title: Monoclonal antibodies for structure-function studies of (R)-3-hydroxybutyrate dehydrogenase, a lipid-dependent membrane-bound enzyme.
abstract: Monoclonal antibodies (mAbs) have been used to study structure-function 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 activity. The purified enzyme (apoBDH, devoid of phospholipid and thereby inactive) can be re-activated with preformed phospholipid vesicles containing 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 conformational epitopes. One mAb was found to be specific for the C-terminal sequence of BDH and served to: (1) map endopeptidase cleavage and epitope sites on BDH; and (2) demonstrate that the C-terminus is essential for the activity of BDH. Carboxypeptidase cleavage of only a few (< or = 14) C-terminal amino acids from apoBDH (as detected by the loss of C-terminal epitope for mAb 3-10A) prevents activation by either bilayer or soluble PC. Further, for BDH in bilayers containing PC, the C-terminus is protected from carboxy-peptidase cleavage, whereas in bilayers devoid of PC the C-terminus is cleaved, and subsequent activation by PC is precluded. We conclude that: (1) the C-terminus of BDH is essential for enzymic activity, consistent with the prediction, 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 cleavage, indicative of a PC-induced conformational change in the enzyme.
authors.affiliation: Department of Molecular Biology, Vanderbilt University, Nashville, TN 37235.
authors.forename: P
authors.initials: P
authors.lastname: Adami
authors.forename: T M
authors.initials: TM
authors.lastname: Duncan
authors.forename: J O
authors.initials: JO
authors.lastname: McIntyre
authors.forename: C E
authors.initials: CE
authors.lastname: Carter
authors.forename: C
authors.initials: C
authors.lastname: Fu
authors.forename: M
authors.initials: M
authors.lastname: Melin
authors.forename: N
authors.initials: N
authors.lastname: Latruffe
authors.forename: S
authors.initials: S
authors.lastname: Fleischer
mesh_terms: D000818:Animals; D000911:Antibodies, Monoclonal; D015153:Blotting, Western; D002268:Carboxypeptidases / Q000378:metabolism; D002417:Cattle; D000939:Epitopes / Q000032:analysis*; D006884:Hydroxybutyrate Dehydrogenase / Q000737:chemistry* / Q000276:immunology / Q000378:metabolism; D007425:Intracellular Membranes / Q000201:enzymology*; D007700:Kinetics; D008081:Liposomes; D008929:Mitochondria, Heart / Q000201:enzymology*; D008930:Mitochondria, Liver / Q000201:enzymology*; D008970:Molecular Weight; D010449:Peptide Mapping; D010743:Phospholipids / Q000494:pharmacology; D011487:Protein Conformation; D051381:Rats
chemical_list: D000911:Antibodies, Monoclonal; D000939:Epitopes; D008081:Liposomes; D010743:Phospholipids; D006884:Hydroxybutyrate Dehydrogenase; D002268:Carboxypeptidases; C022633:serine carboxypeptidase