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X-ADH Is the Sole Alcohol Dehydrogenase Isozyme of Mammalian Brains: Implications and Inferences (Class III Alcohol Dehydrogenase/Tissue-Specific Enzyme) THOMAS B

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X-ADH Is the Sole Alcohol Dehydrogenase Isozyme of Mammalian Brains: Implications and Inferences (Class III Alcohol Dehydrogenase/Tissue-Specific Enzyme) THOMAS B Proc. Natl. Acad. Sci. USA Vol. 82, pp. 8369-8373, December 1985 Biochemistry X-ADH is the sole alcohol dehydrogenase isozyme of mammalian brains: Implications and inferences (class III alcohol dehydrogenase/tissue-specific enzyme) THOMAS B. BEISSWENGER, BARTON HOLMQUIST, AND BERT L. VALLEE* Center for Biochemical and Biophysical Sciences and Medicine, Harvard Medical School, 250 Longwood Avenue, Boston, MA 02115 Contributed by Bert L. Vallee, August 14, 1985 ABSTRACT Class m (X) is the only alcohol dehydrog- occurrence and function of ADH in that tissue has always enase (ADH) in human, equine, bovine, simian, canine, and been of particular interest, but efforts to uncover facts rodent brain and is the first to be identified, purified, and relevant to ethanol have been singularly disappointing. Most characterized from the brain of humans or other vertebrates. of those studies antedate the awareness of the remarkable Like the corresponding isozymes from human placenta and number and diversity of structure and specificity of ADH liver, the X-ADH isozymes purified from mammalian brain are isozymes (4), first noted much earlier (5). neither inhibited by nor do they bind to immobilized pyrazole, Experimental work failed to reveal any significant ADH and they oxidize ethanol only very poorly (Km > 2.5 M). activity in brain (6). The level ofethanol oxidation in rat brain Indeed, it would be incorrect to classify them as "ethanol is so vanishingly low that only assays of exceptionally high dehydrogenases." They contain 4 g.atom of zinc/mol, bind 2 sensitivity could detect it (7). A number of contemporaneous moles of NAD, and readily oxidize long-chain aliphatic and studies employing [14C]ethanol concluded that the perfused aromatic primary alcohols. These findings appear to exclude rat brain does not metabolize [14C]ethanol at all (8, 9). the possibilities that ADH protects the brain of these verte- Immunological work has implied the existence of minuscule brates against ethanol or its metabolic products and that the amounts of class It isozymes in human brain (10). However, brain can generate energy for cerebral function from ADH- none of these or other isozymes have been identified, monitored ethanol metabolism. Thus X-ADH must serve a isolated, purified, or characterized from the brains of either totally different but as yet unknown role. The failure to detect humans or other vertebrates. any ethanol dehydrogenase activity in brain creates an intel- Among all the isozymes now known, the capacity of class lectual dilemma only if it is assumed that such an enzyme has III (X) ADH to oxidize ethanol is minimal. Whereas all other evolved and developed as a protective mechanism for ethanol isozymes are saturated by millimolar or micromolar ethanol, detoxification in that organ, as has been assumed. Tissue and as much as 2.5 M ethanol fails to saturate y-ADH! In contrast, substrate specificities of ADH isozymes are likely to give new the Kms of X-ADH for pentanol and longer-chain aliphatic insight regarding their physiological roles. alcohols are quite analogous to those of all class I and II isozymes for ethanol (11). Oxidation ofethanol has been assumed and generally accept- We now demonstrate that class III (X) ADH is the only ed to be the primary function of human alcohol dehy- ADH isozyme in human brain and that of five other mam- drogenases (ADH; alcohol:NAD' oxidoreductase, EC malian species. We have identified, isolated, purified, and 1.1.1.1), perhaps because an ADH was actually first isolated characterized ADH from each of these sources and proven it and crystallized from yeast (1). The formation of ethanol can identical to those of class III hardly be the function of ADH in mammalian liver. Yet it to have properties virtually seems to have been taken for granted that ethanol was the ADH in human liver and placenta. primary substrate for the enzymes isolated first from the The demonstration that class III is the sole ADH in horse (2) and then from human liver (3). In the ensuing work cerebral tissue cannot answer any questions as to the pres- on these enzymes, differences of rates, specificities, kinetic, ence or absence in brain of any mechanism by which ethanol structural, and functional details, and the reaction product is detoxified. Indeed the isolation of class III from brain favored at equilibrium for various substrates of these en- documents the presence of an alcohol dehydrogenase that is zymes were not given specific emphasis in that regard. not an ethanol dehydrogenase; it must have a different Hence, the tacit premise that the oxidation of ethanol is the function. Minimally, the linguistic inference that "alcohol prime target of human ADH has evolved and persisted to the dehydrogenase" can be considered an "ethanol dehydrog- present. Moreover, most of the metabolic activity of the enase" is tantamount to semantic misunderstanding. Maxi- mammalian ADHs has been assumed to be identical with mally, it has proven to be a fallacious premise for critical those isolated from liver, where they are most abundant and physiological, biochemical, and pharmacological investiga- relatively accessible to isolation and where, in the human, tion of ethanol metabolism in man. from 75 to 90% of ethanol is apparently metabolized. Long- The present data would seem to exclude the possibility that term human pathology due to ethanol (e.g., alcoholic cirrho- X-ADH could protect cerebral tissues against the effects of sis) primarily affects the liver, further focusing attention on ethanol and the metabolic products of its ADH oxidation and the ADH of this organ. Comparatively speaking, minimal generate energy from ADH-monitored ethanol metabolism experimental attention has been given to ADH and its for cerebral function. Rather, the presence of this sole behavior in tissues other than the liver. isozyme in brain (and other tissues) must signal completely On administration of ethanol to humans, the effects on brain are the ones that are apparent most rapidly. Hence, the Abbreviations: ADH, alcohol dehydrogenase; U, unit(s). *To whom reprint requests should be addressed. tClass I comprises the a,3,yfamily of isozymes (jointly also referred The publication costs of this article were defrayed in part by page charge to as "undifferentiated" ADH), class II comprises the ir-ADHs, and payment. This article must therefore be hereby marked "advertisement" class III the X-ADHs, of which at least two forms have been in accordance with 18 U.S.C. §1734 solely to indicate this fact. recognized. 8369 Downloaded by guest on September 25, 2021 8370 Biochemistry: Beisswenger et al. Proc. Natl. Acad Sci. USA 82 (1985) different, albeit as yet unknown, physiological and biochem- ical roles. O0 The failure to detect any ethanol dehydrogenase activity in brain creates an intellectual dilemma only ifit is assumed that the enzyme has evolved and developed as a protective and/or defense mechanism for the detoxification of ethanol in that organ. a - - It would seem to us that the discovery ofthe enzymological Origin-. I- diversity of the ADH isozymes shifts the emphasis now centering on inquiry regarding the properties ofthe isozymes of the liver to those of the brain, placenta, testis, and other organs whose ADH isozyme content is much more selective and tissue-specific. 0) MATERIALS AND METHODS 1 2 3 4 5 6 Human brain was obtained from legal autopsies (with per- FIG. 1. Activity stained (crotyl alcohol substrate) starch gel electrophoregrams of homogenates of equine (lane 1), bovine (lane mission) within 12 hr postmortem and was stored at -70'C. 2), canine (lane 3), rabbit (lane 4), and simian (P. cynocephalus, lane Simian frontal cortex ofMacaca nemestrina, M.fascicularis, 5; M. fascicularis, lane 6) brain. and Papio cynocephalus was obtained frozen from the Regional Primate Research Center (University of Washing- ton, Seattle, WA). Horse brain (frontal cortex) was obtained Two human brains were dissected, and 2-g samples of at slaughter and stored immediately at -70TC, whereas whole anatomically defined sections were homogenized in water. cow, dog, and rat brains were purchased from Pel Freez. After centrifugation, the homogenates were electrophoresed ADH was isolated and purified by the procedure ofWagner in starch gel and stained for activity with both ethanol and et al. (11). ADH activity assays and metal analyses were as pentanol. Fig. 2 shows the results for five anatomically described (12). Retinol (70 1LM) oxidation was measured in distinct samples: right basal ganglia, corpus striatum, vermis, 100 mM sodium phosphate (pH 7.4) containing 0.02% Tween medulla, and right frontal lobe; a liver homogenate is includ- 80 and 2.4 mM NAD' and was monitored by the change of ed for comparison. Staining with ethanol as substrate was absorption at 382 nm. One unit of enzyme activity was entirely negative for ADH activity of all brain samples, defined as the oxidation or reduction of 1 gmol of indicating that class I or II isozymes, present in the liver coenzyme/min. Protein concentrations were measured by homogenate, could not be detected. By comparison, stains the method of Lowry et al. (13), and amino acid analyses with pentanol promptly resulted in bands corresponding to were performed by the phenyl isothiocyanate method (14). class III ADH in all samples. Similarly thalamus, substantia Brain tissue homogenates were electrophoresed in starch nigra, pons, left parietal and posterior cortex, cerebellum, gels (15) and stained for ADH activity using ethanol (100 mM) and the parietal-occipital regions were identical: they con- and pentanol (35 mM), and in some cases crotyl alcohol (70 tained only class III ADH. mM) or cinnamyl alcohol (1.0 mM), as substrates.t The ADH isozymes were isolated and purified from 20 g of NaDodSO4/PAGE was performed by the procedure of brain ofeach species by the procedure devised for liver ADH Laemmli and Favre (16).
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