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Eur J Biochem. 1994 Oct 1;225(1):107-14.

Association of the regulatory subunit of a type II cAMP-dependent protein kinase and its binding proteins with the fibrous sheath of rat sperm flagellum.

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  • 1Department of Molecular Pharmacology, Albert Einstein College of Medicine, Bronx.

Abstract

Demembranated rat sperm flagellar polypeptides capable of binding the regulatory subunit (RII) of a type II cAMP-dependent protein kinase, having apparent subunit molecular masses of 120, 80 and 57 kDa were identified by an RII overlay procedure [Horowitz, J. A., Wasco, W., Leiser, M. & Orr, G. A. (1988) J. Biol. Chem. 263, 2098-2104]. In this study it is shown that all three polypeptides capable of binding RII on a solid-phase blot are tightly associated with the fibrous sheath. Purified fibrous sheath preparations were capable of binding (a) [3H]cAMP and (b) purified catalytic subunits of cAMP-dependent protein kinase forming a functional holoenzyme. The 57-kDa protein was identified as RII by photoaffinity labeling with 8-azido[32P]cAMP. This peptide was phosphorylated by the catalytic subunit of cAMP-dependent protein kinase. RII alpha was also shown to form tight, specific complexes with the fibrous sheath demonstrating the presence of functional RII alpha-binding sites. Truncated RII beta fusion proteins were used to identify the N-terminal amino acids at positions 1-50 as a primary determinant for RII-binding protein interaction. Differential extraction of adult testis with buffers containing Triton X-100, urea and sodium dodecyl sulfate revealed the presence of 80-kDa (major) and 120-kDa (minor) RII-binding proteins in particulate extracts. The 80-kDa polypeptide is only expressed at late stages of spermatogenesis, i.e. during spermiogenesis, suggesting a developmental role for RII anchoring to the sperm flagellar fibrous sheath.

PMID:
7925427
[PubMed - indexed for MEDLINE]
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