Purification, characterization, and molecular cloning of a 60-kDa phosphoprotein in rabbit skeletal sarcoplasmic reticulum which is an isoform of phosphoglucomutase.

Y.S Lee,A.R Marks,N Gureckas,R Lacro,B Nadal-Ginard,D.H Kim

doi:10.1016/s0021-9258(19)36800-0

Abstract

A 60-kDa substrate of calmodulin-dependent protein kinase in rabbit "heavy" skeletal sarcoplasmic reticulum (SR) was characterized by purification and cDNA cloning. Purification was achieved by column chromatography using DEAE-Sephacel, heparin-agarose, and hydroxylapatite in 0.5% 3-[(3-cholamidopropyl)-dimethylammonio]-1-propanesulfonic acid (CHAPS). Analyses of amino acid sequence and composition indicated that the CHAPS-soluble 60-kDa protein is an isoform of phosphoglucomutase (PGM). cDNAs encoding two isoforms of PGM were isolated from rabbit skeletal muscles. The translated amino acid sequences show that the isoforms, PGM1 and PGM2, differ in the N-terminal 77 amino acids and that PGM2 is identical to the 60-kDa protein in the SR. Northern blot analysis showed that the size of the mRNA encoding PGM2 is 2.4 kilobases. The PGM enzyme activity was markedly inhibited in SR membranes, while perturbation of the membranes with CHAPS or guanidine-HCl recovered the enzyme activity. KCl (0.15-1 M) led to a partial recovery of the enzyme activity suggesting that the charge interaction is not the primary force for PGM-SR interaction. PGM is localized in the heavy fraction of SR, where calsequestrin and Ca2+ release channel are enriched. Our results demonstrate that an isoform of PGM localized in junctional skeletal SR is the 60-kDa substrate of calmodulin-dependent protein kinase.

Highlights

A dures” led to little or no phosphorylation of the 60-kDa protein
Addition of purified myosin light chain kinase to the phosphorylation medium did not enhance the phosphorylation of the 60-kDa protein
Two independent sets of experiments showed that the relative amino acid composition of the CHAPS-soluble 60-kDa protein was similar to that of PGM (Ray et al, 1983)

Summary

Introduction

A dures” led to little or no phosphorylation of the 60-kDa protein. Addition of purified myosin light chain kinase to the phosphorylation medium did not enhance the phosphorylation of the 60-kDa protein. The N-terminal amino acid sequence (up to 25 amino acids tested) of the CHAPS-soluble 60-kDa protein (VKIVTVKTKATPDQKPGTSGLRKRV) was identical to that of PGM (Ray et al, 1983). Two independent sets of experiments showed that the relative amino acid composition of the CHAPS-soluble 60-kDa protein was similar to that of PGM (Ray et al, 1983) (data not shown). The isoelectric points (PI) of the protein determinedby two-dimensional gel electrophoresis showed that theprotein has a broad pH profile centered in the pHrange between 6.5 and 7.0, the deduced PI by the cDNA sequence is 7.04. The discrepancy could be due to the different state of phosphorylation and/or oxidation (Dawson and Greene, 1975)

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