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Electrophoretic analyses of alcohol dehydrogenase, aldehyde dehydrogenase, aldehyde reductase, aldehyde oxidase and xanthine oxidase from horse tissues
Comp. Biochem. Physiol. Vol. 78B, No. 1, pp. 131-139, 1984
0305-0491/84 $3.00+0.00 © 1984 Pergamon Press Ltd
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ELECTROPHORETIC ANALYSES OF ALDEHYDE DEHYDROGENASE, ALDEHYDE OXIDASE AND FROM HORSE
ALCOHOL DEHYDROGENASE, ALDEHYDE REDUCTASE, XANTHINE OXIDASE TISSUES
TANYA-LEE SEELEY, PETER B. MATHER and ROGER S. HOLMES School of Science, Griffith University, Nathan, 411 l, Brisbane, Queensland, Australia. Tel: 07-275-7111 (Received 25 October 1983)
Akstract--1. Cellulose acetate zymograms of alcohol dehydrogenase (ADH), aldehyde dehydrogenase (AHD), aldehyde reductase (AHR), aldehyde oxidase (AOX) and xanthine oxidase (XOX) extracted from horse tissues were examined. 2. Five ADH isozymes were resolved: three corresponded to the previously reported class I ADHs (EE, ES and SS) (Theorell, 1969); a single form of class II ADH (designated ADH-C2) and of class III ADH (designated ADH-B2) were also observed. The latter isozyme was widely distributed in horse tissues whereas the other enzymes were found predominantly in liver. 3. Four AHD isozymes were differentially distributed in subcellular preparations of horse liver: AHD-1 (large granules); AHD-3 (small granules); and AHD-2, AHD-4 (cytoplasm). AHD-1 was more widely distributed among the horse tissues examined. Liver represented the major source of activity for most AHDs. 4. A single additional form of NADPH-dependent AHR activity (identified as hexonate dehydrogenase), other than the ADHs previously described, was observed in horse liver. 5. Single forms of AOX and XOX were observed in horse tissue extracts, with highest activities in liver.
INTRODUCTION
Alcohol dehydrogenase (ADH; EC 1.1.1.1) catalyses the reversible oxidation of aliphatic and aromatic primary alcohols to their respective aldehyde derivatives. The products from this reaction have high reactivities and may be highly toxic in living cells (Schaunstein et al., 1977). Aldehydes are also generated in vivo from other oxidative processes derived from amines and lipid hydroperoxides (Schaunstein et al., 1977), and a biological mechanism is required to detoxify these reactive substances. Several enzymes are capable of facilitating this process including aldehyde dehydrogenase (AHD; EC 1.2.1.3), via an NAD-linked irreversible oxidation reaction to the corresponding acid (Racker, 1949); aldehyde reductase (AHR), via the reverse reaction of ADHs and by 'specific' NADPH-dependent reductases (Bosron and Prairie, 1973; Branlant and Biellmann, 1980); as well as aldehyde oxidase (AOX; EC 1.2.3.1) and xanthine oxidase (XOX; EC 1.2.3.2), via direct oxidative processes with molecular oxygen, generating the corresponding acid and hydrogen peroxide (Avis et al., 1956; Gordon et al., 1940; Mahler et al., 1954). Horse liver has served as an important source of mammalian alcohol dehydrogenase and aldehyde dehydrogenase for biochemical studies. Horse liver A D H was first crystallized by Bonnischen and Wassen (1948), and later isolated as three major forms of activity, resulting from the dimerization of two biochemically distinct subunits, E and S (Theorell et al., 1966). The isozymes have been extensively studied and characterized biochemically (see Branden et al., 1975; Pietruszko, 1980). Horse liver aldehyde dehydrogenases have been also studied (Feldman and
Weiner, 1972; Eckfeldt et al., 1976). The latter group has purified and characterized mitochondrial and cytosolic forms of this enzyme, and examined their differential kinetic properties. More recent studies on mammalian liver ADHs and AHDs have indicated that a more extensive multiplicity for these enzymes may exist. Vallee and Bazzone (1982), for example, have established that human liver A D H exists as three classes of enzymes, named I, II and III. Human type I A D H comprises a complex set of isozymes, resulting from the dimeric association of three genetically distinct subunit types, ~t, fl and 7 (Smith et al., 1971; 1973), and represents the major ethanol metabolizing enzymes in the body (Vallee and Bazzone, 1982). Type II (1t-ADH) A D H preferentially oxidizes longer chain and aromatic alcohols, and becomes active with ethanol only at higher ethanol concentrations (Li and Magnes, 1975, Vallee and Bazzone, 1982); while Type III (x-ADH) A D H utilizes medium chain alcohols, and is active with ethanol only at concentrations above 0.5 M (Pares and Vallee, 1981; Vallee and Bazzone, 1982). Algar et al. (1983) have also reported three classes of ADHs in mouse tissues with similar kinetic properties. Subcellular fractionation studies on mammalian liver aldehyde dehydrogenases (AHDs) have demonstrated that the enzyme is localized in at least three major compartments: mitochondria, microsomes and the cytoplasm (Deitrich, 1966; Marjenen, 1972; Koivulo and Koivusalo, 1975) with the A H D (or AHDs) (Nakayasu et al., 1978; Timms and Holmes, 1981; Smolen et al., 1981) isolated from each fraction exhibiting characteristic differences in kinetic properties. Moreover, additional multiplicity has been re-
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TANYA-LEESEELEYet al.
ported in rat and mouse liver with at least two A H D s being found in cytosolic and mitochondrial preparations (Siew et al., 1976; Petersen et al., 1977; Holmes et al., 1983a,b). This communication describes the tissue and subcellular distribution, and electrophoretic properties of alcohol dehydrogenase and aldehyde dehydrogenase from horse tissues, and examines three additional enzymes, aldehyde reductase, aldehyde oxidase and xanthine oxidase, which exhibit catalytic activity with aldehydes ag substrates, in vitro. MATERIALS AND METHODS
Tissues
Tissues were obtained from freshly slaughtered adult horses from a local abattoir and stored on ice immediately for a maximum period of 2 hr, prior to subcellular fractionation and tissue extraction. Chemicals
All substrates, coenzymes and buffer chemicals were obtained from Sigma chemicals (St. Louis, MO, USA) except for trans-2-hexen-l-ol (Aldrich Chemical Co., Wembly, UK), benzaldehyde (Unilab, Sydney, Australia) and acetaldehyde (BDH Chemicals Ltd, Poole, England, UK). Homogenate preparation
Homogenates of liver, kidney, heart, lung, spleen, small intestine, brain, testis and stomach were prepared as previously described (Holmes, 1978a). Final concentrations (w/v) of all tissues was 33°o. Liver and subcellular fractionation
Liver tissue was washed in cold buffered isotonic sucrose (50raM Tris-HC1 pH 7.4; 0.25 M sucrose), blotted dry, weighed, finely minced and homogenized in the same buffer using eight strokes of a Potter-Elvehjem homogenizer in the ratio of 1 g liver in 4 ml buffer. The homogenate was then centrifuged for 10min at 600g to produce a pellet containing most of the whole cells and nuclei, which was discarded. The supernatant (S1) was centrifuged at 9000g for 15 rain to sediment a large granule fraction (consisting predominantly of mitochondria, lysosomes and peroxisomes) (LG). The resulting pellet was washed twice in buffered-sucrose while the supernatant ($2) was centrifuged at 100,000g for 60mins to yield a small granule fraction (consisting of microsomes) (SG) and a cytosolic fraction ($3). LG was resuspended in 10raM sodium phosphate buffer (2 ml/gm liver equivalent), kept on ice for 30 min and vortexed twice before adding sodium deoxycholate (0.2~0 final concn). This was also kept on ice and vortexed twice, for 30min. SG was resuspended in 10mM sodium phosphate buffer containing 0.1% Triton X-100 (2ml/g liver equivalent), and homogenized using an Ultra-Turrax homogenizer. The LG and SG extracts were spun at 25,000g for 20min and the resultant supernatants used in electrophoretic analysis. Cellulose acetate electrophoresis and staining
Homogenate supernatants were subjected to zone electrophoresis on Titan III cellulose acetate plates (94 x 76 mm) (Helena Labs., Texas, USA). Samples (approximately 2/11) were applied 10-15 mm from the cathodal edge of the plate and electrophoresed under the following conditions: alcohol dehydrogenase and aldehyde reductase (Tris-citrate (75 mM) buffer pH 7.0; 150 V for 60 min); aldehyde dehydrogenase, ~ldehyde oxidase and xanthine oxidase (Tris-glycine (44mM) buffer pH 8.5; 200V for 20min). Following electrophoresis, the plates were histochemically stained for enzyme activity using an agar overlay technique
(Holmes, 1978a). The details for the histochemical staining procedures are found in Holmes (1978a~ and Duley and Holmes (1982). RESULTS Figure 1 illustrates a zymogram of alcohol dehydrogenase ( A D H ) isozymes from horse tissues. The isozymes appeared to fall into three distinct classes of A D H activity which are compatible with previous studies of Vallee and Bazzone (1982) on human A D H s and Algar et al. (1983) on mouse A D H s , and they have been designated as such. Class I A D H s are recognized by m M or sub-raM Michaelis constants for ethanol and a high sensitivity to pyrazole inhibition; class II A D H s by high Michaelis constants for ethanol and a low sensitivity to pyrazole inhibition while class III A D H s exhibit total inactivity with ethanol concentrations below 0.5 M and are insensitive to pyrazole inhibition. Differential staining procedures were used to distinguish the A D H s resolved (see Holmes et al., 1983a). Electrophoretic zones of A D H activity were observed following examination of horse tissue extracts. The three class I enzymes (designated EE, ES and SS, based upon the studies of Theorell et al. (1966) and Pietruszko (1980) were found only in liver among the tissues examined: similarly, the class II enzyme (designated ADH-C2 on account of its similarity in properties to this enzyme in the mouse Algar et al., 1983) was found only in liver; while the class lII enzyme (designated A D H - B : ) is widely distributed and exhibited highest activity in kidney. The additional activity zones were shown to exhibit overlapping substrate specificities with three forms of aldehyde dehydrogenase, or to arise from control activities from aldehyde oxidase or another non-specific oxidase. The subcellular localization of horse liver A D H isozymes was examined and the results are illustrated in Fig. 2. All five A D H forms were predominantly localized in the cytosolic fraction, although some activities were also found in washed 'small granular" (microsomes) and 'large granular" (mitochondria, peroxisomes and lysosomes) preparations. F o u r aldehyde dehydrogenase ( A H D ) zones of activity were observed following cellulose acetate electrophoresis and histochemical staining (Fig. 3), which were designated as A H D s 1 to 4, in accordance with the nomenclature used for mouse A H D s (Holmes et al., 1983). Aldehyde oxidase, xanthine oxidase and a 'non-specific' oxidase activity zones were recognized using differential staining techniques for these enzymes (Holmes, 1978a). A H D - I exhibited highest activity in liver but was also widely distributed in other tissues examined. In contrast A H D - 2 and A H D - 4 were predominantly localized in liver and kidney, while A H D - 3 was restricted to liver extracts in this organism. The subcellular distribution of horse liver A H D s was also examined (Fig. 4). A H D - I was found predominantly in the 'large granular' fraction, although significant activity was also observed in the cytosol fraction. This latter activity may represent leakage from the 'large granules' during homogenization. A H D - 2 and A H D - 4 were found in the cytosolic fraction, while A H D - 3 was localized in the 'small granular' microsomal fraction. Aldehyde ox-
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Fig. 1. Cellulose acetate zymogram and diagramatic illustration of the electrophoretic properties of alcohol dehydrogenase (ADH) isozymes from horse tissues. L, liver; K, kidney; H, heart; Lu, lung; Sp, spleen; SI, small intestine; Br brain; T, testis; St, stomach. The zymogram was stained using 10 mM trans-2-hexen-l-ol as substrate. Subunit structures for the previously investigated class I horse liver ADHs (EE, ES and SS) and the proposed class II (ADH-C2) and Class III (ADH-B2) isozymes are given. ADH activity is given as ~ ; aldehyde oxidase activity as 1 ~ ; aldehyde dehydrogenase activity as L;~.; and 'non-specific' oxidase activity
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Fig. 2. Cellulose acetate zymogram and diagrammatic illustration of the electrophoretic properties of alcohol dehydrogenase (ADH) isozymes from subcellular fractions of horse liver. TE, total extract: LG, large granule fraction: SG, small granule fraction: Cy, cytoplasm. The zymogram was stained using 10mM trans-2-hexen-l-ol as substrate. Subunit structures lbr the previously investigated horse liver class l ADHs (EE, ES and SS) and the proposed class II (ADH-C 2) and class III (ADH-B2) isozymes are given. ADH activity is given as m ; aldehyde oxidase activity as ~,~: aldehyde dehydrogenase activity a s ~ : and non-specific oxidase activity as EYY.
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Fig. 3. Cellulose acetate zymogram and diagrammatic illustration of the electrophoretic properties of aldehyde dehydrogenase (AHD) isozymes from horse tissues. L, liver; K, kidney; H, heart; Lu, lung; Sp, spleen; SI, small intestine; Br, brain; T, testis; St, stomach. The zymogram was stained using 20 mM acetyldehyde as substrate. Isozymes are designated as AHD-1, AHD-2, AHD-3 and AHD-4. AHD activity is given as ~ ; aldehyde oxidase activity aslg~l; xanthine oxidase as II1~; and non-specific oxidase aslZ~.
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Fig. 4. Cellulose acetate zymogram and diagrammatic illustration of the electrophoretic properties of aldehyde dehydrogenase (AHD) isozymes from subcellular Fractions of horse liver. TE, total extract; LG, large granule fraction; SG, small granule fraction; Cy, cytoplasm. The zymogram was stained using 20 mM acetaldehyde as substrate. Isozymes are designated as for Fig. 3. AHD activity was given as ~ : aldehyde oxidase activity as ~ N ; xanthine oxidase activity as/IIIIIH:and 'non-specific' oxidase activity asIZ~-
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Fig. 5. Cellulose acetate zymogram and diagrammatic illustration of the electrophoretic properties of aldehyde reductase active enzymes from subcellular fractions of horse liver. TE, total extract; LG, large granule fraction; SG, small granule fraction; Cy, cytoplasm. The zymogram was stained using 5 mM p-nitrobenzaldehyde as substrate. The fine alcohol dehydrogenase isozymes observed in Figs t and 2 all exhibited aldehyde reductase activity and are designated as m , hexonate dehydrogenase activity (reverse reaction) was given a s ~ . Control reductase activity was given a s K .
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TANYA-LEESEELEYet al.
idase and xanthine oxidase activities, which stain as control zones in the absence of the coenzyme (NAD +) in the histochemical agar overlay, were found in the cytosolic fraction (Fig. 4). Figure 5 shows a cellulose acetate zymogram of aldehyde reductase activity from subcellular fractions and a detergent-extracted homogenate of horse liver, using p-nitrobenzaldehyde as substrate and reduced NADP as coenzyme. In addition to the ADH isozymes observed in Fig. 2, an NADPH specific aldehyde reductase was observed which was recognized as hexonate dehydrogenase by differential staining techniques (Duley and Holmes, 1982). Hexonate dehydrogenase was localized in the liver cytoplasmic fraction. DISCUSSION
The present results provide evidence for additional forms of horse liver alcohol dehydrogenase (ADH) and aldehyde dehydrogenase (AHD) to those reported in the available literature. Prior to this study, horse liver ADH isozymes have been investigated and described in terms of three major forms, associated with the random dimerization of E (ethanol active) and S (sterol active) subunits (see review by Branden et al., 1975). Pietruszko (1980) has described further forms of these enzymes but has concluded that these represent polymorphic variants or conformational isomers of the major isozymes, EE, ES and SS. In terms of the classification of mammalian ADH isozymes described by Vallee and Bazzone (1982), these enzymes have properties similar to those of class I ADHs, namely, high affinity for ethanol as substrate and high sensitivity to pyrazole inhibition. The two additional forms of horse liver ADH reported in this paper resemble those of the class II and class III ADHs described by Vallee and Bazzone (1982) for the human ~z and X enzymes, and those described recently for mouse ADHs by Algar et al. (1983). It is likely that these two forms escaped earlier identification since firstly, human class II ADH's have been reported to be quite labile in vivo if not in t'itro at low temp (Li and Magnes, 1975) and secondly class III ADH's would not be evident under normal staining procedures with ethanol. The horse liver ADH-C2 has been purified and characterized biochemically (Seeley and Holmes, 1983) and similar investigations on ADH-B2 are currently being undertaken in this laboratory. ADH-C2 has a low affinity for ethanol as substrate with a Michaelis constant of 42 mM (compared with 1 mM for EE and 11 mM for SS isozymes of Pietruszko, 1975) and is much less sensitive to inhibition by pyrazole, exhibiting a K, of 15 mM as compared to 0.07 #M for the SS isozyme (Theorell, 1966). ADH-B2 is inactive with ethanol as substrate at concentrations less than 0.5M and longer chain alcohols (eg. trans-2-hexen-l-ol) are routinely used as substrate for this isozyme. Both isozymes are dimers with subunit molecular weights similar to those of the class I horse liver ADHs. Additional forms of horse aldehyde dehydrogenase (AHD) have also been reported in this present study. Horse liver AHD was first purified by Feldman and Weiner (1972), although only a single homogeneous
enzyme was described. Eckfeldt et al. (1976) subsequently purified and characterized cytoplasmic and mitochondrial isoenzymes of aldehyde dehydrogenase. These two enzymes (designated F~ and F~ respectively) were reported as tetramers, exhibiting broad aldehyde specificity. Two additional electrophoretic forms of AHD were observed (Figs 3 and 4) which are differentially localized in tissue extracts and liver subcellular fractions. AHD-3 activity was observed in liver microsomal extracts whereas two zones of cytosolic AHD activity were found in horse liver and kidney extracts. These results are directly comparable to those recently reported for mouse liver AHDs. (Holmes, 1978b; Timms and Holmes, 1981: Holmes et al., (1983b) Genetic and biochemical analyses have established that at least 3 genetic loci encode aldehyde dehydrogenase isozymes in the mouse: A h d - l (encoding the mitochondrial isozyme, AHD-I); A h d - 2 (encoding the cytosolic isozyme, (AHD-2) and A h d - 3 (encoding the microsomal isozyme, AHD-3). Moreover, the presence of at least three pools of liver AHD activity in subcellular fractions has been observed in a number of man> malian organisms (Koivula and Koivusalo, 1975: Nakayasu et al., 1978: Timms and Holmes, 1981; Smolen et al., 1981). Human AHDs have also been recently studied and shown to exist as distinct isozymic forms in liver mitochondria, microsomes and cytosolic preparations (Duley et al.. 1983). Electrophoretic analyses of aldehyde oxidase (AOX) and xanthine oxidase (XOX) from horse tissues showed that these enzymes exist as single forms in this species and exhibited highest activity in liver. This is similar to a recent report for these enzymes in human tissues (Duley et al.. 1983) but is in contrast, at least as far as AOX is concerned, with the situation in the mouse. AOX is encoded by two closely linked structural genes, A o x - I and A o x - 2 . in this organism (Holmes, 1979). Hexonate dehydrogenase (one o1" the aldehyde reductases resolved in Fig. 5) also occurs as a single form in horse tissue extracts. This enzyme is defined by its absolute specificity for reduced NADP as coenzyme, which is in contrast to alcohol dehydrogenase isozymes, exhibiting aldehyde reductase activity in the presence of reduced NAD or reduced NADP (Duley and Holmes, 1982). Hexonate dehydrogenase has been purified from human brain by O'Brian and Schofield (1980), and from rat liver and brain by Rivett et al., (1981), and is distinguished by its sensitivity to barbiturate and valproate inhibition. its utilization of D-glucuronate as a substrate, and its low mol. wt (40,000) monomeric subunit structure. In summary, this paper reports an electrophoretic analysis of the tissue distribution, subcellular distribution and multiplicity of enzymes involved with alcohol metabolism in horse tissues, namely alcohol dehydrogenase and aldehyde dehydrogenase. Aldehyde oxidase, xanthine oxidase and aldehyde reductase were also investigated because of their overlapping specificities with these enzymes.
Acknowledgement--This research was supported in part by the Australian Research Grants Scheme.
Electrophoresis of dehydrogenases, reductases and oxidases REFERENCES Algar E. M., Seeley T-L. and Holmes R. S. (1983) Purification and molecular properties of mouse alcohol dehydrogenase isozymes. Eur. J. Biochem. 137, 139-147. Avis P. G., Bergel F., Bray R. C., James D. W. F. and Shotter K. U. (1956) The chemistry of xanthine oxidase, Part II. The homogeneity of crystalline metalloflavoprotein fractions. J. Chem. Soc. 1212-1219. Bonnichsen R. K. and Wassen A. M. (1948) Crystalline alcohol dehydrogenase from horse liver. Archs Biochem. Biophys. 18, 361-363. Bosron W. F. and Prairie R. L. (1973) Reduced triphosphopyridine-linked aldehyde reductase. Archs Biochem. Biophys. 154, 166-174. Branden C-I., Jornvall H., Eklund H. and Furugren B. (1975) Alcohol dehydrogenases. In The Enzymes (Edited by Boyer P. D.), Vol. 11, pp. 103-190. Academic Press, New York. Branlant G. and Biellmann J. F. (1980). Purification and some properties of aldehyde reductases from pig liver. Eur. J. Biochem. 105, 611-621. Deitrich R. A. (1966) Tissue and subcellular distribution of mammalian aldehyde oxidizing capacity. Biochem. Pharmac. 15, 1911-1922. Duley J. A., Harris D. and Holmes R. S. (1983) Analysis of the tissue and subcellular distribution of human alcohol dehydrogenase, aldehyde dehydrogenase, aldehyde oxidase and xanthine oxidase isozymes by electrophoresis and isoelectric focusing. Alcohol. Clin. Exp. Res. (in press). Duley J. A. and Holmes R. S. (1982). Biochemical genetics of aldehyde reductase in the mouse: A h r - l - - a new locus linked to the alcohol dehydrogenase gene complex on chromosome 3. Biochem. Genet. 20, 1067-1083. Eckfeldt J., Mope L., Takio K. and Yonetani T. (1976) Horse liver aldehyde dehydrogenase. Purification and characterization of two isozymes. J. biol. Chem. 251, 236-240. Feldman R. I. and Weiner H. (1972) Horse liver aldehyde dehydrogenase. I. Purification and characterization. J. biol. Chem. 247, 260-266. Gordon A. H., Green D. E. and Subrahmanyan J. (1940) Liver aldehyde oxidase. Biochem. J. 34, 764-774. Holmes R. S. (1978a) Electrophoretic analyses of alcohol dehydrogenase, aldehyde dehydrogenase, aldehyde oxidase, sorbitol dehydrogenase and xanthine oxidase from mouse tissues. Comp. Biochem. Physiol. 61B, 339-346. Holmes R. S. (1978b) Genetics and ontogeny of aldehyde dehydrogenase isozymes in the mouse: localization of Ahd-1 encoding the mitochondrial isozyme on chromosome 4. Biochem. Genet. 16, 1207-1218. Holmes R. S. (1979) Genetics, ontogeny and testosterone inducibility of aldehyde oxidase isozymes in the mouse: evidence for two genetic loci (Aox-I and Aox-2) closely linked on chromosome 1. Biochem. Genet. 17, 517-527. Holmes R. S., Duley J. A. and Burnell J. N. (1983) The alcohol dehydrogenase gene complex on chromosome 3 of the mouse. In lsozymes: Current Topics in Biological and Medical Research, (Edited by Rattozzi M. C., Scandalios J. G. and White G. S.), Vol. 8, pp. 155-174. Alan R. Liss, Inc. New York. Holmes R. S., Duley J. A. and Mather P. B. (1983b) Biochemical and genetic studies on enzymes of alcohol metabolism--the mouse as a model organism for human studies. Aust. N. Z. J. Med. (in press). Koivulo T. and Koivusalo M. (1975) Different forms of rat liver aldehyde dehydrogenase and their subcellular distribution. Biochim. biophys. Acta 397, 9-23.
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Li T.-K. and Magnes L. J. (1975) Identification of a distinctive molecular form of alcohol dehydrogenase in human livers with high activity. Biochim. biophys. Acta. 63, 202-208. Mahler H. R., Mackler B., Green D. E. and Book R. M. (1954) Studies on metalloflavoproteins. III. Aldehyde oxidase: a molybdoflavoprotein. J. biol. Chem. 210, 465-489. Marjenen L. (1972) Intracellular localization of aldehyde dehydrogenase in rat liver. Biochem. J. 127, 633-639. Nakayasu H., Mihara K. and Sato R. (1978) Purification and properties of a membrane-bound aldehyde dehydrogenase from rat liver microsomes. Biochem. biophys. Res. Commun. 83, 697-703. O'Brian M. M. and Schofield P. J. (1980) Polyol pathways of human brain. Biochem. J. 187, 21-30. Petersen D. R., Collins A. C. and Deitrich R. A. (1977) Role of liver cytosolic aldehyde dehydrogenase isozymes in control of blood acetaldehyde concentrations. J. Pharmac. exp. Ther. 201, 471-481. Pietruszko R. (1975) Heterogeneity, polymorphism and substrate specificity of alcohol dehydrogenase from horse liver. In Isozymes, Molecular Structure (Edited by Markert C. L.), Vol. 1, pp. 707-724. Academic Press, New York. Pietruszko R. (1980) Alcohol and aldehyde dehydrogenase isozymes from mammalian liver--their structural and functional differences. In Isozymes: Current Topics in Biological and Medical Research (Edited by Rattazzi M., Scandalios J. G. and Whitt G. S.) Vol. 4, pp. 107-130. Alan R. Liss, New York. Racker E. (1949) Aldehyde dehydrogenase, a diphosphopyridine nucleotide-linked enzyme. J. biol. Chem. 177, 883-892. Rivett A. J., Smith I. L. and Tipton K. F. (1981) Purification of the high-Km aldehyde reductase from rat brain and liver and from ox brain. Biochem. 3. 197, 473-481. Schauenstein E., Esterbaner H. and Zollner H. (1977) Aldehydes in Biological Systems. Their Natural Occurrence and Biological Activities. Pion Ltd, London. Seeley T.-L. and Holmes R. S. (1983) Unpublished results. Siew C., Deitrich R. A. and Erwin V. G. (1976) Localization and characterization of rat liver mitochondrial aldehyde dehydrogenases. Archs Biochem. Biophys. 176, 638-649. Smith M., Hopkinson D. A. and Harris H. (1971) Development changes and polymorphism of human liver alcohol dehydrogenase. Ann. Hum. Genet. 34, 257-263. Smith M., Hopkinson D. A. and Harris H. (1973) Studies on the subunit structure and molecular size of the human alcohol dehydrogenase isozymes determined by the different loci, ADH 1, ADH z and ADH a Ann. Hum. Genet. 36, 401-412. Smolen A., Wayman A-L., Smolen T-N. Petersen D. R. and Collins A. C. (1981) Subcellular distribution of hepatic aldehyde dehydrogenase activity in four inbred mouse strains. Comp. Biochem. Physiol. 69C, 199-204. Theorell H., Taniguchi S., Akeson A. and Skursky L. (1966). Crystallization of a separate steroid-active liver alcohol dehydrogenase. Biochem. biophys. Res. Commun. 24, 603-610. Timms G. P. and Holms R. S. (1981) Genetics of aldehyde dehydrogenase isozymes in the mouse: evidence for multiple loci and localization of Ahd-2 on chromosome 19. Genetics 97, 327-336. Vallee B. L., and Bazzone T. J. (1983) Isoenzymes of human liver alcohol dehydrogenase. In Isozymes, Current Topics in Biological and Medical Research, Vol. 8, pp. 219-244. Alan R. Liss, New York.
0305-0491/84 $3.00+0.00 © 1984 Pergamon Press Ltd
Printed in Great Britain
ELECTROPHORETIC ANALYSES OF ALDEHYDE DEHYDROGENASE, ALDEHYDE OXIDASE AND FROM HORSE
ALCOHOL DEHYDROGENASE, ALDEHYDE REDUCTASE, XANTHINE OXIDASE TISSUES
TANYA-LEE SEELEY, PETER B. MATHER and ROGER S. HOLMES School of Science, Griffith University, Nathan, 411 l, Brisbane, Queensland, Australia. Tel: 07-275-7111 (Received 25 October 1983)
Akstract--1. Cellulose acetate zymograms of alcohol dehydrogenase (ADH), aldehyde dehydrogenase (AHD), aldehyde reductase (AHR), aldehyde oxidase (AOX) and xanthine oxidase (XOX) extracted from horse tissues were examined. 2. Five ADH isozymes were resolved: three corresponded to the previously reported class I ADHs (EE, ES and SS) (Theorell, 1969); a single form of class II ADH (designated ADH-C2) and of class III ADH (designated ADH-B2) were also observed. The latter isozyme was widely distributed in horse tissues whereas the other enzymes were found predominantly in liver. 3. Four AHD isozymes were differentially distributed in subcellular preparations of horse liver: AHD-1 (large granules); AHD-3 (small granules); and AHD-2, AHD-4 (cytoplasm). AHD-1 was more widely distributed among the horse tissues examined. Liver represented the major source of activity for most AHDs. 4. A single additional form of NADPH-dependent AHR activity (identified as hexonate dehydrogenase), other than the ADHs previously described, was observed in horse liver. 5. Single forms of AOX and XOX were observed in horse tissue extracts, with highest activities in liver.
INTRODUCTION
Alcohol dehydrogenase (ADH; EC 1.1.1.1) catalyses the reversible oxidation of aliphatic and aromatic primary alcohols to their respective aldehyde derivatives. The products from this reaction have high reactivities and may be highly toxic in living cells (Schaunstein et al., 1977). Aldehydes are also generated in vivo from other oxidative processes derived from amines and lipid hydroperoxides (Schaunstein et al., 1977), and a biological mechanism is required to detoxify these reactive substances. Several enzymes are capable of facilitating this process including aldehyde dehydrogenase (AHD; EC 1.2.1.3), via an NAD-linked irreversible oxidation reaction to the corresponding acid (Racker, 1949); aldehyde reductase (AHR), via the reverse reaction of ADHs and by 'specific' NADPH-dependent reductases (Bosron and Prairie, 1973; Branlant and Biellmann, 1980); as well as aldehyde oxidase (AOX; EC 1.2.3.1) and xanthine oxidase (XOX; EC 1.2.3.2), via direct oxidative processes with molecular oxygen, generating the corresponding acid and hydrogen peroxide (Avis et al., 1956; Gordon et al., 1940; Mahler et al., 1954). Horse liver has served as an important source of mammalian alcohol dehydrogenase and aldehyde dehydrogenase for biochemical studies. Horse liver A D H was first crystallized by Bonnischen and Wassen (1948), and later isolated as three major forms of activity, resulting from the dimerization of two biochemically distinct subunits, E and S (Theorell et al., 1966). The isozymes have been extensively studied and characterized biochemically (see Branden et al., 1975; Pietruszko, 1980). Horse liver aldehyde dehydrogenases have been also studied (Feldman and
Weiner, 1972; Eckfeldt et al., 1976). The latter group has purified and characterized mitochondrial and cytosolic forms of this enzyme, and examined their differential kinetic properties. More recent studies on mammalian liver ADHs and AHDs have indicated that a more extensive multiplicity for these enzymes may exist. Vallee and Bazzone (1982), for example, have established that human liver A D H exists as three classes of enzymes, named I, II and III. Human type I A D H comprises a complex set of isozymes, resulting from the dimeric association of three genetically distinct subunit types, ~t, fl and 7 (Smith et al., 1971; 1973), and represents the major ethanol metabolizing enzymes in the body (Vallee and Bazzone, 1982). Type II (1t-ADH) A D H preferentially oxidizes longer chain and aromatic alcohols, and becomes active with ethanol only at higher ethanol concentrations (Li and Magnes, 1975, Vallee and Bazzone, 1982); while Type III (x-ADH) A D H utilizes medium chain alcohols, and is active with ethanol only at concentrations above 0.5 M (Pares and Vallee, 1981; Vallee and Bazzone, 1982). Algar et al. (1983) have also reported three classes of ADHs in mouse tissues with similar kinetic properties. Subcellular fractionation studies on mammalian liver aldehyde dehydrogenases (AHDs) have demonstrated that the enzyme is localized in at least three major compartments: mitochondria, microsomes and the cytoplasm (Deitrich, 1966; Marjenen, 1972; Koivulo and Koivusalo, 1975) with the A H D (or AHDs) (Nakayasu et al., 1978; Timms and Holmes, 1981; Smolen et al., 1981) isolated from each fraction exhibiting characteristic differences in kinetic properties. Moreover, additional multiplicity has been re-
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TANYA-LEESEELEYet al.
ported in rat and mouse liver with at least two A H D s being found in cytosolic and mitochondrial preparations (Siew et al., 1976; Petersen et al., 1977; Holmes et al., 1983a,b). This communication describes the tissue and subcellular distribution, and electrophoretic properties of alcohol dehydrogenase and aldehyde dehydrogenase from horse tissues, and examines three additional enzymes, aldehyde reductase, aldehyde oxidase and xanthine oxidase, which exhibit catalytic activity with aldehydes ag substrates, in vitro. MATERIALS AND METHODS
Tissues
Tissues were obtained from freshly slaughtered adult horses from a local abattoir and stored on ice immediately for a maximum period of 2 hr, prior to subcellular fractionation and tissue extraction. Chemicals
All substrates, coenzymes and buffer chemicals were obtained from Sigma chemicals (St. Louis, MO, USA) except for trans-2-hexen-l-ol (Aldrich Chemical Co., Wembly, UK), benzaldehyde (Unilab, Sydney, Australia) and acetaldehyde (BDH Chemicals Ltd, Poole, England, UK). Homogenate preparation
Homogenates of liver, kidney, heart, lung, spleen, small intestine, brain, testis and stomach were prepared as previously described (Holmes, 1978a). Final concentrations (w/v) of all tissues was 33°o. Liver and subcellular fractionation
Liver tissue was washed in cold buffered isotonic sucrose (50raM Tris-HC1 pH 7.4; 0.25 M sucrose), blotted dry, weighed, finely minced and homogenized in the same buffer using eight strokes of a Potter-Elvehjem homogenizer in the ratio of 1 g liver in 4 ml buffer. The homogenate was then centrifuged for 10min at 600g to produce a pellet containing most of the whole cells and nuclei, which was discarded. The supernatant (S1) was centrifuged at 9000g for 15 rain to sediment a large granule fraction (consisting predominantly of mitochondria, lysosomes and peroxisomes) (LG). The resulting pellet was washed twice in buffered-sucrose while the supernatant ($2) was centrifuged at 100,000g for 60mins to yield a small granule fraction (consisting of microsomes) (SG) and a cytosolic fraction ($3). LG was resuspended in 10raM sodium phosphate buffer (2 ml/gm liver equivalent), kept on ice for 30 min and vortexed twice before adding sodium deoxycholate (0.2~0 final concn). This was also kept on ice and vortexed twice, for 30min. SG was resuspended in 10mM sodium phosphate buffer containing 0.1% Triton X-100 (2ml/g liver equivalent), and homogenized using an Ultra-Turrax homogenizer. The LG and SG extracts were spun at 25,000g for 20min and the resultant supernatants used in electrophoretic analysis. Cellulose acetate electrophoresis and staining
Homogenate supernatants were subjected to zone electrophoresis on Titan III cellulose acetate plates (94 x 76 mm) (Helena Labs., Texas, USA). Samples (approximately 2/11) were applied 10-15 mm from the cathodal edge of the plate and electrophoresed under the following conditions: alcohol dehydrogenase and aldehyde reductase (Tris-citrate (75 mM) buffer pH 7.0; 150 V for 60 min); aldehyde dehydrogenase, ~ldehyde oxidase and xanthine oxidase (Tris-glycine (44mM) buffer pH 8.5; 200V for 20min). Following electrophoresis, the plates were histochemically stained for enzyme activity using an agar overlay technique
(Holmes, 1978a). The details for the histochemical staining procedures are found in Holmes (1978a~ and Duley and Holmes (1982). RESULTS Figure 1 illustrates a zymogram of alcohol dehydrogenase ( A D H ) isozymes from horse tissues. The isozymes appeared to fall into three distinct classes of A D H activity which are compatible with previous studies of Vallee and Bazzone (1982) on human A D H s and Algar et al. (1983) on mouse A D H s , and they have been designated as such. Class I A D H s are recognized by m M or sub-raM Michaelis constants for ethanol and a high sensitivity to pyrazole inhibition; class II A D H s by high Michaelis constants for ethanol and a low sensitivity to pyrazole inhibition while class III A D H s exhibit total inactivity with ethanol concentrations below 0.5 M and are insensitive to pyrazole inhibition. Differential staining procedures were used to distinguish the A D H s resolved (see Holmes et al., 1983a). Electrophoretic zones of A D H activity were observed following examination of horse tissue extracts. The three class I enzymes (designated EE, ES and SS, based upon the studies of Theorell et al. (1966) and Pietruszko (1980) were found only in liver among the tissues examined: similarly, the class II enzyme (designated ADH-C2 on account of its similarity in properties to this enzyme in the mouse Algar et al., 1983) was found only in liver; while the class lII enzyme (designated A D H - B : ) is widely distributed and exhibited highest activity in kidney. The additional activity zones were shown to exhibit overlapping substrate specificities with three forms of aldehyde dehydrogenase, or to arise from control activities from aldehyde oxidase or another non-specific oxidase. The subcellular localization of horse liver A D H isozymes was examined and the results are illustrated in Fig. 2. All five A D H forms were predominantly localized in the cytosolic fraction, although some activities were also found in washed 'small granular" (microsomes) and 'large granular" (mitochondria, peroxisomes and lysosomes) preparations. F o u r aldehyde dehydrogenase ( A H D ) zones of activity were observed following cellulose acetate electrophoresis and histochemical staining (Fig. 3), which were designated as A H D s 1 to 4, in accordance with the nomenclature used for mouse A H D s (Holmes et al., 1983). Aldehyde oxidase, xanthine oxidase and a 'non-specific' oxidase activity zones were recognized using differential staining techniques for these enzymes (Holmes, 1978a). A H D - I exhibited highest activity in liver but was also widely distributed in other tissues examined. In contrast A H D - 2 and A H D - 4 were predominantly localized in liver and kidney, while A H D - 3 was restricted to liver extracts in this organism. The subcellular distribution of horse liver A H D s was also examined (Fig. 4). A H D - I was found predominantly in the 'large granular' fraction, although significant activity was also observed in the cytosol fraction. This latter activity may represent leakage from the 'large granules' during homogenization. A H D - 2 and A H D - 4 were found in the cytosolic fraction, while A H D - 3 was localized in the 'small granular' microsomal fraction. Aldehyde ox-
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Fig. 1. Cellulose acetate zymogram and diagramatic illustration of the electrophoretic properties of alcohol dehydrogenase (ADH) isozymes from horse tissues. L, liver; K, kidney; H, heart; Lu, lung; Sp, spleen; SI, small intestine; Br brain; T, testis; St, stomach. The zymogram was stained using 10 mM trans-2-hexen-l-ol as substrate. Subunit structures for the previously investigated class I horse liver ADHs (EE, ES and SS) and the proposed class II (ADH-C2) and Class III (ADH-B2) isozymes are given. ADH activity is given as ~ ; aldehyde oxidase activity as 1 ~ ; aldehyde dehydrogenase activity as L;~.; and 'non-specific' oxidase activity
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Fig. 2. Cellulose acetate zymogram and diagrammatic illustration of the electrophoretic properties of alcohol dehydrogenase (ADH) isozymes from subcellular fractions of horse liver. TE, total extract: LG, large granule fraction: SG, small granule fraction: Cy, cytoplasm. The zymogram was stained using 10mM trans-2-hexen-l-ol as substrate. Subunit structures lbr the previously investigated horse liver class l ADHs (EE, ES and SS) and the proposed class II (ADH-C 2) and class III (ADH-B2) isozymes are given. ADH activity is given as m ; aldehyde oxidase activity as ~,~: aldehyde dehydrogenase activity a s ~ : and non-specific oxidase activity as EYY.
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Fig. 3. Cellulose acetate zymogram and diagrammatic illustration of the electrophoretic properties of aldehyde dehydrogenase (AHD) isozymes from horse tissues. L, liver; K, kidney; H, heart; Lu, lung; Sp, spleen; SI, small intestine; Br, brain; T, testis; St, stomach. The zymogram was stained using 20 mM acetyldehyde as substrate. Isozymes are designated as AHD-1, AHD-2, AHD-3 and AHD-4. AHD activity is given as ~ ; aldehyde oxidase activity aslg~l; xanthine oxidase as II1~; and non-specific oxidase aslZ~.
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Fig. 4. Cellulose acetate zymogram and diagrammatic illustration of the electrophoretic properties of aldehyde dehydrogenase (AHD) isozymes from subcellular Fractions of horse liver. TE, total extract; LG, large granule fraction; SG, small granule fraction; Cy, cytoplasm. The zymogram was stained using 20 mM acetaldehyde as substrate. Isozymes are designated as for Fig. 3. AHD activity was given as ~ : aldehyde oxidase activity as ~ N ; xanthine oxidase activity as/IIIIIH:and 'non-specific' oxidase activity asIZ~-
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Fig. 5. Cellulose acetate zymogram and diagrammatic illustration of the electrophoretic properties of aldehyde reductase active enzymes from subcellular fractions of horse liver. TE, total extract; LG, large granule fraction; SG, small granule fraction; Cy, cytoplasm. The zymogram was stained using 5 mM p-nitrobenzaldehyde as substrate. The fine alcohol dehydrogenase isozymes observed in Figs t and 2 all exhibited aldehyde reductase activity and are designated as m , hexonate dehydrogenase activity (reverse reaction) was given a s ~ . Control reductase activity was given a s K .
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138
TANYA-LEESEELEYet al.
idase and xanthine oxidase activities, which stain as control zones in the absence of the coenzyme (NAD +) in the histochemical agar overlay, were found in the cytosolic fraction (Fig. 4). Figure 5 shows a cellulose acetate zymogram of aldehyde reductase activity from subcellular fractions and a detergent-extracted homogenate of horse liver, using p-nitrobenzaldehyde as substrate and reduced NADP as coenzyme. In addition to the ADH isozymes observed in Fig. 2, an NADPH specific aldehyde reductase was observed which was recognized as hexonate dehydrogenase by differential staining techniques (Duley and Holmes, 1982). Hexonate dehydrogenase was localized in the liver cytoplasmic fraction. DISCUSSION
The present results provide evidence for additional forms of horse liver alcohol dehydrogenase (ADH) and aldehyde dehydrogenase (AHD) to those reported in the available literature. Prior to this study, horse liver ADH isozymes have been investigated and described in terms of three major forms, associated with the random dimerization of E (ethanol active) and S (sterol active) subunits (see review by Branden et al., 1975). Pietruszko (1980) has described further forms of these enzymes but has concluded that these represent polymorphic variants or conformational isomers of the major isozymes, EE, ES and SS. In terms of the classification of mammalian ADH isozymes described by Vallee and Bazzone (1982), these enzymes have properties similar to those of class I ADHs, namely, high affinity for ethanol as substrate and high sensitivity to pyrazole inhibition. The two additional forms of horse liver ADH reported in this paper resemble those of the class II and class III ADHs described by Vallee and Bazzone (1982) for the human ~z and X enzymes, and those described recently for mouse ADHs by Algar et al. (1983). It is likely that these two forms escaped earlier identification since firstly, human class II ADH's have been reported to be quite labile in vivo if not in t'itro at low temp (Li and Magnes, 1975) and secondly class III ADH's would not be evident under normal staining procedures with ethanol. The horse liver ADH-C2 has been purified and characterized biochemically (Seeley and Holmes, 1983) and similar investigations on ADH-B2 are currently being undertaken in this laboratory. ADH-C2 has a low affinity for ethanol as substrate with a Michaelis constant of 42 mM (compared with 1 mM for EE and 11 mM for SS isozymes of Pietruszko, 1975) and is much less sensitive to inhibition by pyrazole, exhibiting a K, of 15 mM as compared to 0.07 #M for the SS isozyme (Theorell, 1966). ADH-B2 is inactive with ethanol as substrate at concentrations less than 0.5M and longer chain alcohols (eg. trans-2-hexen-l-ol) are routinely used as substrate for this isozyme. Both isozymes are dimers with subunit molecular weights similar to those of the class I horse liver ADHs. Additional forms of horse aldehyde dehydrogenase (AHD) have also been reported in this present study. Horse liver AHD was first purified by Feldman and Weiner (1972), although only a single homogeneous
enzyme was described. Eckfeldt et al. (1976) subsequently purified and characterized cytoplasmic and mitochondrial isoenzymes of aldehyde dehydrogenase. These two enzymes (designated F~ and F~ respectively) were reported as tetramers, exhibiting broad aldehyde specificity. Two additional electrophoretic forms of AHD were observed (Figs 3 and 4) which are differentially localized in tissue extracts and liver subcellular fractions. AHD-3 activity was observed in liver microsomal extracts whereas two zones of cytosolic AHD activity were found in horse liver and kidney extracts. These results are directly comparable to those recently reported for mouse liver AHDs. (Holmes, 1978b; Timms and Holmes, 1981: Holmes et al., (1983b) Genetic and biochemical analyses have established that at least 3 genetic loci encode aldehyde dehydrogenase isozymes in the mouse: A h d - l (encoding the mitochondrial isozyme, AHD-I); A h d - 2 (encoding the cytosolic isozyme, (AHD-2) and A h d - 3 (encoding the microsomal isozyme, AHD-3). Moreover, the presence of at least three pools of liver AHD activity in subcellular fractions has been observed in a number of man> malian organisms (Koivula and Koivusalo, 1975: Nakayasu et al., 1978: Timms and Holmes, 1981; Smolen et al., 1981). Human AHDs have also been recently studied and shown to exist as distinct isozymic forms in liver mitochondria, microsomes and cytosolic preparations (Duley et al.. 1983). Electrophoretic analyses of aldehyde oxidase (AOX) and xanthine oxidase (XOX) from horse tissues showed that these enzymes exist as single forms in this species and exhibited highest activity in liver. This is similar to a recent report for these enzymes in human tissues (Duley et al.. 1983) but is in contrast, at least as far as AOX is concerned, with the situation in the mouse. AOX is encoded by two closely linked structural genes, A o x - I and A o x - 2 . in this organism (Holmes, 1979). Hexonate dehydrogenase (one o1" the aldehyde reductases resolved in Fig. 5) also occurs as a single form in horse tissue extracts. This enzyme is defined by its absolute specificity for reduced NADP as coenzyme, which is in contrast to alcohol dehydrogenase isozymes, exhibiting aldehyde reductase activity in the presence of reduced NAD or reduced NADP (Duley and Holmes, 1982). Hexonate dehydrogenase has been purified from human brain by O'Brian and Schofield (1980), and from rat liver and brain by Rivett et al., (1981), and is distinguished by its sensitivity to barbiturate and valproate inhibition. its utilization of D-glucuronate as a substrate, and its low mol. wt (40,000) monomeric subunit structure. In summary, this paper reports an electrophoretic analysis of the tissue distribution, subcellular distribution and multiplicity of enzymes involved with alcohol metabolism in horse tissues, namely alcohol dehydrogenase and aldehyde dehydrogenase. Aldehyde oxidase, xanthine oxidase and aldehyde reductase were also investigated because of their overlapping specificities with these enzymes.
Acknowledgement--This research was supported in part by the Australian Research Grants Scheme.
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