VOLUME REGULATION IN SPIROPLASMA-FLORICOLA - EVIDENCE THAT NA+ IS EXTRUDED BY A NA+ H+ ANTIPORTER/

Marked cell swelling followed by lysis was observed when Spiroplasma f loricola cells were incubated in iso-osmotic solutions of NaCl, KCl, c holine chloride or sorbitol in the absence of an energy source. In the presence of an energy source the cells did not swell suggesting that S. floricola relies on an energy-dependent mechanism(s) for cell volum e regulation. An ammonium chloride dilution procedure was utilized to generate a ph gradient (inside add) across the cell membrane of S. flo ricola cells. The addition of NaCl resulted in an intracellular alkali zation suggesting the presence of a Na+/H+ exchange activity. In Na-22 (+)-loaded cells, glucose-dependent Na-22(-) extrusion was observed at acidic pH in both the presence and absence of Na+ ions. The extrusion was completely inhibited by carbonyl cyanide m-chlorophenylhydrazone (CCCP, 10 mu M) and partially inhibited by dicyclohexylcarbodiimide (D CCD, 100 mu M) indicating that in S. floricola, Na+ movement is driven by the electrochemical gradient of H+ via a Na+/H+ antiporter. The sp ecific ATPase activity of S. floricola membranes was at least twofold higher than that described in other mollicutes. Activity was Mg2+-depe ndent over the ph range (6.5-8.5) tested, but was very little affected by Na+ (up to 100 mM). DCCD (25 mu M) markedly inhibited both membran e-bound and solubilized ATPase activity, whereas orthovanadate (50 mu M) had only a small inhibitory effect. The properties of the enzyme ar e consistent with a F0F1-ATPase. It is suggested that the enzyme opera tes in the direction of hydrolysing ATP formed by glycolysis leading t o the generation of a Delta pH. which is the major driving force for t he Na+/H+ antiport activity.