Abstract
We previously reported the activation of adenylate cyclases from rat brain (Johnson, R. A., Awad, J. A., Jakobs, K. H., and Schultz, G., (1983) FEBS Lett. 152, 11-16) and from human platelets (Jakobs, K. H., Johnson, R. A., and Schultz, G. (1983) Biochim. Biophys. Acta 756, 369-375) by a factor derived from bovine sperm. In this report we describe the conditions for the extraction of the factor from bovine sperm and characteristics of its effects on adenylate cyclase which are consistent with its being a protease. The activating capacity of sperm particles was extracted from previously washed and frozen sperm into a 30,000 X g supernatant fraction by various salts, but not by the nonionic detergent Lubrol-PX. The amount of extracted factor: (a) was greatest with NH4HCO3 greater than NaCl greater than Na acetate; (b) was optimal with 0.5 M salt; (c) was not appreciably affected by the pH of the extraction buffer between pH 5.0 and 8.5; and (d) exhibited the greatest specific activity at the lower pH. The extracted sperm factor could be concentrated without loss by ultrafiltration on Amicon PM-10 membranes. The effect on adenylate cyclase of concentrated and desalted sperm extracted was inhibited 50% by various salts at 10 to 30 mM. The effects of the sperm factor to activate platelet adenylate cyclase, to block its inhibition via the alpha-adrenoceptor, and to block inhibition of stimulated forms of the enzyme by stable guanine nucleotides were prevented by protease inhibitors. A 50% reduction in the sperm factor's activation of platelet adenylate cyclase was caused by 30 nM soybean trypsin inhibitor, 30 nM alpha 2-macroglobulin, 300 nM leupeptin, 1 microM antipain, 15 microM aprotinin, and 100 microM benzamidine. Up to 3 mM phenylmethanesulfonyl fluoride was without effect on activation of the platelet cyclase by the sperm factor. The effects of the sperm factor persisted after its removal by the washing of pretreated platelet membranes and after its inactivation by the subsequent addition of leupeptin. The data strongly support the conclusion that the bovine sperm factor is a trypsin-like protease. alpha-Chymotrypsin, trypsin, and sperm acrosin were comparably effective in stimulating the platelet adenylate cyclase 5- to 8-fold, with concentrations eliciting maximal stimulation being: 200 ng trypsin/ml; 2 micrograms alpha-chymotrypsin/ml; and 2 micrograms acrosin/ml.(ABSTRACT TRUNCATED AT 400 WORDS)
Highlights
Acta 756, 369-37b5y)a factor derived from bovine data suggest that thesperm protease described here is sperm
The effects of the sperm factor to activate platelet adenylate cyclase, to block its inhibition via the aadrenoceptor,andto block inhibition of stimulated zyme which exhibits markedly greater activity with Mn2+ than with Mg2+and is insensitive to stimulation by hormones, guanine nucleotides, fluoride, cholera toxin, or forskolin [14]
The lack of responsiveness of the sperm cyclase suggested the lack of the stimulatory guanine nucleotide regulatory component (Ns)a,nd sperm particles have been used as a test system to which regulatory components from other tissues might be reconstituted [3]
Summary
A-Chymotrypsin, trypsin, and sperm acrosin were Preparation of Human Platelet Membranes-Membranes from hucomparably effective in stimulating the platelet ade- man platelets were prepared as previously described [6]with 5 mM nylate cyclase 5-to %fold, with concentrations elicit-. Protein was determined accordingto Lowry et al [8] NaCl and NH,HCOs. Previously washed and frozen bovine sperm with human serum albumians standard.Experimentswere performed (200 pl; -10s cells/ml) werealiquoted into Eppendorf tubes, were in triplicate with an intra-assay variation of less than 5% of the diluted with 1 ml of 10 mM triethanolamine HCl, pH 7.5, and were means and were repeated at least twice with results comparable to centrifuged at 30,000 X g for 10min. Effects of various salts on adenylate cyclase activated by the sperm extract Sperm factor was derived as described for Fig. 2 from washed-thenfrozen sperm. The extract was concentrated and desalted as described in the text
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