The osmotic lysis of Spiroplasma cells and its use in enzyme studies

A simple and efficient osmotic lysis method was developed for enzyme studies in spiroplasmas. Log phase cells in R2 medium were harvested by centrifugation (19,600 x g for 30 min). Wash buffer supplemented with 0.23 M sucrose maintained the helicity of spiroplasma cells during washing. Osmotic lysis of spiroplasmas was achieved in H buffer that contained no sucrose. Sucrose at concentrations as low as 0.004 M dramatically increased the resistance of the spiroplasmas to osmotic lysis. NADH oxidase, lactate dehydrogenase, and malate dehydrogenase were detected in cell lysates of Spiroplasma floricola (23-6), Spiroplasma citri (R8A2), Spiroplasma apis (SR 3), and Spiroplasma melliferum (AS 576). Citrate synthase, aconitase, isocitrate dehydrogenase, alpha-ketoglutarate dehydrogenase, succinyl coenzyme A synthetase, succinate dehydrogenase, and fumarase were not detected in cell lysates of S. floricola (23-6). NADH oxidase and malate dehydrogenase were found in the cytosol whereas lactate dehydrogenase was loosely associated with the cytomembrane.