Prostaglandin and acyl chain effects on glutamate dehydrogenase activity.

Abstract

AbstractProstaglandins A1 (PGA1), A2, B1, B2, E1, E2, F1α, F2α, and 19 esterified natural fatty acids were tested as effectors of beef liver glutamate dehydrogenase (L‐glutamate: NAD(P)+, oxidoreductase [deaminating], EC 1.4.1.3). All prostaglandins tested are found to activate the enzyme initially, but only PGA2>PGB2≥PGA1 cause a subsequent time‐dependent loss (not inhibition) of NADH oxidation activity. Both PGA1 and PGA2 desensitize glutamate dehydrogenase to allosteric activation by ADP, whereas PGA2 and PGB2 desensitize to allosteric inactivation by GTP. Preincubation of enzyme with diethylstilbestrol prevents the initial activation by the PG. Of the methyl esters, only prostaglandin precursors inactivated the enzyme. Simultaneous desensitization to the ADP and GTP allosteric effects resulted. Multiple esterification to glycerol or phospholipids enhanced the action of linoleoyl and diminished the action of linolenoyl chains. Preincubation of the PGA with glutathione or cysteine prevents the inactivation; i.e., the sulfhydryl binding region of the prostaglandin must be free for enzyme to be inactivated. Sulfhydryl reagents also protect the enzyme from the effects of the unsaturated acyl chains, and pHMB mimics acyl protection against GTP allosteric inactivation. Where the lipid effector is active against sulfhydryl groups, the desensitizations to the ADP and GTP allosteric effectors are reciprocal. The initial activation, subsequent inactivation and desensitization to ADP and GTP are all characteristic of binding in the estrogen‐specific effector site, suggesting this site as the target for PG and acyl action. In the PGA2 activation, the effect is found to be amplified by the cooperativity of the enzyme at 1 PG molecule/6 molecules of GDH. We conclude from the action of the PG and structural analogs that the initial activation of glutamate dehydrogenase is caused by α,β‐unsaturated monoketo cyclopental structures. GTP inhibition is blocked primarily by diketo structures which eventually inactivate the enzyme. ADP activation is blocked by sulfhydryl binding of the unsaturated cyclopental keto structure of the PG. Appearance of a 270 nm absorbance simultaneous to the acyl effects on the enzyme suggests that conjugated unsaturations are responsible for the precursor's qualitatively similar action to that of the PG.