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Biol. 100, 459-472. Proteolytic Activation of the Canine Cardiac Sarcoplasmic Reticulum Calcium Pump+ Madeleine A. Kirchberger,* Douglas Borchman, and Chinnaswamy Kasinathan Department zyxwvutsrq of Physiology and Biophysics, Mount Sinai School of Medicine of the City University of New York, New York, New York 10029 Received January zyxwvutsr 3, 1986; Revised Manuscript Received April 29, 1986 ABSTRACT: Mild trypsin treatment of canine cardiac microsomes consisting largely of sarcoplasmic reticulum vesicles produced a severalfold activation of oxalate-facilitated calcium uptake. The increase in calcium uptake was associated with an increase in ATP hydrolysis. Proteases other than trypsin were also effective although to a lesser degree. Trypsin produced a shift of the Ca2+concentration dependency curve for calcium uptake toward lower Ca2+concentrations, which was almost identical with that produced by phosphorylation of microsomes by cyclic A M P dependent protein kinase when the trypsin and the protein kinase were present at maximally activating concentrations. The Hill numbers (fSD) of the Ca2+dependency after treatment of microsomes with trypsin (1.5 f 0.1) or protein kinase (1.7 f 0.1) were similar and were not significantly different from those for untreated control microsomes (1.6 f 0.1 and 1.8 f 0.1, respectively). Autoradiograms of sodium dodecyl sulfate-polyacrylamide electrophoretic gels indicate that 32Pincorporation into phos- pholamban (Mr 27.3K) or its presumed monomeric subunit (Mr 5.5K) was markedly reduced when tryp- sin-treated microsomes were incubated in the presence of cyclic AMP dependent protein kinase and zy [y- 32P]ATPcompared to control microsomes incubated similarly but pretreated with trypsin inhibitor inactivated trypsin. The activation of calcium uptake by increasing concentrations of trypsin was paralleled by the reduction of phosphorylation of phospholamban. Trypsin treatment of microsomes previously thio- phosphorylated in the presence of cyclic AMP dependent protein kinase and [y-35S]thio-ATP did not result in a loss of 35S label from phospholamban, which suggests that phosphorylation of phospholamban protects against trypsin attack. Trypsin treatment of microsomes prepared from rabbit fast skeletal muscle, which does not contain phospholamban, did not stimulate calcium uptake. However, autoradiograms of gels of rabbit skeletal muscle and canine cardiac microsomes incubated under conditions favorable for the formation of the 100-kilodalton acylphosphoprotein intermediate of the (Ca2+ + Mg2+)-activated ATPase reaction showed an identical pattern of effects of different concentrations of trypsin. These data suggest that trypsin cleaves similar sites on both calcium pump proteins. Therefore, the stimulatory effect of trypsin on cardiac microsomal calcium uptake does not appear to be due to a direct effect on the calcium pump protein. The data are consistent with a model in which a segment of phospholamban is in communication with the cytoplasm. If the segment is cleaved proteolytically, the basal rate of calcium transport is increased. The proteolytic activation of calcium transport would suggest that the presence of this unphosphorylated cytosolic segment has an inhibitory effect on the calcium pump. C a r d i a c muscle relaxation occurs as a result of a decrease in cytoplasmic Ca2+ and consequent dissociation of calcium from troponin C. The decrease in cytoplasmic CaZ+ is brought about by three different mechanisms. The sodium-calcium exchange system (Reuter & Seitz, 1968) and the sarcolemmal calcium pump (Caroni & Carafoli, 1981) transfer Ca2+out of the cell, and the sarcoplasmic reticulum (SR)' calcium This work was supported by Grant H L 15764 from the US. Public Health Service, National Institutes of Health. * Correspondence should be addressed to this author. pump sequesters calcium within the tubular SR network (Michalak, 1985). The relative contributions of these three ' Abbreviations: EGTA, ethylene glycol bis(P-aminoethyl ether)- N,N,N',N'-tetraacetic acid; DTT, dithiothreitol; PMSF, phenyl- methanesulfonyl fluoride; SR, sarcoplasmic reticulum; SDS, sodium dodecyl sulfate; PAGE, polyacrylamide gel electrophoresis; BAEE, so- dium benzoyl-L-arginine ethyl ester; EP, phosphoenzyme intermediate of the sarcoplasmic reticulum (CaZ+ + Mg2+)-activated ATPase protein; Tris, tris(hydroxymethyl)aminomethane; Hepes, N-(2-hydroxyethyl)- piperazine-N'-2-ethanesulfonic acid; TEMED, N,N,N',N'-tetramethyl- ethylenediamine; CAMP, adenosine cyclic 3',5'-phosphate; kDa, kilo- dalton(s). 0006-2960/86/0425-5484$01.50/0 0 1986 American Chemical Society