Chemical modification of a cerebral sodium-plus-potassium ion-stimulated adenosine triphosphatase preparation.
1. A particulate Na(+)+K(+)-stimulated adenosine triphosphatase preparation obtained by treatment of bovine cerebral microsomes with a sodium iodide reagent has been further treated with acid anhydrides likely to convert amino groups into acidic derivatives. 2. The extent of acylation of amino groups was determined by reaction of the remaining amino groups with 2,4,6-trinitrobenzenesulphonic acid. The unmodified preparation contains about 1.2 muequiv. of amino groups/mg of protein of which only about 0.5 muequiv. are accounted for by protein amino groups. Kinetics of the trinitrobenzenesulphonic acid reaction with the unmodified preparation are complex and are altered by ATP or ouabain. 3. The compounds examined cause loss of Na(+)+K(+)-stimulated adenosine triphosphatase activity when relatively few amino groups are modified but ATP was found to afford partial protection against inactivation by methylmaleic anhydride. Na(+)+K(+)-stimulated adenosine triphosphatase activity is partly restored to the dimethylmaleylated preparation by hydrolysis of the dimethylmaleyl-amide bonds but not if more than about 20% of the amino groups have been acylated. 4. Supernatants obtained by high-speed centrifugation of the dimethylmaleylated preparation contained up to 45% of the total protein with less than 10% of the total phospholipid. Methylmaleyl and benzenetricarboxylyl derivatives of the enzyme preparation behaved similarly but tetrafluorosuccinylated material was almost entirely deposited by centrifugation.