Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts:   Target Concepts:
Query: EC:3.4.21.4 (trypsin)
 42,187 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

ATP is obligatory for sperm movement. When sperm from the hamster cauda epididymis were washed and exposed to a demembranation and re-activation medium containing 0.5 or 1 mM ATP, the maximal % motility was attained almost immediately. Increasing the ATP concentration to 3 mM caused a 5 min delay in development of maximal re-activation and a change in the beating pattern as indicated by a decrease in the % of forward progressing sperm, decreased linear velocities but unchanged curvilinear velocities. The inhibition of motility initiation by 3 mM ATP was abolished either by the inclusion of trypsin (50 ng/ml) or by a brief pre-incubation of the sperm in the demembranation and reactivation medium before the addition of ATP. However, the beneficial effect of pre-incubation was prevented if trypsin inhibitor was present. This indicates that the inhibition by ATP acts via a trypsin-sensitive regulatory system, and suggests that an endogenous protease activity is necessary to overcome this. Re-activation of sperm from the caput epididymis by all three concentrations of ATP showed similar lag periods which could also be diminished by pre-incubation or trypsin. The results suggest that, in addition to developing their motile capacity during maturation in the epididymis, sperm exonemes change their sensitivity to ATP concentrations.
 ...
PMID:Temporary inhibition of the initiation of motility of demembranated hamster sperm by high concentrations of ATP. 357 May 32

The rabbit Acrosome Stabilizing Factor (ASF) is a glycoprotein synthesized in the corpus epididymis that reversibly decapacitates sperm. The effects of altering the conformation of ASF were evaluated by using a competitive enzyme-linked immunoabsorption assay (ELISA) with monoclonal antibodies that recognized either sequential or conformational determinants and/or an in vivo decapacitation assay. Heat denaturation (80 degrees C for 30 min) of affinity-purified ASF resulted in destruction of its native conformation concurrent with its loss of biological activity. Acid pH treatment of ASF also resulted in a conformational change in ASF, which caused a shift from the dimeric form (MW = 260,000) to the monomeric form (MW = 130,000). This manipulation allowed the biological activity of the monomeric form of ASF to be assayed separately from the dimer. The monomer was found to be biologically inactive. Proteolysis with trypsin or Staphylococcus-V8 treatment resulted in loss of the native conformation of the molecule, but Staphylococcus-V8 did not destroy the sequential determinant recognized in this analysis. This work indicates that conformation of the ASF macromolecule is important for its biological activity, and also provides a rapid means to evaluate potential decapacitation activity of purified ASF.
 ...
PMID:Correlation of conformational changes in the acrosome stabilizing factor (ASF) with its biological activity. 359 51

A new anionic acrosin inhibitor was found in an acidic extract of boar spermatozoa. The protein was purified by hydrophobic and ion exchange chromatography. According to gel filtration and SDS-electrophoresis the inhibitor preparation shows a molecular mass of Mr approximately 6000-7000 daltons. The isoelectric point is close to pH 4.5. It is an effective inhibitor of boar acrosin and bovine trypsin, but it does not inhibit porcine plasmin and pancreatic kallikrein or bovine chymotrypsin. An inhibitor with identical properties was found in high concentration (97% of the total acrosin inhibiting activity) in the fluid of cauda epididymis and also as a minor acrosin inhibiting component (2% of total acrosin inhibiting activity) in seminal plasma. The results indicate that binding of the inhibitor to spermatozoa may have taken place in the epididymis.
 ...
PMID:Demonstration of an anionic acrosin inhibitor in spermatozoa epididymal fluid and seminal plasma of the boar. 390 26

A method for the isolation and culture of epididymal epithelial cells obtained from pubertal and old adult rats is described. This method permits the establishment of primary cultures of these cells in monolayers from aggregates isolated from whole epididymides and major epididymal anatomical segments (caput, corpus, and cauda) after trypsin and collagenase digestions. A large number of cultured epididymal cells retain a differentiated function as demonstrated by the immunocytochemical and radioimmunoassay finding of acidic epididymal glycoprotein, a spermatozoa-coating protein secreted by the principal cells of rat epididymis. The proliferative potential of cultured epididymal cells obtained from pubertal and old adult donors can be documented by [3H]thymidine labeling and mitotic indices without significant loss of gene expression for acidic epididymal glycoprotein. Results of this study demonstrate that epididymal epithelial cells, consisting of a predominant population of principal cells, can be isolated, cultured, and maintained for up to 3 months.
 ...
PMID:Isolation, culture, and immunocytochemical characterization of epididymal epithelial cells from pubertal and adult rats. 701 41

Rat spermatozoa collected by micropuncture from the cauda epididymidis are immotile when diluted in cell-free cauda epididymidal fluid but are vigorously motile when diluted in 0.154 M NaCl. Removal of proteins from cauda fluid by heat precipitation or enzymatic digestion reduces the ability of cauda fluid to keep cauda sperm immotile. Cauda sperm diluted in trypsin-treated cauda fluid have motility initiation scores that are not significantly different (P less than 0.05) from cauda sperm motility scores after dilution in 0.154 M NaCl. Cell-free lumen fluids from the proximal epididymis have a reduced ability to enforce quiesence on cauda spermatozoa, and rete testis fluid does not keep cauda sperm immotile. However, concentrated rete testis fluid does prevent the initiation of motility. It is proposed that a proteinaceous factor, possibly of testicular origin, is important in preventing the initiation of motility while sperm reside in the distal reproductive tract of the male rat.
 ...
PMID:Sperm motility-inhibiting factor in rat epididymis. 706 14

Rat epididymal spermatozoa were capable of rapid binding to various positively charged beads, but not to beads with negative or no charge. The binding appeared to involve the end-piece and perhaps a portion of the tail. The binding was quite resistant to acid, heat, Triton X-100, sodium dodecyl sulphate and trypsin. The bound spermatozoa could be slowly released from the beads in a concentrated solution of salts, the best among these tested was 1 M-ammonium acetate. The bead-binding property of the spermatozoa was employed to develop, a new, simple and inexpensive method to isolate heads and tails without the use of an ultracentrifuge. The yield of the heads from spermatozoa of the cauda epididymis was 65 to 80% with 2 to 10% of tails contaminating while the yield of the tail was 30 to 40% with no cross contamination.
 ...
PMID:Binding of spermatozoa to positively charged beads as an inexpensive method to isolate sperm heads and tails. 720 82

Previous studies from this laboratory have identified a novel alpha-D-mannosidase on the sperm plasma membranes of several species, including man, which may have a role in fertilization. The polyclonal antibody raised against an isoform of the enzyme purified from rat epididymal fluid was found to cross-react with the alpha-D-mannosidase activity present in the detergent-solubilized spermatozoa and sperm plasma membranes. In the present study, we have used affinity-purified as well as monospecific anti-mannosidase IgG to demonstrate that the sperm mannosidase is an integral plasma membrane component of the rat sperm and is localized on the periacrosomal region of the sperm head. In addition, we demonstrate proteolytic processing of the membrane-bound alpha-D-mannosidase during maturation of spermatozoa. The membrane fractions prepared from testis, and spermatozoa from the caput, corpus, and cauda regions of the epididymis, were solubilized in SDS and resolved by SDS-PAGE. The resolved polypeptides, when subjected to Western blot analysis using affinity-purified anti-mannosidase IgG as the primary antibody, revealed the presence of three specific immunoreactive bands (apparent M(r), 135, 125, and 115 kDa) in the membranes from testis, caput, and corpus spermatozoa. However, the cauda sperm plasma membranes showed only one immunoreactive band of apparent M(r) 115 kDa. The disappearance of the 135-and 125-kDa forms and the appearance of a sharp 115-kDa band on cauda spermatozoa suggests a precursor-product relationship between various molecular forms of the enzyme. Trypsin treatment of testicular and caput sperm membranes largely converted the precursor forms to the mature (115-kDa) form. The in vitro proteolysis resulted in an elevated level of the alpha-D-mannosidase activity in the caput (but not cauda) sperm plasma membrane. Inclusion of trypsin inhibitors (benzamidine and aprotinin) largely prevented the conversion of precursor form to the mature form. These data are consistent with the observed increase in the levels of sperm enzyme activity as spermatozoa move from the caput to the cauda region and suggest that the increase is due to the conversion of enzymatically inactive/less active high molecular weight precursor forms (135 and 125 kDa) into enzymatically active mature form (115 kDa) during sperm maturation.
 ...
PMID:Rat sperm plasma membrane mannosidase: localization and evidence for proteolytic processing during epididymal maturation. 787 80

The binding of N-acetyl-beta-D-glucosaminidase from rat epididymal fluid to the surface of spermatozoa from the cauda epididymis was measured in the presence of sugars, its phosphorylated derivatives, or after treatment of the cells or the enzyme with agents that alter the integrity of proteins or carbohydrates. The binding was saturable, with a Kd in the nanomolar range, was inhibited with phosphorylated derivates of fructose, and did not depend on Ca2+, showing that it is different from the mannose 6-P-recognizing system existing in other tissues for this and other acid hydrolases. Treatment of the cells with sodium periodate or trypsin inhibited the binding, showing that a glycoprotein of the plasmalemma is involved in the affinity site. Fructose or phosphorylated derivates were not detected in the proteins of the epididymal fluid with HPLC. However, with the method used, the presence of these compounds cannot be ruled out, if among the proteins of the fluid there are only a small number of acid hydrolases containing this sugar.
 ...
PMID:Affinity sites for N-acetyl-beta-D-glucosaminidase on the surface of rat epididymal spermatozoa. 800 7

Insectivora are of special interest as the most primitive of the eutherian mammals, but essentially nothing is known of their gamete function. In this respect, the Asian musk shrew (Suncus murinus), investigated in the present study, displays many idiosyncrasies. In the epididymis, the giant acrosome undergoes further stabilization, its unusual resilience being especially evident in a "rim" created by a persistent close alignment of the outer acrosomal and overlying plasma membranes. However, until spermatozoa reached a gland on the vas deferens, no post-testicular change was demonstrable in the sperm head surface, the unusual nature of which was indicated by a dorso-ventral differentiation, by an inability to auto-agglutinate or to bind to the homologous zona pellucida, and by an insensitivity to anti-sperm immunoglobulin IgG in fresh serum. At mating, only about 1 x 10(6) spermatozoa are inseminated as far as the anterior vagina with plug formation. Within the small (6 mm) fallopian tube, the isthmus and ampulla are sharply delineated by their contractile activity and epithelial character; there is evidence of some sperm entry into isthmic crypts and a tendency for ipsilateral ovarian control of sperm transport to the tubal ampulla. The cumulus oophorus does not undergo preovulatory mucification and expansion, is characterized by persistent intercellular gap junctions, and is insensitive to hyaluronidase and trypsin. It is unclear how the compact cumulus is penetrated at fertilization. The giant acrosome contains acrosin and an unusually temperature-dependent cumulytic activity; it is intact in motile ampullary spermatozoa but appears to be discharged before reaching the zona pellucida. Since eggs were not penetrated in the presence of ampullary spermatozoa until 4-10 h after ovulation, Suncus spermatozoa spend a long period in the female before they can fertilize. The determinants of sperm function, including capacitation and the acrosome reaction (AR), may depend on a different set of controls in Suncus, perhaps as a legacy of the resilient giant acrosome. This possibility could be examined in other Crociduran and Soricine shrews selected according to acrosome size.
 ...
PMID:Distinctive features of the gametes and reproductive tracts of the Asian musk shrew, Suncus murinus. 819 63

In the preceding study (Okamura et al., 1992; Biol Reprod 47:1040-1052) we suggested that a 135-kDa protein secreted by porcine epididymis is involved in the sperm maturation. In this work, we have isolated the cDNA clone coding the 135-kDa protein in an effort to investigate its structure and function. The 135-kDa protein was purified from porcine cauda epididymal fluid. Three oligonucleotide probes were synthesized according to the amino acid sequences of N-termini of the native protein and trypsin-digested peptides. A cDNA clone hybridizing with these three probes was isolated from the cDNA library derived from the porcine proximal corpus epididymis. It encodes a novel protein with 1,006 amino acid residues in an open reading frame. Its overall amino acid sequence was significantly homologous (25.7%) to the alpha-mannosidase precursor of Dictiostelium discoideum (P34098). The 135-kDa protein could digest both p-nitro-phenyl-alpha-D-mannoside and high mannose oligo saccharide (Man8-GlcNAc2), strongly suggesting that it is an alpha-mannosidase homologue. The expression of this protein was specific to porcine and was localized to the very narrow parts of epididymis: the border of the caput and corpus epididymis. This protein may serve as a good marker for the functional differentiation in porcine epididymis. A possible role of this protein in the species-specific sperm-egg interaction is discussed.
 ...
PMID:Cloning of complementary DNA encoding a 135-kilodalton protein secreted from porcine corpus epididymis and its identification as an epididymis-specific alpha-mannosidase. 856 59


<< Previous 1 2 3 4 Next >>