Identification of a novel 45 kDa protein (JP-45) from rabbit sarcoplasmic-reticulum junctional-face membrane

Using a biochemical/immunological approach to analyse the protein constituents of skeletal-muscle junctional-face membrane (JFM), we identified a 45 kDa protein. Its N-terminal amino acid was blocked, but the amino acid sequence obtained from several peptides after proteolytic treatment did not sign...

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Bibliographic Details
Published in:Biochemical journal 2000-10, Vol.351 Pt 2 (2), p.537-543
Main Authors: Zorzato, F, Anderson, A A, Ohlendieck, K, Froemming, G, Guerrini, R, Treves, S
Format: Article
Language:English
Subjects:
Age Factors
Animals
Animals, Newborn
Blotting, Western
Catalytic Domain
Chromatography, Agarose
Cyclic AMP-Dependent Protein Kinases - metabolism
Electrophoresis, Polyacrylamide Gel
Immunoblotting
Kidney - metabolism
Liver - metabolism
Membrane Proteins - biosynthesis
Membrane Proteins - chemistry
Membrane Proteins - isolation & purification
Microsomes - metabolism
Muscle, Skeletal - chemistry
Myocardium - metabolism
Peptides - chemistry
Phosphorylation
Precipitin Tests
Rabbits
Sarcoplasmic Reticulum - chemistry
Trypsin - metabolism
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Summary:Using a biochemical/immunological approach to analyse the protein constituents of skeletal-muscle junctional-face membrane (JFM), we identified a 45 kDa protein. Its N-terminal amino acid was blocked, but the amino acid sequence obtained from several peptides after proteolytic treatment did not significantly match that of any protein present in the SwissProt and NCBI (National Center for Biotechnology Information) databases. We synthesized a peptide whose sequence matched that of one of the peptides obtained after CNBr cleavage of the 45 kDa protein; the peptide was conjugated to a carrier and used to raise antibodies. The antiserum was used to study in more detail the biochemical characteristics of the novel 45 kDa protein. Analysis of the proteins present in different subcellular membrane fractions show that the novel 45 kDa polypeptide: (i) is an integral membrane constituent present both in neonatal and adult skeletal-muscle sarcoplasmic reticulum; (ii) is selectively localized in the JFM; (iii) is not present in microsomes obtained from rabbit heart, liver or kidney. Immunoprecitation with anti-(45 kDa protein) antibody indicates that the 45 kDa protein is part of a complex which can be phosphorylated in vitro by the catalytic subunit of protein kinase A.
ISSN:0264-6021
1470-8728
DOI:10.1042/0264-6021:3510537