Evidence for the presence of two distinct membrane ATPases in Spiroplasma citri

Triton X-100 (TX-100) extraction of Spiruplasnta citvi plasma membrane solubilized two types of ATPase differing in their pH of maximum activity. The activity measured at pH 8.5 was inhibited by vanadate and the activity measured at pH 6-5 was not. The vanadate-sensitive ATPase had a relatively basic isoelectric point (8.65) and therefore could be separated from the vanadate-insensitive ATPase using chromatofocusing. Elution of the TX-100 membrane extract in a pH gradient from 9 to 6 generated two peaks of ATPase activity: one in the acidic range, composed of an F,F,-type ATPase, and one in the basic range, corresponding to the vanadate-sensitive activity. Electrophoretic analysis of proteins from the latter peak revealed one major polypeptide of 37 kDa. This peptide was shown to correspond to spot A37 in a two-dimensional protein map of S. citri. Using the gene for the kdp-operon of Escherichia culi as a probe in heterologous hybridization, sequences were detected in the genomic DNA of S. c h i , suggesting that a gene coding for an enzyme related to this P-type ATPase is present in the S. citri genome. We therefore postulate the presense of two distinct kinds of ATPase in S. citrk one of the F-type which is resistant to vanadate inhibition, and one, probably of the P-type, which is vanadate-sensitive.