The interaction of phospholipid membranes and detergents with glutamate dehydrogenase.

1. Both the anionic detergent sodium dodecylsulphate and the cationic detergent cetyltrimethylammonium bromide quenched the protein fluorescence of glutamate dehydrogenase. The anionic compound was more effective and brought about 50% quenching at a detergent concentration of 0.4 mM. The zwitterionic amphiphile, lysolecithin, did not quench the protein fluorescence and neither did the short-chain detergent n-hexylsulphonate, which under the range of concentrations examined (less than 1 mM) does not form micelles. 2. The zwitterionic phospholipid, phosphatidylcholine, did not quench the protein fluorescence but the anionic phospholipids, phosphatidylserine and cardiolipin, induced a reversible quenching of the enzyme fluorescence. These observations confirm the specificity of the phospholipid-enzyme interactions as deduced from the kinetic studies of the preceding paper. The degree of quenching brought about by the phospholipids decreased with increasing ionic strength and increasing pH and could be substantially reduced by basic proteins. An electrostatic contribution to the interaction is inferred from these results. 3. The binding of the anionic phospholipids to the enzyme is manifested in a further enhancement of the fluorescence of a 1-anilinonaphthalene-8-sulphonate-enzyme complex. The presence of substrates and allosteric effectors affect the interaction of the lipids with the enzyme as indicated by the magnitude of this increase in fluorescence. The enhancement of fluorescence of NADH when bound to the enzyme was not affected by the binding of the lipids. 4. The complex formed between the enzyme and phosphatidylserine/phosphatidylcholine can be solubilized in isooctane. The photolability of the aqueous protein when subjected to irradiation at 280 nm is suppressed in the isooctane-soluble complex. 5. Phosphatidylserine brings about a rapid (t 1/2 is about 150 ms at a lipid concentration of 0.75 mM) dissociation of the linear aggregates formed between the enzyme oligomers. 6. A model of the enzyme-lipid-membrane complex, consistent with these results, is proposed. It is suggested that the enzyme is an allotopic protein and that the dissociation of the enzyme in vitro may involve binding sites on the protein which are designed for interaction with the cardiolipin of the inner mitochondrial membrane, when the enzyme is in the mitochondrial matrix.