Horse Liver Alcohol Dehydrogenase

Abstract

The ethanol-active isoenzyme of alcohol dehydrogenase from horse liver has been carboxymethylated with iodo[2-14C]acetate in 6 M guanidine hydrochloride. A study of the tryptic digest of the carboxymethylated enzyme has shown that it contains 14 unique sequences with [2-l4C]-carboxymethylcysteine, two with tryptophan, one .with C-terminal phenylalanine and one with a blocked N-terminal residue which represents the N-terminal sequence in the protein chain. This number of unique residues found in peptides is in each case half the number previously found by direct amino acid analysis (based on a molecular weight of 80000) of the intact enzyme. These results support the view that the native isoenzyme is a dimer consisting of two similar and probably identical polypeptide chains each containing about 370 amino acid residues. Failure to demonstrate the presence of more than one type of chain by chromatography of the carboxymethylated enzyme on DEAE-cellulose provides additional support for the dimer hypothesis. An alternative proposal [I31 that the enzyme is composed of two pairs of dissimilar chains each with a molecular weight of 20000 appears to be incompatible with thcse results.Alcohol dehydrogenase from horse liver is known to exist in the form of isoenzymes which possess different substrate specificities [l-31, and enzyme preparations consisting either of the purified ethanolactive component, or of mixtures of isoenzymes in which this form has been the predominant component, have been extensively studied. This isoenzyme has a molecular weight of about 80000 [4,5] and in its most active form contains 28 free sulphhydry1 (-SH) groups [6]. Inhibition of enzyme activity by iodoacetic acid has been shown to be due to a specific reaction with the SH groups of two cysteine residues, which, moreover, occur in the same sequence in the primary structure of the enzyme protein. At the same time it was established that the number of peptides produced by tryptic digestion was about half the number that would have been expected from its content of lysine and arginine residues. On the basis of these results it was suggested [7,8] that the active enzyme with a molecular weight of 80000 could consist of two similar and possibly identical polypeptide chains and, although no free N-terminal residues could be detected [9,5], the presence in the protein of two moles of C-terminal phenylalanine [ 5 ] , Unusual Abbreviations. CM-, carboxymethyl ; dansyl, dimethylaminonaphthalene 5-sulphonyl chloride.

Blocked α‐amino groups in peptides due to diketopiperazine formation

“…Enzymatic digestions (enzyme to substrate ratio of 1:100, w/w) were performed at 37°C for 4 hr in 0.1 M ammonium bicarbonate with TPCK-trypsin or a-chymotrypsin treated with TLCK [2]. Peptides were purified by exclusion chromatography on Sephadex G-50 fine (Pharmacia, Uppsala, Sweden), high-voltage paper electrophoresis and paper chromatography, as previously described [3]. Compositions were determined with a Beckman 120 B amino acid analyzer fitted with a high-sensitivity chart.…”

Horse Liver Alcohol Dehydrogenase

“…The letters CS, PS and TS refer to chymotryptic, peptic and tryptic peptides, respectively, which are described above and in a previous study [2]; MS refer to tryptic peptides of the maleylated Vol.14, No. 3,1970 and carboxymethylated isoenzyme and BlA, B2, B6 and B7 are cyanogen bromide fragments.…”

“…It separates from the radioactive peptide TS 1 : I (cf. [2]) after a single electrophoresis on paper a t pH 6.5. The recovery, electrophoretic mobility, amino acid composition and N-terminus of TS 1 : 2 are given in Table 3.…”

“…The primary structure around the cysteine residues of the main form within the most ethanol-active group has been determined [2]. It was then shown that this isoenzyme has 14 unique sequences around the cysteine residues and consists of two identical or very similar protein chains, each containing about 370 amino acid residues.…”

Kinetic Transients in the Reduction of Aldehydes Catalysed by Liver Alcohol Dehydrogenase

“…Transient-state kinetic data were analyzed in view of Eqn (l), describing the approach of the absorbance A at time t towards the final absorbance level A , in terms of two exponential transients with amplitudes A1 and A2 and time constants rl and r 2 , or in view of the single-exponential relationship obtained by putting A2 = 0 in Eqn (1).…”