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This Article Full Text Full Text (PDF) All Versions of this Article: 69/5/1572    most recent biolreprod.103.018739v2 biolreprod.103.018739v1 Alert me when this article is cited Alert me if a correction is posted Citation Map Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal My Folders Download to citation manager Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Mishra, S. Articles by Vijayaraghavan, S. Search for Related Content PubMed PubMed Citation Articles by Mishra, S. Articles by Vijayaraghavan, S. Agricola Articles by Mishra, S. Articles by Vijayaraghavan, S.

Binding and Inactivation of the Germ Cell-Specific Protein Phosphatase PP12 by sds22 During Epididymal Sperm Maturation¹

Biological Sciences Department, Kent State University, Kent, Ohio 44242

Testis- and sperm-specific protein phosphatase, PP12, is a key enzyme regulating sperm function. Its activity decreases during sperm maturation in the epididymis. Inhibition of PP12 leads to motility initiation and stimulation. Our laboratory is focused on identifying mechanisms responsible for the decline in PP12 activity during sperm motility initiation in the epididymis. Previously, using immuno-affinity chromatography, we showed that a mammalian homologue of yeast sds22 is bound to PP12 in motile caudal spermatozoa (Huang Z, et al. Biol Reprod 2002; 67:1936–1942). The objectives of this study were to determine: 1) stoichiometry of PP12-sds22 binding and 2) whether PP12 in immotile caput epididymal spermatozoa is bound to sds22. The enzyme from caudal and caput sperm extracts was purified by column chromatography. Immunoreactive PP12 and sds22 from both caudal and caput spermatozoa were found in the flow-through fraction of a DEAE-cellulose column. However, PP12 from caudal spermatozoa was inactive, whereas in caput spermatozoa it was active. The DEAE-cellulose flow-through fractions were next passed through a SP-sepharose column. Caudal sperm sds22 and PP12 coeluted in the gradient fraction. In contrast, caput sperm sds22 and PP12 were separated in the flow-through and gradient fractions, respectively. Further purification through a Superose 6 column showed that PP12-sds22 complex from caudal sperm was 88 kDa in size. Caput sperm sds22 and PP12 eluted at 60 kDa and 39 kDa, respectively. SDS-PAGE of these purified fractions revealed that in caudal sperm, the 88-kDa species is composed of sds22 (43 kDa) and PP12 (39 kDa), suggesting a 1:1 complex between these two proteins. PP12 bound to sds22 in this complex was inactive. Caput sperm sds22 eluting as a 60-kDa species was found to be associated with a 17-kDa protein (p17). This suggests that dissociation of sds22 from p17 or some other posttranslational modification of sds22 is required for its binding and inactivation of PP12. Studies are currently underway to determine the mechanisms responsible for development of sds22 binding to PP12 during epididymal sperm maturation.

¹ Supported by NIH grant RO1 HD38520.

² Correspondence: Srinivasan Vijayaraghavan, Biological Sciences Department, Kent State University, Kent, Ohio 44242-0001. FAX: 330 672 3713; svijayar{at}kent.edu

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