ABSTRACT
The exceptionally high protein concentration in living cells can favor functional protein-protein interactions that can be difficult to detect with purified proteins. In this study we describe specific interactions between mammalian D-glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and L-lactate dehydrogenase (LDH) isozymes from heart and muscle. We use poly(ethylene-glycol) (PEG)-induced coprecipitation and native agarose electrophoresis as two independent methods uniquely suited to mimic some of the conditions that can favor protein-protein interaction in living cells. We found that GAPDH interacts with heart or muscle isozymes of LDH with approximately one-to-one stoichiometry. The interaction is specific; GAPDH shows interaction with two LDH isozymes that have very different net charge and solubility in PEG solution, while no interaction is observed with GAPDH from other species, other NAD(H) dehydrogenases, or other proteins that have very similar net charge and molecular mass. Analytical ultracentrifugation showed that the LDH and GAPDH complex is insoluble in PEG solution. The interaction is abolished by saturation with NADH, but not by saturation with NAD(+) in correlation with GAPDH solubility in PEG solution. The crystal structures show that GAPDH and LDH isozymes share complementary size, shape, and electric potential surrounding the active sites. The presented results suggest that GAPDH and LDH have a functional interaction that can affect NAD(+)/NADH metabolism and glycolysis in living cells.
Subject(s)
Glyceraldehyde-3-Phosphate Dehydrogenases/metabolism , L-Lactate Dehydrogenase/metabolism , Muscle, Skeletal/enzymology , Myocardium/enzymology , Animals , Binding Sites , Glyceraldehyde-3-Phosphate Dehydrogenases/chemistry , L-Lactate Dehydrogenase/chemistry , Protein Structure, Tertiary , Rabbits , SwineABSTRACT
A method for the quantitative assay of mammalian cell micropermeabilization is described. The method is based on the permeabilization-induced loss of endogenous glycolytic cofactors and consequent discontinuation of cellular lactate production. Advantages of the method include sensitivity and precision similar to that of micropermeabilization assays based on the release of 86Rb+ from preloaded cells, avoidance of radioactivity, and simplicity of the measurements and equipment required.
Subject(s)
Cell Membrane Permeability , Lactic Acid/biosynthesis , Animals , Bacterial Toxins/pharmacology , Cell Line, Tumor , Cell Membrane Permeability/drug effects , Glycolysis , Lactic Acid/analysis , Lactic Acid/metabolism , Phosphorus/pharmacology , Rats , Reproducibility of Results , Staphylococcus aureus/metabolismABSTRACT
Four different quaternary ammonium chloride-modified poly(propylenimine) (PPI) dendrimers were synthesized by alkylation of a PPI dendrimer having eight dimethylamino end groups with 1-bromooctane or 1-bromododecane. By varying the mole ratio of alkyl bromide to dendrimer, averages of 4-10 quaternary ammonium groups were formed. The new amphiphilic dendrimers are surface active and are micellar catalysts in water. The dendrimers have critical aggregation concentrations between 8.5 x 10(-4) and 9.0 x 10(-5) M. Decarboxylation of 6-nitrobenzisoxazole-3-carboxylate at 25 degrees C was 650 times faster than in water alone in the presence of a dendrimer quaternized with eight dodecyl chains at a concentration of 2.45 mM in quaternary ammonium groups. The order of the catalytic efficiency of the new dendrimers decreased with the length and number of hydrophobic alkyl groups in the order (C(12))(8) > (C(12))(4) > (C(8))(10) > (C(8))(5). The pseudo-first-order rate constants for basic hydrolysis of p-nitrophenyl hexanoate in pH 9.4 buffer at 30 degrees C using the (C(12))(8) and (C(12))(4) dendrimers were 26 and 13 times higher than those for hydrolysis with no dendrimer. The kinetic data were fit to a single-site binding model to evaluate the contributions of binding constants of reactants to the dendrimers and catalytic rate constants of the bound species to the overall catalytic activity.
Subject(s)
Amines/chemistry , Dendrimers/chemistry , Polypropylenes/chemistry , Quaternary Ammonium Compounds/chemistry , Caproates/chemical synthesis , Catalysis , Dendrimers/chemical synthesis , Hydrolysis , Isoxazoles/chemical synthesis , Molecular Structure , Nitrobenzenes/chemical synthesis , Polypropylenes/chemical synthesisABSTRACT
Evidence for the NADH-modulated formation of a complex between alpha-glycerol-3-phosphate dehydrogenase and l-lactate dehydrogenase was reported [Yong, H., Thomas, G. A., and Peticolas, W. L. (1993) Biochemistry 32, 11124-11131]. This NADH-modulated association suggested a mechanism of potentially great importance to enzyme modulation and the controversial phenomena of direct NADH channeling. In the present paper, we reproduce with additional controls the experiments described by Yong et al. ((1993) Biochemistry 32, 11124-11131). Our results conclusively demonstrate the absence of detectable association between alpha-glycol-3-phosphate dehydrogenase and l-lactate dehydrogenase.
