Purification of Cardiac Sarcolemmal Vesicles: High Sodium Pump Content and ATP-Dependent, Calmodulin-Activated Calcium Uptake1

Abstract

Highly purified vesicles of cardiac sarcolemma were prepared from a homogenate of canine ventricular muscle by density gradient centrifugation. The preparation showed an extremely high content of (Na⁺,K⁺)-ATPase. The steady state levels of Na⁺-dependent phosphoenzyme formation in the presence of Triton X-100 and the specific ouabain binding in the absence of Triton X-100 were, respectively, 773 and 907 pmol.mg⁻¹ under the optimum conditions. On the other hand, the amount of Ca²⁺-dependent phosphoenzyme formed in the absence of Triton X-100 was less than 2 pmol.mg⁻¹. This demonstrates that the preparation was virtually free of contaminant sarcoplasmic reticulum fragments. The preparation showed ATP-dependent Ca²⁺ uptake. Almost all the Ca²⁺ accumulated on the addition of ATP was rapidly released by the subsequent addition of NaCl. This finding gives evidence that the ATP-driven Ca²⁺ pump exists in the cardiac sarcolemma. The Ca²⁺uptake was unaffected by 2 μM digitoxin, 1 μM monesin, and 200 μM dinitrophenol. These results exclude the possibility that transmembrane gradients of Na⁺ and H⁺ were involved in this Ca²⁺ uptake. The Ca²⁺ pump was activated by calmodulin. The concentration of calmodulin giving a half-maximum activation was 0.05 μ.ml⁻¹, which is equivalent to 3 nM. This activation was removed by addition of trifluoperazine, a specific inhibitor of calmodulin.