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Biomedical subjects

G Palmer

Publications and source records attributed to G Palmer.

At least 91 records · Page 5Linked to original sources
PubMed1994

Reaction and free radical kinetics of prostaglandin H synthase with manganese protoporphyrin IX as the prosthetic group.

Prostaglandin H synthase (PGHS) is a hemoprotein with both cyclooxygenase and peroxidase activities. Several aspects of the peroxidase and cyclooxygenase activities and the formation of substrate-induced free radical species were characterized with ovine seminal vesicle PGHS reconstituted with manganese protoporphyrin IX (Mn-PGHS) for comparison with the enzyme-containing heme (Fe-PGHS). Compared to Fe-PGHS, the Km of Mn-PGHS peroxidase for ethyl hydroperoxide was much higher, but that for 15-hydroperoxyeicosatetraenoic acid (15-HPETE) was little changed. The Mn-PGHS peroxidase Vmax value with 15-HPETE was about 4% that with Fe-PGHS. Mn-PGHS oxidized 0.95 +/- 0.05 and Fe-PGHS oxidized 2.06 +/- 0.09 mol of TMPD/mol of 15-HPETE. Reaction of 15-HPETE with Mn-PGHS resulted in approximately equal proportions of two lipid products: 15-hydroxyeicosatetraenoic acid (15-HETE) and a compound identified as 15-ketoeicosatetraenoic acid. Fe-PGHS produced only 15-HETE. Thus, 15-HETE can serve as an efficient two-electron reductant of oxidized Mn-PGHS intermediates. The rate of accumulation of oxidized Mn-PGHS intermediate was dependent on the substrate, decreasing in the following order: 15-HPETE > 11,14-eicosadienoate > arachidonate > EtOOH. The cyclooxygenase specific activity increased in a saturable fashion as the concentration of Mn-PGHS was increased, reaching a value higher than that for Fe-PGHS. Computer simulations of the reaction kinetics indicated that this dependence on the Mn-PGHS level was a consequence of the low rate of formation of oxidized peroxidase intermediate. Incubation of Mn-PGHS with either 15-HPETE or arachidonate resulted in the rapid production of a free radical species. The initial accumulation of radical coincided with the synthesis of PGG2 and PGH2, indicating that the radical was kinetically competent to participate in cyclooxygenase catalysis. Reaction with tetranitromethane, a reagent that selectively nitrates tyrosyl residues, destroyed the cyclooxygenase activity of Mn-PGHS, resulting in a much narrower free radical EPR signal. These effects indicate that tyrosine residues are essential for cyclooxygenase activity and that they influence the radical structure in Mn-PGHS.

Animals↗
PubMed1994

Characterization of the tyrosyl radicals in ovine prostaglandin H synthase-1 by isotope replacement and site-directed mutagenesis.

Several free radical species, attributed to tyrosyl radicals, have been previously observed by electron paramagnetic resonance (EPR) spectroscopy during reaction of pure ovine prostaglandin H synthase-1 (PGHS-1) with hydroperoxide or fatty acid substrates. The identity and location of amino acid residue(s) involved in formation of these radicals has been investigated using wild type and mutant PGHS-1 expressed in transfected COS-1 cells. Upon reaction with hydroperoxide, detergent extracts from microsomes of cells expressing wild type ovine PGHS-1 rapidly produced EPR signals made up of a broad component, resembling either the wide doublet or the wide singlet seen in reactions of the purified ovine enzyme, mixed with a variable proportion of a narrow component. Isotope replacement with perdeuterated tyrosine and phenylalanine in wild type PGHS-1 caused no change in enzymatic activity or capability to form a peroxide-induced radical, but the radical was an isotropic singlet without any wide features. This loss of splitting in the deuterated enzyme establishes that the wide radical signal in PGHS-1 is indeed a tyrosyl radical, validating the earlier assignment based on spectral similarities with a tyrosyl radical in ribonucleotide reductase. A Y385F mutation abolished cyclooxygenase but not peroxidase activity, and the peroxide-induced EPR was narrowed to a singlet essentially identical with that obtained with indomethacin-treated ovine enzyme. Isotopic replacement with perdeuterated tyrosine and phenylalanine in the Y385F mutant led to further narrowing of the peroxide-induced EPR to an isotropic singlet, establishing that this radical also involved a tyrosine residue. A Y355F mutant exhibited some decrease in cyclooxygenase activity, but the peroxidase activity and the peroxide-induced radical characteristics were not significantly different from those of the wild type enzyme, indicating that Tyr355 is not involved in tyrosyl radical formation in PGHS-1.(ABSTRACT TRUNCATED AT 400 WORDS)

Amino Acid Sequence↗
PubMed1994

Application of exciton coupling theory to the structure of mitochondrial cytochrome b.

The circular dichroism of the reduced bis-heme mitochondrial cytochrome b in the Soret region has been analyzed using exciton theory. The theory was applied to a geometric model that reflects the current consensus of the structure of this membrane protein [Degli Esposti, M., De Vries, S., Crimi, M., Ghelli, A., Patarnello, T., & Meyer, A. (1993) Biochim. Biophys. Acta 1143, 243-271]. The analysis suggests that the iron-iron distance is about 19.5 A, with a possible range of 17-23 A; the two hemes appear to be canted at 45 degrees with respect to one another. It appears that the two hemes undergo a relative angular displacement upon reduction, suggesting some flexibility in the protein structure. Some applications of this analysis to other bis-heme proteins are considered.

Animals↗
PubMed1994

Preliminary in vitro investigation of the effects of pulsed Nd:YAG laser radiation on enamel and dentine.

Data available on the effects of pulsed near IR laser energy on dental hard tissue are limited. This study investigates the changes in dental hard tissue when exposed to short duration, pulsed IR wavelength laser light emitted from a Neodymium Yttrium Aluminium Garnet laser (Nd:YAG laser, wavelength 1.06 microns). The results showed that, after treatment with an initiator, there was superficial melting of the enamel. Dentine was altered substantially. The lesions produced in dentine were larger than the cross-section of the delivery fibre optic wand. Considerable inter-sample variation was observed. The temperature rises producing these effects were large and affected the tissue at a distance from the tip.

Aluminum↗
PubMed1994

The diagnosis of periodontal conditions associated with HIV infection.

Objective, reliable, and valid diagnostic criteria are required for studies of HIV-associated periodontal conditions. A set of diagnostic criteria were devised based on a literature review and the clinical experience of the authors. Validity was assessed by comparison with clinical photographs and the criteria were evaluated and refined for ease of use and objectivity. To assess the reliability of the criteria, 9 experienced examiners were shown 20 clinical photographs accompanied by brief vignettes of clinical information. Each examiner was asked to identify signs evident in a particular area of the photograph and to record a diagnosis. Five examiners were then trained and calibrated in the use of the criteria. Finally, all 9 examiners were shown the original 20 photographs and asked to identify signs and record diagnoses. The examiners showed only fair reliability in the recognition of clinical signs, made diagnoses intuitively, and had only fair agreement on the diagnosis of periodontal diseases. The inter-examiner reliability of examiners trained and calibrated in the use of the criteria increased and was greater than among untrained examiners. Rigid diagnostic criteria are essential in epidemiologic studies. Inter-examiner reliability will be increased if examiners are trained in their use and calibrated in the recognition of clinical signs. Diagnostic criteria should be modified as understanding of the diseases they classify increases. The proposed criteria will enhance the value of studies of HIV-associated periodontal changes and will contribute to that understanding.

Chi-Square Distribution↗
PubMed1993

Photodissociation and recombination of carbonmonoxy cytochrome oxidase: dynamics from picoseconds to kiloseconds.

The kinetics of the flash-induced photodissociation and rebinding of carbon monoxide in cytochrome aa3-CO have been studied by time-resolved infrared (TRIR) and transient ultraviolet-visible (UV-vis) spectroscopy at room temperature and by Fourier transform infrared (FTIR) spectroscopy at low temperature. The binding of photodissociated CO to CuB+ at room temperature is conclusively established by the TRIR absorption at 2061 cm-1 due to the C-O stretching mode of the CuB(+)-CO complex. These measurements yield a first-order rate constant of (4.7 +/- 0.6) x 10(5) s-1 (t1/2 = 1.5 +/- 0.2 microseconds) for the dissociation of CO from the CuB(+)-CO complex into solution. The rate of rebinding of flash-photodissociated CO to cytochrome a(3)2+ exhibits saturation kinetics at [CO] > 1 mM due to a preequilibrium between CO in solution and the CuB(+)-CO complex (K1 = 87 M-1), followed by transfer of CO to cytochrome a(3)2+ (k2 = 1030 s-1). The CO transfer from CuB to Fe alpha 3 was followed by CO-FTIR between 158 and 179 K and by UV-vis at room temperature. The activation parameters over the temperature range 140-300 K are delta H++ = 10.0 kcal mol-1 and delta S++ = -12.0 cal mol-1 K-1. The value of delta H++ is temperature independent over this range; i.e., delta Cp++ = 0 for CO transfer. Rapid events following photodissociation and preceding rebinding of CO to cytochrome a(3)2+ were observed. An increase in the alpha-band of cytochrome a3 near 615 nm (t1/2 ca. 6 ps) follows the initial femtosecond time-scale events accompanying photodissociation. Subsequently, a decrease is observed in the alpha-band absorbance (t1/2 approximately 1 microsecond) to a value typical of unliganded cytochrome a3. This latter absorbance change appears to occur simultaneously with the loss of CO by CuB+. We ascribe these observations to structural changes at the cytochrome a3 induced by the formation and dissociation of the CuB(+)-CO complex. We suggest that the picosecond binding of photodissociated CO to CuB triggers the release of a ligand L from CuB. We infer that L then binds to cytochrome a3 on the distal side and that this process is directly responsible for the observed alpha-band absorbance changes. We have previously suggested that the transfer of L produces a transient five-coordinate high-spin cytochrome a3 species where the proximal histidine has been replaced by L. When CO binds to the enzyme from solution, these processes are reversed.(ABSTRACT TRUNCATED AT 400 WORDS)

Carbon Monoxide↗
PubMed1993

Magnetization of fast and slow oxidized cytochrome c oxidase.

Oxidized cytochrome c oxidase can now be prepared either to react rapidly (fast) or to react slowly (slow) with cyanide [Baker, G.M., Noguchi, M., & Palmer, G. (1987) J. Biol. Chem. 262, 595-604]. Slow oxidized cytochrome c oxidase is also characterized by an integer spin g = 12 EPR signal which is absent in the fast form. The saturation magnetization of two samples of both forms of cytochrome oxidase has been studied at four applied magnetic fields (0.3125, 1.25, 2.5, and 5.0 T) over a temperature range from 2 to 200 K using a superconducting susceptometer. The saturation magnetization data of the two preparations are readily distinguished. The data for the coupled cytochrome a3: CuB site of both preparations are most simply interpreted as exhibiting S = 2 paramagnetism with D = +13 cm-1 for the fast and D = -7 cm-1 for the slow forms, respectively. However, there is some indication that the fast form is a mixture of both S = 2 and S = 1 paramagnetic species while the slow form is only S = 2.

Animals↗
PubMed1993

Heme coordination of prostaglandin H synthase.

The heme coordination of ovine prostaglandin H synthase (PGHS) has been characterized by EPR, magnetic circular dichroism, resonance Raman, and optical spectroscopies. The EPR spectrum of ferric PGHS is consistent with an equilibrium mixture of high-spin and low-spin heme species. Both species disappear on reaction of the synthase with hydroperoxides. The high-spin to low-spin interconversion is temperature- and concentration-dependent. Correlation between the axial and rhombic ligand fields of the low-spin heme species suggests that it has bishistidine axial ligation. Magnetic circular dichroism spectra of PGHS also show a temperature-dependent spin transition. Resonance Raman spectra indicate that the enzyme exists as a mixture of six-coordinate low-spin and six-coordinate high-spin ferric heme species. No Raman bands attributable to five-coordinate high-spin heme species are detectable. The magnetic circular dichroism spectra of the fluoride, azide, cyanide, and imidazole derivatives of PGHS resemble those of the corresponding metmyoglobin derivatives and are very different from those of the catalase derivatives. EPR spectra of the imidazole derivative of these three proteins provide additional evidence that the heme coordination structure of PGHS is similar to that of metmyoglobin rather than that of catalase. The midpoint potential of the PGHS Fe(III)/Fe(II) pair is in the range observed for hemeproteins with mono- or bishistidine coordination. These data provide a convincing case that the axial heme ligands of PGHS-1 are a pair of histidine residues, with the distal histidine weakly associated and possibly exchangeable with a weak-field ligand.

Animals↗
PubMed1993

Spectroscopic characterization of the interaction of azide and thiocyanate with the binuclear center of cytochrome oxidase: evidence for multiple ligand sites.

The interactions of azide and thiocyanate with the binuclear center of oxidized cytochrome c oxidase have been characterized by Fourier transform infrared and UV-vis spectroscopy, electron paramagnetic resonance and magnetic and natural circular dichroism. Azide binds in two phases, a high-affinity phase (Kd = 64 microM) in which it is bound as a bridge to the binuclear center and a low-affinity phase (Kd = 20 mM) in which it displaces one of the axial ligands to cytochrome a. Thiocyanate also binds in two phases. The high-affinity phase (Kd = 2.7 mM) involves binding in a terminal mode to CuB; the low-affinity phase is complex and involves both CuA and cytochrome a. In contrast to the recent proposal of Yoshikawa and Caughey [(1990) J. Biol. Chem. 265, 7945-7958], we conclude that cyanide also functions as a bridge between cytochrome a3 and CuB. In the presence of cyanide, azide does not bind to its high-affinity site but thiocyanate does bind to its high-affinity site.

Azides↗
PubMed1993

Steady-state kinetics of ubiquinol-cytochrome c reductase in bovine heart submitochondrial particles: diffusional effects.

In an attempt to establish the relative importance of diffusional and chemical control in the reactivity of the two of the two substrates, ubiquinol and cytochrome c, we have undertaken as extensive characterization of the steady-state kinetics of ubiquinol-cytochrome c reductase (EC 1.10.2.2) when present in open submitochondrial particles from bovine heart. The kinetic pattern follows a Ping Pong mechanism; contrary to the situation found with the isolated enzyme [Speck and Margoliash (1984) J. Biol. Chem. 259, 1064-1072, and confirmed in our laboratory], no substrate inhibition by oxidized cytochrome c was observed with the membrane-bound enzyme. Endogenous oxidized ubiquinone-10 is unable to exert product inhibition under the conditions employed. In the Ping Pong mechanism for this enzyme, the reaction scheme can be clearly divided into two parts, and the Kmin. (kcat./km) value for one substrate is independent of the rate constant for the second substrate. Both ubiquinol-1 and ubiquinol-2 can be used as electron donors reacting with the enzyme from within the lipid bilayer [Fato, Castelluccio, Palmer and Lenaz (1988) Biochim. Biophys. Acta 932, 216-222]; the kmin. values for ubiquinols, when calculated on the basis of their membranous concentrations, are significantly lower than the kmin. for cytochrome c. The temperature-dependence of the kinetic parameters was investigated by titrating each of the substrates under quasi-saturating concentrations of the second substrate. Arrhenius plots of Vmax. extrapolated from both cytochrome c and ubiquinol titrations were linear, when care was taken to verify the quasi-saturating concentrations of the fixed co-substrate. The Arrhenius plots for the kmin. values for both ubiquinol and cytochrome c were linear, but the activation energy was much higher for the former, particularly when calculated for ubiquinol dissolved in the lipid phase; the very low value of activation energy of the kmin. for cytochrome c is strong support for diffusion control being present in the reaction of cytochrome c with the membranous enzyme. In contrast to the soluble enzyme, ubiquinone titrations of submitochondrial particles at low cytochrome c concentrations deviated from hyperbolic behaviour. Changing the medium viscosity with either poly(ethylene glycol) or sucrose had a strong effect on the cytochrome c kmin., whereas the change in the ubiquinol kmin. was much smaller. From the viscosity studies the extent of diffusional control could be calculated, revealing that the reaction with cytochrome c was mostly diffusion-limited. The viscosity of the membrane was changed by incorporating cholesterol; no significant effect on the ubiquinol kmin. ascribable to diffusion control could be recognized.(ABSTRACT TRUNCATED AT 400 WORDS)

Animals↗
PubMed1993

Current issues in the chemistry of cytochrome c oxidase.

Some contemporary issues relevant to the chemistry of mammalian cytochrome c oxidase are discussed. These include the optical properties of heme A and the spectroscopic consequences of the differences in side-chain substitution compared to heme B; a common fallacy concerning the electrostatic exchange interaction between cytochrome a3 and CuB; the question of the number and location of the copper components of the enzyme; and the mode of binding of ligands such as cyanide and azide.

Animals↗
PubMed1993

Weaning. Any old iron.

Gabrielle Palmer takes issue with manufacturers' claims for follow on milks, arguing that they are often 'generous with alarm and economical with the truth'. A balanced weaning diet is the key to preventing iron deficiency anaemia in children.

Anemia, Hypochromic↗
PubMed1992

Prostaglandin H synthase. Kinetics of tyrosyl radical formation and of cyclooxygenase catalysis.

Hydroperoxides are known to induce the formation of tyrosyl free radicals in prostaglandin (PG) H synthase. To evaluate the role of these radicals in cyclooxygenase catalysis we have analyzed the temporal correlation between radical formation and substrate conversion during reaction of the synthase with arachidonic acid. PGH synthase reacted with equimolar levels of arachidonic acid generated sequentially the wide doublet (34 G peak-to-trough) and wide singlet (32 G peak-to-trough) tyrosyl radical signals previously reported for reaction with hydroperoxide. The kinetics of formation and decay of the doublet signal corresponded reasonably well with those of cyclooxygenase activity. However, the wide singlet free radical signal accumulated only after prostaglandin formation had ceased, indicating that the wide singlet is not likely to be an intermediate in cyclooxygenase catalysis. When PGH synthase was reacted with 25 equivalents of arachidonic acid, the wide doublet and wide singlet radical signals were not observed. Instead, a narrower singlet (24 G peak-to-trough) tyrosyl radical was generated, similar to that found upon reaction of indomethacin-treated synthase with hydroperoxide. Only about 11 mol of prostaglandin were formed per mol of synthase before complete self-inactivation of the cyclooxygenase, far less than the 170 mol/mol synthase produced under standard assay conditions. Phenol (0.5 mM) increased the extent of cyclooxygenase reaction by only about 50%, in contrast to the 460% stimulation seen under standard assay conditions. These results indicate that the narrow singlet tyrosyl radical observed in the reaction with high levels of arachidonate in this study and by Lassmann et al. (Lassmann, G., Odenwaller, R., Curtis, J.F., DeGray, J.A., Mason, R.P., Marnett, L.J., and Eling, T.E. (1991) J. Biol. Chem. 266, 20045-20055) is associated with abnormal cyclooxygenase activity and is probably nonphysiological. In titrations of the synthase with arachidonate or with hydroperoxide, the loss of enzyme activity and destruction of heme were linear functions of the amount of titrant added. Complete inactivation of cyclooxygenase activity was found at about 10 mol of arachidonate, ethyl hydrogen peroxide, or hydrogen peroxide per mol of synthase heme; maximal bleaching of the heme Soret absorbance peak was found with 10 mol of ethyl hydroperoxide or 20 mol of either arachidonate or hydrogen peroxide per mol of synthase heme. The peak concentration of the wide doublet tyrosyl radical did not change appreciably with increased levels of ethyl hydroperoxide. In contrast, higher levels of hydroperoxide gave higher levels of the wide singlet radical species, in parallel with enzyme inactivation.(ABSTRACT TRUNCATED AT 400 WORDS)

Animals↗
PubMed1992

Temperature- and pH-dependent changes in the coordination sphere of the heme c group in the model peroxidase N alpha-acetyl microperoxidase-8.

The pH- and temperature-dependent changes in the coordination sphere of the heme c group of N alpha-acetyl microperoxidase-8 (Ac-MP-8) have been studied by examining its optical, resonance Raman, electron paramagnetic resonance, and magnetic circular dichroism spectra. An optical titration indicates that Ac-MP-8 exists in three major ionization forms over the pH 1-12 range that are linked by pK alpha values of approximately 3 and 9. The acid form that is present at pH 1.5 exists as a mixture of five- and six-coordinate high-spin species and most likely has water or buffer ions as axial ligand(s). On titration to pH 7, the His18 residue is deprotonated and becomes the proximal ligand to the iron to give a six-coordinate neutral form that has water as the sixth ligand. This form exists in a thermal high-spin intermediate-spin state equilibrium. On raising the pH to 10, an alkaline form is generated which is predominantly a five-coordinate high-spin species. It is formed by ionization of the proximal His18 residue to its imidazolate form with concomitant dissociation of the water ligand at the sixth site. At concentrations of Ac-MP-8 greater than 10 microM, some six-coordinate low-spin species are formed that are attributed to a dimer in which a His18 residue from a second molecule of Ac-MP-8 coordinates to the sixth site of another to give a bis-His complex. Raising the pH to 11.5 does not produce an appreciable amount of the six-coordinate complex with hydroxide as the sixth ligand. These studies show that Ac-MP-8 is a good water-soluble model for the peroxidases that exhibits minimal aggregation at concentrations below 10 microM in the neutral and alkaline pH regions.

Acetylation↗
PubMed1992

Circuit weight training in early cardiac rehabilitation.

A prospective, randomized study evaluated the feasibility, safety, and efficacy of upper body circuit weight training (CWT) in 25 stable male cardiac patients entering the initial out-of-hospital phase of cardiac rehabilitation. Both groups performed 30 minutes of aerobic exercise only for 6 weeks. The aerobic exercise group (N = 13) continued this regimen for 6 more weeks, during which time the CWT group (N = 12) performed 15 minutes of aerobic exercise followed by CWT (two loops, eight upper body exercises). The only adverse response was in one CWT patient in whom restenosis developed. Peak heart rate during aerobic exercise and CWT was similar, but peak systolic blood pressure during aerobic exercise was significantly greater than during CWT. Peak rate pressure product during aerobic exercise and CWT was similar. Treadmill time increased significantly in both groups. Upper body strength (cumulative pounds lifted) increased significantly only in the CWT group. A coordinated program of CWT and aerobic exercise can be performed safely in stable cardiac patients during phase 2 cardiac rehabilitation, resulting in improved upper body strength and aerobic capacity.

Adult↗