from Azotobacter vinelandii

A method is described for the purification of NAD+ kinase from extracts of Azofobacfer uinelandii (ATCC 9104) cells. The specific activity of the purified enzyme is the highest so far reported. The enzyme requires a metal ion for activity. There is a lack of specificity for the nature of the phosphate donor in the kinase reaction. The Michaelis constants for the enzyme substrates have been determined by a direct spectrophotometric assay method which is described. The enzyme is protected to a remarkable extent from heat inac-tivation by NAD+. The enzyme appears to be a sulfhydryl enzyme; the sulfhydryl groups are protected by NAD+. The biosynthesis of NADP+ occurs by a phosphorylation of NAD+ by adenosine triphosphate (l-3). Since the major path-way for NADP+ synthesis is via NAD+ kinase, it seemed to be of considerable interest to obtain a highly purified enzyme, study its properties, and obtain information which may elucidate the factors which control its activity in living organisms. The most highly purified preparation of the enzyme had previously been obtained by Kornberg (2) from brewer’s yeast cells. This purified enzyme was unstable and somewhat difficult to prepare. In the present communication, a method is described for prepar-ing the enzyme in a highly purified and stable state from Azoto-bacfer vinelandii (ATCC 9104) cells. In addition, some of the properties of this purified enzyme are described