Identification of Ca2+-dependent calmodulin-binding proteins in rat spermatogenic cells as complexes of the heat-shock proteins

Ca2+‐calmodulin (CaM)‐binding proteins in rat testes were characterized by assays for CaM‐binding activity using the CaM‐overlay method on transblots of electrophoresed gels and purification by gel‐filtration, ion exchange, and adsorption chromatographies. A major CaM‐binding protein complex (CaMBP)...

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Bibliographic Details
Published in:Molecular reproduction and development 2004-11, Vol.69 (3), p.316-324
Main Authors: Moriya, Megumi, Ochiai, Masanori, Yuasa, Hajime J., Suzuki, Norio, Yazawa, Michio
Format: Article
Language:English
Subjects:
Amino Acid Sequence
Animals
Biological and medical sciences
Blotting, Western
Ca2+-calmodulin-binding protein
Calcium - metabolism
Calmodulin-Binding Proteins - isolation & purification
Calmodulin-Binding Proteins - metabolism
Fluorescent Antibody Technique
Fundamental and applied biological sciences. Psychology
heat-shock protein
Heat-Shock Proteins - metabolism
Hormone metabolism and regulation
immunofluorescence microscopy
Immunohistochemistry
Male
Mammalian male genital system
Molecular Sequence Data
rat testis
Rats
Spermatids - metabolism
Spermatocytes - metabolism
spermatogenesis
Spermatogenesis - physiology
Vertebrates: reproduction
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Summary:Ca2+‐calmodulin (CaM)‐binding proteins in rat testes were characterized by assays for CaM‐binding activity using the CaM‐overlay method on transblots of electrophoresed gels and purification by gel‐filtration, ion exchange, and adsorption chromatographies. A major CaM‐binding protein complex (CaMBP) was identified and found to be comprised of three proteins with molecular masses 110, 100, and 70 kDa. Amino acid sequence analyses of lysylendopeptidase digests from these proteins indicated that all of the constituents of CaMBP are very similar to the members of the heat‐shock protein family, i.e., the 110‐kDa protein is similar to the APG‐2/94 kDa rat ischemia‐responsive protein, the 100‐kDa protein is similar to the rat counterpart of the mouse APG‐1/94 kDa osmotic stress protein, and the 70‐kDa protein is similar to the rat testis‐specific major heat‐shock protein (HSP70). Immunohistochemistry using anti‐CaMBP and anti‐CaM antibodies demonstrated that CaMBP was co‐localized with CaM in the cytoplasm of pachytene spermatocytes and nuclei of round spermatids. In addition, CaMBP, but not CaM, was localized at a high level in the residual bodies of elongated spermatids. The possible relevance of CaMBP to regulation of cell cycle progression and spermatogenesis is discussed in this paper. Mol. Reprod. Dev. 69: 316–324, 2004. © 2004 Wiley‐Liss, Inc.
ISSN:1040-452X
1098-2795
DOI:10.1002/mrd.20134