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Origin of concurrent ATPase activities in skinned cardiac trabeculae from rat.
Author(s) -
Ebus J P,
Stienen G J
Publication year - 1996
Publication title -
the journal of physiology
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.802
H-Index - 240
eISSN - 1469-7793
pISSN - 0022-3751
DOI - 10.1113/jphysiol.1996.sp021337
Subject(s) - cyclopiazonic acid , chemistry , atp hydrolysis , ouabain , atpase , endoplasmic reticulum , sarcomere , calcium , biophysics , biochemistry , enzyme , endocrinology , myocyte , biology , sodium , organic chemistry
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.