The Interaction of Phospholipid Membranes and Detergents with Glutamate Dehydrogenase

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Abstract
The interaction of beef liver glutamate dehydrogenase with cardiolipin from both beef liver mitochondria and beef heart mitochondria, with phosphatidylcholine from both beef liver mitochondria and egg-yolk and with beef brain phosphatidylserine was investigated by steady-state kinetic methods. The phosphatidylcholine did not inhibit the enzyme under a wide range of conditions. The cardiolipins and phosphatidylserine inhibited the enzyme. The inhibition by these lipids diminished with time if the lipids were prepared and the reaction was studied in either phosphate or Tris buffers, but in zwitterionic buffers these lipids brought about a rapid, reversible inhibition which remained stable with time for at least 150 min. The kinetic type of the inhibition was difficult to determine because of variation between lipid sonicates. Complex mixed types of inhibition were found with cardiolipin, and with phosphatidylserine the inhibition approximated to a non-competitive interaction with Ki(app) values varying between (0.9-6.1) .times. 10-6 M. The extent of inhibition decreased with increasing pH and with increasing ionic strength. Basic proteins, such as cytochrome c, show a higher affinity for the anionic membranes and can dissociate the enzyme .cntdot. lipid complexes. Cosonicates of the cardiolipin and phosphatidylcholine inhibited the enzyme, the extent of inhibition increasing in proportion to the amount of acidic lipid. Sodium dodecylsulfate causes a time-dependent inhibition of the enzyme. The kinetics of this effect and its variation with detergent concentration were studied. The relationship of these observations to the structure and function of the enzyme is discussed. Their apparent regulation of the enzyme by estrogens and other small molecules may be due to their binding in vitro at sites on the enzyme designed for binding cardiolipin, when the enzyme is functioning in vivo. The association of the enzyme oligomer in vitro may, for similar reasons, be an artifact.