated, highly motile spermatozoa were washed in Brackett-Medium. 12.5 to 1000 microns Aprotinin and 50 to 1000 micrograms TLCK, respectively, were added to samples of 1 ml sperm suspension: the specimens were incubated at 37 degrees C. Increasing aprotinin concentrations reduced the gelatinolytic activity of acrosin and the sperm incubation at a concentration of 1000 micrograms Aprotinin/ml sperm. Spermatozoa in all TLCK specimens were entirely immotile 1.5 hours after incubation. The gelatinolytic activity of acrosin was obviously not inhibited at any TLCK concentration. These results suggest that, under these experimental conditions, aprotinin and TLCK may impair primarily the motility spermatozoa.
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PMID:[The effect of the trypsin inhibitor aprotinin (Trasylol) and TLCK on the gelatinolytic activity of acrosin and the motility of rabbit sperm in vitro]. 247 24

The trypsin inhibitors aprotinin (Trasylol) and TLCK (N alpha-p-tosyl-L-lysine chloromethyl ketone) were administered continuously into the lumen of the cervix uteri of sexually mature rabbits by means of surgically implanted osmotic minipumps. The doses were inseminated six days after implantation of the pumps, then sacrificed two to six hours after insemination and their reproductive tracts were prepared for gelatin substrate film test and scanning electron microscopy. At a pumping rate of 50 to 100 micrograms aprotinin/h neither gelatinolytic activity of acrosin nor sperm transport were visibly inhibited. TLCK, at a pumping rate of 10 micrograms/h, did not influence the proteolytic activity of acrosin; however it seemed, presumably for its toxicity, to destroy the fine structure of epithelial surfaces in the vagino-cervical region and to impair sperm transport. These results suggest, that acrosin, under these experimental conditions, is not inhibited by aprotinin and TLCK in vivo and may play no immediate role in sperm transport in the female reproductive tract.
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PMID:[The effect of trypsin inhibitors aprotinin (Trasylol) and TLCK administered locally by osmotic pumps on the gelatinolytic activity of acrosin and the transport of sperm cells in the female reproductive tract of rabbits]. 248 90

In this paper, the characteristics of a highly stable, 34,000 molecular weight form of guinea pig (GP) acrosin are compared with those of acrosins from other mammalian species. GP acrosin, like acrosins from other species, is stable at pH 3.0, has a pH optimum of 8.0, and is inhibited by natural trypsin inhibitors and N-alpha-p-tosyl-L-lysine chloromethyl ketone. Its lack of inhibition by tosyl-phenylalanine chloromethyl ketone indicates that it has a specificity similar to trypsin but not chymotrypsin. The activity of GP acrosin was stimulated by Ca2+ below 75 mM. The enzyme was markedly inhibited by Hg2+, but only weakly inhibited by other metal cations. The disulfide reductants dithiothreitol and 2-mercaptoethanol both inhibited GP acrosin, as did the sulfhydryl reactant, iodoacetic acid. The Michaelis-Menten constant for GP testicular acrosin-catalyzed hydrolysis of the N-benzyloxycarbonyl-L-arginyl amide of 7-amino-4-trifluoromethylcoumarin at pH 8.0 was calculated from Lineweaver-Burk plots to give a value of Km = 2.0 x 10(-5) M with Vmax = 500 mumoles/min/mg protein. The corresponding lysine substrate, the N-benzyloxy-carbonyl L-lysine amide of 7-amino-4-trifluoromethyl-coumarin, had a higher Km = 4.6 x 10(-5) M and lower Vmax = 135 mumoles/min/mg protein, in accord with the substrate preference seen with other mammalian acrosins.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:Guinea pig testicular proacrosin-acrosin system: further characterization of the active enzyme. 249 19


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