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Highly purified sarcoplasmic reticulum (SR) has been prepared from dog hearts and has been incubated with the triplet probe erythrosinyl isothiocyanate to specifically label the Ca2+-stimulated ATPase (Ca2+-ATPase) of the SR. The rotational mobility of the Ca2+-ATPase has been studied in this erythrosin-labelled SR using time-resolved phosphorescence polarization. Qualitatively, the mobility of the cardiac Ca2+-ATPase resembles that of skeletal muscle SR Ca2+-ATPase. Addition of Ca2+ to SR affects the mobility of the Ca2+-ATPase in a way consistent with a segment of the ATPase altering its orientation relative to the plane of the membrane. Phosphorylation of phospholamban in cardiac SR by the purified catalytic subunit of cAMP-dependent protein kinase, which is known to increase the activity of the Ca2+-ATPase by deinhibition, also alters measured anisotropy. The changes observed are not compatible with dissociation of the Ca2+-ATPase from phospholamban after the latter is phosphorylated. The data are more consistent with phospholamban associating with the Ca2+-ATPase following phosphorylation, or more complex models in which only the hydrophilic domain of phospholamban binds with and dissociates from the Ca2+-ATPase.

Phosphorylation of phospholamban in cardiac sarcolemma is implicated in the increased influx of Ca2+ through the slow calcium channel induced by catecholamines. A method is described for the preparation of highly purified sarcolemmal vesicles from rat heart, and this has been used to examine the phosphorylation of phospholamban in 32Pi-perfused rat hearts. Phospholamban phosphorylation is increased 3-fold after 30 s of perfusion with 0.1 microM isoprenaline. The time course of this increase precedes the inotropic response by 5-10 s.

1. Cyclic GMP-dependent protein kinase phosphorylates purified phospholamban. It also phosphorylates phospholamban present in vesicles of cardiac sarcoplasmic reticulum and smooth muscle microsomal fractions, and in transformants of Escherichia coli which contain a plasmid into which a gene encoding phospholamban has been inserted. 2. In vitro the phospholamban present in cardiac sarcoplasmic reticulum membranes is a better substrate for cyclic GMP-dependent protein kinase than for cyclic AMP-dependent protein kinase. 3. Studies using [32P]Pi to label the cellular ATP in intact cardiac or smooth muscle failed to demonstrate that phosphorylation of phospholamban occurs in response to stimuli which increase intracellular cyclic GMP. Possible reasons for this functional separation between increased cyclic GMP and phosphorylation of phospholamban are discussed.