A membrane-bound phosphatidylinositol (PI) kinase (EC 2.7.1.67) was purified by affinity chromatography from bovine brain myelin. This enzyme activity was solubilized with non-ionic detergent and chromatographed on an anion-exchange column. Further purification was achieved by affinity chromatography on PI covalently coupled to epoxy-activated Sepharose, which was eluted with a combination of PI and detergent. The final step in the purification was by gel filtration on an Ultrogel AcA44 column. This procedure afforded greater than 5500-fold purification of the enzyme from whole brain myelin. The resulting activity exhibited a major silver-stained band on SDS/polyacrylamide-gel electrophoresis with an apparent Mr 45,000. The identity of this band as PI kinase was corroborated by demonstration of enzyme activity in the gel region corresponding to that of the stained protein. The purified enzyme exhibited a non-linear dependence on PI as substrate, with two apparent kinetic components. The lower-affinity component exhibited a Km similar to that observed for the phosphorylation of phosphatidylinositol 4-phosphate by the enzyme.

Summary. Rickettsia rickettsii and Rickettsia conorii are the causative agents of two common and serious diseases, Rocky Mountain spotted fever and Mediterranean spotted fever, respectively. In patients naturally infected with either of these organisms, antibodies are produced which cross-react with antigens of the other so extensively that diagnostic tests usually cannot identify the causative agents. The results of this study indicate that serodiagnostic tests with antigen from one of these two organisms could be used to detect antibodies in patients with either of the two rickettsial diseases.

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