ORIGIN OF CONCURRENT ATPASE ACTIVITIES IN SKINNED CARDIAC TRABECULAE FROM RAT

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 activ ity and isometric force production were studied in saponin-skinned tra beculae from rat. ATP hydrolysis was enzymatically coupled to the oxid ation of NADH; the concentration of NADH was monitored photometrically . All measurements were performed at 20 +/- 1 degrees C and pH 7.0. Re sting sarcomere length was adjusted to 2.1 mu m. All solutions contain ed 5 mar caffeine to ensure continuous release of Ca2+ from the SR. 2. The Ca2+-independent ATPase activity, determined in relaxing solution (pCa 9), amounted to 130 +/- 13 mu M s(-1) (mean +/- S.E.M., n = 7) a t the beginning of an experiment. During subsequent measurements in re laxing solution, a decrease in ATPase activity was observed, indicativ e of loss of membrane-bound ATPase activity. The steady-state Ca2+-ind ependent (basal) ATPase activity was 83 +/- 5 mu M s(-1) (n = 66). 3. Treatment of saponin-skinned preparations with Triton X-100 abolished 50 mu M s(-1) (60 %) of the basal ATPase activity. Addition of ouabain (1 mM) suppressed 14 +/- 5% of the basal activity, whereas 8 +/- 3% w as suppressed by 20 mu M cyclopiazonic acid (CPA). It is argued that 3 1 mu M s(-1) of the basal ATPase activity may be associated with MgATP ase from the transverse tubular system. 4. The maximal Ca2+-activated ATPase activity, i.e. the total ATPase activity (determined in activat ing solution, pCa 4.3) corrected for basal ATPase activity, was found to be 409 +/- 15 mu M s(-1) (n = 66). Experiments with CPB indicated t hat at least 9 +/- 6% of the maximal Ca2+-activated ATPase activity or iginates from the sarcoplasmic Ca2+ pump. These experiments indicate t hat the rate of ATP consumption by the SR C2+ transporting ATPase amou nts to at least 37 mu M s(-1) 5. 5. Treatment of preparations with Tri ton X-100 abolished 15 +/- 3% of the maximal Ca2+- activated ATPase ac tivity, indicating that 15 +/- 3 % of the maximal Ca2+-activated ATPas e activity is membrane bound. 6. Variation of free [Ca2+] indicated th at apart from the actomyosin ATPase activity a second Ca2+-dependent A TPase activity contributed to the overall ATP turnover rate. This acti vity was half-maximal at pCa 6.21, and probably reflects the SR Ca2+ t ransporting ATPase. It constituted 18 +/- 3 % of the Ca2+-dependent AT Pase activity, yielding an upper limit for the SR Ca2+ transporting AT Pase activity of 74 mu M s(-1).