Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts:   Target Concepts:
Query: EC:3.4.21.64 (proteinase K)
 4,071 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

The proteolytic enzyme, proteinase K, has been found to destroy all vaginal cells though it does not have the same effect on spermatozoa. In cases of sexual offenses, in which a swab has been used to wipe out the vagina, the female cells and their nuclei on that swab may also contain the heads of spermatozoa. After as short a time as 30 min of proteinase K treatment, the spermatozoa that had separated from the enzymatically destroyed vaginal cells were recovered. This proteinase destruction furnishes some spermatozoa with deformed heads and a somewhat greater number of isolated tails though a sufficient number of spermatozoan heads still remain for a reliable diagnosis. For detection of spermatozoa from a vaginal swab after proteinase K pretreatment, the heads of the spermatozoa are distinctly stained by Oppitz's method. Further, on prior treatment with proteinase K, the ABO blood grouping of the spermatozoa could also be determined on the vaginal swab by using the absorption-elution technique. The resistance of the spermatozoa to proteinase K is the basis for this method.
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PMID:A demonstration of spermatozoa on vaginal swabs after complete destruction of the vaginal cell deposits. 273 65

The functional domains of the glycoproteins of the pig zona pellucida have been analysed using lectin binding, peptide mapping, and immunoblotting in conjunction with analysis by high-resolution two-dimensional polyacrylamide gel electrophoresis (2D-PAGE) and protein detection with the silver-based colour stain. Two of the pig zona pellucida glycoproteins identified in 2D-PAGE were differentially proteolysed within the intact matrix by a variety of enzymes. This proteolysis of specific proteins, however, did not affect the suprastructure of the matrix, or inhibit spermatozoa from adhering to the surface of the zona pellucida. The major glycoprotein appears to be involved in the structural maintenance of the zona pellucida because dissolution of the matrix correlated with proteolysis of this glycoprotein by proteinase K. These glycoproteins were further evaluated by lectin blotting with Ricinus communis agglutinin (RCA) and wheat germ agglutinin (WGA) before and after proteolysis of zona pellucida with trypsin. The lectins bound to all charge species of the three major zona pellucida glycoproteins. Only the most acidic components of the major glycoprotein family, which are not extensively digested, were recognized by these lectins after proteolysis. These studies provide evidence that the major glycoprotein family I of the pig zona pellucida is primarily responsible for maintaining the integrity of the matrix.
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PMID:Evidence for a role of the major glycoprotein in the structural maintenance of the pig zona pellucida. 343 Apr 58

Ejaculated porcine and human spermatozoa, hamster spermatozoa from the cauda epididymidis, isolated hamster sperm heads and hamster cytoplasmic droplets contained activity that hydrolyzed the metalloendoprotease substrate ABZ-Ala-Gly-Leu-Ala-NBA (AAGLAN). Hamster sperm heads were isolated by treating spermatozoa with proteinase K and removing sperm tails with Dowex-50W beads. Hamster sperm activity was characterized using spermatozoa from which cytoplasmic droplets were removed by sonication and centrifugation. Porcine sperm preparations were essentially free of cytoplasmic droplets, while human sperm preparations retained somewhat more droplet material. Activity from all of these sources was inhibited by the metalloendoprotease inhibitors phosphoramidon, 1,10-phenanthroline, CBZ-D-Phe and CBZ-L-Phe but was not competitively inhibited by the metalloendoprotease substrate CBZ-Ser-Leu-amide. The AAGLAN hydrolyzing activity found in intact spermatozoa of all three species had a pH optimum of 6.2, while the optimum of the hamster sperm cytoplasmic droplet activity was 7.0. In addition, hamster sperm preparations were inhibited by ZnCl2 and dithiothreitol, but were not affected by toluene, benzamidine or chymostatin. The AAGLAN hydrolyzing activity of hamster sperm preparations was reduced, but not eliminated, by dialysis. It is concluded that spermatozoa from all three species, hamster sperm heads and hamster cytoplasmic droplets contain metalloendoprotease activity. Furthermore, metalloendoprotease activity found in hamster cytoplasmic droplets is different from that found in spermatozoa.
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PMID:Biochemical studies of metalloendoprotease activity in the spermatozoa of three mammalian species. 354 55

Previous studies from our laboratory have shown that chronic exposure of the father to low doses of cyclophosphamide results in a significant increase in early embryonic death with little effect on the male reproductive system in rats. Such effects on progeny outcome are hypothesized to be mediated by an action of the drug on the nucleus of spermatozoa. The purpose of the present studies was to investigate the effects of cyclophosphamide treatment for 1 or 6 wk on the pattern of decondensation of sperm nuclei and on the sulfhydryl content of sperm nuclear proteins. Adult male rats were treated with cyclophosphamide (6.1 mg/kg/day) daily for 1 or 6 wk. Cauda epididymal spermatozoa were collected, demembranated, and then incubated with dithiothreitol (DTT) and proteinase K. The in vitro decondensation pattern of the nuclei of spermatozoa was divided into two phases: nuclear swelling and nuclear elongation. Spermatozoa from animals treated for 1 wk with cyclophosphamide showed the same decondensation pattern as those treated with vehicle (saline) alone. However, spermatozoa from animals treated for 6 wk with cyclophosphamide showed normal initial nuclear swelling but had a markedly affected nuclear elongation pattern. The changes with time in the decondensation pattern of these spermatozoa were quantitated by morphometric analysis of the head region of the spermatozoa. The nuclear area, curvature, and length of spermatozoa obtained from chronically drug-treated males were all significantly smaller than for those obtained from controls, while cell diameter was not affected.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:Effects of chronic low-dose cyclophosphamide exposure on the nuclei of rat spermatozoa. 771 Nov 81

Follicular fluid alters the physiology and behaviour of spermatozoa by increasing acrosome reaction, accelerating capacitation, attracting the spermatozoon and enhancing vigorous motion of the cell. The objective of this study was to characterize the factor(s) in human follicular fluid that causes vigorous spermatozoa motion. Follicular fluid and its fractions were tested for stimulation of spermatozoa motion using a standardized assay which employs a computerized digital imaging system. Our results show that both follicular fluid and its methanol extract stimulate vigorous spermatozoa motion. To determine the characteristics of the active factor(s), the methanol extract was subjected to molecular weight fractionation, protease digestion, microcrystalline thin-layer chromatography (TLC) and C18 reverse-phase high-pressure liquid chromatography (HPLC). The spermatozoa motion stimulator in the methanol extract was dialysable against a low molecular weight membrane (1000 Da), insensitive to boiling and low pH (3.5) and was largely inactivated by proteinase K digestion. The activity was detected near the solvent front on TLC. Using reverse-phase HPLC monitored at 254 nm (UV), the activity eluted as a single peak of activity at low methanol concentration, indicating that the activity was relatively hydrophilic. The activity in the HPLC peak lost most of its motion-stimulating ability after digestion with proteinase K. The motion stimulator could be a peptide analogous to the egg-associated peptides characterized in echinoderms which stimulate spermatozoa motion, respiration and chemotaxis.
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PMID:Preliminary characterization of a factor in human follicular fluid that stimulates human spermatozoa motion. 798 13

Treatment of male rats with low dosages of cyclophosphamide causes a dramatic increase in early embryo death among their progeny without significantly affecting the general health of the male. It is hypothesized that cyclophosphamide exerts its effects by targeting specific components of spermatozoal nuclei. The purpose of the present studies was to investigate the effects of chronic cyclophosphamide treatment on spermatozoal DNA. Two approaches were pursued. The first was to determine total DNA damage by using the alkaline elution method. The second was to study spermatozoal DNA template function by using an in vitro DNA synthesis system. Adult male rats were treated with saline or cyclophosphamide (6.1 mg/kg/day) daily for 1 or 6 wk. Cauda epididymal spermatozoa were collected and subjected to alkaline elution using DNA-DNA dot hybridization to quantify the fractionated DNA. One week of treatment with cyclophosphamide caused DNA single strand breaks that could be detected only in the presence of proteinase K in the lysis solution; no DNA cross-links were observed in the animals that received 1-wk drug treatment. In contrast, 6 wk of treatment with cyclophosphamide induced a significant increase in both DNA single strand breaks and cross-links in spermatozoal nuclei; the cross-links were attributable primarily to DNA-DNA linkages. The availability of spermatozoal DNA for template function was not affected by 1 wk of treatment with cyclophosphamide but was markedly affected after 6 wk of treatment with this drug. It is proposed that during chromatin transition processes the male genome may be in an open dynamic state with many exposed sites that are vulnerable to alkylating agents. Since there is no DNA repair during spermiogenesis, damage to the genome by alkylation at this stage may be cumulative, resulting in the production of dysfunctional germ cells.
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PMID:Damage to rat spermatozoal DNA after chronic cyclophosphamide exposure. 856 4

Differentiating lysis with proteinase K helps purify the preparations from stains on material evidences and thus permits the detection of more spermatozoa. Quantitative immunofluorescence and the Stat software helps objectively assess the results of the test by mathematical processing and more reliably detect this or that AB0 antigen in the spermatozoa.
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PMID:[The determination of the group classification of sperm by the ABO system by means of the immunofluorescence reaction in a quantitative modification]. 902 72

Reported are 2 autopsy cases in which Y-chromosomal microsatellite short tandem repeats DYS19, DYS389I and II, DYS390, and DYS393 could be haplotyped with vaginal swabs by using a Chelex 100-based DNA extraction method and dual-round polymerase chain reaction. The extraction of DNA from vaginal swabs by using this method was as efficient or more efficient than using proteinase K and phenol-chloroform extraction or the alkaline lysis methods. Y-chromosomal microsatellite short tandem repeats haplotyping based on the dual-round polymerase chain reaction method provided genotypes from all the loci determined. Although amplification of Y-chromosomal microsatellite short tandem repeats loci is not directly involved in the existence of spermatozoa, it is considerably advantageous for male individualization from body fluid mixture stains in criminal cases.
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PMID:Y-chromosomal short tandem repeats haplotyping from vaginal swabs using a chelating resin-based DNA extraction method and a dual-round polymerase chain reaction. 1296 Jun 70

Nuclei of mature mammalian spermatozoa are extraordinarily resistant to chemical and thermal injury. Additionally, decondensation of spermatozoa DNA can be accompanied by little or no visual changes of the sperm head. This study tested whether human spermatozoa could be recovered following several cycles of primer extension preamplification (PEP) and used to achieve fertilization and subsequent development of human oocytes. An attempt was also made to amplify PEP buffer after spermatozoon removal. The results demonstrate that the sperm head can be successfully recovered following treatment with KOH or proteinase K followed by one to four cycles of PEP. It is also shown that following this treatment, the spermatozoa can be injected into the oocytes and will transform into a pronucleus if the oocyte is activated by sperm cytosolic fraction. In some cases, it was also possible to obtain polymerase chain reaction signals using a buffer after sperm cells were removed following several cycles of PEP. Although sperm participation in development was confirmed by fluorescence in-situ hybridization, light microscopy revealed some degree of damage to spermatozoal chromosomes. It is concluded that pre-conceptual analysis of sperm cells may be possible, but more research is necessary to determine the optimal conditions that would preserve sperm DNA integrity while allowing accurate diagnoses.
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PMID:Toward pre-conceptual genetic analysis of human spermatozoa. 1465

Seminal plasma (SP) is known to have immunosuppressive properties in several species. Equine SP has been reported to reduce or inhibit chemotaxis, phagocytosis and complement activity in vitro. The type and amount of the SP component that suppresses sperm-polymorphonuclear neutrophil (PMN) binding in vitro was determined, and the effect of such suppression on the fertility of mares inseminated in the presence of uterine inflammation, was analyzed. Sperm cells were suspended in either SP, semen extender or a mixture of both, and each was mixed with PMN-rich uterine secretions collected at 12 h after artificial insemination (AI). SP reduced binding between spermatozoa and PMNs significantly (P < 0.05). Fertile spermatozoa were suspended in SP or semen extender and used to inseminate mares 12 h after the induction of uterine inflammation. The pregnancy rate was normal (77%) when spermatozoa were suspended in SP, but was dramatically reduced to only 5% when spermatozoa were suspended in extender. The proteins from SP, blood plasma (BP) and a skim-milk-based semen extender (skim milk extender, SME) were precipitated by ammonium sulfate, resuspended in PBS and dialyzed. The effect of the precipitated proteins on sperm-PMN binding was compared with fresh, untreated SP. Both fresh SP, and isolated SP proteins reduced sperm-PMN binding (P < 0.001). Conversely, proteins isolated from either BP or SME did not reduce sperm-PMN binding. The different concentrations of SP proteins used showed a dose-dependent suppression of sperm-PMN binding. Concentrations of 1 mg/ml SP protein significantly reduced sperm-PMN binding and 6 mg/ml reduced the binding to a level similar to that observed with fresh whole SP (P < 0.001). Finally, SP protein digested with proteinase K resulted in the complete loss of SP suppressive activity confirming that the effective component is a proteinaceous substance.
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PMID:Equine seminal plasma reduces sperm binding to polymorphonuclear neutrophils (PMNs) and improves the fertility of fresh semen inseminated into inflamed uteri. 1512 15


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