Kinetic differences in the phospholamban-regulated calcium pump when studied in crude and purified cardiac sarcoplasmic reticulum vesicles

A. Antipenko, Andrew Spielman, M. A. Kirchberger

Research output: Contribution to journalArticle

Abstract

Phospholamban (PLN) phosphorylation contributes largely to the inotropic and lusitropic effects of beta-adrenergic agonists on the heart. The mechanical effects of PLN phosphorylation on the heart are generally attributed solely to an increase in the apparent affinity of the Ca pump in the sarcoplasmic reticulum (SR) membranes for Ca 2+ with little or no effect on V(max(Ca)). In the present report, we compare the kinetic properties of the cardiac SR Ca pump in commonly studied crude microsomes with those of our recently developed preparation of light SR vesicles. We demonstrate that in crude: microsomes, the increase in the apparent affinity of the pump for Ca 2+ is larger, while the increase in V(max(Ca)) is smaller, than in purified vesicles. The greater phosphorylation-induced increase in apparent Ca 2+ affinity in crude microsomes may be further enhanced by an ATP- sensitive inhibitory effect of ruthenium red on the activity of the pump at subsaturating, but not saturating, Ca 2+ concentrations as a result of a greater inhibition in unphosphorylated microsomes. Upon increasing the ATP concentration from 1 to 5 mM, an inhibition by 10 μM ruthenium red is eliminated in phosphorylated microsomes and reduced in control microsomes. Addition of the phosphoprotein phosphatase inhibitor okadaic acid produces a considerable increase in the phosphorylation-induced effects in both crude and purified microsomes. We conclude that the use of purified cardiac SR vesicles is critical for the demonstration of a major increase in V(max(Ca)) in addition to an increase in the pump's apparent affinity for Ca 2+ in response to phosphorylation of PLN by protein kinase A.

Original languageEnglish (US)
Pages (from-to)257-265
Number of pages9
JournalJournal of Membrane Biology
Volume167
Issue number3
DOIs
StatePublished - 1999

Fingerprint

Sarcoplasmic Reticulum
Microsomes
Calcium
Phosphorylation
Ruthenium Red
Adenosine Triphosphate
Okadaic Acid
Adrenergic beta-Agonists
Phosphoprotein Phosphatases
Cyclic AMP-Dependent Protein Kinases
phospholamban
Membranes

Keywords

  • Heart
  • Light sarcoplasmic reticulum

ASJC Scopus subject areas

  • Biophysics
  • Physiology
  • Cell Biology

Cite this

Kinetic differences in the phospholamban-regulated calcium pump when studied in crude and purified cardiac sarcoplasmic reticulum vesicles. / Antipenko, A.; Spielman, Andrew; Kirchberger, M. A.

In: Journal of Membrane Biology, Vol. 167, No. 3, 1999, p. 257-265.

Research output: Contribution to journalArticle

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