The effects of calcium, temperature and phospholamban phosphorylation on the dynamics of the calcium-stimulated ATPase of canine cardiac sarcoplasmic reticulum.
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.
Fowler
C
C
Department of Protein and Molecular Biology, Royal Free Hospital School of Medicine, London, U.K.
Huggins
J P
JP
Hall
C
C
Restall
C J
CJ
Chapman
D
D
D000818:Animals; D002118:Calcium / Q000502:physiology*; D002135:Calcium-Binding Proteins / Q000378:metabolism*; D000252:Calcium-Transporting ATPases / Q000378:metabolism*; D002458:Cell Fractionation / Q000379:methods; D002499:Centrifugation, Density Gradient; D004285:Dogs; D004923:Erythrosine; D005260:Female; D016650:Fluorescein-5-isothiocyanate; D005452:Fluoresceins; D007700:Kinetics; D008297:Male; D009206:Myocardium / Q000201:enzymology*; D010766:Phosphorylation; D012519:Sarcoplasmic Reticulum / Q000201:enzymology*; D013696:Temperature; D013861:Thiocyanates; D000099211:Phospholamban
D002118:Calcium; D002135:Calcium-Binding Proteins; D000252:Calcium-Transporting ATPases; D004923:Erythrosine; D016650:Fluorescein-5-isothiocyanate; D005452:Fluoresceins; D013861:Thiocyanates; D000099211:Phospholamban