Pere Julià

Role of extrahepatic alcohol dehydrogenase in rat ethanol metabolism

Rat alcohol dehydrogenase exhibits three isoenzymes with very different capacities of ethanol oxidation and with characteristic distribution in tissues. ADH-1 (class II isoenzyme, Km = 5 M) is especially concentrated in the most external organs: auditive, bucal, and nasal mucoses, cornea, esophagus, stomach, rectum, penis, and vagina. ADH-2 (class III isoenzyme) is present in all organs but has a poor activity with ethanol. ADH-3 (class I isoenzyme, Km = 1.4 mM) is the main liver isoenzyme, also present in lung, intestine, kidney, and sexual organs. At 33 mM ethanol and pH 7.5, total hepatic activity (3.5 +/- 0.6 units) represents 90% of the whole activity in the male rat, while the remaining 10% is distributed in many organs. The skin is the extrahepatic organ with the highest total activity (88 +/- 15 mU) followed by testis and small intestine. ADH-3 accounts for 96% of total activity (90% hepatic and 6% extrahepatic) and ADH-1 contributes with 4% (extrahepatic). However, in conditions that may be found in the digestive tract mucose after ethanol ingestion (pH 7.5, 1 M ethanol), stomach and small intestine activities represent 10% of the liver activity at 33 mM ethanol. Therefore, oral administration of ethanol will result in a higher contribution of the extrahepatic activity than will intravenous or intraperitoneal administration, because of the great ADH-1 content of the digestive tract. On the other hand, pyrazole inhibition constants at pH 7.5 for ADH-1 (33 mM) and ADH-3 (4.2 microM) are much higher than those at pH 10.0 (0.56 mM and 0.4 microM) and indicate that at the usual concentration of inhibitor only ADH-3 activity will be effectively suppressed. ADH-1 will be, therefore, responsible in part for the residual ethanol oxidation activity in pyrazole-treated rats.

Ocular alcohol dehydrogenase in the rat: Regional distribution and kinetics of the ADH-1 isoenzyme with retinol and retinal

Starch-gel electrophoresis of rat ocular tissues shows two anodic isoenzymes of alcohol dehydrogenase (ADH), designated as ADH-1 and ADH-2, ADH-1 is characteristic of the ocular tissues, and corresponds to more than 95% of all ADH activity in the eye. The well known cathodic forms of rat liver ADH, that we named ADH-3, are not observed in the ocular tissues. ADH-1 is detected in retina, pigment epithelium-choroid, ocular fluid, and cornea but not in the lens. The cornea exhibits the highest ADH activity [200 +/- 59 milliunits (munits) mg-1] followed by the pigment epithelium-choroid (11 +/- 7 munits mg-1). Activity in the retina is very small (0.6 +/- 0.2 munit mg-1) and represents only 0.6% of the total activity in the eye. Most of the rat ocular ADH is localized in the cornea (68%) where it could play a significant role in the detoxication of the alcohols of a broad range of structures. Purified ADH-1 shows a low Km for retinol oxidation (20 microM) and for retinal reduction (30 microM) indicating that this isoenzyme may have a function in the metabolism of retinoids. Ethanol competitively inhibits retinol oxidation, but with a very high apparent inhibition constant (0.6 M) demonstrating that the inhibitory effect is not significantly at the usual concentrations found in the blood during ethanol intoxication.

Structural and Enzymatic Properties of a Gastric NADP(H)- dependent and Retinal-active Alcohol Dehydrogenase*

A class IV-type, gastric alcohol dehydrogenase (ADH) has been purified from frog (Rana perezi) tissues, meaning detection of this enzyme type also in nonmammalian vertebrates. However, the protein is unique among vertebrate ADHs thus far characterized in having preference for NADP+ rather than NAD+. Similarly, it deviates structurally from other class IV ADHs and has a phylogenetic tree position outside that of the conventional class IV cluster. The NADP+ preference is structurally correlated with a replacement of Asp-223 of all other vertebrate ADHs with Gly-223, largely directing the coenzyme specificity. This residue replacement is expected metabolically to correlate with a change of the reaction direction catalyzed, from preferential alcohol oxidation to preferential aldehyde reduction. This is of importance in cellular growth regulation through retinoic acid formed from retinol/retinal precursors because the enzyme is highly efficient in retinal reduction (k cat/K m = 3.4·104 mm −1min−1). Remaining enzymatic details are also particular but resemble those of the human class I/class IV enzymes. However, overall structural relationships are distant (58–60% residue identity), and residues at substrate binding and coenzyme binding positions are fairly deviant, reflecting the formation of the new activity. The results are concluded to represent early events in the duplicatory origin of the class IV line or of a separate, class IV-type line. In both cases, the novel enzyme illustrates enzymogenesis of classes in the ADH system. The early origin (with tetrapods), the activity (with retinoids), and the specific location of this enzyme (gastric, like the gastric and epithelial location of the human class IV enzyme) suggest important functions of the class IV ADH type in vertebrates.

Isoenzymes of alcohol dehydrogenase in retinoid metabolism

Publisher Summary Alcohol dehydrogenase (ADH) is an enzyme widely distributed in animals, plants, and microorganisms. It catalyzes the reversible oxidation of a great variety of alcohols to the corresponding aldehydes and ketones. ADH exhibits several isoenzymes in most species studied. ADH isoenzymes are very active toward long-chain hydrophobic alcohols. This chapter describes the assay method of ADH. ADH isoenzymes are heterogeneously distributed in animal tissues. Prior to purification from a particular organ it is convenient to analyze the isoenzyme composition by starch gel electrophoresis. Procedures for the purification of ADH isoenzymes from a variety of mammalian species have been reported. This chapter describes the purification of the rat ADH isoenzymes. Three ADH isoenzymes are detected in rat tissues that have been named ADH-1, ADH-2, and ADH-3 according to their mobility on starch gel electrophoresis. ADH-1 is the most anodic form, characteristic of the stomach, ocular tissues, and external epithelia. ADH-1 shows K m values of 5 M for ethanol and 20 μM for retinol and it has been considered a class II isoenzyme although no clear correspondence has been found with the human isoenzymes.

Acetylated N-terminal structures of class III alcohol dehydrogenases Differences among the three enzyme classes

The protein chains of mammalian alcohol dehydrogenases typically lack free α‐amino groups. The blocked N‐terminal regions of the class III type of the rat (ADH‐2), human (χχ) and horse enzymes were isolated by digestions with proteases, and characterized by mass‐spectrometry supplemented with chemical analysis of the peptides and their redigestion fragments. Results were confirmed by synthesis of the corresponding peptides, followed by chromatographic comparisons of the native and synthetic products. The N‐terminal regions of the three class III alcohol dehydrogenase subunits are homologous but differ from the class I and II enzymes in both the exact start position and the amino acid sequence, which suggests that different N‐terminal structures are typical for each of the three classes.

Amphibian Alcohol Dehydrogenase

Alcohol dehydrogenase (ADH) catalyses the reversible interconversion of a variety of alcohols and their corresponding aldehydes and ketones, and is widely distributed in organisms. It has been detected in all animals, in plants, and eukaryotic and prokaryotic microorganisms. In vertebrates the ADH system is complex, with at least seven different enzymatic classes (Jornvall and Hoog, 1995, Kedishvili et al., 1997). The study in sub- mammal species is of interest to understand the relationship between the classes found in mammals and the evolutionary origins of the present structures. Moreover, since the enzyme exhibits a wide substrate specificity, the comparative study of the enzymes from distant species may provide valuable information on the function of the enzyme, and how it may change among groups of vertebrates to adapt to different physiological needs.

The Alcohol Dehydrogenase System in the Rat: Comparison with the Human Enzyme

The rat is the most used animal model in studies on the metabolism and pharmacology of ethanol. The research on rat alcohol dehydrogenase (ADH; EC 1.1.1.1) started early (Markovic et al., 1971; Arslanian et al., 1971) and continued more recently (Crabb et al., 1983; Lad and Leffert, 1983, Mezey and Potter, 1983). These reports focused on the liver form that is most active with ethanol. During the last decade a great amount of information has been available on the human enzyme, which is now recognised to be a complex system of isoenzymes. The human ADH isoenzymes were grouped in three classes (I, II, III) according to their kinetic characteristics (Vallee and Bazzone, 1983). The rat, with only one ADH isoenzyme clearly described, appeared to be a model too simple for ethanol metabolism studies. We reinvestigated the rat enzyme in different organs and reported the existence of three distinct isoenzymes: ADH-3 is the liver enzyme with properties similar to human class I; ADH-2, present in all the organs examined, appeared homologous to human class III; ADH-1, isolated from stomach, exhibited characteristics of class II (Julia et al., 1987). Structural studies of classes I and III supported this classification (Julia et al., 1988). In the present report we summarize our work on the rat ADH isoenzymes and compare the rat and the human enzymes, taking into account the new human ADH that we have recently described in human stomach. We use the nomenclature of classes for the rat isoenzymes to allow a clear comparison with the human ADH.