Sodium-plus-potassium ion-activated adenosine triphosphatase in a heavy-membrane fraction isolated from rat skeletal muscle.

for allowing separation of its constituents. These reagents are likely to be specific for amino groups, and the aminoacyl bonds they form (Dixon & Perham, 1968) appeared sufficiently labile to allow their hydrolysis under conditions not deleterious to Na,K-ATPase activity. Microsomal suspensionswere prepared from frozen ox cerebral cortex essentially by the procedure of Schwartz, Bachelard & McIlwain (1962) and then treated with an NaI reagent (Nakao, Tashima, Nagano & Nakao, 1965). Determination of amino groups in this preparation with 2,4,6,-trinitrobenzenesulphonic acid (Habeeb, 1966) showed that lipid amino groups comprise about 55% of the total Reaction with methylmaleic anhydride at pH 8 and 200C causes a loss of Na,K-ATPase activity that is 50% complete when about 5% of the amino groups have been blocked, partial protection against inactivation being afforded by ATP. An 11-fold molar excess of methylmaleic anhydride acylates 90% of the total amino groups, and centrifugation of this acylated material at 150 OOOg for 1 h gives a clear supernatant containing 30% ofthe protein and 5% ofthe phospholipid. Dimethylmaleic anhydride at pH 8.5 acylates the enzyme preparation less effectively than does methylmaleic anhydride at pH8, but loss of Na,K-ATPase activity correlates similarly with the extent of acylation. No more than 43% of the total amino groups are dimethylmaleylated even when the anhydride is in 20or 30-fold molar excess; centrifugation of this material gives a clear supernatant containing 45% of the protein and less than 10% of the phospholipid. Regeneration of amino groups from the methylmaleylated material takes place slowly at pH 6 and 200C, but Na,K-ATPase activity of unmodified material is lost to an extent precluding restoration of enzyme activity. Dimethylmaleyl groups are readily removed at pH 7 and there is partial restoration of Na,K-ATPase activity to the deacylated material provided that not more than 20% of the amino groups were initially acylated.