Monoclonal antibodies for structure-function studies of (R)-3-hydroxybutyrate dehydrogenase, a lipid-dependent membrane-bound enzyme.
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.
Adami
P
P
Department of Molecular Biology, Vanderbilt University, Nashville, TN 37235.
Duncan
T M
TM
McIntyre
J O
JO
Carter
C E
CE
Fu
C
C
Melin
M
M
Latruffe
N
N
Fleischer
S
S
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
D000911:Antibodies, Monoclonal; D000939:Epitopes; D008081:Liposomes; D010743:Phospholipids; D006884:Hydroxybutyrate Dehydrogenase; D002268:Carboxypeptidases; C022633:serine carboxypeptidase