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 constitue nts of skeletal-muscle junctional-face membrane (JFM), we identified a 45 k Da protein. Its N-terminal amino acid was blocked, but the amino acid seque nce obtained from several peptides after proteolytic treatment did not sign ificantly match that of any protein present in the SwissProt and NCBI(Natio nal Center for Biotechnology information) databases. We synthesized a pepti de whose sequence matched that of one of the peptides obtained after CNBr c leavage 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 th e 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 b oth in neonatal and adult skeletal-muscle sarcoplasmic reticulum; (ii) is s electively localized in the JFM; (iii) is not present in microsomes obtaine d from rabbit heart, liver or kidney. Immunoprecitation with anti-(45 kDa p rotein) antibody indicates that the 45 kDa protein is part of a complex whi ch can be phosphorylated ill vitro by the catalytic subunit of protein kina se A.