Nucleotide triphosphate utilization by cardiac and skeletal muscle sarcoplasmic reticulum. Further evidence for an alternative substrate hydrolysis cycle and the effect of calcium NTPase purification.

It was previously observed that the hydrolysis of GTP by cardiac sarcoplasmic reticulum (SR) (in contrast to skeletal muscle SR: (a) was identical in rate with total ATP hydrolysis; (b) gave a similar nonlinear substrate response; (c) was not Ca2+ dependent; and (d) stimulated Ca2+ accumulation but not Ca2+ translocation. Evidence was presented that both the ATPase and GTPase are effected by the same enzyme and represent different hydrolysis cycles (Van Winkle, W. B., Tate, C. A., Bick, R. J., and Entman, M. L. (1981) J. Biol. Chem. 256, 2268-2274). In the present paper, we report that purification of the NTPase from both muscle sources resulted in an alteration in the NTP concentration response compatible with a single high affinity binding site for ATP only in cardiac SR and for both substrates in skeletal muscle SR. As is the case with native skeletal muscle SR, purified skeletal muscle NTPase hydrolyzed GTP in a manner qualitatively similar to ATP (but with no Ca2+-independent NTPase) but with reduced velocity. In contrast, there was no GTPase activity or Ca2+-independent "basic" ATPase activity in the purified cardiac NTPase. Inclusion of oxalate or the ionophore, A23187, in assays with cardiac SR and ATP as the substrate increased the total ATPase activity but had no effect on GTPase activity. Furthermore, the nucleotide-dependent uptake of oxalate by cardiac SR was only apparent with ATP and not with GTP. In the presence of Ca2+, ATP was a potent inhibitor (noncompetitive, Ki of 2-5 microM) of GTPase activity, whereas it was a weaker competitive inhibitor in the absence of Ca2+. We suggest that GTPase and basic ATPase represent similar alternative enzyme cycles for the CaATPase enzyme that are inhibited by the presence of ATP plus Ca2+ but are rendered inactive during the purification of cardiac NTPase.