Evidence for an effect of phospholamban on the regulatory role of ATP in calcium uptake by the calcium pump of the cardiac sarcoplasmic reticulum.
The purpose of this study was to investigate the functional relationship between phospholamban and the nucleotide site of the calcium pump protein of the cardiac sarcoplasmic reticulum. We used control and trypsin-treated cardiac microsomes in which cleavage of the inhibitory cytoplasmic domain of phospholamban is associated with an activation of the calcium pump similar to that produced by protein kinase A catalyzed phospholamban phosphorylation. Phenylglyoxal was shown to inactivate the calcium pump in a pseudo-first-order reaction by binding to a single Arg at the nucleotide binding site. No differences upon trypsin treatment of microsomes were observed in the kinetics of phenylglyoxal inactivation or the ability of millimolar ATP to protect against inactivation. In subsequent kinetic studies, Ca-uptake rates measured at saturating Ca2+ and 5 microM-1 mM MgATP2- were increased 15-32% by trypsin treatment in each of three different microsome preparations. Double-reciprocal plots of the data showed marked downward curvature indicating an acceleratory effect associated with ligand binding to a lower affinity site. At 0.32 microM Ca2+, Ca-uptake rates were lower than at 11 microM Ca2+ but were stimulated to a greater extent by trypsin treatment; control microsomes showed reduced evidence of apparent negative cooperativity. At 0-2 microM MgATP2- and saturating Ca2+, there was a 50% increase in Vmax(app) when the Hill coefficient (N) was 1. At 0-10 microM MgATP2-, second-site binding was evident. At both 0-10 microM and 5 microM-1 mM MgATP2-, trypsin-treated microsomes showed greater activation of Ca uptake attributable to second-site binding than did control microsomes.(ABSTRACT TRUNCATED AT 250 WORDS)
Department of Physiology and Biophysics, Mount Sinai School of Medicine, City University of New York, New York 10029.
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Kirchberger
D000255:Adenosine Triphosphate / Q000494:pharmacology*; D000818:Animals; D001120:Arginine / Q000378:metabolism; D001665:Binding Sites; D017301:Ca(2+) Mg(2+)-ATPase / Q000378:metabolism; D002118:Calcium / Q000378:metabolism*; D002135:Calcium-Binding Proteins / Q000494:pharmacology*; D000252:Calcium-Transporting ATPases / Q000037:antagonists & inhibitors / Q000378:metabolism; D004285:Dogs; D007700:Kinetics; D008861:Microsomes / Q000187:drug effects / Q000378:metabolism; D009206:Myocardium / Q000378:metabolism* / Q000648:ultrastructure; D010658:Phenylglyoxal / Q000378:metabolism / Q000494:pharmacology; D012519:Sarcoplasmic Reticulum / Q000187:drug effects / Q000378:metabolism*; D014357:Trypsin / Q000494:pharmacology; D000099211:Phospholamban
D000255:Adenosine Triphosphate; D001120:Arginine; D017301:Ca(2+) Mg(2+)-ATPase; D002118:Calcium; D002135:Calcium-Binding Proteins; D000252:Calcium-Transporting ATPases; D010658:Phenylglyoxal; D014357:Trypsin; D000099211:Phospholamban