BDH Activity Research Articles

Structure-dependence and metal-dependence on atomically dispersed Ir catalysts for efficient n-butane dehydrogenation

Single-site pincer-ligated iridium complexes exhibit the ability for C-H activation in homogeneous catalysis. However, instability and difficulty in catalyst recycling are inherent disadvantages of the homogeneous catalyst, limiting its development. Here, we report an atomically dispersed Ir catalyst as the bridge between homogeneous and heterogeneous catalysis, which displays an outstanding catalytic performance for n-butane dehydrogenation, with a remarkable n-butane reaction rate (8.8 mol·gIr−1·h−1) and high butene selectivity (95.6%) at low temperature (450 °C). Significantly, we correlate the BDH activity with the Ir species from nanoscale to sub-nanoscale, to reveal the nature of structure-dependence of catalyst. Moreover, we compare Ir single atoms with Pt single atoms and Pd single atoms for in-depth understanding the nature of metal-dependence at the atomic level. From experimental and theoretical calculations results, the isolated Ir site is suitable for both reactant adsorption/activation and product desorption. Its remarkable dehydrogenation capacity and moderate adsorption behavior are the key to the outstanding catalytic activity and selectivity.

In vivo investigation of hyperpolarized [1,3-13C2]acetoacetate as a metabolic probe in normal brain and in glioma

Dysregulation in NAD+/NADH levels is associated with increased cell division and elevated levels of reactive oxygen species in rapidly proliferating cancer cells. Conversion of the ketone body acetoacetate (AcAc) to β-hydroxybutyrate (β-HB) by the mitochondrial enzyme β-hydroxybutyrate dehydrogenase (BDH) depends upon NADH availability. The β-HB-to-AcAc ratio is therefore expected to reflect mitochondrial redox. Previous studies reported the potential of hyperpolarized 13C-AcAc to monitor mitochondrial redox in cells, perfused organs and in vivo. However, the ability of hyperpolarized 13C-AcAc to cross the blood brain barrier (BBB) and its potential to monitor brain metabolism remained unknown. Our goal was to assess the value of hyperpolarized [1,3-13C2]AcAc in healthy and tumor-bearing mice in vivo. Following hyperpolarized [1,3-13C2]AcAc injection, production of [1,3-13C2]β-HB was detected in normal and tumor-bearing mice. Significantly higher levels of [1-13C]AcAc and lower [1-13C]β-HB-to-[1-13C]AcAc ratios were observed in tumor-bearing mice. These results were consistent with decreased BDH activity in tumors and associated with increased total cellular NAD+/NADH. Our study confirmed that AcAc crosses the BBB and can be used for monitoring metabolism in the brain. It highlights the potential of AcAc for future clinical translation and its potential utility for monitoring metabolic changes associated with glioma, and other neurological disorders.

Stereospecificity of Corynebacterium glutamicum 2,3-butanediol dehydrogenase and implications for the stereochemical purity of bioproduced 2,3-butanediol.

The stereochemistry of 2,3-butanediol (2,3-BD) synthesis in microbial fermentations is important for many applications. In this work, we showed that Corynebacterium glutamicum endowed with the Lactococcus lactis genes encoding α-acetolactate synthase and decarboxylase activities produced meso-2,3-BD as the major end product, meaning that (R)-acetoin is a substrate for endogenous 2,3-butanediol dehydrogenase (BDH) activity. This is curious in view of the reported absolute stereospecificity of C. glutamicum BDH for (S)-acetoin (Takusagawa et al. Biosc Biotechnol Biochem 65:1876-1878, 2001). To resolve this discrepancy, the enzyme encoded by butA Cg was produced in Escherichia coli and purified, and the stereospecific properties of the pure protein were examined. Activity assays monitored online by 1H-NMR using racemic acetoin and an excess of NADH showed an initial, fast production of (2S,3S)-2,3-BD, followed by a slow (∼20-fold lower apparent rate) formation of meso-2,3-BD. Kinetic parameters for (S)-acetoin, (R)-acetoin, meso-2,3-BD and (2S,3S)-BD were determined by spectrophotometric assays. V max values for (S)-acetoin and (R)-acetoin were 119±15 and 5.23±0.06μmolmin-1mg protein-1, and K m values were 0.23±0.02 and 1.49±0.07mM, respectively. We conclude that C. glutamicum BDH is not absolutely specific for (S)-acetoin, though this is the preferred substrate. Importantly, the low activity of BDH with (R)-acetoin was sufficient to support high yields of meso-2,3-BD in the engineered strain C. glutamicum ΔaceEΔpqoΔldhA(pEKEx2-als,aldB,butA Cg ). Additionally, we found that the BDH activity was nearly abolished upon inactivation of butA Cg (from 0.30±0.03 to 0.004±0.001μmolmin-1mg protein-1), indicating that C. glutamicum expresses a single BDH under the experimental conditions examined.

Biochemical characterization of unusual meso-2,3-butanediol dehydrogenase from a strain of Bacillus subtilis

meso-2,3-Butanediol dehydrogenase (BDH) catalyzes the redox reaction between (R)-acetoin and meso-2,3-butanediol (meso-BD). The BDHs isolated from bacteria are generally characterized as homo-tetramers. In the present work, an isolated and identified high acetoin (AC)-yielding (41.63g/L) bacteria strain belonging to Bacillus subtilis but without 2,3-butanediol (BD) byproduct during its fermentation was selected. To understand the characteristics of BDH activity from this high AC-yielding strain, we cloned, purified and compared the BDH from Enterobacter aerogenes (CICC10293) (E. a-BDH) with the BDH from this high AC-yielding strain B. subtilis. Sequence alignments indicate a non-conservative amino acid substitution from Asp to Gly at site 194 on the B. s-BDH compared to that of E. a-BDH. Enzymatic analysis of E. a-BDH and D194G B. s-BDH shows D194G B. s-BDH has almost lost its entire enzymatic activity. Moreover, Isothermal titration calorimetry (ITC) measurements detected the substrate binding for the D194G B. s-BDH but no reaction was detected. Circular dicroism (CD) spectroscopy characterization revealed an identical secondary structure of E. a-BDH and D194G B. s-BDH. Remarkably, D194G B. s-BDH is highly susceptible to protease digestion, suggesting that the aspartic acid to glycine substitution might cause the proteolytic susceptibility of D194G B. s-BDH. Furthermore, by homology modeling with meso-2,3-butanediol dehydrogenase from Klebsiella pneumoniae (K. p-BDH) as a template, Gly194 seems to lose the hydrogen bond interactions with the surrounding residues (Gly206, Gly207 and Thr209), resulting in a putative conformational changes of D194G B. s-BDH which might be responsible for the loss of activity.

A new NAD(H)-dependent meso-2,3-butanediol dehydrogenase from an industrially potential strain Serratia marcescens H30

The budC gene coding for a new meso-2,3-butanediol dehydrogenase (BDH) from Serratia marcescens H30 was cloned and expressed in Escherichia coli BL21(DE3), purified, and characterized for its properties. The recombinant BDH with a molecular weight of 27.4 kDa exhibited a reversible transformation between acetoin and 2,3-butanediol. In the presence of NADH, BDH could catalyze the reduction of diacetyl and (3R)-acetoin to (3S)-acetoin and meso-2,3-butanediol, respectively, while (3S)-acetoin as a substrate could be further transformed into (2S, 3S)-2,3-butanediol at pH 9.0. For diol oxidation reactions, (3R)-acetoin and (3S)-acetoin were obtained when meso-2,3-butanediol and (2S,3S)-2,3-butanediol were used as the substrates with BDH and NAD(+). (2R,3R)-2,3-butanediol was not a substrate for the BDH at all. The low K m value (4.1 mM) in meso-2,3-butanediol oxidation reaction and no activity for diacetyl, acetoin, and 2,3-butanediol as the substrates with NADP(+)/NADPH suggested that the budC gene product belongs to a NAD(H)-dependent meso-2,3-BDH. Maximum activities for diacetyl and (3S/3R)-acetoin reduction were observed at pH 8.0 and pH 5.0 while for meso-2,3-butanediol oxidation it was pH 8.0. However, the optimum temperature for oxidation and reduction reactions was about 40 °C. In addition, the BDH activity for meso-2,3-butanediol oxidation was enhanced in the presence of Fe(2+) and for diacetyl and (3S/3R)-acetoin reduction in the presence of Mg(2+) and Mn(2+), while several metal ions inhibited its activity, particularly Fe(3+) for reduction of diacetyl and acetoin. Sequence analysis showed that the BDH from S. marcescens H30 possessed two conserved sequences including the coenzyme binding motif (GxxxGxG) and the active-site motif (YxxxK), which are present in the short-chain dehydrogenase/reductase superfamily.

D-3-hydroxybutyrate oxidation in mitochondria by D-3-Hydroxybutyrate dehydrogenase in Tetrahymena pyriformis

Tetrahymena pyriformis a ciliated protozoan, is considered a good indicator of water pollution. However its energy supply is poorly understood. This work was focused on the metabolism of hydroxybutyrate through the study of the membrane bound mitochondrial NAD+-dependent D-3-hydroxybutyrate dehydrogenase (EC. 1.1.1.30) (BDH), a ketone body catalysing enzyme involvedin the interconversion of D-3-hydroxybutyrate to acetoacetate. Due to lack of informations, the physico-chemical properties and kinetic parameters of the enzyme were examined. The results are the following: 1) D-3-hydroxybutyrate is a good substrate for mitochondria. 2) The enzyme catalytic process follows a bi bi-ordered mechanism where the coenzyme binds first, then allowing the substrate linkage to the active site. 3) Two optimal pH values of 8 and 6.5 corresponding to D-3-hydroxybutyrate oxidation and to acetoacetate reduction respectively. On the other hand, pH changes affect the coenzyme binding to the active site. 4) The BDH activity was found strongly linked to submitochondrial vesicles indicating that the protozoan enzyme is membranous and could require lipids for its function as well as it is for the mammalian enzyme. Moreover, an optimal temperature (40°C) and a break appearing in the Arrhenius plot at 19°C were found. The break suggests a membrane lipid fluidity-dependency of BDH conformational change. 5) Several ligands of the active site including methylmalonate and succinate modulate the BDH activity and are competitive inhibitors toward D-3-hydroxybutyrate. 6) Divalent cations, Mg2+, Mn2+ and Zn2+ protect BDH against thermal inactivation. The protection is the strongest in the presence of Zn2+. Moreover, Ca2+ and Mg2+ are enzyme activators and modulate the substrate binding to the active site. On the other hand, EDTA, a chelating agent, inhibits the enzyme but prevents inhibition by substrate excess. This work provides new insights on the energy metabolism of T. pyriformis wild strain where D-3-hydroxybutyrate is a choice substrate where the properties of BDH have been established especially the activating role of non heavy divalent cations.

The Bacillus subtilis ydjL ( bdhA ) Gene Encodes Acetoin Reductase/2,3-Butanediol Dehydrogenase

Bacillus subtilis is capable of producing 2,3-butanediol from acetoin by fermentation, but to date, the gene encoding the enzyme responsible, acetoin reductase/2,3-butanediol dehydrogenase (AR/BDH), has remained unknown. A search of the B. subtilis genome database with the amino acid sequences of functional AR/BDHs from Saccharomyces cerevisiae and Bacillus cereus resulted in the identification of a highly similar protein encoded by the B. subtilis ydjL gene. A knockout strain carrying a ydjL::cat insertion mutation was constructed, which (i) abolished 2,3-butanediol production in early stationary phase, (ii) produced no detectable AR or BDH activity in vitro, and (iii) accumulated the precursor acetoin in early stationary phase. The ydjL::cat mutation also affected the kinetics of lactate but not acetate production during stationary-phase cultivation with glucose under oxygen limitation. A very small amount of 2,3-butanediol was detected in very-late-stationary-phase (96-hour) cultures of the ydjL::cat mutant, suggesting the existence of a second gene encoding a minor AR activity. From the data, it is proposed that the major AR/BDH-encoding gene ydjL be renamed bdhA.

Purification and characterization of the d-β-hydroxybutyrate dehydrogenase from dromedary liver mitochondria

d-β-Hydroxybutyrate dehydrogenase (BDH) (EC 1.1.1.30), a membrane enzyme, has been purified to homogeneity from dromedary ( Camelus dromedarius) liver mitochondria. Our new purification method consisted of the solubilization of mitochondrial membranes by Triton X 100 and purification of BDH by two steps: DEAE-Sephacel and Phenyl-Sepharose. The molecular mass of the enzyme subunit size was 67 kDa. The purified enzyme is recognized by anti rat liver mitochondrial BDH antibodies. Furthermore, BDH activity was absolutely dependent upon phospholipids. BDH is also characterized by specific enzymatic parameters: an optimum pH of approximately 8 for the oxidation reaction, and approximately 7 for the reduction reaction and kinetic constant (Michaelis and dissociation constants) values of 1.07±0.13 mM for K MBOH, 0.21±0.01 mM for K MNAD + , 1.04±0.20 mM for K DNAD + , 0.29±0.01 mM for K MAcAc, 0.27±0.03 mM K MNADH and 1.12±0.18 mM for K DNADH.

An inducible 1-butanol dehydrogenase, a quinohaemoprotein, is involved in the oxidation of butane by "Pseudomonas butanovora".

Butane-grown "Pseudomonas butanovora" expressed two soluble alcohol dehydrogenases (ADHs), an NAD(+)-dependent secondary ADH and an NAD(+)-independent primary ADH. Two additional NAD(+)-dependent secondary ADHs could be detected when cells were grown on 2-butanol and lactate. The inducible NAD(+)-independent 1-butanol dehydrogenase (BDH) of butane-grown cells was primarily responsible for 1-butanol oxidation in the butane metabolism pathway. BDH was purified to near homogeneity and identified as a quinohaemoprotein, containing, per mol enzyme, 1.0 mol pyrroloquinoline quinone (PQQ) and 0.25 mol haem c as prosthetic groups. BDH was synthesized as a monomer of approximately 66 kDa. It has a broad substrate range, including primary alcohols, secondary alcohols, aldehydes, C(4) diols and aromatic alcohols. It exhibited the lowest K:(m) (7+/-1 microM) and highest k(cat)/K:(m) (72x10(4) M(-1) s(-1)) value towards 1-butanol. BDH exhibited ferricyanide-dependent ADH activity. Calcium ions (up to 10 mM) increased BDH activity substantially. Two BDH internal amino acid sequences showed 73 and 62% identity and 83 and 66% similarity, respectively, when compared with an amino acid sequence of ethanol dehydrogenase from Comamonas testosteroni. The presence of the inducible BDH and secondary ADH may indicate that the terminal and subterminal oxidation pathways are involved in butane degradation of butane-grown "P. butanovora".

Characterization of the NADH-Linked Acetylacetoin Reductase/2,3-Butanediol Dehydrogenase Gene from Bacillus cereus YUF-4.

A 1.4-kbp DNA fragment, including the NADH-linked acetylacetoin reductase/2,3-butanediol dehydrogenase (AACRII/BDH) gene from the chromosomal DNA of Bacillus cereus YUF-4, was cloned in Escherichia coli DH5alpha after its insertion into pUC119, and the resulting plasmid was named pAACRII119. The AACRII/BDH gene had an open reading frame consisting of 1047 bp encoding 349 amino acids. The enzyme exhibited not only AACR activity, but also BDH activity. However, the gene was not located in a 2,3-butanediol (BD) operon, as is the case in the BDH gene of Klebsiella pneumoniae and that of K. terrigena. In addition, there was no BD-cycle-related enzyme gene in the region surrounding the AACRII/BDH gene. The AACR and BDH activities in E. coli DH5alpha/pAACRII119 were 200-fold higher than those in the original B. cereus YUF-4. The characteristics of the AACRII/BDH from E. coli DH 5alpha/pAACRII119 are similar to those of the AACRII/BDH from B. cereus YUF-4. The AACRII/BDH was considered to belong to the NAD(P)- and zinc-dependent long-chain alcohol dehydrogenase (group I ADH) family on the basis of the following distinctive characteristics: it possessed 14 strictly conserved residues of microbial group I ADH and consisted of about 350 amino acids. The enzymatic and genetic characteristics of AACRII/BDH were completely different from those of BDHs belonging to the short-chain dehydrogenase/reductase family. These findings indicated that the AACRII/BDH could be considered a new type of BDH.

Characterization of the NADH-linked acetylacetoin reductase/2,3-butanediol dehydrogenase gene from Bacillus cereus YUF-4

A 1.4-kbp DNA fragment, including the NADH-linked acetylacetoin reductase/2,3-butanediol dehydrogenase (AACRII/BDH) gene from the chromosomal DNA of Bacillus cereus YUF-4, was cloned in Escherichia coli DH5α after its insertion into pUC119, and the resulting plasmid was named pAACRII119. The AACRII/BDH gene had an open reading frame consisting of 1047 bp encoding 349 amino acids. The enzyme exhibited not only AACR activity, but also BDH activity. However, the gene was not located in a 2,3-butanediol (BD) operon, as is the case in the BDH gene of Klebsiella pneumoniae and that of K. terrigena. In addition, there was no BD-cycle-related enzyme gene in the region surrounding the AACRII/BDH gene. The AACR and BDH activities in E. coli DH5α/pAACRII119 were 200-fold higher than those in the original B. cereus YUF-4. The characteristics of the AACRII/BDH from E. coli DH 5α/pAACRII119 are similar to those of the AACRII/BDH from B. cereus YUF-4. The AACRII/BDH was considered to belong to the NAD(P)- and zinc-dependent long-chain alcohol dehydrogenase (group I ADH) family on the basis of the following distinctive characteristics: it possessed 14 strictly conserved residues of microbial group I ADH and consisted of about 350 amino acids. The enzymatic and genetic characteristics of AACRII/BDH were completely different from those of BDHs belonging to the short-chain dehydrogenase/reductase family. These findings indicated that the AACRII/BDH could be considered a new type of BDH.

Characterization and functional role of Saccharomyces cerevisiae 2,3-butanediol dehydrogenase

Using a conserved sequence motif, a new gene ( YAL060W) of the MDR family has been identified in Saccharomyces cerevisiae. The expressed protein was a stereoespecific (2 R,3 R)-2,3-butanediol dehydrogenase (BDH). The best substrates were (2 R,3 R)-2,3-butanediol for the oxidation and (3 R/3 S)-acetoin and 1-hydroxy-2-propanone for the reduction reactions. The enzyme is extremely specific for NAD(H) as cofactor, probably because the presence of Glu223 in the cofactor binding site, instead of the highly conserved Asp223. BDH is inhibited competitively by 4-methylpyrazole with a K i of 34 μM. Yeast could grow on 2,3-butanediol or acetoin as a sole energy and carbon sources, and a 3.6-fold increase in BDH activity was observed when cells were grown in 2,3-butanediol, suggesting a role of the enzyme in 2,3-butanediol metabolism. However, the disruption of the YAL060W gene was not lethal for the yeast under laboratory conditions, and the disrupted strain could also grow in 2,3-butanediol and acetoin. This suggests that other enzymes, in addition to BDH, can also metabolize 2,3-butanediol in yeast.

Sequence analysis of the gene for and characterization of d-acetoin forming meso-2,3-butanediol dehydrogenase of Klebsiella pneumoniae expressed in Escherichia coli

Analysis of the nucleotide sequence of the meso-2,3-butanediol dehydrogenase ( d-acetoin forming) (BDH) gene of Klebsiella pneumoniae IAM1063 revealed that it contains an open reading frame of 768 bp encoding a polypeptide of 256 amino acid residues with a molecular weight of 26,591 Da. The amino acid sequence deduced from the nucleotide sequence of the BDH gene is consistent with the amino-terminal and carboxyl-terminal amino acid sequences, amino acid composition, and molecular weight of purified BDH. The amino acid sequences of BDHs from K. pneumoniae IAM1063 and Klebsiella terrigena VTT-E-74023 showed 92.9% homology in the first 196 amino acid residues, but no homology elsewhere. Moreover, the BDH of K. pneumoniae IAM1063 was 15 amino acid residues longer than that of K. terrigena VTT-E-74023. The characteristics of the BDH from Escherichia coli JM109 pBUD119 (containing the meso-BDH gene) coincided with that of the BDH from the gene source strain ( K. pneumoniae IAM1063). Furthermore, the BDHs from K. pneumoniae IAM1063 and K. terrigena VTT-E-74023 showed similar catalytic properties. This suggests that the downstream of Gln at position 196 in the amino acid sequence is unrelated to BDH activity.

Cloning and expression of a functional rat liver D-β-hydroxybutyrate dehydrogenase-β-galactosidase fusion protein in Escherichia coli

A rat liver bacteriophage lambda expression library was probed using polyclonal antibodies raised to purified rat liver D-beta-hydroxybutyrate dehydrogenase (BDH). A clone was selected that contained a 1.2-kb insert. The insert placed in an expression plasmid was utilized to transform Escherichia coli. These cells were shown to possess phosphatidylcholine-dependent BDH activity. Cells transformed with only the plasmid had no detectable BDH activity in the presence of phosphatidylcholine. The expressed activity in E. coli could be inhibited in a dose-dependent manner by BDH antiserum.

Variations of specific mRNA and polypeptide contents of rat liver d-β-hydroxybutyrate dehydrogenase during an experimental diabetes mellitus

The expression of the rat liver d-β-hydroxybutyrate dehydrogenase (BDH) gene was investigated at different levels: the level of its specific mRNA, the protein content and the enzymatic activity. By using a cDNA probe, we found that the BDH mRNA was about 2kb and we report here that the decrease of BDH activity in diabetic rats is due to a reduction in the content of the enzyme, which is proportional to a diminution in the amount of the BDH mRNA. We also show that insulin is able to reverse this diabetes effect by restoring the level of BDH mRNA, the BDH content and thus its activity. This result indicates that in vivo the control of the expression of the BDH gene by insulin is mainly transcriptional and/or post-transcriptional (mRNA stability).

Concerning the decreased D-3-hydroxybutyrate dehydrogenase activity in the liver and heart of hyperthyroid rats

Whereas in rat liver mitochondria the hyperthyroid state causes an increase both in fatty acid unsaturation and in the Ea of D-3-hydroxybutyrate dehydrogenase and a decrease in phase transition temperature, in hyperthyroid rat heart mitochondria these changes are negligible. D-3-hydroxybutyrate dehydrogenase in both the liver and the heart mitochondria of hyperthyroid rats is reduced by about 35% [12] but this reduction is not due to changes in membrane fluidity in either tissue. Hypothyroidism, on the other hand, affects BDH activity in neither heart nor liver.

Developmental regulation of D-β-hydroxybutyrate dehydrogenase in rat liver and brain

The activities of ketone-metabolizing enzymes in rat brain increase 3- to 5-fold during the suckling period before decreasing to the adult level after weaning. We have observed that a similar developmental pattern also exists for D-β-hydroxybutyrate dehydrogenase (BDH) in rat liver. Utilizing antibodies prepared against the purified protein we determined that the changes in BDH activities in both brain and liver are due to changes in the amount of BDH in the mitochondria. In vitro translations of isolated RNA followed by immunoprecipitation revealed that the increase in BDH activity and content was correlated with an increase in the level of functional BDH-mRNA in both liver and brain. Hydroxybutyrate dehydrogenase, D-β-; Developmental regulation

Kinetic aspects of the role of phospholipids in d-β-hydroxybutyrate dehydrogenase activity

Interactions of phospholipids with d-β-hydroxybutyrate dehydrogenase (BDH), a lecithin-requiring enzyme, have been studied by a kinetic approach. The process of reactivation of BDH by phospholipids, which follows a second-order mechanism, reveals that (1) at least 2 mol of lecithins is essential for the reactivation of the enzyme, and (2) the enzyme contains two dependent binding sites for lecithins. The graphic representation of the time course of reactivation shows a latent phase which decreases when there is an increase in the amount of phospholipids. A Scatchard plot treatment of the reactivation kinetic data reveals the presence of two classes of phospholipid binding sites, which exhibit high and low affinities related to the binding of four and two lecithin molecules, respectively. The effect of temperature on BDH activity and on the inactivation of the apoenzyme with N,N′-dicylohexylcarbodiimide (a specific carboxyl reagent) or with phenylglyoxal (a specific arginine reagent) shows a break at 22–24 °C, indicating a slight structural change in the enzyme-active site around this temperature. In addition, the variations in enzyme kinetic parameters, according to the nature of phospholipids, are in agreement with conformational changes related to the nature and to the fluidity state of phospholipids. However, the apparent NAD + binding constant does not depend on the phospholipid's fluidity.

Basis for decreased d-β-hydroxybutyrate dehydrogenase activity in liver mitochondria from diabetic rats

Liver mitochondria from rats made diabetic with streptozotocin have a reduced level of d-β-hydroxybutyrate dehydrogenase (BDH) activity and decreased ratios of oleic/ stearic and arachidonic/linoleic acids in the phospholipids of the mitochondrial membrane. This altered activity and lipid environment result from insulin deprivation since maintenance of the diabetic rats on insulin leads to normal characteristics ( J. C. Vidal, J. O. McIntyre, P. F. Churchill, and S. Fleischer (1983) Arch. Biochem. Biophys. 224, 643–658). In the present study, the basis for the reduced enzymatic activity of this lipid-requiring enzyme was analyzed using three approaches: (i) Purified d-β-hydroxybutyrate, dehydrogenase was inserted into membranes from mitochondria, submitochondrial vesicles, and mitochondrial lipids extracted therefrom. The activation was the same and optimal irrespective of whether the preparations were derived from normal or diabetic rat liver. Therefore, the decreased activity does not appear to be referable to an altered lipid composition, (ii) BDH activity can be released from the mitochondria by phospholipase A 2 digestion. The released activity was proportional to the endogenous activity in the submitochondrial vesicles from normal and diabetic membranes, (iii) The BDH activity in submitochondrial vesicles was titrated by inhibition with specific antiserum. Less enzyme was found in mitochondria from diabetic rats as compared with those from normal animals. Hence, the lowered enzymatic activity is due to decreased enzyme in the mitochondrial inner membrane and not to the modified lipid environment.