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R M Clegg

Publications and source records attributed to R M Clegg.

48 records · Page 3Linked to original sources
PubMed1985

A comparison of the translational diffusion of a normal and a membrane-spanning lipid in L alpha phase 1-palmitoyl-2-oleoylphosphatidylcholine bilayers.

We have used the fluorescence recovery after photobleaching technique to study the translational diffusion, in L alpha phase multibilayers of 1-palmitoyl-2-oleoylphosphatidylcholine (POPC), of fluorescent derivatives of 1-palmitoyl-2-oleoylphosphatidylethanolamine (NBD-POPE) and a membrane-spanning phosphatidylethanolamine (NBD-MSPE). The latter derivative was prepared from a membrane-spanning glycerol-dialkyl-glycerol tetraether lipid isolated from the thermophilic and acidophilic archaebacterium Sulfolobus solfataricus. The translational diffusion was examined between about 15 degrees and 45 degrees C. It is shown that over this temperature range the translational diffusion coefficient for NBD-MSPE is approximately 2/3 that for NBD-POPE which spans only one monolayer of the bilayer. The result is interpreted in terms of existing models for translational diffusion in lipid membranes.

Archaea↗
PubMed1983

Dynamic evidence for an extended subsite structure of the ligand combining site on wheat germ agglutinin: temperature-jump relaxation with fluorescence detection.

Temperature-jump relaxation methods have been used to study the binding kinetics of fluorescent 4-methylumbelliferyl glycosides of N-acetyl-beta-D-glucosamine and its beta (1 leads to 4)-linked di- and trisaccharides with wheat germ agglutinin. The mono- and disaccharide derivatives yielded biexponential progress curves. The data are consistent with two simple mechanisms in which binding occurs to an extended combining site on the lectin, consisting of at least two different, mutually exclusive, binding subsites. For one model, the bound ligand must slide from one subsite to the other, and the other mechanism requires the dissociation of the bound ligand from the protein before it can combine to the other subsite. Binding of 4-methylumbelliferyl monosaccharide to nonequivalent sites is improbable. The underlying kinetic and equilibrium parameters were obtained for the proposed subsites. The binding kinetics of the 4-methylumbelliferyl trisaccharide derivative are more complicated and may result from ligand-mediated linking reactions between molecules of the lectin. This study emphasizes that binding studies at equilibrium should take into account that the data result from an average of different binding configurations of all the ligands.

Acetylglucosamine↗
PubMed1981

Properties of P3 esters of nucleoside triphosphates as substrates for RNA polymerase from Escherichia coli.

P3-[(2,4-Dinitrophenyl)amino]ethyl (DNPNHEt) and P3-methyl phosphate esters of nucleoside 5'-triphosphates have been synthesized. Their properties as substrates in the initiation and elongation steps of transcription have been examined by using RNA polymerase from Escherichia coli and poly[d(A-T)] or T7 DNA as templates. It is shown that transcription can be initiated by ATP-EtNHDNP and that 2,4-dinitrophenyl residues are incorporated at the 5' end of the RNA molecules. Steady-state kinetic experiments of abortive initation on promoters A1 and A3 of T7 DNA revealed that ATP-EtNHDNP, ADP-EtNHDNP, and ATP-OCH3 have lower Km values and markedly reduced Vmax values compared to those of ATP. The two classes of esters, NTR-EtNHDNP and NTP-OCH3, were found to differ regarding their utilization as substrates for elongation. Both ATP-OCH3 and UTP-OCH3 are substrates for transcription. However, only the pyrimidine derivatives of NTP-EtNHDNP are elongation substrates which release DNPNHEt-PP upon utilization. This dramatic difference between the purine and pyrimidine derivatives of NTP-EtNHDNP reflects a selective process in the transcriptional complex for purines and pyrimidines.

DNA-Directed RNA Polymerases↗
PubMed1981

Binding kinetics of methyl alpha-D-mannopyranoside to concanavalin A: temperature-jump relaxation study with 4-methylumbelliferyl alpha-D-mannopyranoside as a fluorescence indicator ligand.

The binding of methyl alpha-D-mannopyranoside and methyl alpha-D-glucopyranoside to concanavalin A has been investigated by the temperature-jump relaxation kinetic technique using the competitive inhibitor 4-methylumbelliferyl alpha-D-mannopyranoside as an indicator of the binding reaction. The analysis shows that these saccharides bind to concanavalin A in a single bimolecular step. The binding parameters are compared to those of derivatized carbohydrates which have previously been used to study the binding of saccharides to concanavalin A. The similarity of the association rate constants indicates that a common process is involved in the binding of all carbohydrates to concanavalin A. The different affinities of saccharides for the lectin are primarily due to the different dissociation rate constants. A discussion of the proposed mechanism is given under the Appendix to clarify the fact that one of the observed relaxation times is faster than is possible with only the kinetic indicator reaction.

Binding, Competitive↗
PubMed1977

Binding of 4-methylumbelliferyl alpha-D-mannopyranoside to tetrameric and unmodified or derivatized dimeric concanavalin A: equilibrium studies.

The binding of 4-methylumbelliferyl alpha-D-mannopyranoside (MUM) and concanavalin A, composed of intact polypeptide chains, was studied by equilibrium dialysis, difference spectroscopy, and fluorescence titration (Dean, B.R., and Homer, R.B. (1973), Biochim. Biophys. Acta. 322, 141-144), measured either at a fixed wavelength or above 350 nm. Dimeric and tetrameric concanavalin A samples were used under conditions of apparently full metal saturation. The results are consistent with a single carbohydrate-specific site per protomer, without interaction between sites; no indication for additional unspecific binding could be obtained. The values of the association constant are independent of the method or of the saturation range used and 4-methylumbelliferyl alpha-D-mannopyranoside, bound at a fractional saturation of 0.91 can be totally displaced by methyl alpha-D-mannopyranoside. The thermodynamic binding parameters for acetylated or succinylated concanavalin A, composed of intact polypeptide chains, were obtained by titration of total MUM fluorescence in the temperature range 9-39 degrees C. For unmodified dimeric concanavalin A at 25.0 degrees C, the values are K = (3.36 +/- 0.04) 10(4) M-1 with delta H degrees = -8.3 +/- 0.1 kcal mol-1 and delta S degrees = 7.2 +/- 0.3 eu; for tetrameric concanavalin A, the affinity is increased by 25% and within experimental error the values of delta H degrees and delta S degrees are identical to those for the dimeric protein. Derivatized concanavalin A shows binding characteristics that are entirely comparable to those of the native protein.

Binding Sites↗
PubMed1977

Binding of 4-methylumbelliferyl alpha-D-mannopyranoside to dimeric concanavalin A: fluorescence temperature-jump relaxation study.

The kinetics of saccharide binding to the dimer form of concanavalin A (con A) has been studied at pH 5.5 with the fluorescence temperature-jump method. 4-Methylumbelliferyl alpha-D-mannopyranoside, a fluorescent carbohydrate derivative which is quenched upon binding to con A, was used as the ligand. Three relaxation effects were seen. The major relaxation (r = 20-400 ms) was investigated at four different temperatures. The behaviour of this relaxation as a function of reactant concentrations is consistent with a simple one-step bimolecular association reaction. These conclusions result from the analysis of both the relaxation times and amplitudes, and from the comparison of the kinetically determined equilibrium parameters (Kass = 3.5 x 10(4) M-1 at 18.5 degrees C, delta H degrees = -(6-7) kcal/mol) to those obtained from a parallel series of equilibrium experiments (Loontiens, F.G., Clegg R.M., and Jovin, T.M. (1977), Biochemistry 16, preceding paper in this issue). The association and dissociation rate constants are in the range of (6-15) x 10(4) M-1 s-1 and (1.5 - 5.6) s-1, respectively, within a temperature range of 13.5-28.1 degrees C. The activation energies for the forward and reverse reactions are approximately 10 and approximately 15 kcal/mol, respectively. The two additional relaxations which are also present in the absence of saccharides result from changes in the protein fluorescence and are attributed to protein conformational changes which are not affected by the binding of saccharides. These effects were further studied using succinylated, acetylated, and demetallized con A. The faster relaxation (13 ms at 18.5 degrees C) was independent of the concentration of the protein and was not present in the derivatized con A samples. The two derivatized forms of con A show almost identical carbohydrate binding parameters as the underivatized protein. A limited series of stopped-flow experiments yielded results which were fully compatible with those from the relaxation measurements.

Acetates↗
PubMed1977

Binding of 4-methylumbelliferyl alpha-D-mannopyranoside to tetrameric concanavalin A Fluorescence temperature-jump relaxation study.

The kinetics of saccharide binding to the treatment form of concanavalin A have been studies at pH 7.2 with the temperature-jump method. 4-Methylumbelliferyl alpha-D-mannopyranoside was used as a ligand; its fluorescence is totally quenched upon binding. A single relaxation of ligand fluorescence (tau = 20-400 ms) was observed and was investigated at three different temperatures, using kinetic titration and dilution types of experiments. The concentration dependence of the relaxation time and amplitude was consistent with a single-step bimolecular association and independent binding sites. In the temperature range 13-24 degrees C the association and dissociation rate parameters are in the range (6-10) X 10(4) M-1 s-1 and (1.4-3.2)s-1 respectively, corresponding to activation energies for the forward and reverse reactions equal to approx. 13 and 8 kcal/mol (54 and 33 kJ/mol) respectively. Two additional relaxations of protein fluorescence (3 ms and larger than 1 s at 25 degrees C) were unaffected by carbohydrate binding. Tetrameric concanavalin A shows carbohydrate binding parameters that are almost identical to those of native or derivatized dimeric concanavalin A.

Calorimetry↗