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Biomedical subjects

D G Priest

Publications and source records attributed to D G Priest.

At least 73 records · Page 4Linked to original sources
PubMed1980

Thermodynamics and stoicheiometry of the binding of substrate analogues to uricase.

The subunit composition, metal content, substrate-analogue binding and thermal stability of Aspergillus flavus uricase were determined. A. flavus uricase is a tetramer and contains no copper, iron or any other common prosthetic group. Analytical-gel-filtration and equilibrium-dialysis experiments showed one binding site per subunit for urate analogues. The free energy of xanthine binding was -30.5 kJ (-7.3 kcal)/mol of subunit by equilibrium dialysis and -30.1 kJ (-7.2 kcal)/mol of subunit by microcalorimetry. The enthalpy change for xanthine binding was -15.9 kJ (-3.8 kcal)/mol of subunit when determined from the temperature-dependence of the equilibrium constant and -18.0 kJ (-4.3 kcal)/mol of subunit when measured microcalorimetrically. The thermal inactivation rate of A. flavus uricase increases as protein concentration is decreased. This concentration-dependent instability is not due to subunit dissociation.

Aspergillus flavus↗
PubMed1980

Non-classical inhibition of uricase by cyanide.

The interactions of Aspergillus flavus uricase with the substrates O2 and urate and the inhibitors xanthine, cyanide, periodate and hydroxylamine were investigated. Under equilibrium conditions O2 does not bind directly to the enzyme, and the absence of O2 had no effect on either the binding stoicheiometry or binding constant for xanthine, as measured by equilibrium dialysis and microcalorimetry. Cyanide, periodate and hydroxylamine inhibit uricase in a non-classical manner. A decrease in initial velocity to a steady-state inhibited velocity can be observed on a time scale of minutes. The time-dependence, which is unaltered by prior incubation with the inhibitors, is consistent with a first-order transition. Rate constants for induction of inhibition are linearly dependent on inhibitor concentration, but independent of urate and O2 concentrations. Radioactively labelled urate forms a stable but reversible complex with uricase in the presence of cyanide and O2. These results were used to deduce the nature of enzyme-bound intermediates and thus for the proposal of a novel mechanism for cyanide inhibition.

Aspergillus flavus↗
PubMed1980

Electrophoretic identification of poly-gamma-glutamate chain-lengths of 5,10-methylenetetrahydrofolate using thymidylate synthetase complexes.

A method has been developed to characterize the poly-gamma-glutamates of 5,10-methylenetetrahydrofolate. Incorporation of 5,10-methylenetetrahydrofolates into a ternary complex with L. casei thymidylate synthetase and 5-fluoro-2-deoxy[3H]uridylate stabilizes the reduced folate against oxidation, loss of the one carbon moiety, and poly-gamma-glutamate degradation. The covalent ternary complexes, containing 5,10-methylenetetrahydrofolate polyglutamates, were resolved electrophoretically. Electrophoretic mobility was shown to be a linear function of polyglutamate chain-length. The method can potentially be applied to analysis of chemically prepared folate polyglutamates, the monitoring of enzyme-mediated interconversions of polyglutamates and characterization of tissue extract polyglutamates.

Chemical Phenomena↗
PubMed1979

Purification of uricase from mammalian tissue.

A simple, rapid procedure for the purification of uricase from mammalian tissue is reported. The procedure is based on the precipitation of mammalian uricase under certain dialysis conditions, and on its low solubility near neutral pH. Exceptionally high yields of homogeneous enzyme are obtained.

Animals↗
PubMed1978

Interaction of pyridoxal 5-phosphate with apo-serine hydroxymethyltransferase.

The interaction of pyridoxal 5-phosphate with beef liver serine hydroxymethyltransferase (5,10-methylenetetrahydrofolate:glycine hydroxymethyltransferase, EC 2.1.2.1) has been investigated using sedimentation velocity, kinetic and equilibrium techniques. No evidence for an aggregating system could be found in sedimentation velocity experiments in the presence or absence of pyridoxal 5-phosphate. Reassociation of pyridoxal 5-phosphate with apoenzyme and reacquisition of enzymic activity follow identical kinetics. An initial fast step is followed by a second order process with a rate constant of 66 M-1. s-1. A dissociation constant of 27.5 micrometer was obtained from equilibrium studies. No interaction of binding sites was exposed by altering pH or in the presence of glycine or folate. Maxima observed in pH profiles with both binding and reactivation are interpreted as the composite fo two overlapping processes, one of which is ionization of the pyridinium nitrogen of pyridoxal 5-phosphate and the other a functional group on the apoenzyme. Evidence is presented to indicate the necessity for the formation of an enzyme . pyridoxal 5-phosphate Schiff's base complex during catalytic turnover.

Apoenzymes↗
PubMed1978

Serine transhydroxymethylase. Equilibrium binding of folate analogs as active site probes.

Formation of a quinoid-like structure within the glycyl-pyridoxal phosphate moiety of serine transhydroxymethylase (5,10-methylenetetrahydrofolate: glycine hydroxymethyltransferase, EC 2.1.2.1) is dependent upon the dissociation of the 2-S hydrogen of glycine which in turn requires the presence of tetrahydrofolate or analogs thereof. Equilibrium binding studies with the series folate, dihydrofolate, and tetrahydrofolate showed that reduction of the pteridine ring enhances both quinoid formation and binding. A 5,8-deazafolate series showed that modifications in the 4 position, 10 position and the glutamyl position yield interrelated alterations of quinoid formation which could not be correlated with binding.

Binding Sites↗