Adenylate kinase is tightly bound to axonemes of Tetrahymena cilia

Cilia of Tetrahymena thermophila possess adenylate kinase ATP:AMP phosphotransferase, EC 2.7.4.3] activity. More than 95% of the total activity was recovered in the axonemal fraction when cilia were demembranated with 0.2% Nonidet P-40. There was no loss of the specific activity of adenylate kinase when axonemes were thoroughly washed with HMEK solution (10 mM HEPES, 5 mM MgCl2, 0.1 mM EDTA, and 0.1 M KCl, pH 7.4). These results suggest that adenylate kinase is tightly bound to axoneme. Solubilization of adenylate kinase was markedly increased when axonemes were incubated in HME buffer (10 mM HEPES, 1 mM MgCl2, 0.1 mM EDTA, pH 7.4) containing concentrations of NaCl (or KCl) exceeding 1 M. Therefore, routine isolation of adenylate kinase from axonemes involved pre-extracting axonemes with 0.5 M NaCl in HME buffer followed by extraction in HME buffer containing 1.5 M NaCl. Native-gel electrophoresis of the high salt extract revealed two protein bands (band I and band III). An active staining for adenylate kinase showed a single active band corresponding to the position of band III. Two-dimensional gel electrophoresis using native-gel electrophoresis in the first dimension and SDS-PAGE in the second dimension suggests that band III protein contains at least nine polypeptides ranging from 21 to 110 kDa.