Structure and Function Relationships in Isoenzymes of Horse Liver Alcohol Dehydrogenase

Pietruszko, Regina; Ringold, Howard J.; Li, T.-K.; Vallée, Bert L.; Åkeson, Åke; Theorell, Hugo

doi:10.1038/221440a0

Cited by 65 publications

(25 citation statements)

References 15 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…Starch gel electrophoresis of crude liver extracts reveals two additional bands between isoenzyme III and IV which we are naming IVa and IVb. Similar electrophoretic patterns showing up to nine fractions have recently been described by Pietruszko et al [8].…”

Section: Resultssupporting

confidence: 64%

See 1 more Smart Citation

Subunit Composition of horse liver alcohol dehydrogenase isoenzymes

Lutstorf

Wartburg

1969

FEBS Letters

View full text Add to dashboard Cite

Section: Resultssupporting

confidence: 64%

“…However, in this species more than three enzymatically active fractions can be distinguished by electrophoresis and chromatography [6,7]. Starch gel electrophoresis reveals up to nine fractions [8,9]. The present communication reports on the subunit composition of these horse liver ADH fractions.…”

Section: Introductionmentioning

confidence: 99%

Subunit Composition of horse liver alcohol dehydrogenase isoenzymes

Lutstorf

Wartburg

1969

FEBS Letters

View full text Add to dashboard Cite

“…Dissociation/reassociation studies [34,35] of the isoenzymes of horse liver alcohol dehydrogenase also indicate a dimeric structure. Studies involving the determination of the complete amino acid sequence of the protein chain of the ethanol-active isoenzyme, as well as of the slightly different chain occurring in the other two homogeneous and hybrid groups of isoenzymes, are in progress [32,36] and it is hoped that these studies in conjunction with X-ray d8rac-tion analysis of the crystalline enzymes will lead to the elucidation of the mechanism of catalysis.…”

Section: Discussionmentioning

confidence: 99%

Horse Liver Alcohol Dehydrogenase

Jörnvall

Harris

1970

European Journal of Biochemistry

View full text Add to dashboard Cite

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.

show abstract

“…Pure isoenzyme 111 [16], i.e. enzyme molecules constituted of two identical ethanol-active subunits [17], was prepared either from crude liver according to [16] or from commercial preparations with a simplified procedure [18]. The enzyme activity was assayed in 0.1 M sodium phosphate pH 8.8, 1.9 mM NAD+, 7.4 mM semicarbazide .…”

Section: Materials a N D Methodsmentioning

confidence: 99%

“…1 represents data from several experiments, which were all performed on pure isoenzyme I11 (or EE) [16,17] in pseudo-first-order conditions. Inactivation rates increased with the inactivator concentrations, and a maximum rate was observed between 30-60 pM clac3PdAD+; for higher concentrations, the inactivation was biphasic, a first fast phase being added to the previous saturated one (see Fig.1).…”

Section: Enzyme Inactivation Under Binary Complex Formmentioning

confidence: 99%

Affinity Laeling of Horse‐Liver Alcohol Dehydrogenase by 3‐Chloroacetylpyridine – Adenine Dinucleotide

Foucaud¹,

Biellmann²

1981

European Journal of Biochemistry

View full text Add to dashboard Cite

3-Chloroacetyl-pyridine -adenine dinucleotide (clac3PdAD+) is a reactive NAD+ analogue with the highest possible conservation of the coenzyme structure on its pyridine moiety; it has been designed to alkylate an enzyme catalytic group from the active site. Its interactions with the horse liver alcohol dehydrogenase as well as the interaction of structurally related compounds with this enzyme have been investigated for their specificity and their selectivity. The following results have been obtained. (1) clac'PdAD' is active as coenzyme and its isosteric analogue 3-propionylpyridine-adenine dinucleotide (pp3PdAD+) does not display cooperative properties in its activity and binding with the horse liver alcohol dehydrogenase. (2) The horse liver alcohol dehydrogenase is irreversibly inactivated by clac3PdAD+ subsequently to the formation of a specific reversible binary complex, with a Kd of 11 3 KM; NAD+ protects the enzyme in a strictly competitive way, with a K d of 67 pM at pH 7.5, 22 "C. No inactivation occurs with 3-chloroacetylpyridine and/with 4-chloroacetylpyridineadenine dinucleotide (clac4PdADf). (3) The pH dependence of the rate of the enzyme inactivation by clac3PdAD+ reflects the variation with pH of the dissociation constant of the coenzyme analogue; this variation is similar to that of NAD'. (4) The formation of ternary complexes with ethanol and pyrazole increases the rate of enzyme inactivation, consistently with the enzyme mechanism. From this effect, their dissociation constant could be calculated and compared to literature data related to other ternary complexes. In contrast, iinidazole is shown to decrease the interaction between clac3PdAD+ and the horse liver alcohol dehydrogenase. (5) The inactivation of the enzyme is biphasic at high clac3PdADf concentration, and this biphasicity does not result from a heterogeneity of the preparation of the enzyme or coenzyme analogue. Similarities between this biphasicity and a substrate-induced biphasicity under certain conditions of inactivation of the enzyme are discussed. It is concluded that clac3PdAD+ is highly specific, and a valuable tool for structural and mechanistic studies of the horse liver alcohol dehydrogenase.

show abstract

Structure and Function Relationships in Isoenzymes of Horse Liver Alcohol Dehydrogenase

Cited by 65 publications

References 15 publications

Subunit Composition of horse liver alcohol dehydrogenase isoenzymes

Subunit Composition of horse liver alcohol dehydrogenase isoenzymes

Horse Liver Alcohol Dehydrogenase

Affinity Laeling of Horse‐Liver Alcohol Dehydrogenase by 3‐Chloroacetylpyridine – Adenine Dinucleotide

Contact Info

Product

Resources

About