Origin of concurrent ATPase activities in skinned cardiac trabeculae from rat.

Abstract

1. To determine the rate of ATP turnover by the sarcoplasmic reticulum (SR) Ca2+ pump in cardiac muscle, and to assess the contributions of other ATPase activities to the overall ATP turnover rate, ATPase activity and isometric force production were studied in saponin-skinned trabeculae from rat. ATP hydrolysis was enzymatically coupled to the oxidation of NADH; the concentration of NADH was monitored photometrically. All measurements were performed at 20 +/- 1 degrees C and pH 7.0. Resting sarcomere length was adjusted to 2.1 microns. All solutions contained 5 mM caffeine to ensure continuous release of Ca2+ from the SR. 2. The Ca(2+)-independent ATPase activity, determined in relaxing solution (pCa 9), amounted to 130 +/- 13 microM s-1 (mean +/- S.E.M., n = 7) at the beginning of an experiment. During subsequent measurements in relaxing solution, a decrease in ATPase activity was observed, indicative of loss of membrane-bound ATPase activity. The steady-state Ca(2+)-independent (basal) ATPase activity was 83 +/- 5 microM s-1 (n = 66). 3. Treatment of saponin-skinned preparations with Triton X-100 abolished 50 microM s-1 (60%) of the basal ATPase activity. Addition of ouabain (1 mM) suppressed 14 +/- 5% of the basal activity, whereas 8 +/- 3% was suppressed by 20 microM cyclopiazonic acid (CPA). It is argued that 31 microM s-1 of the basal ATPase activity may be associated with MgATPase from the transverse tubular system. 4. The maximal Ca(2+)-activated ATPase activity, i.e. the total ATPase activity (determined in activating solution, pCa 4.3) corrected for basal ATPase activity, was found to be 409 +/- 15 microM s-1 (n = 66). Experiments with CPA indicated that at least 9 +/- 6% of the maximal Ca(2+)-activated ATPase activity originates from the sarcoplasmic Ca2+ pump. These experiments indicate that the rate of ATP consumption by the SR Ca2+ transporting ATPase amounts to at least 37 microM s-1. 5. Treatment of preparations with Triton X-100 abolished 15 +/- 3% of the maximal Ca(2+)-activated ATPase activity, indicating that 15 +/- 3% of the maximal Ca(2+)-activated ATPase activity is membrane bound. 6. Variation of free [Ca2+] indicated that apart from the actomyosin ATPase activity a second Ca(2+)-dependent ATPase activity contributed to the overall ATP turnover rate. This activity was half-maximal at pCa 6.21, and probably reflects the SR Ca2+ transporting ATPase. It constituted 18 +/- 3% of the Ca(2+)-dependent ATPase activity, yielding an upper limit for the SR Ca2+ transporting ATPase activity of 74 microM s-1.