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Biomedical subjects

E H Creaser

Publications and source records attributed to E H Creaser.

15 recordsLinked to original sources
PubMed1990

Protein engineering of alcohol dehydrogenases: effects of amino acid changes at positions 93 and 48 of yeast ADH1.

By protein engineering we have investigated changes to two amino acid residues (Trp93 and Ser48) in the substrate pocket of yeast alcohol dehydrogenase 1. Upon changing Thr48 to serine we produced an enzyme which has markedly greater activity towards aliphatic alcohols with chain length up to 8, together with a general increase in catalytic activity (V/K). Changes at position 93 were less pronounced, with the Phe enzyme being more active than the parent towards the range of alcohols but with the alanine enzyme showing very little difference from the wild-type. Enzymes with the double changes at 48 and 93 showed increased activity towards alcohols with 3-8 carbons but the increases were not additive over the single changes. The enzymes with changes at the two positions would metabolize both stereoisomers of 2-octanol whereas the parent ADH would attack only one of them. None of the engineered enzymes would attack cyclohexanol or aromatic alcohols. The results are in general agreement with the prediction that reducing the size of amino acids in the substrate pocket would enhance the ability to oxidize alcohols larger than ethanol.

Alcohol Dehydrogenase↗
PubMed1977

Peptide separations using fluorescence detection.

The formation of fluorophores by the action of o-phthalaldehyde with amino acids and peptides has provided a highly sensitive assay for these compounds. A relatively simple system for the analysis and separation of peptides, in the range 5 nmole to 10 micromole, normally derived from enzymic digestion of proteins, is described. The system comprises a gradient-generating device feeding volatile pyridine buffers via a pump to a column of cation-exchange resin. Eluate from the column is fed through a proportioning pump to a fluorocolorimeter, output from which is displayed on a recorder. For analytical runs the eluate is mixed with o-phthalaldehyde in borate buffer containing Brij 35 and 2-mercaptoethanol prior to its passage into the detector. For preparative work the eluate stream is split, one reacting with 0-phthalaldehyde, the other for collection. Results on the analysis and preparation of tryptic peptides derived from cytochrome c and Salmonella histidinol dehydrogenase are discussed.

Alcohol Oxidoreductases↗
PubMed1977

Purification and properties of histidinol dehydrogenases from psychrophilic, mesophilic and thermophilic bacilli.

As a first step in elucidating one molecular mechanism of adaptation to life at extreme temperatures, we purified and characterized the enzyme histidinol dehydrogenase (EC 1.1.1.23) from a number of bacilli whose growth temperatures range from 5 degrees t to 90 degrees C. The enzymes were purified by (NH4)2SO4 precipitation, ion-exchange chromatography on Sephadex, affinity chromatography on histamine- or histidine-Sepharose and preparative gradient gel electrophoresis. All had similar mol.wts. (29200), sedimentation coefficients (S20,w 2.56S), affinities for histidinol and NAD+ (Km = 48 micron and 0.2 mM respectively) and all had pH optima at 9.6. Marked differences were observed in stability with respect to temperature and the temperature at which the initial velocity for histidinol dehydrogenation was optimal. These optima range from 25 degrees C for the enzyme from the psychrophilic species through to 41 degrees C for the mesophiles to 85-92 degrees C for the extreme thermophiles. It is concluded that the ability of the enzymes to operate at their various optimum temperatures is an intrinsic property of their amino acid sequences.

Alcohol Oxidoreductases↗
PubMed1977

Improved chromatographic identification of coloured amino acid thiohydantoins.

A new N-terminal reagent for peptides and proteins, 4-N,N-dimethylamino-naphthylazobenzene-4'-isothiocyanate, is described which gives purple thiohydantoin derivatives; chromatographic separation of 24 amino acid thiohydantoins is reported. Such standard purple derivatives can be used as markers in the separation of the red 4-N,N-dimethylaminoazobenzene-4'-thiohydantoins. Conversely, standard red thiohydantoins can be used as markers in the separation of unknown purple amino acid thiohydantoins. In two-dimensional thin-layer chromatography, the precision of identifying the unknowns can be greatly improved by running markers with a colour different from the unknowns on the same side of the sheet.

Amino Acids↗
PubMed1976

A novel manual method for protein-sequence analysis.

A novel manual method for protein-sequence analysis is described. Three peptides, the hexapeptide (Leu-TRP-Met-Arg-Phe-Ala), insulin A chain and glucagon were used to test this technique. Peptides (1 or 2 nmol) were hydrolysed with acid and their qualitative amino acid compositions were confirmed by reacting with 4-NN-dimethylaminoazobenzene-4'-sulphonylchloride and 4-NN-dimethylaminoazobenzene 4'-isothiocyanate. Sequence determination of 20-200 nmol of peptide was then performed by the combined use of phenyl isothiocyanate and 4-NN-dimethylaminoazobenzene 4'-isothiocyanate, a new procedure that is analogous to the dansyl-Edman method with the replacement of dansyl chloride by 4-NN-dimethylaminoazobenzene 4'-isothiocyanate as the N-terminal residue determination reagent. On t.l.c. this new N-terminal reagent gave brightly coloured 4-NN-dimethylaminoazobenzene-4-thiohydantoins of amino acids and showed the following advantages: (1) the detection sensitivity is in the pmol range; (2) u.v. observation is not required; (3) there is no destruction of acid-labile amino acids; (4) two-dimensional t.l.c. separation is adequate to identify 24 amino acids, except leucine and isoleucine (this pair of amino acids can be resolved by using 4-NN-dimethylaminoazobenzene-4'-sulphonyl chloride); (5) the determination of a new N-terminal residue (from coupling to t.l.c. identification) takes only 3 h; (6) the colour difference beteen isothiocyanate, thiocarbamoyl and thiohydantoin derivatives facilitates the identifications.

Amino Acid Sequence↗
PubMed1976

4-NN-dimethylaminoazobenzene 4'-isothiocyanate, a new chromophoric reagent for protein sequence analysis.

4-NN-Dimethylaminoazobenzene 4'-isothiocyanate was synthesized for the purpose of improving the ease and sensitivity of peptide sequence analysis. The method of 4-NN-dimethylaminoazobenzene 4'-isothiocyanate synthesis, the preparation of 24 4-NN-dimethylaminoazobenzene-4'-thiohydantoins of amino acids and their t.l.c. separation are described. All the thiohydantoins, except those of leucine and isoleucine, could be satisfactorily separated by chromatography on a two-dimensional polyamide sheet. The sensitive azo group permits the detection of 4-NN-dimethylaminoazobenzene-4'-thiohydantoins of amino acids as red spots down to pmol amounts directly on the sheet. A simple sensitive method for sequencing dipeptides and the first two or three N-terminal amino acids of proteins is also reported. The colour change of the spots from purple to blue to red after being exposed to HCl vapour, corresponding to the chemical change from 4-NN-dimethylaminoazobenzene-4' isothiocyanate to the 4-NN-dimethylaminoazobenzene-4'-thiocarbamoyl amino acid derivative to the 4-NN-dimethylaminoazobenzene-4'-thiohydantoin amino acid derivative, reveals a very interesting and valuable feature of this reagent.

Amino Acid Sequence↗
PubMed1975

Analysis of an L-histidinol-utilizing mutant of Pseudomonas aeruginosa.

Transductional analysis was applied to the Pseudomonas aeruginosa mutant PAO14 (hnc-1). This mutant can utilize L-histidinol as sole source of carbon and nitrogen and has a 60-fold increased histidinol dehydrogenase (HDH) content (Dhawale, Creaser & Loper, 1972). Transductional analysis was carried out using 18 histidine-requiring mutants to see where the hnc-1 locus maps in relation to the structural genes of histidine biosynthesis. The hnc-1 marker cotransduced with group IV genes at 97 to 100 % and not at all with group I, which is known to be the structural gene for HDH. The data obtained in the studies of Km (histidinol) and Km (NAD), and the effect of pH and temperature on the HDH activity from PAO1 and PAO14 are in full agreement with the genetic data that the hnc-1 mutation is not in the structural gene for HDH. It is suggested that hnc-1 may be a mutation in a regulatory gene affecting HDH synthesis in PAO14 and may map close to his-IV whose function in histidine biosynthesis is not known.

Alcohol Oxidoreductases↗