Comparison of 3-hydroxybutyrate dehydrogenase from bovine heart and rat liver mitochondria

Jo, McIntyre; Latruffe, Norbert; Sc, Brenner; Fleischer, Sidney

doi:10.1016/0003-9861(88)90171-3

Cited by 22 publications

(28 citation statements)

References 34 publications

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“…All four mAbs produced against bovine heart BDH are the IgG1 type and one of the mAbs produced against rat liver BDH is IgM type. Five of the six mAbs show cross-species reaction with BDH, consistent with the similarity of bovine heart and rat liver BDH (McIntyre et al, 1988). Western blots demonstrate that mAbs 3-IOA, 4-4D and 36-4-1 are highly specific in detecting BDH among other mitochondrial proteins ( Figure 1).…”

Section: Characterization Of Mabs Against Bdhsupporting

confidence: 70%

“…These results suggest that inhibition of BDH-MPL by mAb 3-2C does not significantly alter substrate Km values, but decreases the limiting catalytic rate of the enzyme. For native BDH in SMV, no significant inhibition was observed even with mAb 3-2C, although polyclonal antibodies have been found previously to inhibit BDH in SMV (Churchill et al, 1983b;McIntyre et al, 1988). Although BDH in SMV was not significantly inhibited by mAb 3-2C, we found on the basis of immunoprecipitation studies that the epitope for this mAb is at least partially accessible.…”

Section: Epitopecontrasting

confidence: 53%

“…SMV from bovine heart and rat liver were isolated from mitochondria as described by Fleischer et al (1974). Bovine heart and rat liver BDHs were purified from the respective mitochondria as described (McIntyre et al, 1988). Stocks of apoBDH were stored in buffer containing 5 mM Hepes/NaOH, pH 7.5, 5 mM dithiothreitol (DTT) and LiBr (0.15 M for the rat liver enzyme and 0.4 M for the bovine heart enzyme).…”

Section: Preparation Of Membranes Enzymes and Phosphollpid Vesiclesmentioning

confidence: 99%

“…It is localized at the matrix face of the mitochondrial inner membrane (McIntyre et al, 1978a;Fleischer et al, 1983). The enzyme has been purified from bovine heart and rat liver mitochondria (Bock and Fleischer, 1974;McIntyre et al, 1988) and more recently from rat brain (Zhang and Churchill, 1990). Purified BDH is devoid of lipid or detergent and is referred to as apoBDH.…”

Section: Introductionmentioning

confidence: 99%

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Monoclonal antibodies for structure-function studies of (R)-3-hydroxybutyrate dehydrogenase, a lipid-dependent membrane-bound enzyme

Adami

Duncan

McIntyre

et al. 1993

Biochemical Journal

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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.

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Section: Characterization Of Mabs Against Bdhsupporting

confidence: 70%

Section: Epitopecontrasting

confidence: 53%

Section: Preparation Of Membranes Enzymes and Phosphollpid Vesiclesmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

See 2 more Smart Citations

Monoclonal antibodies for structure-function studies of (R)-3-hydroxybutyrate dehydrogenase, a lipid-dependent membrane-bound enzyme

Adami

Duncan

McIntyre

et al. 1993

Biochemical Journal

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show abstract

“…Purified BDH, devoid of lipid (apo-BDH), was isolated from bovine heart mitochondria as described in [25] and stored at -196 0C in 0.4 M LiBr/5 mM Hepes/NaOH, pH 7.0, containing 5 mM dithiothreitol. Vesicles of mitochondrial phospholipids (MPL) were prepared as described previously [8,10,24].…”

Section: Purification Of Bdh and Reconstitution With Phospholipid Vesmentioning

confidence: 99%

Effect of selective thiol-group derivatization on enzyme kinetics of (R)-3-hydroxybutyrate dehydrogenase

Dalton

McIntyre

Fleischer

1993

Biochemical Journal

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(R)-3-Hydroxybutyrate dehydrogenase (BDH) is a phosphatidylcholine-requiring tetrameric enzyme with two thiol groups (SH-1 and SH-2) per protomer. By first protecting the more rapidly reacting thiol group (SH-1) with diamide [1,1'-azobis-(NN'-dimethylformamide), DM] to form DM(SH-1)BDH, SH-2 can be selectively derivatized by reaction with maleimide reagents such as 4-maleimido-2,2,6,6-tetramethyl-piperidine-N-oxyl (MSL), which gives DM(SH-1)MSL(SH-2)BDH. Reduction with dithiothreitol (DTT) regenerates SH-1, yielding MAL(SH-2)BDH (where MAL is the diamagnetic reduction product of MSL-BDH and DTT). The enzymic activity of DM(SH-1)BDH is decreased to approx. 4% relative to the native purified enzyme, and the apparent Km for substrate, KmBOH, is increased approx. 100-fold. Reduction of DM(SH-1)BDH with DTT regenerates SH-1 and restores normal enzymic function. Modification of SH-2 with piperidinylmaleimide [MAL(SH-2)BDH] diminishes enzymic activity to approx. 35% of its original value, but has no significant effect on apparent KmBOH. The doubly derivatized enzyme, DM(SH-1)MSL(SH-2)BDH, has lower enzymic activity [about half that for DM(SH-2)BDH] and a yet higher apparent KmBOH than DM(SH-1)BDH. Derivatization of SH-2 with different maleimide reagents results in diminished activity approximately proportional to the size of the maleimide substituent, suggesting that this inhibition is steric. Whereas modification of SH-1 results in marked changes in kinetic parameters (increased apparent Km and reduced apparent Vmax), derivatization of SH-2 has a lesser effect on enzymic function. Thus SH-1 is postulated to be closer to the active centre than is SH-2, although neither is involved in catalysis, since: (1) the activity of the derivatized enzyme is not abolished; and (2) activity can be enhanced by increasing substrate (and cofactor) concentrations.

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Kinetic characterization of a cytosolic L-β-hydroxybutyrate dehydrogenase from hepatopancreas of a terrestrial snail,Cepaea nemoralis

Stuart

Ballantyne

1997

J. Exp. Zool.

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Comparison of 3-hydroxybutyrate dehydrogenase from bovine heart and rat liver mitochondria

Cited by 22 publications

References 34 publications

Monoclonal antibodies for structure-function studies of (R)-3-hydroxybutyrate dehydrogenase, a lipid-dependent membrane-bound enzyme

Monoclonal antibodies for structure-function studies of (R)-3-hydroxybutyrate dehydrogenase, a lipid-dependent membrane-bound enzyme

Effect of selective thiol-group derivatization on enzyme kinetics of (R)-3-hydroxybutyrate dehydrogenase

Kinetic characterization of a cytosolic L-β-hydroxybutyrate dehydrogenase from hepatopancreas of a terrestrial snail,Cepaea nemoralis

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