Proacrosin Activation Research Articles

Phospholipid vesicle stimulation of proacrosin activation.

Aqueous dispersions of synthetic phospholipids, in the form of anionic, single bilayer vesicles, were observed to stimulate the appearance of acrosin esterase activity from its zymogen precursor, proacrosin. Enzymatic activity measurements, in parallel with polyacrylamide disc gel electrophoresis in the presence of sodium dodecyl sulfate, indicated that the enzymatic activity produced had resulted from the conversion of proacrosin to acrosin (EC 3.4.21.10), and not from the direct stimulation of a possible proacrosin esterase activity. It is suggested that such bilayer lipid vesicles can be used as a model membrane system to study the activation of proacrosin in vitro.

Effect of polyamines on the activity of acrosin and the activation of proacrosin.

Polyamines, especially spermine, were demonstrated to stimulate both the esterase activity and proteinase activity of acrosin, a critical enzyme in the fertilization process. The stimulation was demonstrated to result from an increase in the maximal velocity, with little effect on the affinity of the enzyme for substrate. Spermine also protected acrosin against autoproteolytic degradation. There was, however, no stimulation or stabilization of trypsin activity. Although polyamines stimulated the activity of acrosin, they inhibited the autocatalytic activation of proacrosin, the zymogen form of the enzyme. Spermine produced the greatest stimulation of proteinase activity and the greatest inhibition of proacrosin activation. This is the first report of polyamine stimulation of a proteolytic enzyme and inhibition of zymogen activation. These results suggest that polyamines could serve as in vivo modulators of the proteolytic activity of acrosin and the activation of proacrosin.

Boar proacrosin. Purification and preliminary activation studies of proacrosin isolated from ejaculated boar sperm.

Two forms of proacrosin have been purified from ejacualted boar spermatozoa. The isolation method utilized benzamidine to inhibit the premeture activation of the zymogen and included pH precipitation, ammonium sulfate fractionation, and sodium chloride precipitation. Further purification was achieved by Sephadex G-200 FILTRATION OF THE PREPARATION AFTER IT WAS TREATED WITH 8 M urea. The overall proacrosin yield was 58% with a specific acitivity of 253 units/mg of protein. The molecular weights of the proacrosins determined by sodium dodecyl sulfate disc gel electrophoresis were 55,000 and 53,000. Proacrosin autoactivation followed the classical S-shaped activation curve and calcium was not required to obtain full activation. Time course activation studies in 0.1 M Tris/HCl, pH 8.4, at 0 degrees were monitored with sodium dodecyl sulfate-disc gel eletrophoresis and anlytical gel electrophoresis with staining techniques for protein and enzymatic activity. Under the conditions used, the zymogens were sequentially degraded to three different active specise of acrosin (alpha, beta, and gamma). The approximate molecualr weights of the acrosins were 49,000, 39,000, and 25,000 for the alpha, beta, and gamma forms, respectively. The autoactivation is concentration-dependent and can be proteolytically stimulated with either alpha- or beta-acrosin and trypsin, indicating the activation of proacrosin can via a bimolecular process.

Benzamidine as an inhibitor of proacrosin activation in bull sperm

Epididymal and ejaculated sperm contain a zymogen form of acrosin (acrosomal proteinase, EC 3.4.21.10) which is converted to active enzyme prior to fertilization. Benzamidine at concentrations greater than 10 mM has been shown to inhibit the conversion of proacrosin to acrosin. Based on this inhibition, a procedure was developed for extracting and quantitating the proacrosin content of bull sperm. Sperm were isolated from semen and washed by centrifugation through 1.3 M sucrose and the outer acrosomal membrane removed by homogenization. When 25 mM benzamidine was added to the semen and wash solutions, 98% or more of the acrosin activity in the sperm homogenate was present as proacrosin. Proacrosin can be extracted from the sperm homogenate by dialysis at pH 3, which solubilized the proenzyme and removed benzamidine. Benzamidine has been useful in isolating proacrosin and provides a new method for studying the activation of proacrosin in intact sperm. Neutralization of sperm extracts, after removal of benzamidine, resulted in rapid activation of proacrosin with a pH optimum of 8.5, and activation was complete within 15 min over a pH range of 7.0 to 9.5. Rapid activation also occurred during the washing of sperm in the absence of benzamidine, and this activation correlated with a swelling of the acrosomal membrane. This rapid activation appears to result from a small amount of acrosin activity consistently present in the sperm extract. These results indicate an autocatalytic conversion of proacrosin to acrosin and suggest that disruption of the acrosomal membrane may trigger this activation.

Acrolysin, the aminoproteinase catalyzing the initial conversion of proacrosin to acrosin in mammalian fertilization

Extracts of mammalian sperm acrosomes and testes contain a proteinase, acrolysin, that hydrolyzes the aminopeptide bonds of hydrophobic aminoacyl residues. Acrolysin has hydrolytic specificity and properties similar to the proteinase, thermolysin (E.C. 3.4.24.4), that also catalyzes the conversion of proacrosin to acrosin (E.C. 3.4.21.10). Acrolysin is the apparent initiator of proacrosin activation in mammalian fertilization.

Occurrence of multiple forms of bull and ram acrosin during proenzyme activation and inhibition of activation by p-nitrophenyl p'-guanidinobenzoate.

Proacrosin was extracted from freshly ejaculated bull and ram spermatozoa with acidic buffer solution pH 3.0, and was partially purified by gel filtration to remove acrosin inhibitors. The occurrence of multiple active forms during proacrosin activation at pH 7.8 was monitored by active enzyme staining of samples after polyacrylamide gel electrophoresis at pH 3.8. For comparison, the protein pattern of such activation samples was also determined after sodium dodecylsulfate-polyacrylamide gel electrophoresis. Proacrosin activation was completely prevented in the presence of 10(-4) M p-nitrophenyl p'-guanidinobenzoate.