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)

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

The antibodies of keratin and vimentin were used as the histochemical probes determined by the immuno-fluorescent technique to recognize the rat epididymal epithelial cells in the different ages from the connective tissue both in intact epididymides and in isolated cultured cells. It also showed that an enriched suspension of epididymal epithelial cells could be obtained by sequential digestion with 0.05% trypsin and 0.1% collagenase. The morphological characteristics were appeared during the cells in culture. Therefore the epididymal epithelial cells isolated and cultured by present methods could be used as a research model to study their functions.
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PMID:[The recognition of rat epididymal epithelial cells]. 170 22

We have studied the binding of [125I-iodo]androgen-binding protein (ABP) and of [3H]delta 6-testosterone photoaffinity-labelled ABP to receptors in the plasma membrane of rat epididymal cells in three ways: ABP binding to a Triton X-100-solubilized membrane extract, ABP binding to isolated epithelial cells in suspension and autoradiography of segments of dissected epididymides after in-vitro intraluminal injection of labelled ABP. The binding of iodinated ABP to the receptor was similar to that of photoaffinity-labelled ABP in gel filtration. The ABP-receptor complex was eluted from Superose 6 gels as an aggregate, with a molecular mass of 2000 kDa. It was separated into two peaks by sucrose gradient ultracentrifugation, with respective sedimentation coefficients of 18.4 and 9.0 s. The activity of the receptor (ABP-binding capacity/mg protein) was tenfold higher in the caput than in the cauda. The binding of ABP to the receptor was pH dependent, being almost abolished at pH less than 4. The binding at 4 degrees C of photoaffinity-labelled ABP to epithelial cells corresponded to two types of binding sites. The numbers of high-affinity and low-affinity sites per cell were 1600 and 7700 respectively; the association constants of these sites were 67.9 and 2.8 litres/nM respectively. The binding was decreased by treatment of the cells with trypsin or incubation in the presence of EDTA. The binding in vitro of labelled ABP to the epididymis epithelium reached a maximum after about 20 min at 4 degrees C. In the autoradiographic study the tracer was found to be closely associated with coated pits, coated vesicles, endosomes and pale multivesicular bodies. Treatment of rats with cycloheximide significantly reduced the uptake of the tracer. Perfusion in vitro of epididymides with chloroquine produced a fourfold increase of the tracer in endosomes and multivesicular bodies.
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PMID:Evidence that androgen-binding protein endocytosis in vitro is receptor mediated in principal cells of the rat epididymis. 193 Jun 25

Cauda epididymal guinea pig spermatozoa are arranged in rouleaux, with the sperm heads stacked one on top of the other; the plasma membranes over the apical segment of the acrosomes of adjacent sperm are linked and form non-fusigenic "junctional" zones. A complex structural and temporal sequence of membrane fusions occurs during the acrosome reaction of guinea pig sperm in rouleaux. In this study, we have devised a procedure for dispersing the rouleaux and isolating a population of single, motile guinea pig sperm, and have investigated the ultrastructural features of the acrosome reaction in single sperm to determine if the pattern of membrane fusions is different from sperm in rouleaux. The rouleaux were dispersed using trypsin, and damaged cells were removed by passing the sperm suspension through a glass bead column; a population of 70-90% motile, acrosome-intact, single sperm was obtained. Sperm were then induced to undergo lysolecithin-mediated, "synchronous" acrosome reactions, and processed for transmission electron microscopy. The acrosome reaction involved a complex sequence of membrane fusions between the plasma membrane (PM) and outer acrosomal membrane (OAM). On the convex surface of the apical segment, sheets of hybrid membrane and parallel arrays of hybrid membrane tubules formed; filaments were associated with the luminal surface of the residual OAM in these regions. Hybrid membrane vesicles were produced on the concave surface of the apical segment, but fusion was delayed relative to the convex surface. In the principal segment, branching arrays of hybrid membrane tubules formed and later vesiculated.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:Ultrastructural analysis of the acrosome reaction in a population of single guinea pig sperm. 201 5

A previous study has characterized the major 47 kDa anti-sticking factor (ASF-I) from goat cauda-epididymal plasma (Roy, N., and Majumder, G.C., Biochim. Biophys. Acta, 991:114-122, 1989). This study reports the purification and characterization of ASF-II, another anti-sticking factor from the goat epididymal plasma. ASF-II was purified to apparent homogeneity by using concanavalin A-agarose affinity chromatography, DEAE-cellulose chromatography, alumina gel adsorption, and isoelectric focussing techniques. It showed a single protein band by both non-denaturing and SDS-polyacrylamide gel electrophoresis. ASF-II showed a molecular weight of 36,000 and a sedimentation constant of 2.4S. ASF-II is largely stable to heat treatment and it is a specific glycoprotein having high affinity and specificity for its anti-sticking action. At saturating concentration (1 nM) it inhibited adhesion of nearly 50% of spermatozoa to the glass surface of the haemocytometer counting chamber. Both the protein and sugar parts of the factor are essential for the anti-sticking activity since it lost its activity completely when treated with trypsin, L-fucosidase, or mannosidase. ASF-II does not coat the glass surface and it binds to spermatozoa. Data are consistent with the view that ASF-II has not been derived from the larger ASF-I molecule due to its enzymic modifications. Both ASF-I and -II had no effect on sperm forward motility as evidenced by spectrophotometric motility assays, indicating thereby the suitability of the factors to improve the existing sperm motility assays by eliminating the possibility of cell-sticking artifacts.
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PMID:Characterization of anti-sticking factor-II from goat epididymal plasma. 209 69

Several lines of evidence have demonstrated conclusively the presence of cAMP-dependent protein kinase (ecto-RC) activity on the external surface of goat cauda-epididymal intact spermatozoa. The intact-cell ecto-kinase that caused transfer of the terminal phosphate of exogenous ATP to the serine and threonine residues of exogenous histone was specifically activated by cAMP. As well, the ecto-kinase caused phosphorylation of the synthetic peptide Kemptide. The isolated spermatozoa, before or after incubation with reaction mixture for ecto-kinase assays, were approximately 99.5% viable, as shown by the analyses of ethidium bromide fluorescence and the cytosolic marker enzymes lactic dehydrogenase and 3-phosphoglycerate kinase. The ecto-kinase activity was not due to contamination of epididymal plasma and damaged cells or to protein kinase that may have leaked from the cells. There was little uptake of ATP and histone by the cells. The intact-cell kinase activity was strongly (80-90%) inhibited by treatment with membrane nonpenetrating surface probes: p-chloromercuriphenylsulfonic acid (2 microM), diazonium salt of sulfanilic acid (DSS, 0.5 mM), and proteases such as trypsin, chymotrypsin, and pronase (each 125 micrograms/mL). Disruption of sperm plasma membrane by sonication or Triton X-100 (0.2%) caused about a fivefold increase of the intact sperm kinase activity. Highly purified sperm plasma membrane (PM) possessed ecto-kinase activity that was resolved into type I and II kinases by DEAE-cellulose chromatography, the type I isoenzyme being the major (approximately 70%) enzymic species. Treatment of the intact spermatozoa with DSS prior to isolation of PM caused a marked loss of the activities of both the isoenzymes, indicating thereby the "ecto" nature of the PM-bound type I and II kinases. Preparations of vigorously forward-motile spermatozoa with 100% intactness had approximately fourfold higher specific activity of the ecto-kinase than the "composite" cells from which the former cells were isolated. However, the profiles of the type I and II ecto-kinases of the composite, as well as forward-motile spermatozoa, were nearly identical. The data are consistent with the view that ecto-kinases may have role in the regulation of flagellar motility.
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PMID:Type I and II cAMP-dependent ecto-protein kinases in goat epididymal spermatozoa and their enriched activities in forward-motile spermatozoa. 216 Aug 33

Plasma membranes were purified from flagella of porcine cauda epididymal sperm and proteolytic regulation of bicarbonate-sensitive adenylate cyclase was studied. It was found that the epididymal sperm plasma membrane contained a trypsin-like proteinase which inactivated adenylate cyclase. Bicarbonate activates adenylate cyclase as reported previously, but, at the same time, the anions enhance the inactivation of the enzyme by the membrane-bound trypsin-like proteinase. This phenomenon is not due to the direct activation of the proteinase, but closely related to the activation of adenylate cyclase by bicarbonate. It was also found that seminal proteinase inhibitors blocked the inactivation of adenylate cyclase and maintained the bicarbonate activation of the enzyme at high level. Actually, bicarbonate keeps adenylate cyclase fully active in ejaculated sperm, because membrane-bound proteinase is completely inhibited by the seminal proteinase inhibitors. These results suggest that the interactions between membrane-bound proteinase and seminal proteinase inhibitor are involved in the regulation of the bicarbonate-sensitive adenylate cyclase system.
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PMID:Effects of a membrane-bound trypsin-like proteinase and seminal proteinase inhibitors on the bicarbonate-sensitive adenylate cyclase in porcine sperm plasma membranes. 216 77

Buffalo blood serum is a potent source of antisticking factor (ASF) that inhibits with high affinity adhesion of goat epididymal spermatozoa to the glass surface of hemocytometer counting chamber. The serum is also capable of inhibiting glass-sticking of spermatozoa of the buffalo, ram, and bull. The serum ASF activity is nondialyzable and stable to heat treatment at 100 degrees C for two minutes. The activity of the serum ASF was lost completely when treated with trypsin (50 micrograms/ml) at 37 degrees C for thirty minutes indicating the polypeptide nature of the ASF. Serum ASF activity consists of at least two factors (A and B) as shown by concanavalin A-agarose affinity chromatography. ASF-A and -B represent nearly 75% and 25% of the total serum ASF activity. ASF-B is a glycoprotein as it binds with high affinity to concanavalin A. The sera of species such as man, goat, and rat possess ASF activity.
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PMID:Antisticking protein factors in buffalo blood serum. 222 76

On terminally differentiated sperm cells, surface proteins are segregated into distinct surface domains that include the anterior and posterior head domains. We have analyzed the formation of the anterior and posterior head domains of guinea pig sperm in terms of both the timing of protein localization and the mechanism(s) responsible. On testicular sperm, the surface proteins PH-20, PH-30 and AH-50 were found to be present on the whole cell (PH-20) or whole head surface (PH-30, AH-50). On sperm that have completed differentiation (cauda epididymal sperm), PH-20 and PH-30 proteins were restricted to the posterior head domain and AH-50 was restricted to the anterior head domain. Thus these proteins become restricted in their distribution late in sperm differentiation, after sperm leave the testis. We discovered that the differentiation process that localizes these proteins can be mimicked in vitro by treating testicular sperm with trypsin. After testicular sperm were treated with 20 micrograms/ml trypsin for 5 min at room temperature, PH-20, PH-30, and AH-50 were found localized to the same domains to which they are restricted during in vivo differentiation. The in vitro trypsin-induced localization of PH-20 to the posterior head mimicked the in vivo differentiation process quantitatively as well as qualitatively. The quantitative analysis showed the process of PH-20 localization involves the migration of surface PH-20 from other regions to the posterior head domain. Immunoprecipitation experiments confirmed that there is protease action in vivo on the sperm surface during the late stages of sperm differentiation. Both the PH-20 and PH-30 proteins were shown to be proteolytically cleaved late in sperm differentiation. These findings strongly implicate proteolysis of surface molecules as an initial step in the mechanism of formation of sperm head surface domains.
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PMID:Evidence that proteolysis of the surface is an initial step in the mechanism of formation of sperm cell surface domains. 222 75

Due to the central role the acrosomal region plays in sperm-egg interactions, monoclonal antibodies (mAbs) were used to identify components of this domain in mouse sperm. Several sperm proteins that localize specifically to the anterior acrosomal region are described here in terms of electrophoretic mobility, susceptibility to proteolytic degradation, and post-translational modification during epididymal transit. Six different mAbs were used, each recognizing a distinctive antigen (Ag) or set of Ags in cauda epididymal mouse sperm: a doublet of 185/200 Kd (M42 mAb); 150-160 Kd (M5 mAb); 105 Kd (W71 mAb); 21, 35, and 60 Kd (M41 mAb); 27 and 33 Kd (W33 mAb); and 57 and 86 Kd (W108 mAb). Previously reported work implicates two of these, M42 Ag and M5 Ag, as participants in sperm-zona interaction (Saling and Lakoski: Biol Reprod 33:527-536, 1985; Saling: Dev Biol 117:511-519, 1986; and Lakoski et al.: Biol Reprod 38:221-233, 1988). Recognition of some (M42, M5, W108), but not all (W33), of the Ags by their corresponding mAbs was affected by sperm incubation with proteases (trypsin or collagenase). Evidence of post-translational modification during epididymal maturation was suggested by altered electrophoretic mobility of several of the Ags (M42, M5, W33, and W108) accompanying sperm transit from proximal to distal epididymis. Retention of sperm within the caput epididymis prevented structural alterations for the four proteins examined, indicating that spatial rather than temporal factors are critical for Ag modification in maturing mouse sperm.
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PMID:Proteins of the acrosomal region in mouse sperm: immunological probes reveal post-testicular modifications. 254 83


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