Further purification and characterization of the succinyl-CoA:3-hydroxy-3-methylglutarate coenzyme A transferase from rat-liver mitochondria

Succinyl-CoA:3-hydroxy-3-methylglutarate coenzyme A transferase, previously identified in rat-liver mitochondria (Deana et al. (1981), Biochim. Biophys. Acta 662, 119-124), was purified to near homogeneity and further characterized. After the last purification steps consisting of Ultrogel AcA-44 filtration and agarose-hexane-coenzyme A chromatography, the enzyme was apparently tetrameric with a mass of 48-52 kDa determined by gel filtration on Sephadex G-75, ultracentrifugation through a sucrose gradient and SDS-gel electrophoresis. By means of a HPLC technique developed for measuring the CoA esters we could determine the enzyme activity in both forward and reverse directions and show that the kinetic constants, i.e., Km of reactants and Vmax, are not too different for the two reactions. Double-reciprocal plots of the enzyme velocities versus the concentration of one substrate at different fixed concentrations of the other substrate gave families of straight lines converging below the substrate-abscissa for both forward and backward reactions, indicating a kinetic mechanism of rapid equilibrium random Bi-Bi type. The competitive inhibition of the product succinate with respect to both reactants, 3-hydroxy-3-methylglutarate and succinyl-CoA, as well as the Haldane relationships are consistent with this conclusion. An inhibitory effect on CoA transferase activity by acetate, acetoacetate, acetyl-CoA, acetoacetyl-CoA, coenzyme A, carnitine, ZnCl2 and high concentrations of the monovalent anions ClO4-, F-, I- and Cl- was also found.     

A new plot for multiple enzyme inhibition

A new graphical method is described for analyzing the results of multiple inhibition experiments. It is applicable to either single- or multi-substrate enzyme systems obeying Michaelis-Menten kinetics and is valid irrespective of the type of inhibition (competitive, noncompetitive, uncompetitive, mixed). According to this method, mutually exclusive inhibitor binding gives rise to lines that converge on the vertical axis, whereas mutually nonexclusive inhibitors yield lines that intersect to the left of the vertical axis. It has been pointed out that the inhibitor interaction factor can be determined directly from multiple inhibition experiments only if at least one of the inhibitors is noncompetitive. When this is the case, the present plot provides a very simple way of determining the inhibitor interaction factor from the coordinates of the intersection point.     

Activation of (Na+ + K+)-dependent ATPase by lipid vesicles of negative phospholipids.

1. Kidney (Na+ + K+)-stimulated ATPase was depleted of phospholipids by extraction with lubrol and inserted in lipid structures of known composition. Both ouabain-sensitive ATPase and phosphatase reactions could be partially restored by lipid replacement. 2. Lipid vesicles of natural and synthetic negative phospholipids proved to be effective. The low activity of uncharged liposomes was increased when negative charges were included into the bilayer structure. 3. Reactivation by negative phospholipids was accompanied by spontaneous re-assembly of a stable lipid-protein complex. By contrast, the interaction of lipid deficient ATPase complex with uncharged lamellae was possible only after sonication of lipid-protein suspension. Reactivation did not ensue. 4. The ouabain-sensitive ATPase reactivated by synthetic dioleoylphosphatidylglycerol yielded curvilinear Arrhenius plots. The same pattern was seen with the original undepleted microsomal preparation. A discontinuity close to the temperature of fluid-order transition was found with dimyristoyl phosphatidylglycerol. 5. It is concluded that reassembly of lipid-deficient (Na+ + K+)-stimulated ATPase requires the addition of diacylphospholipids with fluid acyl-chains and negatively charged polar heads able to assemble in an expanded lamellar configuration.