Characterization of (Na +, K +)-ATPase isolated from embryonic chick hearts and cultured chick heart cells

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The (Na +, K +)-ATPase obtained from 14–16-day-old embryonic chick hearts (ventricles) was compared with that from trypsin-dissociated and denervated cultured chick embryonic heart cells. Two methods of enzyme preparation were studied: (1) isolation in mannitol, and (2) NaI extraction. Electron microscopy showed that the NaI preparation (100 000 × g fraction) consisted of membranes organized as vesicles. Ouabain produced 92% inhibition of the total ATPase activity in the NaI preparation compared with a 34% inhibition of the mannitol preparation. For the intact heart preparation, half-maximal inhibition occurred at 2.7·10 −6 M ouabain; K m was about 4.6·10 −4 M ATP, and ν max was 91 μmoles P i per h per mg protein. The (Na +, K +)-ATPase isolated from the cultured heart cells had about a 10-fold lower specific activity, but otherwise it had virtually identical characteristics. The optimal MgCl 2 concentration was 3 mM in the presence of 3 mM ATP, and the optimal K + concentration was 8 mM. Specific activity increased with Na + concentration and reached maximum at 50 mM. Li + did not substitute for Na +. The optimal pH was 7.0–8.0, with marked depression of activity at acid pH. Ca 2+, Sr 2+, Mn 2+, and Ni 2+ (2 mM) partially depressed the transport ATPase, and Ba 2+ and Zn 2+ almost completely depressed it. Tetracaine (2 mM) depressed the (Na +, K +)-ATPase. Tempterature studies showed breaks in the Arrhenius plots at 25° and 15°; the Q 10 values averaged 2.2 (25–35°) and 3.9 (15–25°) (activation energies of 14.5 and 23.1 kcal/mole, respectively). Many of these characteristics of the (Na +, K +)-ATPase are reflected in the electrical properties of the myocardial cell.