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G C Roberts

Publications and source records attributed to G C Roberts.

At least 181 records · Page 10Linked to original sources
PubMed1978

Ultraviolet difference-spectroscopic studies of substrate and inhibitor binding to Lactobacillus casei dihydrofolate reductase.

The u.v. difference spectra generated when methotrexate, trimethoprim or folate bind to Lactobacillus casei dihydrofolate reductase were analysed. The difference spectrum producted by methotrexate binding is shown to consist of three components: (a) one closely resembling that observed on protonation of methotrexate, reflecting an increased degree of protonation on binding; (b) a pH-independent contribution corresponding to a 40 nm shift to longer wavelengths of a single absorption band of methotrexate: (c) a component arising from perturbation of tryptophan residue(s) of the enzyme. Quantitative analysis of the pH-dependence of component (a) shows that pK of methotrexate is increased from 5.35 to 8.55 (+/-0.10) on binding. In contrast, folate is not protonated when bound to the enzyme at neutral pH. At pH7.5, where methotrexate is bound 2000 times more tightly than folate, one-third of the difference in binding energy between the two compounds arises from the difference in chaarge stage. A similar analysis of the difference spectra generated on trimethoprim binding demonstrates that this compound, too, shows an increase in pK on binding but only from 7.22 to 7.90 (+/-0.10), suggesting that its 2,4-diaminopyrimidine ring does not bind to the enzyme in precisely the same way as the corresponding moiety of methotrexate.

Folic Acid↗
PubMed1977

Origins of specificity in the binding of small molecules to dihydrofolate reductase.

Dihydrofolate reductase is the target for the therapeutically important 'anti-folate' drugs such as methotrexate and trimethoprim. Methotrexate is a powerful inhibitor of the enzyme, binding up to 10,000 times more tightly than the structurally similar substrate, folate. Two contributions to this striking difference in affinity have been identified: the two ligands bind in different charge states, and there are conformational differences between the two complexes. The origins of the tight binding of methotrexate have been explored further by studying the binding of 2,4-diaminopyrimidine and p-aminobenzoyl-L-glutamate, which may be considered as 'fragments' of methotrexate. These two compounds bind simultaneously but also cooperatively, the binding of one 'fragment' leading to a 50-fold increase in the affinity for the other. Studies of structural analogues of these fragments show that the specificity as well as the strength of binding can be altered by the presence of the other 'fragment'; both positive and negative cooperativity are observed. The relation of these observations to methotrexate binding, and the notion of intramolecular cooperativity in ligand binding are discussed.

Hydrogen-Ion Concentration↗
PubMed1976

Latent homology of murine lymphoid antigens revealed through the complement mediated absorption of xenogeneic antiserum.

Xenoantiserum against C57BL/6 mouse spleen cells was produced by immunization of rabbits. Such antiserum displayed cytotoxic activity for several strains of mice. Standard "in vitro" absorption removed species specific antibody revealing antibody directed against the C57BL/6 strain. When the xenoantiserum was absorbed with DBA/2 spleen cells in the presence of complement, the binding of anti-C57BL/6 was increased. Such data suggest that xenoantibodies directed against spleen cells possess a broader capacity to react with cross-strain lymphoid antigens than previously described.

Animals↗
PubMed1976

Large-scale purification and characterization of dihydrofolate reductase from a methotrexate-resistant strain of Lactobacillus casei.

Dihydrofolate reductase has been purified from a methotrexate-resistant strain of Lactobacillus casei NCB 6375. By careful attention to growth conditions, up to 2.5 g of enzyme is obtained from a 400 litre culture. The purification procedure, involving poly-ethyleneimine treatment, DEAE-cellulose chromatography and affinity chromatography on methotrexate-aminohexyl-Sepharose, operates on the gram scale, with overall yields of 50-60%. Elution of the affinity column by reverse (upward) flow was used, as it led to recovery of the enzyme in a much smaller volume. The enzyme obtained appears to be more than 98% pure, as judged by gel electrophoresis, isoelectric focusing, and gel filtration. It has a mol.wt. of approx. 17900 and a turnover number of 4s-1 (50mM-triethanolamine/400mM-KCl, pH 7.2, 25 degrees C) with dihydrofolate and NADPH as substrates. The turnover number for folate is 0.02s-1. Michaelis constants for a variety of substrates have been measured by using a new fluorimetric assay (0.36 muM-dihydrofolate; 0.78 muM-NADPH), and binding constants determined by using the quenching of protein fluorescence (dihydrofolate, 2.25 X 10(6)M-1; NADPH, greater than 10(8)M-1). The pH/activity profile shows a single maximum at pH 7.3; at this pH, marked activation by 0.5M-NaCl is observed.

Amino Acids↗