Search PubMed⌕ Search

Biomedical subjects

J I Harris

Publications and source records attributed to J I Harris.

At least 37 records · Page 2Linked to original sources
PubMed1976

Iodination of glyceraldehyde 3-phosphate dehydrogenase from Bacillus stearothermophilus.

The reaction of iodine with glyceraldehyde 3-phosphate dehydrogenase from Bacillus stearothermophilus was investigated. The active-site thiol group of the cysteine residue homologous with cysteine-149 in the pig muscle enzyme was protected by reaction with tetrathionate. The apoenzyme was readily inhibited by KI3 solution at pH8, but the coenzyme, NAD+, protected the enzyme against inhibition and decreased the extent of iodination. At pH 9.5, ready inhibition of both apo- and holo-enzyme was observed. Tryptic peptides containing residues iodinated at pH 8 were isolated and characterized. One of the most reactive residues in both holo- and apo-enzymes was a tyrosine homologous with tyrosine-46 in the pig muscle enzyme, and this residue was iodinated without loss of enzymic activity. Other reactive tyrosine residues in the apoenzyme were in positions homologous with residues 178, 273, 283 and 311 in the pig muscle enzyme, but they were not readily iodinated in the holoenzyme. Histidine residues in both holo- and apo-enzymes were iodinated at pH 8 in sequence positions homologous with residues 50, 162 and 190 in the pig muscle enzyme. The inhibition of the enzyme was not correlated with the iodination of a particular residue. The results are discussed in relation to a three-dimensional model based on the structure of the lobster muscle enzyme and demonstrate that conformational changes affecting the reactivity of several tyrosine residues most probably occur on binding of the coenzyme.

Amino Acid Sequence↗
PubMed1976

Metal-replacement studies in Bacillus stearothermophilus aldolase and a comparison of the mechanisms of class I and class II aldolases.

A comparison of the product-inhibition patterns during cleavage of D-fructose 1,6-diphosphate by aldolases from yeast, rabbit muscle and Bacillus stearothermophilus shows an ordered reaction sequence for all three enzymes, with dihydroxyacetone phosphate the last-leaving product. Addition of Zn2+, Co2+, Fe2+, Mn2+ or Cd2+ ions to the inactive apo-(Bacillus stearothermophilus aldolase) restores activity to different extents, whereas Ni2+, Mg2+ or Cu2+ ions have no effect. The cleavage activity of this aldolase is not enhanced by added K+ ion. The effects of metal replacement on thermal stability, Km and Vmax. are given and the possible role of the metal is discussed in the light of these results.

Animals↗
PubMed1976

Succinylation of glyceraldehyde-3-phosphate dehydrogenase from Bacillus stearothermophilus. A reactive threonine residue in the apoenzyme.

1. Glyceraldehyde-3-phosphate dehydrogenase from bacillus stearothermophilus can be extensively succinylated in the presence of substrates and coenzyme without appreciable loss of activity. 2. The apoenzyme in the absence of substrates is rapidly inhibited by small amounts of succinic anhydride. NAD+, glyceraldehyde-3-phosphate and inorganic phosphate all afford protection from inhibition, and inhibition is slowly reversed in the presence of pyrophosphate at pH 8.5. 3. Kinetic and spectral studies have shown that the specific inhibition is associated with the succinylation of the aliphatic hydroxyl group of a serine or threonine residue. 4. The residue specifically succinylated has been identified as one of the two threonine residues, most probably Thr-150, adjacent to the activ-site cysteine residue in the primary structure. Its unusual reactivity is discussed in relation to the three-dimensional structure of the enzyme. 5. A second residue, a lysine homologous with Lys-212 in the pig muscle enzyme, can be succinylated in both holoenzyme and apoenzyme with no detectable effect upon the enzymic activity.

Amino Acid Sequence↗
PubMed1976

The subunits of rabbit-muscle phosphofructokinase. A search for sequence repetition.

Rabbit muscle phosphofructokinase uniformly carboxymethylated with iodo[2-14C]acetate consists of subunits with a molecular weight of 80 000 +/- 5000. The subunit polypeptide chain contains 16 and 52 residues respectively of cysteine and arginine and, contrary to previous results, peptide mapping experiments gave no indication that phosphofructokinase chains yield fewer than the expected numbers of cysteine and arginine containing peptides. To test further for the possible occurrence of repeat sequences within a single subunit chain, cysteine-containing peptides were isolated and sequenced from tryptic and thermolytic digests of s-[2-14C]carboxymethylated phosphofructokinase. In all, 15 different cysteine sequences (comprising a total of 104 residues) were identified, showing that not more than one of an expected 16 cysteine-containing sequences is repeated, and that the subunits of phosphofructokinase are of unique sequence along their entire length. The near quantitative isolation of several cysteine-containing peptides shows further that all subunits are of similar if not identical sequence.

Amino Acid Sequence↗
PubMed1975

The binding of nicotinamide-adenine dimucleotide to glyceraldehyde 3-phosphate dehydrogenase from Bacillus stearothermophilus.

The binding of NAD+ to glyceraldehyde 3-phosphate dehydrogenase (EC 1.2.1.12) from Bacillus stearothermophilus has been studied by measurement of protein fluorescence quenching. Slight negative co-operativity was observed in the binding of the third and fourth coenzyme molecules to the tetrameric enzyme. The first two coenzyme molecules were tightly bound. In this respect the enzyme resembles that from sturgeon muscle rather than that from yeast.

Apoenzymes↗
PubMed1975

Hybridization of glyceraldehyde-3-phosphate dehydrogenase.

1. Glyceraldehyde-3-phosphate dehydrogenases [EC 1.2.1.12] from rabbit, pig, lobster, yeast, E. coli, and B. stearothermophilus have been subjected to hybridization in 3M NaCL. 2. Suitable mixtures of electrophoretically distinct glyceraldehyde-3-phosphate dehydrogenases were found to give five-membered by electrophoresis on cellulose acetate. 3. The thermophile enzyme did not form hybrids with any of its mesophile counterparts, presumably because it does not dissociate under the conditions used. 4. Hybridization of pig enzyme with lobster enzyme that had been inactivated by selective carboxymethylation of Cystetramer containing only one active pig enzyme subunit. Individual subunits would thus appear to express their activity independently even within hybrid tetramers formed with subunits of another species. 5. The successful hybridization of glyceraldehyde-3-phosphate dehydrogenases from evolutionarily distant sources suggests that the tertiary and quaternary structures of the enzyme have been highly conserved.

Amino Acid Sequence↗
PubMed1974

Triose phosphate isomerase from the coelacanth. An approach to the rapid determination of an amino acid sequence with small amounts of material.

The preparation and purification of cyanogen bromide fragments from [(14)C]carboxymethylated coelacanth triose phosphate isomerase is presented. The automated sequencing of these fragments, the lysine-blocked tryptic peptides derived from them, and also of the intact protein, is described. Combination with results from manual sequence analysis has given the 247-residue amino acid sequence of coelacanth triose phosphate isomerase in 4 months, by using 100mg of enzyme. (Two small adjacent peptides were placed by homology with the rabbit enzyme.) Comparison of this sequence with that of the rabbit muscle enzyme shows that 207 (84%) of the residues are identical. This slow rate of evolutionary change (corresponding to two amino acid substitutions per 100 residues per 100 million years) is similar to that found for glyceraldehyde 3-phosphate dehydrogenase. The reliability of sequence information obtained by automated methods is discussed.

Amino Acid Sequence↗