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.
Department of Plant Pathology, University of Georgia, Griffin 30223.
J
J
Chen
C J
CJ
Chang
D001433:Bacteriolysis; D007770:L-Lactate Dehydrogenase / Q000378:metabolism; D008291:Malate Dehydrogenase / Q000378:metabolism; D009097:Multienzyme Complexes / Q000378:metabolism; D009247:NADH, NADPH Oxidoreductases / Q000378:metabolism; D009997:Osmotic Pressure; D013149:Spiroplasma / Q000201:enzymology* / Q000502:physiology
D009097:Multienzyme Complexes; D007770:L-Lactate Dehydrogenase; D008291:Malate Dehydrogenase; C023107:NADH oxidase; D009247:NADH, NADPH Oxidoreductases