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Biomedical subjects

G C Roberts

Publications and source records attributed to G C Roberts.

At least 145 records · Page 8Linked to original sources
PubMed1983

Use of transferred nuclear Overhauser effect measurements to compare binding of coenzyme analogues to dihydrofolate reductase.

Transferred nuclear Overhauser effect measurements have been made on complexes of NADP+ and thioNADP+ with Lactobacillus casei dihydrofolate reductase to provide information about the glycosidic bond conformations in these complexes. Both NADP+ and thioNADP+ are shown to have very similar anti conformations about their adenine glycosidic bonds when bound to the enzyme. However, their nicotinamide glycosidic bond conformations are very different: while NADP+ binds in an exclusively anti conformation, thioNADP+ binds with a distribution of syn/anti conformations very similar to that observed in nicotinamide mononucleotides in free solution (approximately 50:50). Thus for thioNADP+, binding to the enzyme does not significantly perturb the potential function for rotation about the nicotinamide glycosidic bond. Earlier NMR studies [Hyde, E. I., Birdsall, B., Roberts, G. C. K., Feeney, J., & Burgen, A. S. V. (1980) Biochemistry 19, 3738] had indicated that large downfield 1H shifts of the nicotinamide ring protons (0.61-1.36 ppm) are detected on binding NADP+ while only very small shifts (less than 0.1 ppm) are observed in complexes with thioNADP+. The chemical shift and conformational findings are best explained if the thionicotinamide ring extends into solution making essentially no contacts with the enzyme.

Binding Sites↗
PubMed1983

Mobility and active-site coupling in 2-oxo acid dehydrogenase complexes.

The 2-oxo acid dehydrogenase complexes consist of multiple copies of each of three enzymes, 2-oxo acid decarboxylase (E1), lipoate acetyltransferase (E2) and lipoamide dehydrogenase (E3), which catalyse successive steps in the overall reaction. The complexes are based on a structural core made up of the E2 chains, which also contain lipoic acid residues covalently attached to lysine residues. These lipoic acid residues are involved in transferring the substrate between the different active sites. A combination of limited proteolysis and 1H NMR experiments has shown that the E2 component has an unusual structure, having a substantial segment of polypeptide chain in the form of a highly flexible random coil. This flexibility allows the lipoyl-lysine residues to move rapidly over considerable distances, and provides a mechanism for the system of active-site coupling observed in these complexes.

Binding Sites↗
PubMed1983

Occupational exposure in medicine--a review of radiation doses to hospital staff in north-west England.

The personal monitoring service operated by the Regional Physics Department at the Christie Hospital and Holt Radium Institute, Manchester, monitors staff involved with the uses of ionising radiations at all hospitals and clinics administered by the North Western Region Health Authority in England. Monitoring results relating principally to exposure during 1981 have been collated and examined. The analysis indicates that the doses received by staff are for the most part very low and provide little reason for concern. The only area of work in which worthwhile and cost-effective dose reductions could probably be achieved is that involving the use of pre-loaded applicators in gynaecological intra-cavitary therapy. Some relatively high staff exposures result from the use of this technique, and very significant reductions in these doses are confidently expected from a programme which has now commenced for the increasing use of remotely-controlled after-loading equipment housed in shielded treatment rooms.

Environmental Exposure↗
PubMed1983

Ab initio molecular orbital calculations of electron distribution in tetramethylammonium ion.

Ab initio molecular orbital calculations of electron distribution in tetramethylammonium ion and its uncharged isoelectronic analogue, neopentane, have been carried out. Comparison of the two compounds permits a detailed description of the delocalization of the positive charge of tetramethylammonium ion. The van der Waals surface of this ion is found to be characterized by "patches" of positive charge associated with the methyl groups, interspersed with essentially neutral regions. The consequences of this nonspherical charge distribution for interaction with anions have been explored by calculations of the interaction energy of fluoride ion with tetramethylammonium ion and neopentane in several mutual orientations. The lowest-energy orientation is found to be one in which the anion approaches a "face" of the tetrahedral cation (opposite to a C--N bond direction). The origins of this preference and the electron redistribution produced by the interaction with fluoride are discussed. The tetramethylammonium ion is clearly not a featureless positively charged sphere but will have appreciable geometrical specificity in its interaction with a presumed anionic group on the acetylcholine receptor.

Electrons↗
PubMed1982

A 31P-nmr study of the intact liver fluke Fasciola hepatica.

31P-NMR techniques offer a useful method of studying changes in the metabolism of intact parasitic worms. The liver flukes, Fasciola hepatica, provide good quality 31P high resolution NMR spectra for at least 6 h under anaerobic conditions. The levels of ATP remain constant throughout this period. There is no signal for phosphocreatine or phosphoarginine. In contrast to the findings in mammalian tissues, there is a distinct peak for the terminal phosphate of ADP. A number of signals are observed in the phosphodiester region of the spectrum the largest of which is identified as L-alpha-glycerophosphoryl choline. Serotonin (5-hydroxytryptamine) causes an appreciable increase in the levels of sugar phosphates when the flukes are incubated in the absence of glucose. The addition of glucose also causes a marked increase in the signals for the hexose phosphate.

Adenosine Triphosphate↗
PubMed1982

Hydrogen-1, carbon-13, and phosphorus-31 nuclear magnetic resonance studies of the dihydrofolate reductase-nicotinamide adenine dinucleotide phosphate-folate complex: characterization of three coexisting conformational states.

The Lactobacillus casei dihydrofolate reductase-folate-NADP+ complex is shown by 1H and 13C NMR to exist in three interconverting conformational states, I, IIa, and IIb. The proportions of the three states, as estimated from the intensities of the three separate 13C resonances observed in the complex containing [3-carboxamido-13C]NADP+, are pH dependent. State I predominates at low pH and states IIa and IIb predominate at high pH; the ratio IIa:IIb is pH independent. The pH dependence of the interconversion of states I and IIa + IIb can be explained by a model in which a group on the enzyme has a pK of less than 5 in state IIa + IIb and greater than 7 in state I. 1H, 13C, and 31P NMR has been used to characterize the structural differences between the three states of the complex. As judged by the 1H and 13C chemical shifts of the bound coenzyme, states I and IIa are similar to one another but quite different from state IIb. This difference appears to be a localized one, since only the nicotinamide 2 and 4 protons, nicotinamide 3-carboxamide 13C, and pteridine 7 proton show differences in chemical shift between these states. These differences are, however, large--up to 1.4 ppm for 1H and 2 ppm for 13C. The remaining coenzyme protons, as well as the three 31P nuclei, are unaffected. Studies of the C2 proton resonances of the seven histidine residues show that the ionizable group responsible for the interconversion of states I and IIa + IIb is not a histidine (although two histidines show slight differences in environment between states IIa and IIb); the possible identity of this ionizable group and the nature of the conformational differences between the states are discussed.

Carbon Isotopes↗
PubMed1982

Cross-linking and 1H n.m.r. spectroscopy of the pyruvate dehydrogenase complex of Escherichia coli.

The pyruvate dehydrogenase complex of Escherichia coli was treated with o-phenylene bismaleimide in the presence of the substrate pyruvate, producing almost complete cross-linking of the lipoate acetyltransferase polypeptide chains as judged by sodium dodecyl sulphate/polyacrylamide-gel electrophoresis. This took place without effect on the catalytic activities of the other two component enzymes and with little evidence of cross-links being formed with other types of protein subunit. Limited proteolysis with trypsin indicated that the cross-links were largely confined to the lipoyl domains of the lipoate acetyltransferase component of the same enzyme particle. This intramolecular cross-linking had no effect on the very sharp resonances observed in the (1)H n.m.r. spectrum of the enzyme complex, which derive from regions of highly mobile polypeptide chain in the lipoyl domains. Comparison of the spin-spin relaxation times, T(2), with the measured linewidths supported the idea that the highly mobile region is best characterized as a random coil. Intensity measurements in spin-echo spectra showed that it comprises a significant proportion (probably not less than one-third) of a lipoyl domain and is thus much more than a small hinge region, but there was insufficient intensity in the resonances to account for the whole lipoyl domain. On the other hand, no evidence was found in the (1)H n.m.r. spectrum for a substantial structured region around the lipoyl-lysine residues that was free to move on the end of this highly flexible connection. If such a structured region were bound to other parts of the enzyme complex for a major part of its time, its resonances might be broadened sufficiently to evade detection by (1)H n.m.r. spectroscopy.

Cross-Linking Reagents↗
PubMed1982

Limited proteolysis and proton NMR spectroscopy of Bacillus stearothermophilus pyruvate dehydrogenase multienzyme complex.

The pyruvate dehydrogenase multienzyme complex of Bacillus stearothermophilus was treated with chymotrypsin at pH 7 and 0 degrees C. Loss of the overall catalytic activity lagged behind the rapid cleavage of the lipoate acetyltransferase polypeptide chains, whose apparent Mr fell from 57 000 to 45 000 as judged by sodium dodecylsulphate/polyacrylamide gel electrophoresis. The inactive chymotrypsin-treated enzyme had lost the lipoic-acid-containing regions of the lipoate acetyltransferase chains, yet remained a highly assembled structure. Treatment of this chymotryptic core complex with trypsin at pH 7.0 and 0 degrees C caused a further shortening of the lipoate acetyltransferase polypeptide chains to an apparent Mr of 28 000 and was accompanied by disassembly of the complex. The lipoic-acid-containing regions are therefore likely to be physically exposed in the intact complex, protruding from the structural core formed by the lipoate acetyltransferase component between the subunits of the other component enzymes. Proton nuclear magnetic resonance spectroscopy demonstrated that the enzyme complex contains large regions of polypeptide chain with remarkable intramolecular mobility, most of which were retained after excision of the lipoic-acid-containing regions with chymotrypsin. It is likely that the highly mobile regions are in the lipoate acetyltransferase component and facilitate movement of the lipoic acid residues. Such polypeptide chain mobility provides the molecular basis of a novel system of active-site coupling in the 2-oxo acid dehydrogenase multienzyme complexes.

Bacterial Proteins↗
PubMed1981

Negative cooperativity between folinic acid and coenzyme in their binding to Lactobacillus casei dihydrofolate reductase.

The binding of folinic acid (5-formyl-5,6,7,8-tetrahydrofolate) to Lactobacillus casei dihydrofolate reductase has been measured. The natural 6S, alpha S diastereoisomer has a binding constant of 1.3 (+/- 0.6) X 10(8) M-1 at pH 6.0, 25 degrees C; the 6R, alpha S diastereoisomer binds approximately 10(4)-fold more weakly. The natural diastereoisomer of folinic acid binds negatively cooperatively with the coenzymes NADP+ and NADPH, binding 3 times more weakly in the presence of NADP+ and 600 times more weakly in the presence of NADPH than to the enzyme alone. Negative cooperativity has been unequivocally distinguished from competition by measurements of coenzyme binding as a function of folinic acid concentration, of the effects of folinic acid on the 1H and 31P chemical shifts of the bound coenzyme, and of the effects of folinic acid on the coenzyme dissociation rate constant. The latter experiments also give evidence for the coexistence of two slowly interconverting conformational forms of the ternary enzyme-coenzyme-folinic acid complex. Small changes in structure of the oxidized coenzymes have substantial effects on the cooperativity with folinic acid, with the thionicotinamide analogue showing positive rather than negative cooperativity. The changes in environment of the bound coenzyme produced by folinic acid, as revealed by 1H and 31P NMR, demonstrate clearly that the negative cooperativity shown by NADP+ and NADPH, respectively, arises by two structurally distinct mechanisms.

Calorimetry↗
PubMed1981

Limited proteolysis and proton n.m.r. spectroscopy of the 2-oxoglutarate dehydrogenase multienzyme complex of Escherichia coli.

The 2-oxoglutarate dehydrogenase multienzyme complex of Escherichia coli was treated with trypsin at pH 7.0 at 0 degrees C. Loss of the overall catalytic activity was accompanied by rapid cleavage of the lipoate succinyltransferase polypeptide chains, this apparent Mr falling from 50 000 to 36 000 as judged by sodium dodecyl sulphate/polyacrylamide-gel electrophoresis. A slower shortening of the 2-oxoglutarate decarboxylase chains was also observed, whereas the lipoamide dehydrogenase chains were unaffected. The inactive trypsin-treated enzyme had lost the lipoic acid-containing regions of the lipoate succinyltransferase polypeptide chains, yet remained a highly assembled structure, as judged by gel filtration and electron microscopy. The lipoic acid-containing regions are therefore likely to be physically exposed in the complex, protruding from the structural core formed by the lipoate succinyltransferase component between the subunits of the other component enzymes. Proton nuclear magnetic resonance spectroscopy of the 2-oxoglutarate dehydrogenase complex revealed the existence of substantial regions of polypeptide chain with remarkable intramolecular mobility, most of which were retained after removal of the lipoic acid-containing regions by treatment of the complex with trypsin. By analogy with the comparably mobile regions of the pyruvate dehydrogenase complex of E. coli, it is likely that the highly mobile regions of polypeptide chain in the 2-oxoglutarate complex are in the lipoate succinyltransferase component and encompass the lipoyl-lysine residues. It is clear, however, that the mobility of this polypeptide chain is not restricted to the immediate vicinity of these residues.

Electrophoresis, Polyacrylamide Gel↗