The interaction of glutamate dehydrogenase and malate dehydrogenase with phospholipid membranes.

Abstract

The interaction between sonicated dispersions of purified phospholipids, corresponding to those found in the inner mitochondrial membrane, and the enzymes was studied by kinetic and spectroscopic methods.With glutamate dehydrogenase the charge type of the lipid head group determines the extent of lipid‐protein complexing. Phosphatidylcholine did not significantly alter the activity of the enzyme at lipid concentrations up to 1 mM, and the nuclear magnetic resonance (NMR) spectrum of the lipid (1%, w/v) was not altered in the presence of enzyme (up to 20% w/v). Phosphatidylethanolamine did not affect the activity in the oxidation of glutamate, but interfered with the assay in the reverse direction, possibly owing to Schiff‐base formation with 2‐oxoglutarate. The negatively charged cardiolipin, which is found uniquely in the inner mitochondrial membrane, strongly and reversibly inhibited the enzyme (50% inhibition with 0.1–20 μM lipid, depending on conditions). Another anionic phospholipid, phosphatidylserine (which does not occur in mitochondria), also inhibited the enzyme, but less effectively (50% inhibition at about 8 μM). The lipid‐enzyme interaction decreases with increasing ionic strength, indicating an electrostatic contribution to the binding. NMR studies suggest that there is also an apolar interaction involving the lipid chains.The studies with malate dehydrogenase concentrated on detecting possible stabilization of this enzyme through interaction with lipids. A clear‐cut stabilization was found with lysophosphatidylcholine. Under conditions such that the enzyme alone lost 50% on the initial activity in about 2 h, lysophosphatidylcholine gave a slight (20%) activation and this activity remained constant for several hours. NMR studies indicated that, as with the related enzyme 3‐hydroxybutyrate dehydrogenase, both the polar choline head group and the hydrocarbon chains are involved in the binding of the lipid to the protein.These specific lipid · protein complexes are probably examples of allotopic interactions. This form of complexing may explain aspects of the behaviour of these enzymes in vivo.