RESUMO
All of the acid (pH 4.0) extracted proacrosin from porcine epididymal spermatozoa was found to be tightly associated with a specific protein referred to as the binding protein. A combination of gel filterations and gel electrophoresis revealed that the binding protein is composed of a major 28 kd and a minor 29 kd protein. Both of the proteins were shown to be nonproteolytic by gelatin SDS-PAGE analysis and the amino acid composition analysis of the purified 28 kd protein revealed that it is not related to the proteolytic component of the proacrosinacrosin system.
Assuntos
Acrosina/metabolismo , Proteínas de Transporte/isolamento & purificação , Precursores Enzimáticos/metabolismo , Espermatozoides/metabolismo , Aminoácidos/análise , Animais , Proteínas de Transporte/metabolismo , Cromatografia em Gel , Eletroforese em Gel de Poliacrilamida , Epididimo , Masculino , Peso Molecular , SuínosRESUMO
A cell-free translation system was used to determine the molecular mass of the protein component of precursor(s) to boar proacrosin. Poly(A)(+)-mRNA was extracted from freshly excised boar testis into phenol/chloroform, precipitated in chilled (-20 degrees C) ethanol, then translated in a cell-free, reticulocyte lysate system with Tran 35S-label. Analysis of the resulting products by SDS-PAGE followed by autoradiography demonstrated multiple bands of translated proteins. Both Western blotting and immunoprecipitation with a specific polyclonal antibody to boar proacrosin yielded a single major band with a relative molecular weight of approximately 64,000. These results suggest that proacrosin (Mr = 53,000-55,000), which contains both protein and carbohydrate moieties, results from the cellular processing of a proacrosin precursor molecule.
Assuntos
Acrosina/metabolismo , Precursores Enzimáticos/metabolismo , Acrosina/genética , Acrosina/isolamento & purificação , Animais , Western Blotting , Sistema Livre de Células , Precursores Enzimáticos/genética , Precursores Enzimáticos/isolamento & purificação , Técnicas In Vitro , Masculino , Peso Molecular , Poli A/genética , Poli A/metabolismo , Testes de Precipitina , Biossíntese de Proteínas , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Suínos , Testículo/metabolismoRESUMO
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.
Assuntos
Isoflurofato , Peptídeo Hidrolases/metabolismo , Espermatozoides/enzimologia , Acrosina/imunologia , Anticorpos/imunologia , Autorradiografia , Radioisótopos de Carbono , Eletroforese em Gel de Poliacrilamida , Precursores Enzimáticos/imunologia , Feminino , Fertilização/efeitos dos fármacos , Fertilização/fisiologia , Humanos , Hidrólise , Imuno-Histoquímica , Masculino , Interações Espermatozoide-Óvulo/efeitos dos fármacos , Interações Espermatozoide-Óvulo/fisiologia , Espermatozoides/citologia , Espermatozoides/fisiologiaRESUMO
Biochemical and immunochemical methods have been used to examine the proacrosin-acrosin system of human and boar spermatozoa. Marked biochemical similarities including the relative molecular weights of proacrosin (approx. 55,000), alpha-acrosin (45,000-49,000) and beta-acrosin (34,000-38,000) were observed for both species. In addition, the time course of proacrosin autoconversion between 0 to 60 min revealed that the purified proacrosin from both species autoconverted to alpha-acrosin and then to beta-acrosin at approximately the same time intervals. Despite these apparent biochemical similarities, distinct immunological differences between the human and boar proacrosin-acrosin systems were observed. The human proacrosin antibody immunoreacted with purified human proacrosin and alpha-acrosin but not with beta-acrosin. The antibodies to boar proacrosin cross-reacted with the purified boar proacrosin, alpha-acrosin and beta-acrosin. The antibodies to human proacrosin also cross-reacted with boar proacrosin and to a weak extent with boar alpha-acrosin but not with the boar beta-acrosin. While antibodies to boar proacrosin did not react with any of the components of the human proacrosin system. Additionally, in the non-purified sperm extracts the human proacrosin antibody preparation reacted with several proteins larger than proacrosin and one with a molecular weight of approximately 34,000. In the non-purified boar sperm extracts, the antibodies to boar proacrosin only cross-reacted with the known components of the proacrosin-acrosin system suggesting a high degree of specificity. Thus, immunochemical evidence is presented that indicates there are specific structural differences which occur in the proacrosin-acrosin system of mammalian sperm.
Assuntos
Acrosina/biossíntese , Precursores Enzimáticos/biossíntese , Precursores Enzimáticos/metabolismo , Serina Endopeptidases/biossíntese , Espermatozoides/enzimologia , Acrosina/imunologia , Animais , Anticorpos Monoclonais/imunologia , Eletroforese em Gel de Poliacrilamida , Precursores Enzimáticos/imunologia , Humanos , Masculino , Espermatozoides/metabolismo , SuínosRESUMO
A recently recognized non-proacrosin zymogen referred to as sperminogen has been purified from human spermatozoa, and several of its properties have been determined. The purification procedure included acid extraction of washed ejaculated sperm at pH 3.0, followed by gel filtration of the solubilized extract over a Sephadex G-75 superfine column. The sperminogen eluted from the column in a single band that was completely separated from the proacrosin band. This separation was confirmed by a gelatin-sodium dodecyl sulfate-polyacrylamide gel electrophoresis (gelatin-SDS-PAGE) zymograph. This zymograph also demonstrated that the final sperminogen preparation contained four forms of zymogen, with molecular weights between 32,000 and 36,000. At neutral pH, the sperminogen was converted into spermin, its enzymatically active form, yielding a sigmoidal curve typical of zymogen autoactivation. The effects of several factors on the rate of this autoconversion indicate specific differences between sperminogen and proacrosin. Spermin hydrolyzed N-alpha-benzoyl-L-arginine ethyl ester (BzArgOEt), and was inhibited by lima bean trypsin inhibitor, pancreatic trypsin inhibitor, N-acetyl-L-leucyl-L-leucyl-L-argininal (leupeptin), and tosyl-L-lysine chloromethyl ketone, indicating that the enzyme has a trypsin-like specificity and probably belongs to the class of trypsin-like enzymes. Since acrosin is generally believed to be the only trypsin-like enzyme in mammalian sperm, the demonstration of human sperminogen and spermin necessitates further inquiry into the functions and the relationships between sperm proteinase systems.