MONOCLONAL-ANTIBODIES FOR STRUCTURE-FUNCTION STUDIES OF (R)-3-HYDROXY-BUTYRATE DEHYDROGENASE, A LIPID-DEPENDENT MEMBRANE-BOUND ENZYME

Monoclonal antibodies (mAbs) have been used to study structure-functio n relationships of (R)-3-hydroxybutyrate dehydrogenase (BDH) (EC 1.1.1 .30), a lipid-requiring mitochondrial membrane enzyme with an absolute and specific requirement for phosphatidylcholine (PC) for enzymic act ivity. The purified enzyme (apoBDH, devoid of phospholipid and thereby inactive) can be re-activated with preformed phospholipid vesicles co ntaining PC or by short-chain soluble PC. Five of six mAbs cross-react with BDH from bovine heart and rat liver, including two mabs to confo rmational epitopes. One mAb was found to be specific for the C-termina l sequence of BDH and served to: (1) map endopeptidase cleavage and ep itope sites on BDH; and (2) demonstrate that the C-terminus is essenti al for the activity of BDH. Carboxypeptidase cleavage of only a few (l ess-than-or-equal-to 14) C-terminal amino acids from apoBDH (as detect ed by the loss of C-terminal epitope for mAb 3-10A) prevents activatio n by either bilayer or soluble PC. Further, for BDH in bilayers contai ning PC, the C-terminus is protected from carboxy-peptidase cleavage, whereas in bilayers devoid of PC the C-terminus is cleaved, and subseq uent activation by PC is precluded. We conclude that: (1) the C-termin us of BDH is essential for enzymic activity, consistent with the predi ction, from primary sequence analysis, that the PC-binding site is in the C-terminal domain of BDH; and (2) the allosteric activation of BDH by PC in bilayers protects the C-terminus from carboxypeptidase cleav age, indicative of a PC-induced conformational change in the enzyme.