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Abstract.: The partially purified 57-kDa protein of Spiroplasma melliferum was autophosphorylated when incubated with ATP in the presence of ZnCl2. Autophosphorylation was also apparent by showing the in situ phosphorylation of the 57-kDa protein band separated by polyacrylamide gel electrophoresis under nondenaturing conditions. The autophosphorylation was affected neither by the pH of the reaction mixture nor by the presence of NaF. The steady state level of the phosphorylated 57-kDa protein remained constant for up to 15 min, suggesting the absence of a phosphoprotein phosphatase activity in the preparation. As the initial phosphorylation rate did not decrease upon a 100-fold dilution of the 57-kDa protein under constant substrate concentration, it is suggested that the autophosphorylation is an intramolecular process.

Covalent modification ofSpiroplasma floricola membrane proteins by fatty acids was determined by in vivo labeling of the cells with radioactive fatty acids followed by separation on one-dimensional SDS-polyacrylamide gels and visualization by autoradiography. Approximately 25 different proteins were found to be labeled with [3H]-palmitate, whereas almost none were labeled with [3H]-oleate. The radioactivity could not be removed from the palmitoylated membrane proteins by boiling in SDS or by exhaustive extraction with chloroform-methanol (2:1). Nevertheless, treating the palmitoylated proteins with a 0.1N KOH solution removed approximately 70% of the bound [3H]-palmitate. The major protein-bound fatty acid species were identified, following their release from the protein by chemical cleavage, as palmitic acid and stearic acid (83% and 7.5%, respectively).