Biochemical characterization of the mechanism of binding of a regulatory protein to the spermatozoa- specific protein phosphatase in Swiss Albino Rat

Objective : The germ cell/tissue- specific Protein phosphatase, PP172, plays a pivotal role in sperm function. Its activity gets reduced during sperm maturation in the epididymis. Inhibition of PP172 results in motility initiation and stimulation. The aim of the present study was to determine the biochemical mechanism of PP172-sds22 binding in reference to motility status of Swiss albino rat spermatozoa. Methodology and results: The enzyme from caudal and caput sperm extracts was purified by column chromatography. PP172 from caudal spermatozoa was inactive, whereas in caput spermatozoa it was active. The DEAE-cellulose flow through fractions was next passed through an SP-sepharose column. Caudal sperm sds22 and PP172 were co-eluted in the gradient fraction. In contrast, caput sperm sds22 and PP172 were separated in the flow-through and gradient fractions, respectively. Further purification through a Superose 6 column showed that PP172-sds22 complex from caudal sperm was about 80 kDa in size. Caput sperm sds22 and PP172 were observed to be eluted at 65 kDa and 39 kDa, respectively. SDSPAGE of these purified fractions revealed that in caudal sperm, the 80 kDa species is composed of sds22 (43 kDa) and PP172 (39 kDa), suggesting an equal ratio complex between these two key proteins. PP172 bound to sds22 in this complex was observed to be inactive. Conclusion and application of findings : The results indicate that dissociation of sds22 from apparently 22 kDa protein is required for its binding and inactivation of PP172. Further studies to determine the mechanisms responsible for development of sds22 binding to PP172 during epididymal sperm maturation are in progress.