Phospholipid vesicle stimulation of proacrosin activation.

Abstract

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.Acrosin (EC 3.4.21.10) is an acrosomal proteinase thought to digest a path for the spermatozoon through the zona pellucida of the ovum, a process that is required for fertilization (1). However, freshly ejaculated spermatozoa contains greater than 95% of the potential acrosin activity as proacrosin, the enzymatically inactive zymogen (2, 3). Interest in this enzyme has been enhanced by the observations that acrosin inhibitors prevented fertilization both in vitro (4, 5) and in vivo (6, 7). Since fertilization does not occur in the absence of enzymatically active acrosin, the conversion of proacrosin to acrosin is of critical importance in the reproductive process.In a previous communication (8), possible mechanisms for proacrosin activation were examined. The results suggested that for highly purified boar proacrosin, the zymogen appeared to have the capability to autocatalytically generate acrosin from proacrosin. However, factors that influence the activation of the zymogen have received only minimal attention. Calcium (9), zinc (9), and polyamines (10) have been demonstrated to inhibit the activation process in varying degrees. Immunological (11) and biochemical fractionation techniques (12, 13) indicated that enzymatically active acrosin was bound to the inner acrosomal membrane, suggesting that proacrosin was also membrane bound and that the activation of proacrosin occurred on the membrane. However, the effect of membranes on the activation process has not been evaluated. In this communication we demonstrate that aqueous phospholipid dispersions, in the form of anionic single bilayer vesicles, enhanced the autocatalytic conversion of highly purified boar proacrosin to acrosin. It appears that phospholipid bilayer vesicles may be used as a model membrane system to study the mechanism of proacrosin activation in vitro. MATERIALS AND METHODSProacrosin was isolated from ejaculated boar spermatozoa by extraction at pH 3.0 followed by pH 5.6 fractionation, ammonium sulfate and sodium chloride precipitation, and Sephadex G-75 chromatography in the presence of 8 M urea (8). TheThe costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U. S. C. §1734 solely to indicate this fact. 149 purity of the proacrosin preparation was es...

Fertilization: A Uterine Glycosaminoglycan Stimulates the Conversion of Sperm Proacrosin to Acrosin

“…3.2.1.31) is induced by androgens such as testosterone (4, 6) when normal pituitary function is available (7). The response occurs in the epithelial cells of the proximal part of the convoluted tubule (8), and results from a dramatic increase in the rate of enzyme synthesis (4).…”

“…Anionic phospholipid vesicles also stimulate proacrosin conversion to acrosin (7). However, the uterine factor is not associated with subcellular organelles, nor is it a phospholipid, for it remains in the supernatant fluid after ultracentrifugation (2 hours at 150,000g), is not ether-extractable, and is resistant to digestion with phospholipase C (E.C.…”

Acrosin, the peculiar sperm-specific serine protease

“…The affinity of acrosin to artifical and natural membranes (Brown and Hartree 1976;Parrish et al 1978;Straus et al 1981) plus the capacity of phospholipid vesicles to promote the autoactivation of the zymogen proacrosin (Parrish et al 1978) indicate an association of the protease with the acrosomal membranes. Further evidence for this topography has been given by the immunohistochemical localization of acrosin activity at the surface of acrosome-reacted spermatozoa, implying the attachment of acrosin to the inner acrosomal membrane (Schill and Wolff 1974;Green and Hockaday 1978).…”

Association of proacrosin with phospholipid membranes

“…Anionic liposomes have also been shown to cause a marked acceleration in the spontaneous autoproteolytic conversion of proacrosin into acrosin (Parrish et al, 1978). Presumably this stimulation of zymogen conversion involves an electrostatic binding interaction between the zymogen and membrane surface similar to that observed with acrosin.…”

“…Direct measurements of proacrosin-liposome binding are complicated by the extreme instability of the zymogen in the presence of anionic phospholipids (Parrish et al, 1978). Accordingly, a sedimentation technique was developed to rapidly assess protein-liposome binding and was used in conjunction with benzamidine, an inhibitor of proacrosin conversion (Zahler & Polakoski, 1977;).…”