Purification and characterization of phospholamban from canine cardiac sarcoplasmic reticulum.

M Inui,M Kadoma,M Tada

doi:10.1016/s0021-9258(19)83681-5

Abstract

Phospholamban, a putative regulator of the Ca2+-dependent ATPase of cardiac sarcoplasmic reticulum (SR), was purified from canine cardiac SR membranes. Cardiac SR was extracted with deoxycholate and fractionated with ammonium sulfate followed by gel permeation high performance liquid chromatography in the presence of the nonionic detergent, octa-ethylene glycol mono-n-dodecyl ether (C12E8), and KI. Further purification was achieved with CM-Sepharose CL 6B column chromatography in the presence of C12E8. The purified phospholamban showed a single band of 22,000 daltons on neutral sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis (Weber, K., and Osborn, M. (1969) J. Biol. Chem. 244, 4406-4412) and 27,000 daltons on alkaline SDS gels (Laemmli, U. K. (1970) Nature (Lond.) 227, 680-685). Boiling of phospholamban in 2% SDS produced total conversion into the lower molecular weight component on SDS gels (11,000 on Laemmli gel and 10,500 on Weber and Osborn gel). The apparent molecular weight of phospholamban on SDS gels was slightly increased by cAMP-dependent phosphorylation. The extent of phosphorylation catalyzed by cAMP-dependent protein kinase in the purified phospholamban preparations was about 42 nmol of phosphate/mg of protein when the protein concentration was determined by the method of Lowry et al. (Lowry, O. H., Rosebrough, N. J., Farr, A. L., and Randall, R. J. (1951) J. Biol. Chem. 193, 265-275), or 138 nmol/mg of protein based on the protein concentration estimated by the dye absorption method. Rabbit antisera were prepared against purified phospholamban. The obtained antisera were found to bind to purified phospholamban as well as that in cardiac SR. No reaction was detected in fast skeletal muscle SR by immunofluorescent staining of Western blots. The present preparation of purified phospholamban and the antisera should facilitate further understanding of the regulatory action of phospholamban on the calcium pump ATPase.

Highlights

From the Division of Cardiology, the First Departmentof Medicine and the Departmenotf Pathophysiology, Osaka University School of Medicine, Osaka 55$-Japan
We previously indicated that phosphorylation of a 22,000dalton protein, phospholamban, of cardiac SR catalyzed by CAMP-dependent proteinkinaseaugmentsCa2+-dependent ATPase activitythrough anenhancement of therates of formation and decomposition of the phosphorylated interpurified phospholambanshowed a singleband of mediate, EP, of the ATPase [3, 4]
Fractionation of Phospholamban from CardiaScR-In order to purify phospholamban from cardiac SR, we examined the conditions where phospholamban could be effectively separated from other SR proteins

Summary

Introduction

From the Division of Cardiology, the First Departmentof Medicine and the Departmenotf Pathophysiology, Osaka University School of Medicine, Osaka 55$-Japan. The phospholamban-enriched fractions (about 20 ml) determined by SDS-polyacrylamide gel electrophoresis werepooled and dialyzed overnight against 4 liters of 50 mMKC1, 0.1% Cl,E8, 5 mM 2-mercaptoethanol, 20 mM Tris/HCl, pH 7.5 (Buffer B), at 4 "C.

Results

Conclusion