Electrophoretic analyses of alcohol dehydrogenase, aldehyde dehydrogenase, aldehyde oxidase, sorbitol dehydrogenase and xanthine oxidase from mouse tissues.

1. Cellulose acetate zymograms of alcohol dehydrogenase (ADH), aldehyde dehydrogenase, sorbitol dehydrogenase, aldehyde oxidase, "phenazine" oxidase and xanthine oxidase extracted from tissues of inbred mice were examined. 2. ADH isozymes were differentially distributed in mouse tissues: A2--liver, kidney, adrenals and intestine; B2--all tissues examined; C2--stomach, adrenals, epididymis, ovary, uterus, lung. 3. Two NAD+-specific aldehyde dehydrogenase isozymes were observed in liver and kidney and differentially distributed in other tissues. Alcohol dehydrogenase, aldehyde oxidase, "phenazine" oxidase and xanthine oxidase were also stained when aldehyde dehydrogenase was being examined. 4. Two aldehyde oxidase isozymes exhibited highest activities in liver. 5. "Phenazine oxidase" was widely distributed in mouse tissues whereas xanthine oxidase exhibited highest activity in intestine and liver extracts. 6. Genetic variants for ADH-C2 established its identity with a second form of sorbitol dehydrogenase observed in stomach and other tissues. The major sorbitol dehydrogenase was found in high activity in liver, kidney, pancreas and male reproductive tissues.     

Isoelectric focusing studies of aldehyde dehydrogenases from mouse tissues: variant phenotypes of liver, stomach and testis isozymes

Isoelectric focusing techniques (IEF) were used to examine the tissue distribution and genetic variability of aldehyde dehydrogenases (AHDs) from inbred strains of mice. Twelve zones of AHD activity were resolved which were differentially distributed between tissues. Liver extracts exhibited highest activity for most enzymes, with the exception of isozymes found in stomach (AHD-4) and testis (AHD-4 and AHD-6). Genetic variants for AHD-1 (liver mitochondrial isozyme) and AHD-4 (stomach isozyme) were examined from inbred strains and F1 hybrid animals. The results were consistent with dimeric subunit structures (designated as A2 and D2 isozymes respectively). IEF patterns for activity variants of testis-specific AHD-6 were identical, with 3-banded phenotypes being observed. pI values for the AHD forms as well as for aldehyde oxidase and xanthine oxidase isozymes, which stain in the absence of coenzyme, were reported.     

Isoelectric focusing studies of aldehyde dehydrogenases, alcohol dehydrogenases and oxidases from mammalian anterior eye tissues

1. Isoelectric focusing (IEF) and zymogram methods were used to examine the tissue distribution, multiplicity and substrate specificities of alcohol dehydrogenases (ADHs), aldehyde dehydrogenases (ALDHs) and ocular oxidases (EOXs) from mammalian anterior eye tissues. 2. Baboon, cattle, pig and sheep corneal extracts exhibited high ALDH activities; the corneal ALDHs were distinct from the major liver ALDHs and distinguished by their preference for medium-chain aldehydes. 3. Baboon and pig corneal extracts also showed high ADH activities, by comparison with ovine and bovine samples. Moreover, the ADHs were distinct from the major liver isozymes in pI value and substrate specificity. 4. Mammalian lens extracts exhibited significant ALDH activity of a form corresponding to the major liver cytosolic isozyme. Minor activity of the corneal enzyme was also observed in some species. 5. Lens ADH phenotypes were species-specific, and consisted of either Class II activity (baboon and sheep), Class III ADH activity (pig), or activities of both ADH classes (cattle). 6. Lens extracts also exhibited a complex pattern of ocular oxidase (EOX) activities following IEF. 7. A role in peroxidatic aldehyde detoxification is proposed for these enzymes in anterior eye tissues.     

Biochemical genetics of aldehyde dehydrogenase isozymes in the mouse: Evidence for stomach- and testis-specific isozymes

Electrophoretic and activity variants have been observed for stomach and testis aldehyde dehydrogenases, respectively, among inbred strains of the house mouse (Mus musculus). Genetic evidence was obtained for two new loci encoding these isozymes (designated Ahd-4 and Ahd-6, respectively, for the stomach and testis isozymes) which segregated independently of a number of mouse gene markers, including Ahd-1 (encoding mitochondrial aldehyde dehydrogenase) on chromosome 4, ep (pale ears), a marker for chromosome 19, on which Ahd-2 (encoding liver cytosolic aldehyde dehydrogenase) has been previously localized, and Adh-3 (encoding the stomach-specific isozyme of alcohol dehydrogenase) on chromosome 3. Recombination studies have indicated, however, that Ahd-4 and Ahd-6 are distinct but closely linked loci on the mouse genome. An extensive survey of the distribution of Ahd-1, Ahd-2, Ahd-4, and Ahd-6 alleles among 56 strains of mice is reported. No variants have been observed, so far, for the microsomal (AHD-3) and mitochondrial/cytosolic (AHD-5) isozymes previously described. This study, in combination with previous investigations on mouse aldehyde dehydrogenases, provides evidence for six genetic loci for this enzyme.     

Mouse mitochondrial aldehyde dehydrogenase isozymes: purification and molecular properties

Aldehyde dehydrogenase isozymes (AHD-1 and AHD-5) have been isolated in a highly purified state from extracts of mouse liver mitochondria. The enzymes have distinct subunit sizes, as determined by SDS/polyacrylamide gel electrophoresis: AHD-1, 63,000; AHD-5, 49,000. Gel exclusion chromatography, using sephadex G-200, indicated that both isozymes are dimers, although AHD-1 may also exist as a monomeric form as well. The enzymes exhibited widely divergent kinetic characteristics. The purified allelic forms of AHD-1, AHD-1A (C57BL/6J mice) and AHD-1B (CBA/H mice), exhibited high Km values with acetaldehyde as substrate, 1.4 mM and 0.78 mM respectively, whereas AHD-5 exhibited a low Km value with acetaldehyde of 0.2 microM. In addition, the isozymes exhibited distinct pH optima for catalysis (AHD-1, pH range 6.5-7.5; AHD-5, pH range 8.5-10.0), and were differentially sensitive towards disulphuram inhibition, with 50% inhibition occurring 13 and 0.1 microM for the AHD-1 and AHD-5 isozyme respectively. Based upon the kinetic characteristics, it is suggested that AHD-5 may be the primary enzyme for oxidizing mitochondrial acetaldehyde during ethanol oxidation in vivo.     

Liver aldehyde oxidase and xanthine oxidase genetics in the mouse

A 'null' activity variant for the major liver isozyme of aldehyde oxidase (AOX-1) in adult male mice and an electrophoretically distinct, high activity variant of the second liver isozyme (AOX-2) were used to examine the segregation of the genetic loci encoding these enzymes (Aox-1 and Aox-2 respectively) in breeding studies. A single recombinant between these loci was observed among the 147 backcross progeny examined, which confirms a previous report (Holmes, 1979) for close linkage and genetic distinctness of the two loci. An activity variant for mouse liver xanthine oxidase (XOX) is also reported which behaved as though controlled by codominant alleles at a single locus (designated Xox-1 ). Genetic analyses showed that the Xox-1 locus segregated independently of the multiple- A ox loci.     

Genetics of alcohol dehydrogenase and aldehyde dehydrogenase from Monodelphis domestica cornea: further evidence for identity of corneal aldehyde dehydrogenase with a major soluble protein

A didelphid marsupial, the gray short-tailed opossum (Monodelphis domestica), was used as a model species to study the biochemical genetics of alcohol dehydrogenases (ADHs) and aldehyde dehydrogenase (ALDH) in corneal tissue. Isoelectric point variants of corneal ALDH (designated ALDH3) and a major soluble protein in corneal extracts were observed among eight families of animals used in studying the genetics of these proteins. Both phenotypes exhibited identical patterns following PAGE-IEF and were inherited in a normal Mendelian fashion, with two alleles at a single locus (ALDH3) showing codominant expression. The data provided evidence for genetic identity of corneal ALDH with this major soluble protein, and supported biochemical evidence, recently reported for purified bovine corneal ALDH, that this enzyme constitutes a major portion of soluble corneal protein (Abedinia et al. 1990). Isoelectric point variants for corneal ADH were also observed, with patterns for the two major forms (ADH3 and ADH4) and one minor form (ADH5) being consistent with the presence of two ADH subunits (designated gamma and delta), and variant phenotypes existing for the gamma subunit. The genetics of this enzyme was studied in the eight families, and the results were consistent with codominant expression of two alleles at a single locus (designated ADH3). It is relevant that a major detoxification function has been proposed for corneal ADH and ALDH, in the oxidoreduction of peroxidic aldehydes induced by available oxygen and UV-B light (Holmes & VandeBerg, 1986a). In addition, a direct role for corneal ALDH as a UV-B photoreceptor in this anterior eye tissue has also been proposed (Abedinia et al. 1990).     

Purification and molecular properties of a Class II alcohol dehydrogenase (ADH-C2) from horse liver

An alcohol dehydrogenase (ADH) from horse liver was purified by ion-exchange and affinity chromatography. The enzyme (designated ADH-C2), is a dimer with a similar subunit size (47,300 mol. wt), as determined by SDS-polyacrylamide gel electrophoresis, to other mammalian ADHs. Zinc analyses and 1,10 phenanthroline inhibition studies indicated that each subunit contained 2 g atoms of zinc, with at least one involved catalytically. The enzyme exhibited similar kinetic properties to human pi-ADH and mouse ADH-C2, previously classified as class II ADHs [Vallee and Bazzone (1983) Isozymes, Vol. 8, pp. 219-244; Algar et al. (1983) Eur. J. Biochem. 137, 139-147] but differed in most respects from the extensively investigated horse Class I ADHs; EE, ES and SS. Horse ADH-C2 exhibited a Km value for ethanol of 42 mM and a broad substrate specificity, with Km values decreasing dramatically with an increase in chain length. The enzyme was much less sensitive to pyrazole inhibition (by at least 3 orders of magnitude) as compared with the Class I ADHs.     

Alcohol dehydrogenase: multiplicity and relatedness in the solvent-producing clostridia

Abstract: Alcohol dehydrogenase (ADH) is a key enzyme for the production of butanol, ethanol, and isopropanol by the solvent-producing clostridia. Initial studies of ADH in extracts of several strains of Clostridium acetobutylicum and C. beijerinckii gave conflicting molecular properties. A more coherent picture has emerged because of the following results: (i) identification of ADHs with different coenzyme specificities in these species; (ii) discovery of structurally conserved ADHs (type 3) in three solvent-producing species; (iii) isolation of mutants with deficiencies in butanol production and restoration of butanol production with a cloned alcohol/aldehyde dehydrogenase gene; and (iv) resolution of various ' C. acetobutylicum ' cultures into four species. The three ADH isozymes of C. beijerinckii NRRL B592 have high sequence similarities to ADH-1 of Clostridium sp. NCP 262 (formerly C. acetobutylicum P262) and to the ADH domain of the alcohol/aldehyde dehydrogenase of C. acetobutylicum ATCC 824/DSM 792. The NADH-dependent activity of the ADHs from C. beijerinckii NRRL B592 and the BDHs from C. acetobutylicum ATCC 824 is profoundly affected by the pH of the assay, and the relative importance of NADH and NADPH to butanol production may be misappraised when NAD(P)H-dependent activities were measured at different pH values. The primary/secondary ADH of isopropanol-producing C. beijerinckii is a type-1 enzyme and is highly conserved in Thermoanaerobacter brockii (formerly Thermoanaerobium brockii ) and Entamoeba histolytica . Several solvent-forming enzymes (primary ADH, aldehyde dehydrogenase, and 3-hydroxybutyryl-CoA dehydrogenase) are very similar between C. beijerinckii and the species represented by Clostridium sp. NCP 262 and NRRL B643. The realization of such relationships will facilitate the elucidation of the roles of different ADHs because each type of ADH can now be studied in an organism most amenable to experimental manipulations.     

Aldehyde oxidase and alcohol dehydrogenase genetics in the mouse. New alleles for the Aox-2 and Adh-3 loci

The genetic variability of one of the liver isozymes of aldehyde oxidase (AOX-B₂ or AOX-2) and the stomach isozyme of alcohol dehydrogenase (ADH-C₂) has been examined among strains of mice. Evidence is presented for a fourth allele of Aox-2 and a third allele of Adh-3 . The hybrid allozyme pattern for mouse liver AOX was consistent with a dimeric subunit structure for this enzyme.     

Acetaldehyde utilization and toxicity in Drosophila adults lacking alcohol dehydrogenase or aldehyde oxidase

Metabolic utilization and toxicity of acetaldehyde were studied in flies lacking alcohol dehydrogenase (ADH), aldehyde oxidase (AO), or both functions. Prior to the experiments, mutant alleles Adhn4 and mal were transferred to the same genetic background by 10 successive backcrosses. By comparison with wild-type flies, various deleterious, pleiotropic effects could be attributed to the mal allele but not to Adhn4. Of the four genotypes studied (mal, Adhn4, mal Adhn4, and wild), all were able to use acetaldehyde as a resource in a similar way. In spite of its high toxicity, acetaldehyde appeared a better resource than ethanol. Flies treated with intermediate acetaldehyde concentrations (around 0.5%) exhibited a very high interindividual heterogeneity which could reflect a physiological adaptation occurring as a consequence of the aldehyde treatment. Toxicity tests showed that ADH-negative flies were more sensitive to acetaldehyde than wild type, but this is most likely explained by the transformation of the aldehyde into alcohol. Our results show that the aldehyde metabolizing enzyme (AME) system in Drosophila is neither ADH nor AO. The existence of an aldehyde dehydrogenase is plausible.     

Rabbit liver alcohol dehydrogenase: isolation and characterization of class I isozymes

Livers of rabbits contain three classes of alcohol dehydrogenase (ADH) isozymes which are highly analogous to the human classes. Class I ADHs migrate toward cathode on starch gel and are very sensitive to 4-methylpyrazole (4-MePz) inhibition. Class II ADH migrates slowly toward anode and is less sensitive to 4-MePz. Class III ADH migrates rapidly toward anode and is insensitive to 4-MePz. There are one class II, one class III and at least three class I ADH isozymes present in the rabbit liver. The three class I isozymes purified to homogeneity are all dimers with subunit molecular weight of 41700. Two are heterodimers composed of A-, C-chains and B-, C-chains, respectively. The third one is a homodimer, contains only the C-chain. These results indicate that among all the mammals examined, rabbit ADH bears the greatest resemblance to the human enzyme.     

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.     

Genetics and ontogeny of aldehyde dehydrogenase isozymes in the mouse: Localization of Ahd-1 encoding the mitochondrial isozyme on chromosome 4

Electrophoretic variants for the mitochondrial isozyme of aldehyde dehydrogenase (AHD) have been observed in inbred strains and in Harwell linkage testing stocks of Mus musculus. F₁ (LVC×C57BL/Go) mice showed a codominant allele three-banded phenotype, which suggests a dimeric subunit structure (designated AHD-A₂). The anodal-migrating supernatant isozyme of AHD was electrophoretically invariant among the 23 inbred strains and stocks examined. The genetic locus encoding AHD-A₂ (suggested name Ahd-1) is localized on chromosome 4 and was mapped close to je (jerker) and Gpd-1 (encoding the liver and kidney isozyme of glucose-6-phosphate dehydrogenase). Ontogenetic analyses demonstrated that both AHD isozymes exhibited low activity in late fetal and early neonatal liver and kidney extracts, and reached adult levels within 3 weeks of birth.     

Opossum alcohol dehydrogenases: Sequences, structures, phylogeny and evolution: evidence for the tandem location of ADH genes on opossum chromosome 5

BLAT (BLAST-Like Alignment Tool) analyses and interrogations of the recently published opossum genome were undertaken using previously reported rat ADH amino acid sequences. Evidence is presented for six opossum ADH genes localized on chromosome 5 and organized in a comparable ADH gene cluster to that reported for human and rat ADH genes. The predicted amino acid sequences and secondary structures for the opossum ADH subunits and the intron-exon boundaries for opossum ADH genes showed a high degree of similarity with other mammalian ADHs, and four opossum ADH classes were identified, namely ADH1, ADH3, ADH6 and ADH4 (for which three genes were observed: ADH4A, ADH4B and ADH4C). Previous biochemical analyses of opossum ADHs have reported the tissue distribution and properties for these enzymes: ADH1, the major liver enzyme; ADH3, widely distributed in opossum tissues with similar kinetic properties to mammalian class 3 ADHs; and ADH4, for which several forms were localized in extrahepatic tissues, especially in the digestive system and in the eye. These ADHs are likely to perform similar functions to those reported for other mammalian ADHs in the metabolism of ingested and endogenous alcohols and aldehydes. Phylogenetic analyses examined opossum, human, rat, chicken and cod ADHs, and supported the proposed designation of opossum ADHs as class I (ADH1), class III (ADH3), class IV (ADH4A, ADH4B and ADH4C) and class VI (ADH6). Percentage substitution rates were examined for ADHs during vertebrate evolution which indicated that ADH3 is evolving at a much slower rate to that of the other ADH classes.     

Mouse aldehyde dehydrogenase genetics: Positioning of Ahd-1 on chromosome 4

Electrophoretic variants of mitochondrial aldehyde dehydrogenase (AHD-A₂) are widely distributed among inbred strains of Mus musculus and have been used to localize the gene encoding AHD-A₂(Ahd-1) at the non-centromeric end of chromosome 4. In the mouse (Mus musculus), aldehyde dehydrogenase (AHD; E.C.1.2.1.3) exists as at least three isozymes which are differentially distributed in liver subcellular fractions (designated A₂, B₄ and Cy* for the mitochondrial, soluble and microsomal isozymes respectively) and in various tissues of this animal (Holmes, 1978a; 1978b; Timms & Holmes, 1981). Electrophoretic variants have been previously reported for the A₂ and B₄ isozymes among inbred strains of mice, and the genetic loci (designated Ahd-1 and Ahd-2) have been localized on chromosomes 4 and 19 respectively (Holmes, 1978b; Timms & Holmes, 1980). This paper describes further genetic analyses of AHD-A₂ enabling Ahd-1 to be positioned at the non-centromeric end of chromosome 4. Forty-three inbred strains of Mus musculus were used in these studies (Table 1). Two series of matings were carried out. 1) Female SM/J mice and male NZC/B1 mice were mated to obtain F, female offspring which were backcrossed to male NZC/B1 mice. These progeny were used to examine the segregation and linkage relationship of b (brown), Pgm-2 (encoding phosphoglucomutase B) and Ahd-1 (Table 2). 2) Female C57BL/6J mice and male SM/J. mice were mated to obtain F, female offspring which were backcrossed to male SM/J mice. The segregation and linkage relationship of Pgm-2, Gpd-1 (encoding the liver and kidney isozyme of hexose-6 phosphate dehydrogenase) and Ahd-1 were examined for these backcross progeny (Table 3). Methods for preparing liver and kidney extracts and the cellulose acetate electrophoresis procedure for typing Ahd-1, Pgm-2 and Gpd-1 have been previously described (Holmes, 1978b). A previous study has described the electrophoretic patterns for allelic variants for mitochondria1 AHD and of the hybrid phenotype for this enzyme (Holmes, 1978b). The three-allelic isozyme pattern for hybrid animals was consistent with a dimeric subunit structure: AHD-A¹A², AHD-A¹A² and AHD-3, with the A1 and A2 subunits being encoded by separate alleles at a single locus, designated Ahd-1 (Ahd-1^oand Ahd-1^brespectively). The distribution of these alleles among 43 inbred strains of mice is given in Table 1. The allelic variants were approximately equally distributed among the inbred strains examined and no divergence of phenotype was observed among the 6 substrains of C57BL mice (Ahd-1^aallele) and 5 substrains of BALB/c (Ahd-1^ballele) mice examined. Genetic variants for phosphoglucomutase-B (PGM-B) have been reported by Shows, Ruddle and Roderick (1969) and the gene (Pgm-2) was subsequently localized on chromosome 4 near b (brown) by Chapman, Ruddle and Roderick (1970). Table 2 illustrates the results of a three-point cross between b, Pgm-2 and Ahd-1. Variation from the expected 1:1:1:1:1:1 ratio for unlinked loci was significant(x²= 73.15; 7 df; P < 1 × 10^-5), indicating that the three loci are linked. Recombination frequency data are consistent with the gene order: b - Pgm-2 - Ahd-1 The second cross examined the segregation of Pgm-2, Ahd-1 and Gpd-1 loci (Table 3). The latter locus has been previously positioned on chromosome 4 (linkage group VIII) by Hutton & Roderick (1970) and Chapman (1975), and has been used to localize Ahd-1 in this region (Ahd-1 and Gpd-1 exhibit a recombination frequency of 10.3 ± 3.7 %) (Holmes, 1978b). The data from Table 3 is consistent with a gene order of Pgm-2 - Ahd-1 - Gpd-1. The recombination frequency data of Ahd-1 with Gpd-1, Pgm-2 and b also supports the proposal that Ahd-1 is localized between Pgm-2 and Gpd-1 (Tables 2 and 3; Holmes, 1978b). Recent metabolic studies have indicated that mitochondria1 aldehyde dehydrogenase (AHD) plays a very important role in the metabolism of acetaldehyde derived from ethanol, ensuring a low concentration of acetaldehyde in the blood leaving the liver (Grunnet, 1973; Parilla et al., 1974; Corral1 et al., 1976). Moreover, genetic variation of this isozyme in human livers has been recently reported (Harada et al., 1978), and this polymorphism has been proposed as the molecular basis for individual and racial differences in alcohol sensitivity (Goedde et al., 1979). Consequently, genetic analyses of mitochondria1 AHD are of particular significance to studies on the genetic control of alcohol metabolism in mammals. In summary, this report confirms previous studies which demonstrated that the genetic locus encoding mitochondrial aldehyde dehydrogenase in the mouse (Ahd-1) is on chromosome 4 (Holmes, 1978b), and positions the gene with respect to b (brown), Pgrn-2 (encoding phosphoglucomutase B) and Gpd-1 (encoding the liver and kidney isozyme of hexose-6-phosphate dehydrogenase). In addition, the distribution of the 2-allelic phenotypes for this isozyme has been examined among 43 in- bred strains of mice.     

Purification and immunochemical characterization of aldehyde dehydrogenase from 2-acetylaminofluorene-induced rat hepatomas.

1. A series of aldehyde dehydrogenase isozymes (aldehyde:NAD (P)+ oxidoreductase, EC 1.2.1.5), has been purified from hepatomas induced in Sprague-Dawley rats by 2-acetylaminofluorene. 2. The functional hepatoma-specific aldehyde dehydrogenase isozymes exist as 105 000-dalton dimers composed to two subunits of 53 000 daltons. Isoelectric points of the purified isozymes are 6.9-7.2. 3. Antiserum to these purified hepatoma-specific aldehyde dehydrogenases has been produced and the immunological relationships of these isozymes to their normal liver counterpart have been studied. Results of Ouchterlony double diffusions, agar-gel immunoelectrophoresis and polyacrylamide gel and agar immunoelectrophoresis indicate that anti-hepatoma aldehyde dehydrogenase antiserum cross-reacts with normal liver aldehyde dehydrogenase.     

Molecular evolution of mammalian class I alcohol dehydrogenase

Phylogenetic relationship and the rates of evolution of mammalian alcohol dehydrogenases (ADHs) have been studied by using the amino acid sequences from the human (ADH alpha, ADH beta, and ADH gamma), rat, mouse, and horse (ADH E and ADH S). With the maize ADH1 and ADH2 used as references, the patterns of the amino acid replacements in the beta-sheets, alpha-helices, and random coils in each of the catalytic and coenzyme-binding domains were analyzed separately. The phylogenetic trees based on the different sets of amino acid substitutions consistently showed that (1) multiple ADHs in human and horse have arisen after mammalian radiation, (2) the common ancestor of human ADHs alpha and beta diverged from the ancestor of ADH gamma first and the former two ADHs diverged from each other more recently, and (3) the human ADHs are more closely related to the rodent ADHs than to the horse ADHs. Furthermore, the estimated branch lengths showed that the rodent ADHs are evolving faster than the other ADHs. This difference in evolutionary rate between the two groups of organisms is explainable either in terms of the difference in the number of cell generations per year or in terms of reduction of functional constraints.     

A shortened synthesis of 4-(3-aminopropyl) pyrazole, an affinity ligand for alcohol dehydrogenase purification

The most efficient, specific and rapid procedures for alcohol dehydrogenase (ADH) purification utilize immobilized 4-(3-aminopropyl) pyrazole to which pyrazole sensitive ADHs, i.e. class I isozymes, bind. Because of the length of the reported synthesis of this affinity resin, we synthesized the 4-(3-aminopropyl) pyrazole ligand by a new method in two steps from commercially available nicotinaldehyde. The ligand synthesized by this simplified procedure was directly coupled to the chain-extended support, Activated CH-Sepharose 4B, to yield the same ligand-spacer combination as reported by L.G. Lange and B.L. Vallee (Biochem. 15: 4681-4686, 1976). Human and hamster class I ADHs purified using this resin were homogeneous by SDS-PAGE followed by silver staining. Specific activity and recovery of human class I ADH were comparable to those previously reported.     

Human aldehyde dehydrogenase. Activity with aldehyde metabolites of monoamines, diamines, and polyamines

Two isozymes (E1 and E2) of human aldehyde dehydrogenase (EC 1.2.1.3) were purified to homogeneity 13 years ago and a third isozyme (E3) with a low Km for gamma-aminobutyraldehyde only recently. Comparison with a variety of substrates demonstrates that substrate specificity of all three isozymes is broad and similar. With straight chain aliphatic aldehydes (C1-C6) the Km values of the E3 isozyme are identical with those of the E1 isozyme. All isozymes dehydrogenate naturally occurring aldehydes, 5-imidazoleacetaldehyde (histamine metabolite) and acrolein (product of beta-elimination of oxidized polyamines) with similar catalytic efficiency. Differences between the isozymes are in the Km values for aminoaldehydes. Although all isozymes can dehydrogenate gamma-aminobutyraldehyde, the Km value of the E3 isozyme is much lower: the same appears to apply to aldehyde metabolites of cadaverine, agmatine, spermidine, and spermine for which Km values range between 2-18 microM and kcat values between 0.8-1.9 mumol/min/mg. Thus, the E3 isozyme has properties which make it suitable for the metabolism of aminoaldehydes. The physiological role of E1 and E2 isozymes could be in dehydrogenation of aldehyde metabolites of monoamines such as 3,4-dihydroxyphenylacetaldehyde or 5-hydroxyindoleacetaldehyde; the catalytic efficiency with these substrates is better with E1 and E2 isozymes than with E3 isozyme. Isoelectric focusing of liver homogenates followed by development with various physiological substrates together with substrate specificity data suggest that aldehyde dehydrogenase (EC 1.2.1.3) is the only enzyme in the human liver capable of catalyzing dehydrogenation of aldehydes arising via monoamine, diamine, and plasma amine oxidases. Although the enzyme is generally considered to function in detoxication, our data suggest an additional function in metabolism of biogenic amines.