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Biomedical subjects

P F Knowles

Publications and source records attributed to P F Knowles.

At least 55 records · Page 3Linked to original sources
PubMed1987

The organic cofactor in plasma amine oxidase: evidence for pyrroloquinoline quinone and against pyridoxal phosphate.

Plasma amine oxidases (EC 1.4.3.6) are classified as containing the organic cofactor pyridoxal phosphate. Biochemical and bioassays on the pig plasma amine oxidase fail to reveal the presence of pyridoxal phosphate and 31P n.m.r. evidence is also inconsistent with pyridoxal phosphate in the enzyme. Resonance Raman spectral studies on phenylhydrazone derivatives of the pig and bovine plasma enzymes have been carried out and comparisons made with the corresponding derivatives of pyridoxal phosphate and pyrroloquinoline quinone (PQQ). The resonance Raman evidence indicates that the cofactor in both plasma amine oxidases is PQQ or a closely related species and not pyridoxal phosphate. The results substantiate earlier reports concerning the identity of the organic cofactor.

Amine Oxidase (Copper-Containing)↗
PubMed1986

Electron nuclear double-resonance (ENDOR) spectroscopy of amine oxidase from pig plasma.

Electron nuclear double-resonance ('ENDOR') spectroscopic studies on pig plasma amine oxidase have been carried out at 15 K. Deuterium-exchange studies show the presence of two sets of exchangeable protons, probably from two water molecules; from the magnitude of their hyperfine couplings, one is assigned to be equatorially, and the other axially, co-ordinated. Only one 14N hyperfine coupling is observed, suggesting that the bonding of all amino acid (histidine) or organic cofactor ligands is similar. Upon addition of azide, a further hyperfine coupling to nitrogen is observed which is smaller than that observed for the native enzyme; the hyperfine couplings to the remaining nitrogens are slightly altered.

Amine Oxidase (Copper-Containing)↗
PubMed1985

Association of spin-labelled cardiolipin with dimyristoylphosphatidylcholine-substituted bovine heart cytochrome c oxidase. A generalized specificity increase rather than highly specific binding sites.

The endogeneous lipid of bovine heart cytochrome c oxidase has been replaced by dimyristoylphosphatidylcholine using cholate-mediated exchange. The lipid-substituted preparation contained less than 1 mole cardiolipin per mole enzyme and possessed full oxidative activity. The association of spin-labelled cardiolipin with such lipid-substituted cytochrome oxidase preparations has been assayed using ESR spectroscopy. An average relative association constant 5.4-times that for phosphatidylcholine is obtained for cardiolipin. Measurements on preparations with increasing contents of unlabelled cardiolipin, introduced during lipid exchange, reveal that this selectivity corresponds to a generalized increase in specificity for all lipid association sites on the protein.

Animals↗
PubMed1984

Direct structural information for the copper site of dopamine beta-mono-oxygenase obtained by using extended X-ray-absorption fine structure.

Copper K-edge e.x.a.f.s (extended X-ray-absorption fine structure) was measured for dopamine beta-mono-oxygenase in aqueous solution. Comparison with the Cu K-edge e.x.a.f.s. of bovine erythrocyte superoxide dismutase shows a close resemblance. Detailed analysis of the e.x.a.f.s. indicates that the copper atom is bound to four imidazole groups at 0.201 nm with one or two oxygen atoms at 0.23 nm.

Binding Sites↗
PubMed1984

The kinetics of ammonia release during the catalytic cycle of pig plasma amine oxidase.

The time course of NH3 release during the catalytic cycle of pig plasma amine oxidase was followed by using the quenched-flow technique in conjunction with a sensitive assay for NH3. These studies were made under both air and O2-saturating conditions. The results establish unequivocally that NH3 is released in the step whereby a reduced enzyme species is re-oxidized by molecular O2 rather than in the step leading to the reduced enzyme. It is concluded that the catalytic cycle of the enzyme conforms to an aminotransferase mechanism rather than one in which an imine is an intermediate.

Amine Oxidase (Copper-Containing)↗
PubMed1984

An extended-X-ray-absorption-fine-structure study of bovine erythrocyte superoxide dismutase in aqueous solution. Direct evidence for three-co-ordinate Cu(I) in reduced enzyme.

Copper and zinc K-edge e.x.a.f.s. (extended X-ray-absorption fine structures) were measured for the metal sites of oxidized and reduced bovine superoxide dismutase in aqueous solution. Detailed analysis of the spectra indicates that the copper site of the enzyme changes on reduction and is most probably co-ordinated to three imidazole groups at a shorter distance Cu-N(alpha) = 0.194 nm (1.94 A) in the reduced form compared with a co-ordination of four imidazole groups at 0.199 nm (1.99 A) and an oxygen atom from solvent water at 0.224 nm (2.24 A) in the oxidized form. Examination of the edge, near-edge structure and e.x.a.f.s. of the zinc sites indicates that the stereochemical changes at copper that accompany reduction introduce minimal perturbation on the stereochemistry at zinc.

Animals↗
PubMed1983

An extended X-ray-absorption-fine-structure study of the copper and zinc sites of freeze-dried bovine superoxide dismutase.

Copper and zinc K-edge-extended X-ray-absorption fine structures were measured for the metal sites of freeze-dried bovine superoxide dismutase and the model compounds tetrakis(imidazole)cupric nitrate and tetrakis(imidazole)zinc perchlorate. Detailed simulation of the spectra indicates that the copper site of the enzyme is best fit by co-ordination of four imidazole groups with Cu-N(alpha) distances of 0.198 nm (1.98 A). The zinc site is best fit by three imidazole groups at 0.201 nm (2.01 A) and an oxygen (from aspartate) at 0.203 nm (2.03 A).

Animals↗
PubMed1982

ESR spin-label studies of lipid-protein interactions in membranes.

Lipid spin labels have been used to study lipid-protein interactions in bovine and frog rod outer segment disc membranes, in (Na+, K+)-ATPase membranes from shark rectal gland, and in yeast cytochrome oxidase-dimyristoyl phosphatidylcholine complexes. These systems all display a two component ESR spectrum from 14-doxyl lipid spin-labels. One component corresponds to the normal fluid bilayer lipids. The second component has a greater degree of motional restriction and arises from lipids interacting with the protein. For the phosphatidylcholine spin label there are effectively 55 +/- 5 lipids/200,000-dalton cytochrome oxidase, 58 +/- 4 mol lipid/265,000 dalton (Na+, K+)-ATPase, and 24 +/- 3 and 22 +/- 2 mol lipid/37,000 dalton rhodopsin for the bovine and frog preparations, respectively. These values correlate roughly with the intramembrane protein perimeter and scale with the square root of the molecular weight of the protein. For cytochrome oxidase the motionally restricted component bears a fixed stoichiometry to the protein at high lipid:protein ratios, and is reduced at low lipid:protein ratios to an extent which can be quantitatively accounted for by random protein-protein contacts. Experiments with spin labels of different headgroups indicate a marked selectivity of cytochrome oxidase and the (Na+, K+)-ATPase for stearic acid and for cardiolipin, relative to phosphatidylcholine. The motionally restricted component from the cardiolipin spin label is 80% greater than from the phosphatidylcholine spin label for cytochrome oxidase (at lipid:protein = 90.1), and 160% greater for the (Na+, K+)-ATPase. The corresponding increases for the stearic acid label are 20% for cytochrome oxidase and 40% for (Na+, K+)-ATPase. The effective association constant for cardiolipin is approximately 4.5 times greater than for phosphatidylcholine, and that for stearic acid is 1.5 times greater, in both systems. Almost no specificity is found in the interaction of spin-labeled lipids (including cardiolipin) with rhodopsin in the rod outer segment disc membrane. The linewidths of the fluid spin-label component in bovine rod outer segment membranes are consistently higher than those in bilayers of the extracted membrane lipids and provide valuable information on the rate of exchange between the two lipid components, which is suggested to be in the range of 10(6)-10(7) s-1.

Animals↗
PubMed1981

Steady-state kinetic studies on benzylamine oxidase from pig plasma.

Steady-state kinetic studies on the enzyme benzylamine oxidase from pig plasma are described. Eadie-Hofstee plots with benzylamine as the varying substrate are non-linear; examination of this data indicates that the observed effects are probably due to the amine substrate participating in at least two reactions with enzyme. Ammonia and imidazole modify the activity of the enzyme; under specified conditions of pH or modifier concentration, the effect on the activity can be either activation or inhibition. Eadie-Hofstee plots of the data establish that the modifier also participates in at least three reactions with the enzyme. Eadie-Hofstee plots at pH 9 with oxygen as the varying substrate are linear, which allows kinetic parameters to be determined. From studies on the effect of ammonia and imidazole on these parameters, information has been derived on how these modifiers affect component steps of the catalytic cycle.

Ammonium Chloride↗
PubMed1981

A catalytic mechanism for benzylamine oxidase from pig plasma. Stopped-flow kinetic studies.

1. Ammonium ion is shown to decrease the rate constants for Schiff's base formation and formation of a reduced intermediate during the catalytic cycle of benzylamine oxidase from pig plasma. The rat constant for reoxidation of the reduced intermediate is also inhibited whilst the rate constant for conversion of the oxidised enzyme form back to native enzyme is stimulated by ammonium ion. 2. Ammonium ion changes the electron paramagnetic resonance spectrum of the cupric centres in the enzyme, indicating that ammonia binds to the copper. 3. A catalytic mechanism for benzylamine oxidase is proposed on the basis of these and other results. This mechanism includes a novel step in which a hydroxyl coordinated to copper acts as a nucleophyle to facilitate hydride ion transfer to oxygen during the reoxidation process.

Ammonium Chloride↗
PubMed1979

Properties of cupric ions in benzylamine oxidase from pig plasma as studied by magnetic-resonance and kinetic methods.

Benzylamine oxidase from pig plasma has been studied by a variety of chemical and physical techniques. 1. Analytical ultracentrifugation, gel electrophoresis and isoelectric-focusing studies suggest that the enzyme is composed of two subunits with closely similar primary structures. 2. E.s.r. and n.m.r. measurements show that the enzyme contains two well-separated (greater than 0.6 nm) Cu2+ ions at chemically distinct sites. Each Cu2+ ion is coordinated by two water molecules, one 'axial' and the other 'equatorial'. Both water molecules undergo fast exchange (10(5)--10(8) s-1) with solvent and are deprotonated in the pH range 8--9, but only the equatorial water molecule is displaced by the inhibitors N3- and CN-. 3. Kinetic and e.s.r. measurements show that azide and cyanide compete against O2 binding and also make the two Cu2+ sites identical. It is concluded that Cu2+ must participate in the re-oxidation of reduced enzyme by molecular O2.

Animals↗
PubMed1979

Properties of the cupric sites in bovine superoxide dismutase studied by nuclear-magnetic-relaxation measurements.

Proton nuclear-relaxation rates have been measured as a function of frequency, temperature, pH and cyanide concentration in aqueous solutions of superoxide dismutase from bovine erythrocytes. The results show that, whereas for pH less than or equal to 9 only one water molecule is bound to each Cu2+ ion, at higher pH a second co-ordination site for OH- becomes available; it is proposed that this involves cleavage of the bond between Cu2+ and the histidine residue that bridges to Zn2+.

Chemical Phenomena↗
PubMed1978

Morphology of egg phosphatidylcholine-cholesterole single-bilayer vesicles, studied by freeze-etch electron microscopy.

Homogeneous, small, single-bilayer vesicles were prepared from egg phosphatidylcholine with various concentrations of cholesterol by ultrasonic dispersion in 0.1 M KCl, 0.01 M Tris, pH 8.0, buffer, followed by gel chromatography. The shape and size distributions of the fractionated vesicles were investigated for preparations with cholesterol compositions from 0 to 50 moles/100 moles, using freeze-etch electron microscopy. The size distribution was estimated from the shadow width of vesicles which were exposed by etching and the vesicle shape was checked by comparing the images obtained by tilting the replicas. The widths of the vesicle diameter distributions were relatively broad, corresponding to standard deviations in the range 60--90 A, but showing no systematic variation with cholesterol composition. In all cases it was found that 70% of the vesicle diameters lay within 150 A of the modal value. The apparent vesicle diameters remained constant for cholesterol compositions up to 20 moles/100 moles (modal diameter = 330 +/- 20 A, mean diameter = 350 +/- 3 A), but there was a sharp net increase in diameter at 30 moles cholesterol/100 moles reaching a model diameter of 430 +/- 20 A (mean diameter = 430 +/- 3 A) at 50 moles cholesterol/100 moles. Using the tilted microscope stage it was found that all vesicles were spherical at all cholesterol compositions studied, including those above 30 moles cholesterol/100 moles. The molecular mechanism by which cholesterol controls the vesicle size is discussed in terms of the asymmetric distribution of cholesterol across the vesicle bilayer.

Cholesterol↗