Several studies suggest that acrosin, an acrosomal trypsin-like serine proteinase, plays a role in fertilization. The enzyme is present in an enzymatically inactive precursor form, called proacrosin and is believed to be converted to the enzymatically active form(s) through one/multiple physiological event(s) prior to the sperm penetration of the zona pellucida. Although, the proacrosin-acrosin system of several species has been well documented, the study of the enzyme system in bovine caput and cauda epididymis (where the maturation of spermatozoa occurs) has not been characterized. The present study demonstrates the quantification and partial characterization of the proacrosin-acrosin proteinase system in unpurified acrosomal extracts of bovine caput and cauda epididymal sperm. Proacrosin activation followed the sigmoidal type of activation curve. Activation experiments demonstrate that almost 80-90y0 of this protein exists in zymogen (proacrosin) form either in ejaculated or caput and cauda epididymal spermatozoa. Time-course activation studies showed that the zymogen in isolated spermatozoa was completely converted to active non-zymogen form in 3 and 5 h after removal from the cauda and caput regions, respectively, at p H 8.0 at 25 "C. This conversion was markedly inhibited by calcium in a dose dependent manner and the inhibition was reversible. O n the other hand, calcium has a stimulatory effect on the hydrolytic activity of acrosin. These studies reveal that the proacrosinacrosin system can be identified in crude extracts of bull epididymal and ejaculated sperm.

An acrosomal fraction, termed the ALM (acrosomal lamina complex) was isolated from hamster cauda epididymal spermatozoa that contain specific domains of the acrosomal matrix and an adherent detergent-insoluble complex, termed the acrosomal lamina, which is derived from the outer acrosomal membrane. By SDS-PAGE, the ALM fraction exhibited two major polypeptides of Mr=29,000 (ALM29) and Mr=22,000 (ALM22). We have also shown that an ALM complex binds both acrosin and N-acetylglucosaminidase (NAGA) in a dose-dependent manner and acrosin binds to the ALM29 polypeptide only. The objective of the present study is to investigate the binding efficiency of acrosin to the V8 protease-generated peptides of ALM29 and ALM22 polypeptides, to examine the presence of phosphate groups on both ALM29 and ALM22 polypeptides and to identify the binding competency of dephosphorylated ALM29 polypeptide to acrosin. Diagonal gel electrophoresis was performed to investigate whether both ALM29 and ALM22 polypeptides are joined by disulfide bridge(s). Both polypeptides migrate on a diagonal line with a comparable Rf in both non-reduced and reduced conditions suggesting that both polypeptides are not joined by disulfide bridge(s). All V8 protease-digested peptides of ALM29 and ALM22 showed immunoreactive bands when stained with anti-ALM22 antibody suggesting that all peptides are antigenically related family. Both ALM29 and ALM22 polypeptides were stripped out of the ALM complex by high pH (pH-11) extraction and were treated with alkaline phosphatase followed by immunoblot analysis. Both ALM29 and ALM22 polypeptides showed a reduction in size (~3 kDa) by alkaline phosphatase treatment. A sedimentation assay was employed to determine whether alkanine phosphate treated ALM29 polypeptide possesses acrosin binding. Dephosphorylated ALM29 polypeptide revealed a significant reduction (~50%) in acrosin binding in comparison with acrosin to a native ALM29 polypeptide. Our studies conclude that both ALM29 and ALM22 polypeptides are phosphorylated and demonstrate the role of phosphate group of ALM29 polypeptide in acrosin binding.