The separation and partial purification of membrane-bound (Na+ + K+)-dependent Mg2+-ATPase and (Na+ + K+)-independent Mg2+-ATPase from frog skeletal muscle

• 1.1. Two distinct, membrane-bound, ATP-hydrolysing enzymes have been isolated from the same homogenate of frog skeletal muscle. Both are stimulated by Mg²⁺; one shows virtually no activity without Na⁺ and K⁺ and the other is unresponsive. to the monovalent cations. We have tentatively concluded that the (Na⁺ + K⁺)-dependent ATPAse is bound to fragments of plasma membrane and the (Na⁺ + K⁺)-independent ATPase to fragments of sarcomplasmic reticulum.
• 2.2. The (Na⁺ + K⁺)-stimulated Mg²⁺-ATPase ((Na⁺ + K⁺)-Mg²⁺-ATPase) was obtained by exposing a low-speed pellet (900 × g) from a coarse homogenate to high salt concentrations, followed by extensive washing and by differential centrifugation procedures. The enzyme found in the final pellet (10500 × g) was purified many-fold by density gradient centrifugation.
• 3.3. The highest specific activity of the purified (Na⁺ + K⁺)-Mg²⁺-ATPase was 600 μmoles of P_i liberated per mg nitrogen per h. Ouabain inhibited the activity of this preparation by 94%. The ATPase activity was synergistically stimulated by Na⁺ + K⁺, the optimum concentrations being 105 mM Na⁺ and 45 mM K⁺. A broad optimum pH range of 7.3–7.7 was found. Ca²⁺ at an added concentration of 2 mM inhibited the total ATPase activity by about 50 %.
• 4.4. The (Na⁺ + K⁺)-independent Mg²⁺-ATPase was found in a microsomal fraction (44000 × g). After purification on a density gradient the enzyme had a specific activity of 300–400 μmoles of P_i liberated per mg nitrogen per h. Ca²⁺ activated the ATPase to an even greater degree than Mg²⁺ did.
• 5.5. There was no inhibition by ouabain and little or no stimulation by Na⁺ and/or K⁺ of this Mg²⁺ (or Ca²⁺) stimulated ATPase (Mg²⁺ (or Ca²⁺)-ATPase). Attempts were made to decrease the activity of the Mg²⁺-ATPase and thereby accentuate any (Na⁺ + K⁺)-Mg²⁺-ATPase activity which might be present. The Mg²⁺-ATPase was unaffected by deoxycholate but was markedly reduced by urea, NaN₃ and high salt concentrations; there was no evidence whatsoever of the (Na⁺ + K⁺)-Mg²⁺-ATPase.
• 6.6. Examination with the electron microscope showed that the (Na⁺ + K⁺)-Mg²⁺-ATPase and the Mg²⁺ (or Ca²⁺)-ATPase preparations were entirely membranous and were indistinguishable. There was little evidence in either preparation of mitochondria, inner or outer mitochondrial membranes, rough endoplasmic reticulum, collagen, actin or myosin fibrils.
• 7.7. When treated with saponin both preparations showed the hexagonal pattern characteristic of artificial and natural membranes which have an appreciable cholesterol content. The cholesterol and phospholipid concentrations were determined.
• 8.8. Cytochrome c oxidase was not present in the purified (Na⁺ + K⁺)-Mg²⁺-ATPase preparation but might have been a minor contaminant of two of the three bands containing the purified Mg²⁺ (or Ca²⁺)-ATPase.
• 9.9. Both preparations showed some NADH-diaphorase activity and 5′-nucleotidase activity. Hence these enzymes did not distinguish between the two types of membranes.