Determination of genotypes of human aldehyde dehydrogenase ALDH2 locus

Virtually all Caucasians have two major aldehyde dehydrogenase isozymes, ALDH1 and ALDH2, in their livers, while approximately 50% of Japanese and other Orientals are "atypical" in that they have only ALDH1 and are missing ALDH2. We previously demonstrated the existence of an enzymatically inactive but immunologically cross-reactive material (CRM) in atypical Japanese livers. Among 10 Japanese livers examined, five had ALDH1 but not ALDH2 isozyme. These are considered to be homozygous atypical at the ALDH2 locus. Four had both ALDH1 and ALDH2 components detected by starch gel electrophoresis, that is, they are apparently usual. However, biochemical and immunological studies revealed that three of these four livers contained CRM. These three livers should be heterozygous atypical in the ALDH2 locus, that is, genotype ALDH2(1)/ALDH2(2). A Japanese liver, as well as control Caucasian livers, had no CRM, and they must be homozygous usual ALDH2(1)/ALDH2(1). Although the number of liver specimens examined is limited, the frequencies of three genotypes determined in this study are compatible with the values calculated based on the genetic model that two common alleles ALDH2(1) and ALDH2(2) for the same locus are codominantly expressed in Orientals. The remaining liver had only ALDH2 isozyme and was missing ALDH1. This type was not previously found in Caucasians and Orientals. The two-dimensional crossed immunoelectrophoresis revealed the existence of a CRM corresponding to ALDH1 in this liver. The abnormality can be considered to be due to structural mutation at the ALDH1 locus producing a defective ALDH1 molecule, although other possibilities such as post-translational modifications are not ruled out.     

Structural mutation in a major human aldehyde dehydrogenase gene results in loss of enzyme activity.

Most Caucasians have two major liver aldehyde dehydrogenase isozymes, ALDH1 and ALDH2, while approximately 50% of Orientals have only ALDH1 isozyme, missing the ALDH2 isozyme. A remarkably higher frequency of acute alcohol intoxication among Orientals than among Caucasians could be related to the absence of the ALDH2 isozyme, which has a low apparent Km for acetaldehyde. Examination of liver extracts by two-dimensional crossed immunoelectrophoresis revealed that an atypical Japanese liver, which had no ALDH2 isozyme, contained an enzymatically inactive but immunologically cross-reactive material corresponding to ALDH2, beside the active ALDH1 isozyme. Therefore, the absence of ALDH2 isozyme in atypical Orientals is not due to regulatory mutation, gene deletion, or nonsense mutation, but must be due to a structural mutation in a gene for the ALDH2 locus, resulting in synthesis of enzymatically inactive abnormal protein.     

Liver alcohol dehydrogenase and aldehyde dehydrogenase in the Japanese: isozyme variation and its possible role in alcohol intoxication.

Forty autopsy livers from Japanese individuals were studied concerning alcohol dehydrogenase (ADH) and aldehyde dehydrogenase (ALDH) isozymes using electrophoretic and enzyme assay methods. A remarkably high frequency (85%) was found for the atypical ADH phenotype. The gene frequencies of ADH22 and ADH32 were .625 and .05, respectively. The usual ALDH phenotype showed two major isozyme bands, a faster migrating (low Km for acetaldehyde) and a slower migrating isozyme (high Km for acetaldehyde). Fifty-two percent of the specimens had an unusual phenotype of ALDH, which showed only the slower migrating isozyme. The usual phenotype was inhibited about 20%--30% by disulfiram and the unusual type up to 90%. Such a high incidence in the Japanese of the unusual phenotype, which lacks in the low Km isozyme, suggests that the initial intoxicating symptoms after alcohol drinking in these subjects might be due to delayed oxidation of acetaldehyde rather than its higher-than-normal production by typical or atypical ADH.     

Liver alcohol and aldehyde dehydrogenase isozymes in a Chinese population in Hong Kong

The distribution of alcohol dehydrogenase (ADH) and aldehyde dehydrogenase (ALDH) isozymes in the livers of a Chinese population in Hong Kong was examined. Among the 90 livers examined, 7 were typical ADH phenotype consisting the normal beta 1-type isozymes and 83 were atypical phenotype consisting the beta 2-type isozymes. Livers of 48 subjects were of deficient type in ALDH containing ALDH-II alone and 42 were of normal type with both ALDH-I and ALDH-II. When the combination of ADH and ALDH isozymes is considered, the Chinese population in Hong Kong falls into 4 subgroups. For each group, the rates of ethanol and acetaldehyde clearance have a distinct and characteristic potential which is directly related to its particular combination of isozymes.     

CuZn-Superoxide Dismutases in Rice: Occurrence of an Active, Monomeric Enzyme and Two Types of Isozyme in Leaf and Non-Photosynthetic Tissues

Rice leaves and seed embryos contain four isozymes of CuZn-superoxidedismutase (SOD) and two isozymes of Mn-SOD. CuZn-SOD I is amajor enzyme in leaves, but not in embryos or etiolated seedlings.CuZn-SODs II,III and IV were found in the embryos but were alsofound as minor isozymes in leaves. CuZn-SODs I, II and IV were purified to homogeneity from riceleaves. CuZn-SODs I and II had similar properties with respectto molecular weight, dimeric structure, absorption spectrumand metal content, but their amino acid compositions differedfrom each other. The absorption spectrum of CuZn-SOD IV wassimilar to that of isozymes I and II, but this enzyme was amonomer with a molecular mass of 1.7 kDa. Antibody against CuZn-SODI from rice did not cross-react with isozymes II and IV. Antibodiesagainst CuZn-SOD from spinach leaves cross-reacted with isozymeI but not with isozymes II, III and IV. By contrast, the antibodiesagaist CuZn-SOD from spinach seeds cross-reacted with isozymesII, III and IV but not with isozyme I. Thus, the isozyme thatis expressed mainly in leaves (CuZn-SOD I) and the isozymesexpressed mainly in non-photosynthetic tissues (CuZn-SODs II,III, IV) are immunologically distinct. (Received October 7, 1988; Accepted January 27, 1989)     

Differential effects of Mg2+ ions on the individual kinetic steps of human cytosolic and mitochondrial aldehyde dehydrogenases

Although the structures of mammalian cytosolic and mitochondrial ALDH have been determined, several differences, mainly functional, between these two 70% identical isozymes remain unexplained. A major difference is the differential effect of Mg(2+) ions that inhibits the cytosolic and activates the mitochondrial isozyme. Here, we have investigated the effect of Mg(2+) ions on each individual kinetic step of ALDH1 and ALDH2. The metal ions were found not to affect either acylation or hydride transfer for either isozyme. The lack of a Mg(2+) ion effect on hydride transfer was further demonstrated with an E399Q mutant of ALDH1 whose rate-limiting step had been changed from NADH dissociation to hydride transfer. The other steps, however, were affected by Mg(2+) ions for both isozymes. The metal ions inhibited NADH dissociation, the rate-limiting step for ALDH1, and enhanced deacylation, the rate-limiting step for ALDH2. Our results indicated that, with both isozymes, Mg(2+) ions tightened the binding of NADH, and by binding to the coenzyme, they increased the nucleophilicity of the nucleophile Cys302. The inhibition of ALDH1 and activation of ALDH2 at pH 7.4 are due to their different rate-limiting steps. Mg(2+) ions affected similarly the NADH activation of the esterase reaction for both isozymes. In contrast, the metal ions affected only the NAD(+) activation of ALDH1. This latter finding and other features described here can be rationalized on the basis of the known three-dimensional structures of the isozymes.     

Plant triose phosphate isomerase isozymes : purification, immunological and structural characterization, and partial amino Acid sequences

We report the first complete purifications of the cytosolic and plastid isozymes of triose phosphate isomerase (TPI; EC 5.3.1.1) from higher plants including spinach (Spinacia oleracea), lettuce (Lactuca sativa), and celery (Apium graveolens). Both isozymes are composed of two isosubunits with approximate molecular weight of 27,000; in spinach and lettuce the plastid isozyme is 200 to 400 larger than the cytosolic isozyme. The two isozymes, purified from lettuce, had closely similar amino acid compositions with the exception of methionine which was four times more prevalent in the cytosolic isozyme. Partial amino acid sequences from the N-terminus were also obtained for both lettuce TPIs. Nine of the 13 positions sequenced in the two proteins had identical amino acid residues. The partial sequences of the plant proteins showed high similarity to previously sequenced animal TPIs. Immunological studies, using antisera prepared independently against the purified plastid and cytosolic isozymes from spinach, revealed that the cytosolic isozymes from a variety of species formed an immunologically distinct group as did the plastid isozymes. However, both plastid and cytosolic TPIs shared some antigenic determinants. The overall similarity of the two isozymes and the high similarity of their partial amino acid sequences to those of several animals indicate that TPI is a very highly conserved protein.     

Acetaldehyde oxidation in rat liver mitochondria, action of Mg2+, ATP and rotenone on acetaldehyde oxidation in intact mitochondria, and on some purified aldehyde dehydrogenase isozymes

In intact rat liver mitochondria acetaldehyde is oxidized by three functionally distinct dehydrogenase systems. Two of these reduce intramitochondrial nicotinamide adenine dinucleotide (NAD): one is operative with micromolar acetaldehyde concentrations and is stimulated by Mg2+, the other is operative with millimolar acetaldehyde concentrations and is stimulated by adenosine 5'-triphosphate (ATP). The third system reduces added NAD and is stimulated by rotenone. Connected to these systems, three aldehyde dehydrogenase isozymes (ALDH) have been purified: a low-Km ALDH activated by Mg2+, a high-Km ALDH activated by ATP and Mg2+, a high-Km ALDH activated by rotenone. The properties of some isozymes are affected by detergents. Thus, deoxycholate augments the stimulation of low-Km isozyme by Mg2+ and confers sensitivity to Mg2+ and ATP on one of the high-Km isozymes. A fourth isozyme has been purified. Its affinity for acetaldehyde is so low that it is very unlikely that acetaldehyde is the physiological substrate.     

AMP deaminase isozymes in human tissues

In human, there are four AMP deaminase (AMP aminohydrolase, EC 3.5.4.6) isozymes: E1, E2, M and L. Chromatographic, electrophoretic and immunological studies showed the existence of isozymes E1 and E2 in erythrocytes, isozyme M in muscle and isozyme L in liver and brain. The tissues such as heart, kidney and spleen contained isozymes E1, E2 and L. Isozymes E1, M and L were isolated as apparently homogeneous preparations. The three isozymes were all tetramers composed of identical subunits, but differing slightly in molecular weight; isozyme E1 showed a subunit molecular weight of 80 000, isozyme M 72 000 and isozyme L 68 000. They were immunologically different from one another. The antisera precipitated only the corresponding enzyme and did not precipitate any other isozyme. The three isozymes were also different in kinetic and regulatory properties. Isozyme E2 was very similar to isozyme E1 in immunological and kinetic properties, although isozyme E2 could be separated from isozyme E1 by phosphocellulose chromatography, and zonal electrophoresis.     

Stomach and duodenal alcohol and aldehyde dehydrogenase isozymes in Chinese

Alcohol dehydrogenase (ADH) and aldehyde dehydrogenase (ALDH) isozyme phenotypes were determined in surgical and endoscopic biopsies of the stomach and duodenum by agarose isoelectric focusing. gamma-ADH was found to be the predominant form in the mucosal layer whereas beta-ADH was predominant in the muscular layer. Low-Km ALDH1 and ALDH2 were found in the stomach and duodenum. High-Km ALDH3 isozymes occurred only in the stomach but not in the duodenum. The isozyme patterns of gastric mucosal ALDH2 and ALDH3 remained unchanged in the fundus, corpus, and antrum. The stomach ALDH3 isozymes exhibited a Km value for acetaldehyde of 75 mM, and an optimum for acetaldehyde oxidation at pH 8.5. Since the Km value was high, ALDH3 contributed very little, if any, to gastric ethanol metabolism. The activities of ALDH in the gastric mucosa deficient in ALDH2 were 60-70% of that of the ALDH2-active phenotypes. These results indicate that Chinese lacking ALDH2 activity may have a lower acetaldehyde oxidation rate in the stomach during alcohol consumption.     

Electrophoretic and biochemical studies of human aldehyde dehydrogenase isozymes in various tissues

Four major ALDH isozymes have been identified in human tissues using starch gel electrophoresis and isoelectric focusing. The isozyme bands have been termed as ALDH I, II, III and IV according to their decreasing electrophoretic migration and increasing isoelectric point. The isozymes have been partially purified via preparative isoelectric focusing. Kinetic characteristics of ALDH I and II were found to be quite similar to ALDH enzyme 2 and enzyme 1 described earlier by Greenfield and Pietruszko (Biochem Biophys Acta, $\text{483}$ 35–45 1977). ALDH III and IV showed a very high Km for propionaldehyde (1.0–1.5 mM at pH 9.5) and were not inhibited by disulfiram at pH 9.5. A variant phenotype of ALDH which lacked in isozyme I was detected in various tissues from Japanese individuals. Comparative kinetic properties of normal and variant enzyme are given.     

Differential expression of alpha, mu and pi classes of isozymes of glutathione S-transferase in bovine lens, cornea, and retina

Isozyme characterization of glutathione S-transferase (GST) isolated from bovine ocular tissue was undertaken. Two isozymes of lens, GST 7.4 and GST 5.6, were isolated and found to be homodimers of a Mr 23,500 subunit. Amino acid sequence analysis of a 20-residue region of the amino terminus was identical for both isozymes and was identical to GST psi and GST mu of human liver. Antibodies raised against GST psi cross-reacted with both lens isozymes. Although lens GST 5.6 and GST 7.4 demonstrated chemical and immunological relatedness, they were distinctly different as evidenced by their pI and comparative peptide fingerprint. A corneal isozyme, GST 7.2, was also isolated and established to be a homodimer of Mr 24,500 subunits. Sequence analysis of the amino-terminal region indicated it to be about 67% identical with the GST pi isozyme of human placenta. Antibodies raised against GST pi cross-reacted with cornea GST 7.2. Another corneal isozyme, GST 8.7, was found to be homodimer of Mr 27,000 subunits. Sequence analysis revealed it to have a blocked amino-terminus. GST 8.7 immunologically cross-reacted with the antibodies raised against cationic isozymes of human liver indicating it to be of the alpha class. Two isozymes of retina, GST 6.8 and GST 6.3, were isolated and identified to be heterodimers of subunits of Mr 23,500 and 24,500. Amino-terminal sequence analysis gave identical results for both retina GST 6.8 and GST 6.3. The sequence analysis of the Mr 23,500 subunit was identical to that obtained for lens GSTs. Similarly, sequence analysis of the Mr 24,500 subunit was identical to that obtained for the cornea GST 7.2 isozyme. Both the retina isozymes cross-reacted with antibodies raised against human GST psi as well as GST pi. The results of these studies indicated that all three major classes of GST isozymes were expressed in bovine eye but the GST genes were differentially expressed in lens, cornea, and retina. In lens only the mu class of GST was expressed, whereas cornea expressed alpha and pi classes and retina expressed mu and pi classes of GST isozymes.     

Isolation and properties of four alpha-amylase isozymes from human submandibular saliva

Four isoamylases have been isolated from human submandibular secretions by gel filtration and isoelectric focusing. The isozymes (1A, 1B, 2A, 2B) were each purified about 8-fold and each yielded one major band on disc gel electrophoresis. In all cases the major protein band contained more than 95% of the protein and amylase activity recovered. The isoenzymes, in order of their relative positions on the polyacrylamide gels (from the anodal end), their isoelectric points, and percentage distribution in the submandibular secretion are as follows: isozyme 2A, pH 5.9, 9%; isozyme 1A, pH 5.9, 18%; isozyme 2B, pH 6.4, 63%; isozyme 1B, pH 6.4, 10%. Amino acid analyses showed that the protein compositions of the four isoamylases were essentially the same. Possible differences were noted in aspartic acid, serine, glutamic acid, and proline contents. Molecular weights, determined by SDS disc gel electrophoresis, were 57,000 for 1A and 1B, and 54,000 for 2A and 2B. This molecular weight difference is attributed mainly to the presence of bound carbohydrate on isozymes 1A and 1B. Gas Chromatographic analysis was used for determining the carbohydrate compositions. Molar ratios of sugars were similar for both glycoprotein amylases (moles sugar/mole enzyme): glucosamine, 3; mannose, 3; galactose, 2; fucose, 3. Isoamylase 1A, which had more carbohydrate than 1B, also contained about 2 moles of N-acetylneuraminic acid. Sialic acid was not detected in isozyme 1B.     

Distribution of AMP-deaminase isozymes in rat tissues

1. The distribution of AMP deaminase isozymes in rat tissues was analyzed by electrophoresis on cellulose acetate membrane, by chromatography on phosphocellulose column, and by the application of immunological technique employing specific antisera against three parental AMP deaminases (isozymes A, B and C). Skeletal muscle extracts and diaphragm extracts contain a single identical isozyme, isozyme A. The major isozyme species of liver, kidney and testes are also identical and they are isozyme B. Heart extracts contains isozyme C exclusively. Extracts of brain, lung and spleen contain five isozymes, presumably a complete set of five B-C hybrids. 2. Developmental patterns of AMP deaminase isozyme were studied. In early postnatal life, extracts of heart, liver, kidney and lung contain five isozymes similar to those observed in adult brain. During postnatal development, a shift to isozyme C occurs in heart, whereas a shift to isozyme B occurs in liver and kidney. Five isozymes in lung remain throughout development. In brain a shift of B to five isozymes is observed during development. Isozyme A is the predominant form in muscle throughout postnatal development. 3. AMP deaminase in the regenerating liver was analyzed, but the data indicated that there was no change of isozyme distribution during hepatic regeneration.     

The subcellular location of isozymes of NADP-isocitrate dehydrogenase in tissues from pig, ox and rat

Antibodies against purified NADP-isocitrate dehydrogenase from pig liver cytosol and pig heart were raised in rabbits. The purified enzymes from these sources are different proteins, as demonstrated by differences in electrophoretic mobility and absence of crossreactivity by immunotitration and immunodiffusion. The NADP-isocitrate dehydrogenase in the soluble supernatant homogenate fraction from pig liver, kidney cortex, brain and erythrocyte hemolyzate was identical with the purified enzyme from pig liver cytosol, as determined by electrophoretic mobility and immunological techniques. The enzyme in extracts of mitochondria from pig heart, kidney, liver and brain was identical with the purified pig heart enzyme by the same criteria. However, the 'mitochondrial' isozyme was the major component also in the soluble supernatant fraction of pig heart homogenate. The 'cytosolic' isozyme accounted for only 1-2% of total NADP-isocitrate dehydrogenase in pig heart, as determined by separation of the isozymes with agarose gel electrophoresis and immunotitration. The mitochondrial isozyme was also the predominant NADP-isocitrate dehydrogenase in porcine skeletal muscle. The ratio of cytosolic/mitochondrial isozyme for porcine whole tissue extract, determined by immunotitration, was about 2 for liver and 1 for kidney cortex and brain. The distribution of isozymes in cell homogenate fractions from ox and rat tissues corresponded to that observed in organs of porcine origin. The mitochondrial and cytosolic isozymes from ox and rat tissues exhibited crossreactivity with the antibodies against the pig heart and pig liver cytosol enzyme, respectively, and the electrophoretic migration patterns were similar qualitatively to those found for the isozymes in porcine tissues. Nevertheless, there were species specific differences in the characteristics of each of the corresponding isozymes. NAD-isocitrate dehydrogenase was not inhibited by the antibodies, confirming that the protein is distinct from that of either isozyme of NADP-isocitrate dehydrogenase.     

A chemical and enzymological account of the multiple forms of human liver aldehyde dehydrogenase. Implications for ethnic differences in alcohol metabolism

Three major low-pI zones of aldehyde dehydrogenase (aldehyde:NAD+ oxidoreductase, EC 1.2.1.3) may be visualized with specific histochemical staining after starch gel electrophoresis at pH 7.4 of Caucasian human liver extracts, whereas about 50% of Chinese human liver extracts show only two such zones. The three zones of activity were purified to apparent homogeneity from Caucasian liver. The substrate specificity of each form was investigated by double reciprocal plots using 13 aldehydes of various chemistries. The acetaldehyde-preferring isozyme I lacking in 50% of Chinese livers had a slightly lower native and subunit molecular weight than the "universal' isozymes IIa and IIb. All forms were highly sensitive to disulfiram inhibition. This inhibition could be protected against, or reversed, by dithiothreitol. 2,2'-Dithiodipyridine was a slower inhibitor of isoenzyme I. All three purified forms of the enzyme, as well as crude extracts of normal and isozyme I-deficient Chinese livers, showed positive immunoreactivity to antibodies prepared in rabbits against type I enzyme. Tryptic peptide maps of forms IIa and IIb were almost identical, whereas that of form I, although showing some similarities, was clearly different. These results provide a consistent explanation for the acetaldehyde-mediated extreme sensitivity to moderate alcohol ingestion shown normally by about 50% of oriental subjects and during disulfiram (Antabuse) therapy by all subjects.     

Purification and characterization of PLC-beta m, a muscarinic cholinergic regulated phospholipase C from rabbit brain membrane

Two isozymes of phosphoinositide-specific phospholipase C were isolated and purified from salt-washed rabbit brain membranes. The membranes were extensively washed with isotonic, hypertonic and hypotonic buffers prior to solubilization with sodium cholate. Two isozymes (PLC-IV and PLC-beta m) were purified by a combination of DEAE-Sephacel, AH-Sepharose, heparin-Sepharose, AcA-34 gel filtration and mono-Q FPLC chromatographies. The major activity (PLC-beta m) was purified to homogeneity and had an estimated molecular weight of 155,000 on sodium-dodecyl sulfate-polyacrylamide gels (SDS-PAGE). This isozyme was immunologically identified as PLC-beta, an isozyme previously characterized in bovine brain cytosol and 2 M KCl membrane extracts. A second isozyme, PLC-IV, was immunologically distinct from PLC-beta and PLC-gamma and was purified to a stage where three protein bands (Mr 66,000, 61,000 and 54,000) on SDS-PAGE correlated with enzyme activity. The catalytic properties of the isozymes were studied and found to be very similar. The specific activities for PIP2 were greater than those obtained when PI was used. Both PLC-IV and PLC-beta m were Ca2(+)-dependent; near maximal stimulation for PI and PIP2 hydrolysis was observed at 0.5 microM free Ca2+. Sodium pyrophosphate and sodium fluoride stimulated phospholipase C activity of both isozymes. Polyclonal antibodies raised against PLC-beta m were able to inhibit carbachol and GTP gamma S stimulated phospholipase C activity in 2 M KCl washed rabbit cortical membranes. This suggests that in rabbit brain muscarinic cholinergic stimulation regulates PLC-beta m.     

A Comparison of Two Sucrose Synthetase Isozymes from Normal and shrunken-1 Maize

The maize sucrose synthetase isozyme (SS2) present in sh1 endosperm, sh1 seedlings, and in suspension culture cells was purified to homogeneity from each of these tissues by sequential ammonium sulfate fractionation, diethylaminoethyl-cellulose chromatography, gel filtration chromatography, and affinity elution with UTP from a carboxymethyl-cellulose column. Cyanogen bromide digests were used to demonstrate that the SS2 isozymes in these different tissues are structurally identical and are therefore the product of the same gene. The sucrose synthetase produced by the Sh1 gene (SS1) was purified by modification of the SS2 procedure and was used in comparative analyses of the two isozymes. Ouchterlony assays demonstrated that SS1 and SS2 have partial antigenic identity. The two isozymes have similar enzyme kinetics in the sucrose cleavage reaction but differ in their relative activities with ADP and TDP. The amino acid compositions of SS1 and SS2 are similar, and proteolytic digests revealed that they share limited structural homologies.     

Isozymes of isocitrate dehydrogenase from an obligately psychrophilic bacterium, Vibrio sp. strain ABE-1: purification, and modulation of activities by growth conditions

Each of the two isozymes, which are different in thermostability and quaternary structure, of isocitrate dehydrogenase (NADP+) [IDH: EC 1.1.1.42] was purified to an electrophoretically homogeneous state from an obligately psychrophilic marine bacterium, Vibrio sp. strain ABE-1. Hydrophobic chromatography was an efficient procedure to separate the two isozymes from each other. The isoelectric points of isozyme I (IDH-I; a dimer, Mr 88,100) and isozyme II (IDH-II; a monomer, Mr 80,500) were found to be pH 4.9 and 5.2, respectively. The two isozymes were similar in amino acid compositions, though there were slight differences in the contents of nonpolar and hydroxyl amino acids. However, their NH2-terminal amino acid sequences and immunochemical properties were clearly different from each other. The NH2-terminal amino acid sequence analysis also indicated that the subunits of IDH-I are chemically identical or highly homologous. Non-immuno-crossreactivity between the isozymes enabled us to measure the intracellular contents of the isozymes. IDH-I and -II were found to be differentially regulated in vivo by various growth conditions. IDH-I was induced by acetate, while IDH-II remained almost unchanged.