Subunit Composition of horse liver alcohol dehydrogenase isoenzymes

Lutstorf, Ursala; Wartburg, J. P. von

doi:10.1016/0014-5793(69)80332-7

Cited by 43 publications

(12 citation statements)

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“…The high absorption at 290 nm of cinnamaldehyde was a very sensitive tool to study such an activity with this enzyme. The reduction of this aldehyde with horse liver alcohol dehydrogenase has been described [22], so the oxidation of the corresponding alcohol was not surprising. The oxidation of cinnamyl alcohol has also been recently described by other authors [23].…”

Section: Discussionmentioning

confidence: 95%

Preparation and Properties of 3-Halopyridine-adenine Dinucleotides, NAD + Analogues and of Model Compounds

et al. 1976

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The preparation of model compounds 1-(2',6'-dichlorobenzyl)-3-halogenopyridiniurn and the study of their properties were achieved. Their chemical reduction to the corresponding 1,4-dihydropyridines is proved by spectroscopic analysis. 3-Iodopyridine-adenine dinucleotide was prepared by enzymic transglycosidation while the 3-chloro, 3-bromo and 3-iodo pyridine-adenine dinucleotides were synthesized from 3-amino-pyridine-adenine dinucleotide. The 3-halogenopyridine -adenine dinucleotides were proved to be active as hydrogen acceptors with alcohol as a substrate. The absorption band at 290 nm of cinnamaldehyde appeared to be a very sensitive tool for studying the enzymic reaction. With the alcohol dehydrogenase from yeast, only slight activity was detected. 3-Halogenopyridine -adenine dinucleotides are competitive inhibitors with respect to nicotinamide--adenine dinucleotide with alcohol dehydrogenase from yeast, lactate dehydrogenase and malate dehydrogenase. The use of 3-iodopyridine -adenine dinucleotide as a heavy-atom derivative for X-ray structure det6rmination is proposed.Although the analogues of NAD+ with structural modification at the pyridinium ring are numerous, their differences with NAD' are only slight considering the similarity of the group at C-3. We thought that halo3PdAD+, where the amide group is replaced by a halogen, may be interesting analogues for the study of the NAD+ binding site of dehydrogenases.The carbon-halogen distances vary from 2.05 A for iodine [l] to 1.35 A for fluorine [2]. Moreover the possibility of using io3PdAD+ as a heavy-atom derivative for the X-ray structure determination of some dehydrogenases and as a photochemical label [3] seemed interesting.

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Section: Discussionmentioning

confidence: 95%

Preparation and Properties of 3-Halopyridine-adenine Dinucleotides, NAD + Analogues and of Model Compounds

et al. 1976

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“…Thcir purity was checked by electrophoresis on starch gel [9] using NAD+, ethanol, tetrazolium blue and phenazine methosulfate for staining [lo]. The isoenzyme concentrations were determined by the absorbance at 280 nmusing a value of 0.455 for a I-mg/ml protein solution [Ill.…”

Section: Purification Of the Zsoenzynze Of Liver Alcohol Dehydrogerzasementioning

confidence: 99%

5-Methylnicotinamide-adenine Dinucleotide. Kinetic Investigation with Major and Minor Isoenzymes of Liver Alcohol Dehydrogenase and Structural Determination of Its Binary Complex with Alcohol Dehydrogenase

1981

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5-Methylnicotinamide -adenine dinucleotide and 3-cyano-5-methylpyridine -adenine dinucleotide were prepared from 5-methylthionicotinamide -adenine dinucleotide by chemical conversion. The 5-methylthionicotinamide -adenine dinucleotide was obtained by enzymic transglucosidation. Model compounds ascertained the structure. None of the dinucleotides rnethylated at C-5 was active with the major isoenzyme EE of horse liver alcohol dehydrogenase, but activity with 5-methylnicotinaniide -adenine dinucleotide was measured with the minor isoenzymes. The binding of 5-methylnicotinamide -adenine dinucleotide to liver alcohol dehydrogenase, investigated by X-ray diffraction methods to 0.37-nm resolution, occurs with the pyridinium ring away from the active site as previously described for 3-iodopyridine -adenine and pyridine -adenine dinucleotides. A general conclusion on the use of inhibitors as tools for exploration of the active site is drawn.The X-ray structure determination of the orthorhombic apoenzyme of the alcohol dehydrogenase from horse liver [I] prompted the study of the binding of coenzyme or coenzyme analogues and substrates to clarify the binding mode and the reaction mechanism. It has been shown previously that when an oxidized or a reduced coenzyme is soaked into these crystals, they crack and dissolve. Crystallisation of ternary eiizyme-NADH-substrate complexes gives triclinic or monoclinic crystals with a different conformation of the enzyme molecule [2]. During a screening of coenzyme analogues it was found that 5-methylnicotinamide-adenine dinucleotide (m5NAD)+ did not induce cracking of the orthorhombic crystals in spite of earlier reports that this analogue showed activity as an hydride acceptor [3]. This apparent discrepancy between the activity and absence of crystal cracking prompted us to carry out a more thorough chemical and biochemical study of this analogue and of model compounds. We report here the results of our chemical and biochemical investigations and the structure determination at 0.37-nm resolution of the orthorhombic binary complex alcohol dehydrogenase-m5NAD+. A study of m5NAD+ with lactate dehydrogenase has been reported recently [4]. Enzymes. Alcohol dehydrogenase (EC 1.1.1.1); lactate dehydrogenase (EC 1 .I .1.27); NAD(P)+ glycohydrolase (EC 3.2.2.6); glyceraldehyde-3-phosphate dehydrogenase (EC 1.2.1.12). MATERIALS AND METHODS 5- MODEL COMPOUNDS3-Cyano-5-methylpyridine (1). A suspension of 5-methylnicotinamide (3 g) in phosphorous oxychloride ( 5 ml) was heated under reflux for 45 min. After cooling, the excess of reagent was removed under reduced pressure and the mixture poured into a concentrated sodium carbonate solution and extracted thrice with chloroform. The extract was dried over sodium sulfate. Removal of the solvent under reduced pressure gave a solid which was sublimed (65°C under 1.33 Pa). The 3-cyano-5-methyl pyridine (I) was obtained as white crystals (1.93 g, yield 74%): m.p. 82-84°C (cf. 83-84°C [6]). ' H NMR (C'HCI,): 6 = 2.43 ppm (3H), 7.81 ppm (IH) and 8.69 p...

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“…Two polypeptide chains of the horse liver enzyme, E and S (ethanol-active and steroid-active, respectively), combine randomly to form the homodimeric isoenzymes, EE and SS (with EE apparently the most abundant form), as well as the heterodimeric form, ES (2,3). The human liver ADH isoenzyme pattern is more complex.…”

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confidence: 99%

Human alcohol dehydrogenase: structural differences between the beta and gamma subunits suggest parallel duplications in isoenzyme evolution and predominant expression of separate gene descendants in livers of different mammals.

Bühler¹,

Hempel²,

Kaiser³

et al. 1984

Proc. Natl. Acad. Sci. U.S.A.

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ABSTRACTlibman alcohol dehydrogenase (ADH; alcohol:NAD+ oxidoreductase, EC 1.1.1.1) occurs in multiple forms, which exhibit distinct electrophoretic mobilities and enzymatic properties. The homogeneous isoenzymes I3P, and y1yi were isolated from livers of Caucasians with "tyrpical" ADH phenotype by double ternary complex affinity chromatography and ion exchange chromatography. The differences between the PI and Vi subunits were determined by structural analysis of all tryptic peptides from the carboxymethylated proteins. The human f3I and Vi chains differ at 21 of the 373 positions (5.6%). Ten tryptic peptides account for the differences. All residue substitutions are compatible with one-base mutations and result in largely unaltered properties, but five lead to charge differences. Sixteen substitutions are at positions corresponding to the catalytic domain of the well-known horse enzyme; five correspond to the coepzyme-binding domain. Substitutions adjacent to important regions may correlate with differences in coenzyme binding, substrate specificities, and active-site relationships. The residue replacements between the PI and yV subunits of human ADH are not identical to the known substitutions between ethanol-active (E) and steroid-active (S) subunits of horse ADH. Thus, the duplication leading to human pi and yi subunits is separate and different from that leading to equine E and S subunits. Both duplications are likely to have occurred after the ancestral separation of human and equine ADH. Of the 21 residues that are different between P1I/ yi, 13 in y'but only 6 in flu are identical to those of the horse E chain. This suggests a closer relationship between yV and E, although .81 in mah and E in the horse are the subunits recovered in highest yield from liver ADH preparations. Consequently, in these two mammalian species, relative activities of genes for an isoenzyme family appear to be different.Human and horse alcohol dehydrogenases (ADH; alcohol:

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Subunit Composition of horse liver alcohol dehydrogenase isoenzymes

Cited by 43 publications

References 12 publications

Preparation and Properties of 3-Halopyridine-adenine Dinucleotides, NAD + Analogues and of Model Compounds

Preparation and Properties of 3-Halopyridine-adenine Dinucleotides, NAD + Analogues and of Model Compounds

5-Methylnicotinamide-adenine Dinucleotide. Kinetic Investigation with Major and Minor Isoenzymes of Liver Alcohol Dehydrogenase and Structural Determination of Its Binary Complex with Alcohol Dehydrogenase

Human alcohol dehydrogenase: structural differences between the beta and gamma subunits suggest parallel duplications in isoenzyme evolution and predominant expression of separate gene descendants in livers of different mammals.

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