Limited Proteolysis and a Reactive Cysteine Residue Define Accessible Regions in the Native Conformation of the Adenovirus Hexon Protein

Jörnvall, Hans; Philipson, Lennart

doi:10.1111/j.1432-1033.1980.tb04421.x

Cited by 103 publications

(23 citation statements)

References 18 publications

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“…Liquid phase sequencer degradataions were performed in a Beckman 890D sequencer with a 0.1 M Quadrol peptide program and with glycine-precycled polybrene as carrier [22]. Phenylthiohydantoin (PTH) derivatives were analyzed by RP-HPLC [23], supplemented at the positions corresponding to the exchanged residue by amino acid analysis after back hydrolysis in 6 M HCI, containing 0.1% SnClz, for independent residue confirmation.…”

Section: Structural Analysismentioning

confidence: 99%

Atypical human liver alcohol dehydrogenase: the β₂‐Bern subunit has an amino acid exchange that is identical to the one in the β₂‐Oriental chain

et al. 1984

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Section: Structural Analysismentioning

confidence: 99%

Atypical human liver alcohol dehydrogenase: the β₂‐Bern subunit has an amino acid exchange that is identical to the one in the β₂‐Oriental chain

et al. 1984

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“…In all cases, degradations were carried out in the presence of glycine-precycled polybrene with a 0.1 M quadrol peptide program [9]. Phenylthiohydantoin derivatives were identified by high-performance liquid chromatography [lo] using a Hewlett-Packard 1084B instrument, supplemented where applicable with thin-layer chromatography [Ill and with 14Canalysis in a scintillation counter.…”

Section: Structural Unalysismentioning

confidence: 99%

Sorbitol dehydrogenase

Jeffery

Cederlund

Jörnvall

1984

European Journal of Biochemistry

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The first primary structure for a sorbitol dehydrogenase has been determined by analysis of the tetrameric enzyme from sheep liver. The ['4C]carboxymethylated protein was cleaved with CNBr and proteolytic enzymes. Peptides were purified by several methods, often utilizing exclusion chromatography for pre-fractionation and reverse-phase high-performance liquid chromatography for final purification. Different methods of sequence analysis complemented each other, mainly the manual dimethylaminoazobenzene isothiocyanate method and the use of liquidphase sequencer degradations.All eight major CNBr fragments were purified and form the basis of the work. Three minor CNBr fragments derived from an acid cleavage and from a partly resistant Met-Thr bond were also obtained, as well as evidence for a contaminating homologous polypeptide. Most of the tryptic peptides were purified, including all with methionine residues, thus overlapping the CNBr fragments. Combined, all data permit the deduction of a 354-residue amino acid sequence for the polypeptide chain of sorbitol dehydrogenase. The N terminus is acyl-blocked, the C terminus is formed by a proline residue, tryptophan is the least common residue (two, at positions 50 and 301) and there are 10 cysteine residues, including the residue previously shown to be especially reactive (at postion 43). Similarities to 'long' alcohol dehydrogenases have functional implications.Liver sorbitol dehydrogenase was recently found to be similar to zinc-containing alcohol dehydrogenases [I], constituting an apparent structural intermediate [2] between the distantly related alcohol dehydrogenases from yeast and horse liver [3,4]. The results suggested wide and parallel evolutionary relationships between groups of dehydrogenases [5] and emphasized relationships in a metabolic pathway from glucose [6]. The complete primary structure of a sorbitol dehydrogenase is necessary for further correlations.In the present work, the amino acid sequence of the entire protein chain of sheep liver sorbitol dehydrogenase was determined. The work was centered on purification and analysis of all CNBr fragments, which were overlapped by identification of the methionine-containing tryptic peptides. Throughout, [14C]carboxymethylation of cysteine residues was used to obtain suitable markers in several peptides of all digests. A 354-residue primary structure for the protein chain was deduced. Evidence was obtained for the occurrence of a minor contaminating component which appeared distantly homologous to sorbitol dehydrogenase in one region. Otherwise the preparations are homogeneous, suggesting all subunits in the tetramer to be identical. Results to support the amino acid sequence are given in this study, together with aspects of methodological interest. Further correlations in a protein family of zinc-containing alcohol/polyol dehydrogenases are given in an accompanying paper [7].

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“…Liquidphase sequencer degradations were performed in a Beckman 890D sequencer as well as in a model 890C instrument fitted with a cold trap, new valves, and a conversion cell [34]. A 0.1 M quadrol peptide program was used for degradations in the presence of glycine-precycled polybrene [35] and phenylthiohydantoins were identified by HPLC [36].…”

Section: Sequence Analysismentioning

confidence: 99%

Aldehyde dehydrogenase from human liver

Hempel

Bahr‐Lindström

Jörnvall

1984

European Journal of Biochemistry

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166

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Analysis of CNBr fragments and other peptides from human liver cytoplasmic aldehyde dehydrogenase enabled determination of the complete primary structure of this protein. The monomer has an acylated amino terminus and is composed of 500 amino acid residues, including I1 cysteine residues. N o evidence of any microheterogeneity was obtained, supporting the concept that the enzyme is a homotetramer. The disulfiram-sensitive thiol in the protein, earlier identified through its reaction with iodoacetamide, is contributed by a cysteine residue at position 302, while the cysteine which in horse liver mitochondrial aldehyde dehydrogenase is reactive with coenzyme analogs appears to correspond to either Cys-455 or Cys-463. Analysis of glycine distribution and prediction of secondary structures to localize PEP regions typical for coenzyme-binding are not fully unambiguous, but suggest a short region around position 245 as a likely segment for this function. In this region, sequence similarities to parts of a bacterial aspartate-P-semialdehyde dehydrogenase and a mammalian alcohol dehydrogenase were noted. Otherwise, no extensive similarities were detected in comparisons with characterized mammalian enzymes of similar activity or subunit size as aldehyde dehydrogenase (glyceraldehyde-3-phosphate dehydrogenase and glutamate dehydrogenase, respectively).Aldehyde dehydrogenases have a wide substrate specificity and participate in ethanol metabolism through clearance of acetaldehyde. In human liver, two major isoenzymes of aldehyde dehydrogenase are present and have been ascribed to the cytoplasm and mitochondria, as in the livers of other mammalian species [ 1 -51. Structurally, the isoenzymes behave like different proteins in the sense that hybrid molecules are not (but see [6]) usually found [7]. Furthermore, a comparison of short segments from horse cytoplasmic and mitochondrial aldehyde dehydrogenases indicated a high extent of substitutions (about 50%) between the two forms [8]. Immunologically, however, they appear to be homologous [5,9] and the two forms are also similar in many enzymic properties. One notable exception is the much greater sensitivity of the cytoplasmic isoenzyme to inactivation by disulfiram [I, 5, 10, 111. This compound is also used clinically for alcohol aversion therapy, acting through the effects of acetaldehyde accumulation [12].Only recently have detailed molecular aspects of aldehyde dehydrogenase been studied. In the human cytoplasmic isoenzyme, a cysteine-containing segment has been implicated in the reaction with disulfiram because reaction of one of its cysteine residues with iodoacetamide is blocked by disulfiram [I 31. Another segment from horse mitochondrial aldehyde dehydrogenase, also containing a reactive cysteine residue, has been implicated in NAD(H) binding from its reaction with a coenzyme analog [14]. Although not finally proven, the possi- bility of a position close to the active site for one of these cysteine residues appears likely [I 31; this would be compatible with the ...

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Limited Proteolysis and a Reactive Cysteine Residue Define Accessible Regions in the Native Conformation of the Adenovirus Hexon Protein

Cited by 103 publications

References 18 publications

Atypical human liver alcohol dehydrogenase: the β₂‐Bern subunit has an amino acid exchange that is identical to the one in the β₂‐Oriental chain

Atypical human liver alcohol dehydrogenase: the β₂‐Bern subunit has an amino acid exchange that is identical to the one in the β₂‐Oriental chain

Sorbitol dehydrogenase

Aldehyde dehydrogenase from human liver

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Limited Proteolysis and a Reactive Cysteine Residue Define Accessible Regions in the Native Conformation of the Adenovirus Hexon Protein

Cited by 103 publications

References 18 publications

Atypical human liver alcohol dehydrogenase: the β2‐Bern subunit has an amino acid exchange that is identical to the one in the β2‐Oriental chain

Atypical human liver alcohol dehydrogenase: the β2‐Bern subunit has an amino acid exchange that is identical to the one in the β2‐Oriental chain

Sorbitol dehydrogenase

Aldehyde dehydrogenase from human liver

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Atypical human liver alcohol dehydrogenase: the β₂‐Bern subunit has an amino acid exchange that is identical to the one in the β₂‐Oriental chain

Atypical human liver alcohol dehydrogenase: the β₂‐Bern subunit has an amino acid exchange that is identical to the one in the β₂‐Oriental chain