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.

Poly-3-Hydroxybutyrate Degradation in Rhizobium (Sinorhizobium) meliloti : Isolation and Characterization of a Gene Encoding 3-Hydroxybutyrate Dehydrogenase

“…3). The R. meliloti BdhA and the B. subtilis YxjF do not contain the C-terminal domain found in the mammalian Bdh which has been shown to be involved in binding phosphatidylcholine and which is essential for enzyme activity (1,38,52). This is consistent with the observation that the bacterial Bdh does not demonstrate a requirement for phosphatidylcholine for activity.…”

Lactococcus lactis, an Alternative System for Functional Expression of Peripheral and Intrinsic Arabidopsis Membrane Proteins

“…Based on sequence similarity, the Arabidopsis ceQORH protein belongs to the short-chain dehydrogenases/reductases (SDR) family [40] . SDRs are known to use artificial electron acceptors like nitroblue tetrazolium (NBT) as substrates, and activity of heterologously expressed and purified SDR requires conditions mimicking a lipid environment [41] – [43] . Since the native ceQORH protein is associated with the inner membrane of the chloroplast envelope [16] , we tried to recover this dehydrogenase activity in combining the purified recombinant ceQORH protein with a soybean lipid extract as previously described [43] .…”

Calcium regulation of chloroplast protein translocation is mediated by calmodulin binding to Tic32

“…The fact that calmodulin regulates protein import by binding to Tic32 is especially intriguing because the protein has been shown to be a vital component of the Tic translocon (14). In contrast, Tic32 itself does not require the Tic translocon for insertion into the inner envelope membrane (15) and thus is itself not inhibited in its translocation by calmodulin or calcium (8).…”

“…SDRs are known to accept nonspecific substrates like nitroblue tetrazolium (NBT) or FeCN for their reaction, but activity of heterologously expressed or purified protein sometimes requires conditions mimicking a lipid environment (14). Because the native Tic32 is tightly associated with the inner envelope membrane (5), we tried to recover dehydrogenase activity by combining purified heterologously expressed Tic32 with phosphatidylcholine (PC) lipids (Fig.…”