Abstract
The binding of porcine heart mitochondrial malate dehydrogenase and beta-hydroxyacyl-CoA dehydrogenase to bovine heart NADH:ubiquinone oxidoreductase (complex I), but not that of bovine heart alpha-ketoglutarate dehydrogenase complex, is virtually abolished by 0.1 mM NADH. The malate dehydrogenase and beta-hydroxyacyl-CoA enzymes compete in part for the same binding site(s) on complex I as do the malate dehydrogenase and alpha-ketoglutarate dehydrogenase complex enzymes. Associations between mitochondrial malate dehydrogenase and bovine serum albumin were observed. Subtle convection artifacts in short-time centrifugation tests of enzyme association with the Beckman Airfuge are described. Substrate channeling of NADH from both the mitochondrial and cytoplasmic malate dehydrogenase isozymes to complex I and reduction of ubiquinone-1 were shown to occur in vitro by transient enzyme-enzyme complex formation. Excess apoenzyme causes little inhibition of the substrate channeling reaction with both malate dehydrogenase isozymes in spite of tighter equilibrium binding than the holoenzyme to complex I. This substrate channeling could, in principle, provide a dynamic microcompartmentation of mitochondrial NADH.
Highlights
From the Department of Biochemistry, Oklahoma State University, Stillwater, Oklahoma 74078-0454 and the TDepartment of Biochemistry, Indiana University School of Medicine, Northwest Center for Medical Education, Gary, Indiana 46408
( 7 )raises thepossibility that substrate channelingof NADH can occur to complex I, thereby providing a dynamic microcompartmentation of NADH within the mitochondrial matrix. One would expect such substrate channelingto occur by the stableenzyme-enzyme complexes described( 7 ) .we report binding and substrate channeling studies with malate dehydrogenases that demonstrate that NADHabolishes the measured binding of the mitochondrial enzymes, but an efficient substrate channelingof NADH by transient enzymeenzyme complexes occurs in vitro
Measurements of the V, and K, of complex I for NADH were made by conventional initial velocity methods on a Hitachi 100-80 spectrophotometer and analyzed by nonlinear least squares analysis with weights based on a standard deviation of 10% of measured velocities
Summary
No evidence of interaction between the malate andhydroxyacyl-CoA dehydrogenases was obtained in separate fluorescence polarization measurements withfluorescein-derivatized malate dehydrogenase.Confidence thatthesepolarization cence titrations (to determine the dissociation constant of NADH) data correctly indicate negligible interactions between the or for the substratechanneling measurements.Complex I was assayed in solutions of mM potassium phosphate, pH 8.0, 2 mM sodium azide, 0.1 mM ubiquinone-11,0.1 mM NADH, and a nominal asolectin concentration of 0.22 mg/ml for the ubiquinone assay This nominal asolectin concentration isthat calculated assuming completreecovery of the asolectin during the preparation of liposomes [11];the true asolectin concentration is considerably lower since only the lighter malate and hydroxyacyl-CoA dehydrogenases was provided by measurementswiththis fluorescein-derivatized malate dehydrogenase,which demonstrated 1) the previously published interaction between malate dehydrogenase and citrate synthase [21] and2) substantial interactionbetween fluorescein-derivatized malate dehydrogenase and bovine serum alliposomes are collected in ]:his procedure.
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