Studies on the rabbit proacrosin-acrosin system

Abstract

A single molecular form ( M r = 68,000 approx) of a homogeneous preparation of rabbit testis proacrosin (S. K. Mukerji and S. Meizel (1979) J. Biol. Chem. 254, 11721–11728) was initially converted by autoactivation into an acrosin ( M r = 68,000); both gave a single activity and protein bands with similar electrophoretic mobilities ( R m = 0.25) when subjected to polyacrylamide disc gel electrophoresis on 7.5% gel at pH 4.5. Two additional bands ( R m values of 0.395 – 0.412 and 0.497 – 0.519, respectively) were noticeable only when proacrosin was activated further after attaining maximum activity. The slowest- and the fastest-moving bands were separated into two acrosin activity peaks by Sephadex G-100 gel-filtration chromatography on a calibrated column. The molecular weights of the two proteins, determined by rechromatography on the same column, was estimated to be 68,000 and 34,000, respectively. Also, sodium dodecyl sulfate-polyacrylamide disc gel electrophoresis of three acrosins gave protein bands which corresponded to molecular weights of approximately 68,000, 52,000, and 34,000, respectively. Electrophoresis data suggest that the loss of acrosin activity generally observed following prolonged activation of proacrosin is caused by self-aggregation of the M r 34,000 form of acrosin. This property was not shown by M r 68,000 acrosin. Initial acrosin ( M r = 68,000) was activated by divalent cations such as Ca 2+ and Mg 2+. The enzyme was inhibited by Zn 2+, Fe 2+, Hg 2+, and sulfhydryl blockers such as 5,5′-dithiobis(2-nitrobenzoic acid), p-hydroxymercuribenzoate, and iodoacetate, apparently due to their reaction with one out of six titratable sulfhydryl groups per mole of acrosin. Probably Zn 2+ is involved in acrosomal stabilization. The initial rabbit acrosin ( M r = 68,000) appears to be the major and most stable form, and is generated from proacrosin with little structural alteration. This may be the functionally active form which plays an essential role in mammalian fertilization.