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Atpases in plasma membrane enriched fractions from Dictyostelium discoideum MacDonald, James Innes Scott


Evidence is presented for the existence of an ATPase activity in D. discoideum plasma membranes. This activity was distinct from the mitochondrial ATPase in that it was insensitive to azide and oligomycin. The ATPase was stimulated by Mg⁺² and to a lesser extent by Ca⁺² and was not affected by equimolar Na⁺/K⁺ or ouabain. Vanadate, DES, thimerosal and DCCD all proved to be partially inhibitory. Enzyme activity was solubilized with a wide variety of detergents, with lysolecithin giving the best results. Concomitant with solubilization was a partial loss of DES sensitivity which was shown to be due to the presence of a labile DES sensitive ATPase in addition to a stable DES insensitive activity. The DES sensitive ATPase was stimulated by Mg⁺² but only somewhat by Ca⁺² whereas the DES insensitive enzyme was stimulated equally by either. The DES sensitive enzyme also displayed Michaelis-Menten kinetics when enzyme activity was measured as a function of the ATP concentration while the DES insensitive ATPase displayed kinetics which were indicative of a sigmoidal relationship between substrate concentration and enzyme activity. Fractionation of solubilized plasma membranes by ion exchange chromatography resolved two DES insensitive ATPase activities, designated peaks I and II. Peak I was insensitive to vanadate and expressed optimum activity with pyrophosphate. Optimal activity was at alkaline pH values. Peak II was purified by two different procedures. The first involved an initial separation on DEAE-Sephace1 followed by centrifugation through a linear glycerol gradient. The second involved an initial chromatographic fractionation by Sephacryl S-300 gel filtration followed by DEAE-Sephacel ion exchange chromatography of the ATPase containing fractions. Both procedures resulted in preparations that contained a single major component of apparent molecular weight 6 4 kDa, as assessed by SDS-polyacrylamide gel electrophoresis. The purified ATPase was sensitive to vanadate and fluoride but insensitive to DCCD , thimerosal and N-ethylmaleimide. The enzyme was activated equally by either Mg⁺² or Ca⁺² in millimolar concentrations. ATP hydrolysis was also stimulated by millimolar concentrations of Mn⁺², Zn⁺² or Cu⁺². The ATPase displayed sigmoidal kinetics when assayed as a function of ATP concentration in the absence of any divalent cation. Addition of 1 or 10 mM Mg⁺² or Ca⁺² increased the substrate affinity of the enzyme, while 100 mM divalent cation proved inhibitory. The enzyme was not stimulated by low concentrations of Ca⁺² or by Ca⁺²-calmodulin, suggesting that it was probably not a Ca⁺² pump.

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