Diversity of Vertebrate Class I Alcohol Dehydrogenase

Abstract

Class I alcohol dehydrogenase has been characterized from ostrich liver in order to evaluate enzyme variability between two independent lines, mammalian forms of class I alcohol dehydrogenase as a group, and a sufficient number of the enzyme from the most recent animal class (Aves, birds) as another. Between the two enzyme groups, patterns are consistent and mutually similar. This indicates conserved metabolic and catalytic properties of class I alcohol dehydrogenase, suggesting its metabolic role to be distinct, in spite of its protein variability. The new structure has a microheterogeneity (position 112, Arg/Cys) in a variable Zn-binding loop. In addition, it also establishes further native variants at active-site positions, including one thus far unique residue at the inner part of the substrate-binding pocket (Ala141), and a replacement at position 271 (giving His271), which is also the site of a human alcohol dehydrogenase y,Iy2 isozyme variability. The data correlate with functional differences in catalytic properties, the ostrich enzyme having a comparatively high K , for ethanol (5.9 mM at pH lo), and emphasize the importance of single positions in substrate and coenzyme binding, paralleling isozyme variability with protein variability within the class I enzymes.

The vertebrate alcohol dehydrogenase system: variable class II type form elucidates separate stages of enzymogenesis.

“…This dating has been deduced from estimates of molecular changes (10), the apparent absence of class I in lower life forms (4), and the MATERIALS AND METHODS Protein Purification. Alcohol dehydrogenase from ostrich liver, obtained from Kolm'arden Zoological Park, Sweden, was purified by ion-exchange chromatography on DEAESepharose, affinity chromatography on AMP-Sepharose, and exclusion chromatography on Sephadex G-100, essentially as described for the class I enzyme from this source (12). The ethanol class I alcohol dehydrogenase of this liver binds to DEAE-Sepharose (12), but, unexpectedly, we found that much ethanol dehydrogenase activity did not bind.…”

“…Alcohol dehydrogenase from ostrich liver, obtained from Kolm'arden Zoological Park, Sweden, was purified by ion-exchange chromatography on DEAESepharose, affinity chromatography on AMP-Sepharose, and exclusion chromatography on Sephadex G-100, essentially as described for the class I enzyme from this source (12). The ethanol class I alcohol dehydrogenase of this liver binds to DEAE-Sepharose (12), but, unexpectedly, we found that much ethanol dehydrogenase activity did not bind. We therefore applied the flow-through fraction to AMP-Sepharose for purification.…”

“…Alcohol dehydrogenase purification from liver usually involves a DEAE ion-exchange chromatography step, an affinity step, and an exclusion chromatography step (4,10,12,18). By using this protocol to purify alcohol dehydrogenases from ostrich liver, we noticed that recovery of total alcohol dehydrogenase activity was low at the intermediate AMP-Sepharose step (37%) after completion of the gradient of NADI from 0-10 mM [in 50 mM Tris HCl (pH 8.0)].…”

Alcoholytic deblocking of N‐terminally acetylated peptides and proteins for sequence analysis

“…The polypeptides employed were obtained from natural sources [6][7][8][9][10][11] or prepared synthetically [15]. Samples for deacetylation were either recovered in solution from column separations and lyophilized in glass tubes, directly spotted onto sequencer filters, or electroblotted to sequencer filters after SDS-polyacrylamide gel separation [16].…”

Class 2 aldehyde dehydrogenase. Characterization of the hamster enzyme, sensitive to daidzin and conserved within the family of multiple forms

“…The protein, purified as described [11], was dissolved in 6 M guanidinium chloride, 0.4 M Tris, pH 8.15, 2 mM EDTA, and carboxymethylated by treatment with neutralized iodo [2][3][4][5][6][7][8][9][10][11][12][13][14] C]acetate after reduction with dithiothreitol [12]. The carboxymethylated protein was cleaved in separate batches with Lys-C, Glu-C, and Asp-N specific proteases, trypsin, and chymotrypsin at protease:substrate ratios of 1:10-1:150 for 4-20 h at 37°C in 0.1 M ammonium bicarbonate, pH 8.1, with up to 2.2 M urea for solubilization.…”