Search PubMed⌕ Search

Biomedical subjects

C E Cooper

Publications and source records attributed to C E Cooper.

At least 73 records · Page 4Linked to original sources
PubMed1995

Interconversion of fast and slow forms of cytochrome bo from Escherichia coli.

The fully oxidized fast form of cytochrome bo from Escherichia coli is shown to convert spontaneously to a slow form when stored at -20 degrees C in 50 mM potassium borate, pH 8.5, containing 0.5 mM potassium EDTA. Evidence for the conversion, and that the form produced is analogous to the slow form of bovine heart cytochrome c oxidase, comes from (a) decreases in the extents of fast (k = 1-2 x 10(3) M-1 s-1) H2O2 binding and fast (k = 20-30 M-1 s-1) cyanide binding; (b) changes in the optical spectrum that are like those induced by formate, i.e., a blue shift in the Soret absorption band, loss of absorbance in the alpha and beta bands, and a red shift in the "630 nm" charge-transfer band; (c) changes in the EPR spectrum that are like those induced by formate, i.e., disappearance of signals at g = 8.6 and g = 3.71, and appearance of signals at g approximately 13, g = 3.14, and g = 2.58; and (d) appearance of a slow phase of reduction of heme o by dithionite. The mutant enzyme E286Q also converts to a slow form under the same conditions, as shown by (a) a decrease in the extent of fast H2O2 binding; (b) changes in the optical spectrum like those seen with wild-type enzyme; and (c) changes in the EPR spectrum that are like those induced by formate, i.e., disappearance of signals at g = 7.3 and g = 3.6 and appearance of signals at g approximately 13, g = 3.18, and g = 2.59.(ABSTRACT TRUNCATED AT 250 WORDS)

Borates↗
PubMed1995

Performance comparison of several published tissue near-infrared spectroscopy algorithms.

We have collected multiwavelength near-infrared (NIR) attenuation spectra on human forearm muscle, the adult rat head, and newborn piglet head to compare the changes in chromophore concentration derived from these data using published algorithms from four groups. We find differences between the results from the algorithms on each data set, particularly in their estimation of cytochrome oxidase (cyt-aa3) redox changes. We also find some differences when applying the same algorithm to the three data sets, suggesting possible difficulties in transferring algorithms between different physiological systems (e.g., Kurth, C. D., Steven, J. M., Benaron, D., and Chance, B. (1993) J. Clin. Monit. 9, 163-170). We have also compared the algorithms using simulated data generated using measured hemoglobin absorption spectra and a diffusion model for light transport in tissue. We find that while the algorithms from three groups are in broad agreement, that published by Piantadosi (Piantadosi, C. A. (1993) Methods Toxicol. 2, 107-126) produces significantly different results for cyt-aa3 and HbO2. Either the hemoglobin spectra used to produce the simulated data are inaccurate or the modeling is incorrect, or this algorithm is erroneous.

Adult↗
PubMed1995

Mild hypothermia after severe transient hypoxia-ischemia ameliorates delayed cerebral energy failure in the newborn piglet.

Severely birth-asphyxiated human infants develop delayed ("secondary") cerebral energy failure, which carries a poor prognosis, during the first few days of life. This study tested the hypothesis that mild hypothermia after severe transient cerebral hypoxia-ischemia decreases the severity of delayed energy failure in the newborn piglet. Six piglets underwent temporary occlusion of the common carotid arteries and hypoxemia. Resuscitation was started when cerebral [phosphocreatine (PCr)]/[inorganic phosphate (Pi)] as determined by phosphorus magnetic resonance spectroscopy had fallen almost to zero and [nucleotide triphosphate (NTP)]/[exchangeable phosphate pool (EPP)] had fallen below about 30% of baseline. Rectal and tympanic temperatures were then reduced to 35 degrees C for 12 h after which normothermia (38.5 degrees C) was resumed. Spectroscopy results over the next 64 h were compared with previously established data from 12 piglets similarly subjected to transient cerebral hypoxia-ischemia, but maintained normothermic, and six sham-operated controls. The mean severity of the primary insult (judged by the time integral of depletion of [NTP]/[EPP]) was similar in the hypothermic and normothermic groups. In the normothermic group, [PCr]/[Pi] and [NTP]/[EPP] recovered after the acute insult and then fell again. Minimum values for these variables observed between 24 and 48 h were significantly higher in the hypothermic group and not significantly different from the control values (p < 0.05, analysis of variance). A large reduction in secondary energy failure relative to the extent of the primary insult was shown and no further fall in either [PCr]/[Pi] or [NTP]/[EPP] took place up to 64 h in the hypothermic piglets.(ABSTRACT TRUNCATED AT 250 WORDS)

Acute Disease↗
PubMed1994

Nanomolar concentrations of nitric oxide reversibly inhibit synaptosomal respiration by competing with oxygen at cytochrome oxidase.

Nitric oxide (NO) reversibly inhibited oxygen consumption of brain synaptosomes. Inhibition was reversible, occurred at the level of cytochrome oxidase, and was apparently competitive with oxygen, with half-inhibition by 270 nM NO at oxygen concentrations around 145 microM and by 60 nM at around 30 microM O2. Isolated cytochrome oxidase was inhibited by similar levels of NO. These levels of NO are within the measured physiological and pathological range for a number of tissues and conditions, suggesting that NO inhibition of cytochrome oxidase and the competition with oxygen may occur in vivo.

Animals↗
PubMed1994

Brain-metabolite transverse relaxation times in magnetic resonance spectroscopy increase as adenosine triphosphate depletes during secondary energy failure following acute hypoxia-ischaemia in the newborn piglet.

The adenosine triphosphate (ATP)-dependent sodium/potassium pump extrudes intracellular sodium in exchange for extracellular potassium. Low ATP causes pump dysfunction increasing both intracellular sodium and water thereby enhancing metabolite mobility. This should be detectable by proton magnetic resonance spectroscopy (MRS) as increased metabolite transverse relaxation times (T2s). During secondary cerebral energy failure in the newborn piglet, proton and phosphorus MRS showed large increases in the T2s of choline, creatine, N-acetylaspartate, and lactate that correlated with ATP depletion. These results provide insight into factors affecting metabolite T2s and show that T2s may be useful for studying cellular oedema.

Adenosine Triphosphate↗
PubMed1994

The analysis of rate limitation within enzymes: relations between flux control coefficients of rate constants and unidirectional rates, rate constants and thermodynamic parameters of single isolated enzymes.

The extent to which a rate constant or step within an enzyme mechanism limits the net enzyme rate in a particular condition can be quantified as a flux control coefficient. We derive here a number of relations between the control coefficients and the unidirectional rates, rate constants, and thermodynamic parameters of the enzyme. These and other relations are used to suggest a number of methods for experimentally measuring control coefficients within enzymes.

Enzymes↗
PubMed1994

Transport of K+ and other cations across phospholipid membranes by nonesterified fatty acids.

The rate of change of internal pH and transmembrane potential has been monitored in liposomes following the external addition of various cation salts. Oleic acid increases the transmembrane movement of H+ following the imposition of a K+ gradient. An initial fast change in internal pH is seen followed by a slower rate of alkalinization. High concentrations of the fatty acid enhance the rate comparable to that seen in the presence of nigericin in contrast to the effect of FCCP (carbonyl cyanide p-(tri-fluoromethoxy)phenyl hydrazone) which saturates at an intermediate value. The ability of nonesterified fatty acids to catalyze the movement of cations across the liposome membrane increases with the degree of unsaturation and decreases with increasing chain length. Li and Na salts cause a similar initial fast pH change but have less effect on the subsequent slower rate. Similarly, the main effect of divalent cation salts is on the initial fast change. The membrane potential can enhance or inhibit cation transport depending on its polarity with respect to the cation gradient. It is concluded that nonesterified fatty acids have the capability to complex with, and transport, a variety of cations across phospholipid bilayers. However, they do not act simply as proton/cation exchangers analogous to nigericin nor as protonophores analogous to FCCP. The full cycle of ionophoric action involves a combination of both functions.

Fatty Acids, Nonesterified↗
PubMed1994

Absolute quantification of deoxyhaemoglobin concentration in tissue near infrared spectroscopy.

We describe a simple technique for non-invasively determining the absolute concentration of deoxyhaemoglobin (Hb) in living tissue using near infrared spectroscopy (NIRS). The technique uses second-differential spectroscopy to determine the relative concentration of Hb to tissue water by fitting of the spectral features of these two chromophores in the 710 nm to 840 nm region of the near infrared (NIR) spectrum. Since the concentration of tissue water is generally known with accuracies of a few per cent, one can then obtain the absolute concentration of Hb ([Hb]). The validity and likely accuracy of the technique is assessed by applying it to artificially generated NIRS data. Some clinical validation is presented by comparing, during an in vivo study, changes in [Hb] obtained by this method and those calculated using more conventional techniques of relative quantification. Finally, we discuss the likely clinical significance of the measurement of absolute Hb concentration.

Algorithms↗
PubMed1994

The effect of ferredoxin(BED) overexpression on benzene dioxygenase activity in Pseudomonas putida ML2.

The benzene dioxygenase from Pseudomonas putida ML2 is a multicomponent complex comprising a flavoprotein reductase, a ferredoxin, and a terminal iron-sulfur protein (ISP). The catalytic activity of the isolated complex shows a nonlinear relationship with protein concentration in cell extracts, with the limiting factor for activity in vitro being ferredoxin(BED). The relative levels of the three components were analyzed by using 125I-labelled antibodies, and the functional molar ratio of ISP(BED), ferredoxin(BED), and reductase(BED) was shown to be 1:0.9:0.8, respectively. The concentration of ferredoxin(BED) was confirmed by quantitative electron paramagnetic resonance spectroscopy of the 2Fe-2S centers in ferredoxin(BED) and ISP(BED) of whole cells. These results demonstrate that the ferredoxin(BED) component is a limiting factor in dioxygenase activity in vitro. To determine if it is a limiting factor in vivo, a plasmid (pJRM606) overproducing ferredoxin(BED) was introduced into P. putida ML2. The benzene dioxygenase activity of this strain, measured in cell extracts, was fivefold greater than in the wild type, and the activity was linear with protein concentration in cell extracts above 2 mg/ml. Western blotting (immunoblotting) and electron paramagnetic resonance spectroscopic analysis confirmed an elevated level of ferredoxin(BED) protein and active redox centers in the recombinant strain. However, in these cells, the increased level of ferredoxin(BED) had no effect on the overall rate of benzene oxidation by whole cells. Thus, we conclude that ferredoxin(BED) is not limiting at the high intracellular concentration (0.48 mM) found in cells.

Benzene↗
PubMed1994

A hydrogen-donating monohydroxamate scavenges ferryl myoglobin radicals.

The addition of 25 microM hydrogen peroxide to 20 microM metmyoglobin produces ferryl (FeIV = O) myoglobin. Optical spectroscopy shows that the ferryl species reaches a maximum concentration (60-70% of total haem) after 10 minutes and decays slowly (hours). Low temperature EPR spectroscopy of the high spin metmyoglobin (g = 6) signal is consistent with these findings. At this low peroxide concentration there is no evidence for iron release from the haem. At least two free radicals are detectable by EPR immediately after H2O2 addition, but decay completely after ten minutes. However, a longer-lived radical is observed at lower concentrations that is still present after 90 minutes. The monohydroxamate N-methylbutyro-hydroxamic acid (NMBH) increases the rate of decay of the fenyl species. In the presence of NMBH, none of the protein-bound free radicals are detectable; instead nitroxide radicals produced by oxidation of the hydroxamate group are observed. Similar results are observed with the trihydroxamate, desferrioxamine. "Ferryl myoglobin" is still able to initiate lipid peroxidation even after the short-lived protein free radicals are no longer detectable (E.S.R. Newman, C.A. Rice-Evans and M.J. Davies (1991) Biochemical and Biophysical Research Communications 179, 1414-1419). It is suggested that the longer-lived protein radicals described here may be partly responsible for this effect. The mechanism of inhibition of initiation of lipid peroxidation by hydroxyamate drugs, such as NMBH, may therefore be due to reduction of the protein-derived radicals, rather than reduction of ferryl haem.

Animals↗
PubMed1993

Slow ('resting') forms of mitochondrial cytochrome c oxidase consist of two kinetically distinct conformations of the binuclear CuB/a3 centre--relevance to the mechanism of proton translocation.

We have purified slow ('resting') cytochrome oxidase from bovine heart, free of contamination with fast ('pulsed') enzyme. This form of the enzyme shows two kinetic phases of reduction of haem a3 by dithionite (k = 0.020 +/- 0.005 s-1 and k = 0.005 +/- 0.002 s-1). The presence of ligands that bind to the oxidized or reduced binuclear centre (formate or carbon monoxide respectively) has no effect on these rates. Varying the dithionite concentration also has no effect on either phase, although at low dithionite concentrations a lag phase is observed as the rate of haem a reduction is slower. The results are consistent with a model for reduction of the slow enzyme where the rate of electron transfer to the binuclear centre is the limiting step, rather than an equilibrium model where the haem a3 redox potential is low. Increasing the pH decreases the rate of the slower phase of dithionite reduction, but has no effect on the faster phase. EPR studies show that the slow phase (only) correlates with the disappearance of the g' = 12/g' = 2.95 signals, with the same pH dependence; again the presence of formate has no effect on these results. Deconvolution of the oxidized optical spectra shows that the enzyme reduced in the slow phase has a blue-shifted Soret band, relative to that reduced in the faster phase. Incubation of the oxidized enzyme at high pH causes a line-broadening of both the g' = 12 and g' = 2.95 EPR signals with no obvious effect on the amount of signal. The results are interpreted in a model where the presence of a carboxylate bridge between haem a3 and CuB defines the slow enzyme. It is suggested that the two rates of dithionite reduction are the result of different ligation to CuB--where water is the ligand the binuclear centre is FeIV/CuI (EPR-silent) and where hydroxide is the ligand the binuclear centre is FeIII/CuII (g' = 12/g' = 2.95 EPR signals).

Animals↗
PubMed1993

Control analysis applied to single enzymes: can an isolated enzyme have a unique rate-limiting step?

Control analysis is used to analyse and quantify the concept of a rate-limiting step within an enzyme. The extent to which each rate constant within the enzyme limits the steady-state rate of the enzyme and the levels of enzyme intermediate species are quantified as flux and concentration control coefficients. These coefficients are additive and obey summation theorems. The control coefficients of triose phosphate isomerase, carbamate kinase and lactate dehydrogenase are calculated from literature values of the rate constants. It is shown that, contrary to previous assumption, these enzymes do not have a unique rate-limiting step, but rather flux control is shared by several rate constants and varies with substrate, product and effector concentrations, and with the direction of the reaction. Thus the general assumption that an enzyme will have a unique rate-limiting step is unjustified.

Enzymes↗
PubMed1993

Kinetics of inhibition of purified and mitochondrial cytochrome c oxidase by psychosine (beta-galactosylsphingosine).

1. Psychosine (beta-galactosylsphingosine) is the toxic agent in Krabbe's disease (globoid cells leukodystrophy). It inhibits purified bovine heart mitochondrial cytochrome c oxidase; there is a rapid phase of inhibition (complete within 10-15 s) and a slower phase (complete within 10-15 min). Both phases are also seen in rat liver mitochondria. IC50 is about 200 microM psychosine in the purified enzyme and less than 20 microM in mitochondria. Psychosine inhibition is due to binding to cytochrome oxidase, not cytochrome c. 2. Bovine heart submitochondrial particles show inhibition similar to rat liver mitochondria. However, although proteoliposomes containing bovine heart cytochrome oxidase show an identical fast phase, they have no noticeable slow phase of inhibition. Addition of phospholipid liposomes to submitochondrial particles relieved the majority of psychosine inhibition, consistent with the removal of those molecules binding in the slow phase. Psychosine can inhibit cytochrome oxidase molecules facing in either direction in proteoliposomes and submitochondrial particles, suggesting that it can rapidly interact with both sides of a membrane when added externally. 3. At high ionic strength, the presence of psychosine decreases the Vmax. of cytochrome oxidase with little effect on the Km for cytochrome c. This non-competitive inhibition suggests that the psychosine-enzyme complex is kinetically inactive and not labile over the time course of the assay. Psychosine does not inhibit the reduction of haem a or haem a3 by artificial electron donors, but does inhibit the reduction of haem a by cytochrome c.

Animals↗