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 radioassay for tosyl arginine methyl esterase activity in spermatozoa was used to compare trypsin-like activity between spermatozoa to mice and rabbits. The assay was sensitive down to 10 ng trypsin. Spermatozoa suspensions showed 10-fold lower activities in mouse sperm. The relevance of using mice for fertility studies in discussed in light of the lack of significant tryptic-like activity in mouse spermatozoa.
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PMID:Demonstration of the lack of acrosin in mouse spermatozoa by a radioassay. 444 85

Spermatozoa from several mammalian species have been dissected by chemical methods to yield free heads, tails with attached midpieces, and tails from which the mitochondrial components of the midpiece were removed. Mouse and rat spermatozoa were cleaved by brief treatment with trypsin to yield free heads and tails, while human, guinea pig, and rabbit spermatozoa were cleaved by trypsin only after incubation with 2-mercaptoethanol or dithiothreitol. Spermatozoa were also cleaved at the junction of the head and the tail by treatment with acid and base. Mitochondria were removed from intact spermatozoa or isolated tails by mechanical shear after treatment with 2-mercaptoethanol or dithiothreitol. The dissected components of spermatozoa were fractionated with good yield and high purity by density gradient centrifugation. Ultrastructural analysis indicates that proteolytic cleavage to yield separated heads and tails occurs at a specific location in the neck of the spermatozoon, leaving the basal plate attached to the head of the cell. In contrast, after acid cleavage the basal plate remains with the midpiece. Proteolytic treatment has no apparent effect on any other spermatozoan structures, whereas acid or base treatment results in damage to the plasma membrane, the acrosome, and other structures. The specificity of the proteolytic cleavage suggests that a particular protein or group of proteins may be responsible for the linkage between the sperm head and tail.
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PMID:Chemical dissection of mammalian spermatozoa. 474 21

The demembranation and reactivation of ejaculated rabbit spermatozoa have been studied. ATP, Mg, glutamate, dithiothreitol (DTT), and Tris-HCl were found to be essential for a good reactivation. With this experimental model, we investigated the effects of protease inhibitors on the reinitiation of movement by ATP and on the movement of already motile spermatozoa. Soybean trypsin inhibitor (STI) prolonged the length of reactivation by 7- to 8-fold, whereas pepstatin, antithrombin III, phenylmethylsulfonyl fluoride (PMSF), and alpha-1-antitrypsin had no significant effect. Aprotinin (1.5 micrograms/ml) and leupeptin (50 micrograms/ml) completely prevented the reinitiation of movement by ATP; aprotinin at the same concentration even blocked the movement of motile spermatozoa. A tissue-specific seminal plasma factor could also prevent both the reinitiation of movement and the movement of motile spermatozoa. However, it took 2-3 times the amount of seminal plasma to stop the movement than to prevent the reinitiation of movement. The inhibitor in the seminal plasma is most probably not a protease or an aprotinin-like protease inhibitor since a partially purified preparation of the seminal plasma inhibitor does not hydrolyze a trypsin substrate, is not inhibited by protease inhibitors and has no significant capacity to inhibit trypsin. The data suggest that aprotinin and the seminal plasma inhibitor block movement through different mechanisms. Aprotinin and the seminal plasma inhibitor represent two new tools to study the regulation of sperm movement.
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PMID:Aprotinin and a seminal plasma factor inhibit the motility of demembranated reactivated rabbit spermatozoa. 619 May 16

Exposure of the spheroidal spermatozoa of Ascaris suum to an extract of the male accessory gland causes their transformation into ameboid cells. We have investigated the mechanism of this transformation, also termed activation, by labeling the proteins of accessory gland extracts with fluorescein isothiocyanate (FITC) or [125I], followed by qualitative localization of the sperm activating substances (SAS) and quantitative measurements of [125I]-SAS binding. Fluorescent patches of FITC-conjugated SAS were localized at the spermatozoan surface and were concentrated primarily at the posterior region. Few fluorescent patches were detectable in the region of the newly formed pseudopodia following transformation. Although spermatozoan transformation occurs within 2-5 min after exposure to SAS, the fluorescent patches became more distinct after a minimum of 8 min and reached maximum density at 15-30 min. Spermatozoa activated with [125I]-SAS became radioactively labeled in direct proportion to the amount of available [125I]-SAS until a saturation level was reached. SDS-polyacrylamide gel electrophoresis combined with autoradiography indicated that the cells bind two SAS components, of small (9,000 MW) and large (56,000 MW) sizes. These same two components were also detectable in a membrane fraction, obtained by differential centrifugation, of the spermatozoa after incubation with [125I]-SAS. binding of the two SAS components was not inhibited by preincubation of the spermatozoa with trypsin or Concanavalin A; however, the 56,000 MW component of SAS was not detectable in autoradiograms of spermatozoa incubated with periodic acid (1.6-10 mM) treated SAS. Such cells also failed to transform into ameboid spermatozoa. These results indicate that the two components of SAS that bind to the spermatozoan surface are possibly responsible for inducing the cell transformations associated with activation.
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PMID:Surface receptors: are they involved in transformation of spermatozoa of Ascaris? 626 Mar 62

Sialoglycoproteins of rat epididymal fluid and spermatozoa were radiolabelled by the NaIO4/KB3H4 method. At least 10 sialoglycoproteins of the epididymal fluids could be consistently demonstrated by polyacrylamide gel electrophoresis in sodium dodecyl sulphate. Two major ones (Mr 21000 and 66000) were present in the fluids of the caput and cauda epididymidis. Two (Mr 28000 and 40000) were found only in the former and two (Mr 32000 and 42000) only in the latter. There were at least 11 sialoglycoproteins bound to the epididymal spermatozoa. During epididymal transport, 8 sialoglycoproteins on the spermatozoa decreased, one (Mr 48000) remained constant and one (Mr 31000) increased in amount. During sperm maturation, some sperm-bound sialoglycoproteins, especially 3 of small molecular weights, became more resistant to the treatments with neuraminidase, trypsin and Triton X-100.
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PMID:Characterization of sialoglycoproteins of rat epididymal fluid and spermatozoa by periodate-tritiated borohydride. 629 82

Bound sialic acids on rat spermatozoa were assayed by oxidation with 1 mM-NaIO4 at 0 degree C, liberating C-9 as formaldehyde which was further quantitated using 3-methyl-2-benzothiazolinone. The mean +/- s.d. (n = 20) content of bound sialic acids of spermatozoa from the caput and cauda epididymidis was 50.9 +/- 8.0 and 25.2 +/- 3.8 nmol/10(8) spermatozoa respectively. About 85% of the former and 75% of the latter could be extracted by 1% Triton X-100 and 2 mM-dithiothreitol. About 70% of the former and 20% of the latter were released by neuraminidase from Vibrio cholerae. About 40% of the former and 30% of the latter were sensitive to trypsin. During sperm maturation, the decrease in the total bound sialic acids was due to the decrease in the neuraminidase-sensitive but not the neuraminidase-resistant sialic acids.
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PMID:Direct assay of bound sialic acids on rat spermatozoa from the caput and cauda epididymidis. 630 Mar 82

Two types of trypsin-like proteases, spermosin and acrosin, have been highly purified from spermatozoa of the ascidian (Prochordata) Halocynthia roretzi by a procedure including diethylaminoethylcellulose chromatography, Sephadex G-100 gel filtration, and soybean trypsin inhibitor-immobilized Sepharose 4B chromatography. Each purified preparation was judged to be homogeneous on the basis of chromatographic analysis and sodium dodecyl sulfate-gel electrophoresis. The molecular weights of spermosin and acrosin were estimated to be 27,000 and 32,000-34,000, respectively, by gel electrophoresis in sodium dodecyl sulfate. The isoelectric point of the former was 6.5, while that of the latter was 5.5. Non-ionic detergents, e.g. Brij 35, showed marked stabilizing effects on the purified enzymes. Both of these enzymes had pH optima between 8.5 and 9.0, and their activities were enhanced by the addition of calcium chloride. The enzymes were inhibited by diisopropyl fluorophosphate, phenylmethanesulfonyl fluoride, leupeptin, antipain, soybean trypsin inhibitor, aprotinin, ovomucoid, valyl-prolyl-arginyl-chloromethane, glycyl-valyl-arginyl-chloromethane, p-aminobenzamidine, benzamidine, zinc chloride, and mercuric chloride. Lima bean trypsin inhibitor and tosyl-lysyl-chloromethane strongly inhibited acrosin, but not spermosin. While the substrate specificity of acrosin was rather broad, that of spermosin was very narrow; the latter enzyme hydrolyzed only t-butyloxycarbonyl-valyl-prolyl-arginine 4-methylcoumaryl-7-amide among 12 peptidyl-arginine (or lysine) 4-methylcoumaryl-7-amides tested. Thus, the ascidian spermatozoa possess at least two proteases, acrosin and spermosin; the former shows the properties closely related to those of mammalian acrosin (EC 3.4.21.10), but the latter is a unique type of acrosin-like enzyme in respect to the substrate specificity and inhibitor susceptibility.
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PMID:Purification and characterization of two types of trypsin-like enzymes from sperm of the ascidian (Prochordata) Halocynthia roretzi. Evidence for the presence of spermosin, a novel acrosin-like enzyme. 636 18

Ten kinds of argininal-containing compounds were examined for their inhibitory effects on the fertilization of the solitary ascidian and on the activities of acrosin and spermosin, trypsin-like proteases isolated from spermatozoa of this animal. Benzyloxycarbonyl-Val-Pro-argininal (I) and benzyloxycarbonyl-Phe-Leu-argininal (II) showed the strongest inhibition on the fertilization. Leupeptin (acetyl-Leu-Leu-argininal, III) was ranked next (I, II greater than III). The activity of ascidian acrosin was susceptible to most of the compounds, among which II was the best inhibitor and followed with I and III (II greater than I, III). Spermosin suffered significant inhibition only with I and II (I greater than II). These results suggest that not only acrosin but also spermosin is involved in fertilization of the ascidian.
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PMID:Evidence for the participation of two sperm proteases, spermosin and acrosin, in fertilization of the ascidian, Halocynthia roretzi: inhibitory effects of leupeptin analogs on enzyme activities and fertilization. 638 Nov 75

Follicular fluid from different mammalian species possesses two factors responsible for the induction of capacitation: a sperm-stimulating factor and an acrosomal reaction-inducing factor. The glycosamineglycan-sulfate (GAGs) extracted from pig follicular fluid induce acrosome reaction in pig spermatozoa which exhibit no morphological difference between the GAGs-induced reaction and the natural one. Acrosomal reaction commenced 30 min after the addition of GAGs and depended on GAGs concentration reaching 80% of acrosomal reacted spermatozoa after 6 hr of incubation with 7 mg of GAGs/ml. Chemical composition differs with the chemical data that characterize them as proteoglycans since those we obtained were practically protein free (2%). Another difference resides in the uronic acid content, which is almost twofold higher (59%). Electron microscope observations of the acrosomal reacted spermatozoa revealed that the addition of 10 mg/ml of trypsin soybean inhibitor did not interfere with any of the acrosomal reaction steps. The active capacitating factors may also originate from the follicular fluid released into the genital tract during ovulation.
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PMID:Glycosamineglycan sulfate as acrosomal reaction-inducing factor of follicular fluid. 650 28

The ejaculated porcine spermatozoa were fractionated into the cytosol, membrane, midpiece plus tail (flagella) and head fractions, and their adenylate cyclase activities were measured. About 65% of the total activity was located in the flagella fraction. For all the fractions, Mn2+-dependent adenylate cyclase activity was about 20 times higher than Mg2+-dependent activity, and guanine nucleotides, fluoride, and other reagents tested did not activate adenylate cyclase. The results suggest that the GTP-dependent regulatory subunit is absent in porcine spermatozoa. The porcine seminal plasma was found to stimulate the adenylate cyclase activity in spermatozoa. The stimulating factor in porcine seminal plasma was partially purified by gel filtration and the molecular weight of the factor appeared to be between 200 and 300. The partially purified factor is heat stable and is not inactivated by treatment with Pronase, trypsin, phospholipase A2 or D but is inactivated by acid hydrolysis. It is easily soluble in water, partially soluble in methanol, and insoluble in ethanol, ethyl ether, chloroform, or acetone. The activation of sperm adenylate cyclase by the factor occurred without a time lag. The activating effect was dose-dependent, saturated at high dose, and ascribed to the increase of the maximal velocity (Vmax). The effect of the factor appears to be limited to adenylate cyclase in spermatozoa; the factor activated adenylate cyclase both in porcine and bovine spermatozoa but failed to activate those in other porcine tissues. The factor was shown to activate the enzyme not only in the ejaculated spermatozoa but also in the epididymal sperm. The factor was also found to elevate the cAMP level in the intact porcine spermatozoa. The factor enhanced the motility of corpus and cauda epididymal spermatozoa. These findings indicate the possibility that this factor initiates the spermatozoan motility upon ejaculation through directly activating adenylate cyclase.
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PMID:Activation of spermatozoan adenylate cyclase by a low molecular weight factor in porcine seminal plasma. 663 Feb 19


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