A combined n.m.r. and molecular biological approach to studying enzymes in vivo.
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Adenosine Triphosphate↗
Biomedical subjects
K M Brindle
Publications and source records attributed to K M Brindle.
31P-NMR studies of glucose and glutamine metabolism in cultured mammalian cells.
31P-NMR measurements of the concentrations of phosphorus-containing metabolites in mammalian cells immobilised and perifused with glucose and glutamine as sole carbon source have shown that the intracellular Pi concentration is significantly higher in cells perifused with glutamine than with glucose. The data are consistent with the proposal that the rate of glutamine utilisation may be controlled by the activity of phosphate-activated glutaminase.
Animals↗
31P NMR magnetization-transfer measurements of ATP turnover during steady-state isometric muscle contraction in the rat hind limb in vivo.
31P NMR magnetization-transfer measurements have been used to measure the flux between ATP and inorganic phosphate during steady-state isometric muscle contraction in the rat hind limb in vivo. Steady-state contraction was obtained by supramaximal sciatic nerve stimulation. Increasing the stimulation pulse width from 10 to 90 ms, at a pulse frequency of 1 Hz, or increasing the frequency of a 10-ms pulse from 0.5 to 2 Hz resulted in an increase in the flux which was an approximately linear function of the increase in the tension-time integral. The flux showed an approximately linear dependence on the calculated free cytosolic ADP concentration up to an ADP concentration of about 90 microM. The data are consistent with control of mitochondrial ATP synthesis by the cytosolic ADP concentration and indicate that the apparent Km of the mitochondria for ADP is at least 30 microM.
Adenosine Triphosphate↗
Phospholipid bilayer contribution to 31P NMR spectra in vivo.
The magnetic field-dependent phosphodiester (PDE) signal found in 31P NMR spectra of liver and brain has been studied using saturation transfer and proton decoupling techniques. This PDE component, which accounts for as much as 45% of the signal in vivo, has been identified as primarily phospholipid bilayer with a small contribution from a motionally averaged macromolecule(s).
Animals↗
Determination of buffering capacity of rat myocardium during ischemia.
To determine the buffering capacity of ischemic rat myocardium, lactate production was altered by glycogen depletion prior to total global ischemia. Lactate production was monitored by 1H-NMR spectroscopy in perfused rat hearts and determined by enzymatic assay of freeze-clamped tissue extracts. Intracellular pH was measured by 31P-NMR spectroscopy. The relationship between total lactate produced and pH varied considerably, depending on the final pH reached. At pH greater than 6.4 this relationship is linear with a total buffering capacity (delta lactate/delta pH) of 25 mumol H+/g wet weight per pH unit. At lower pH values (pH less than 6.4), the total buffering capacity increases progressively. Since ischemia is invariably accompanied by ATP and phosphocreatine (PCr) hydrolysis, the proton production/consumption during high-energy phosphate hydrolysis must be considered when evaluating the intrinsic buffering capacity of the myocardium against proton loads produced by lactate production from glucose and glycogen. Schemes are presented which allow an estimation of the contribution of ATP and PCr hydrolysis and the buffering by the CO2/HCO3- system during ischemia. At pH greater than 6.4, the majority (about 60%) of buffering is due to hydrolysis of adenosine triphosphate, phosphocreatine in the heart, and neutralization of sodium bicarbonate in the perfusate. At pH less than 6.4 an increasing proportion of cardiac buffering is from intrinsic cardiac buffers, most likely from intracellular proteins. After correction for these contributions to the observed total cardiac buffering capacity, the intrinsic buffering capacity of the myocardium can be accounted for by a high capacity (170 mumol/g wet weight) but low pKa (5.2) buffering system.
Adenosine Triphosphate↗
31P NMR magnetization-transfer measurements of flux between inorganic phosphate and adenosine 5'-triphosphate in yeast cells genetically modified to overproduce phosphoglycerate kinase.
31P NMR magnetization-transfer measurements were used to measure flux between inorganic phosphate and ATP in the reactions catalyzed by phosphoglycerate kinase and glyceraldehyde-3-phosphate dehydrogenase in anaerobic cells of the yeast Saccharomyces cerevisiae. Flux between ATP and Pi and glucose consumption and ethanol production were measured in cells expressing different levels of phosphoglycerate kinase activity. Overexpression of the enzyme was obtained by transforming the cells with a multicopy plasmid containing the phosphoglycerate kinase coding sequence and portions of the promoter element. Fluxes were also measured in cells in which the glyceraldehyde-3-phosphate dehydrogenase activity had been lowered by limited incubation with iodoacetate. These measurements showed that both enzymes have low flux control coefficients for glycolysis but that phosphoglycerate kinase has a relatively high flux control coefficient for the ATP----Pi exchange catalyzed by the two enzymes. The Pi----ATP exchange velocities observed in the cell were shown to be similar to those displayed by the isolated enzymes in vitro under conditions designed to mimic those in the cell with respect to the enzyme substrate concentrations.
Adenine Nucleotides↗
31P-NMR saturation transfer studies of aerobic Escherichia coli cells.
31P-NMR measurements of saturation transfer have been used to measure the flux between Pi and ATP in Escherichia coli cells respiring on an endogenous carbon source. Measurements were made in the wild type and in cells genetically modified to give a 5-fold higher concentration of the F1F0-ATP synthase. The flux in the two cell types was not significantly different. This, together with studies using inhibitors specific for the glycolytic enzyme, glyceraldehyde-3-phosphate dehydrogenase and the ATP synthase, suggests that the observed flux arises predominantly from glycolytic rather than ATP synthase activity. Although this conclusion is in disagreement with previous experiments on E. coli, it is in agreement with recent experiments on yeast.
Adenosine Triphosphate↗
31P NMR measurements of myocardial pH in vivo.
A 31P NMR magnetization transfer method for measuring myocardial pH in vivo is demonstrated in the lamb, dog and cat. The method involves measuring the difference in chemical shift between the resonances of phosphocreatine and inorganic phosphate in magnetization transfer difference spectra in which the gamma-phosphate resonance of ATP has been saturated. The method has been verified by measuring the chemical shift difference between the resonances of 2-deoxyglucose 6-phosphate and phosphocreatine following infusion of the animals with 2-deoxyglucose. The measured pH values are significantly lower than those obtained in previous studies on the heart in vivo.
Animals↗
31P-NMR saturation transfer measurements of exchange between Pi and ATP in the reactions catalysed by glyceraldehyde-3-phosphate dehydrogenase and phosphoglycerate kinase in vitro.
Recent 31P-NMR saturation transfer measurements of flux between Pi and ATP in the perfused rat heart (Kingsley-Hickman, P., Sako, E.Y., Andreone, P.A., St. Cyr, J.A., Michurski, S., Foker, J.E., From, A.H.L., Petein, M. and Ugurbil, K. (1986) FEBS Lett. 198, 159-163) have given a P/O ratio (mols ATP synthesised/atoms oxygen consumed) which was close to 6. This anomalously high value was attributed to exchange in the reaction catalysed by the mitochondrial F1F0-ATP synthase. We show here that this exchange could also be catalysed by the glycolytic enzymes glyceraldehyde-3-phosphate dehydrogenase and phosphoglycerate kinase. 31P-NMR saturation transfer measurements of the exchange catalysed by these enzymes in vitro, under conditions designed to mimic those present in the perfused rat heart, have shown that they could catalyse a quantitatively significant Pi-ATP exchange in vivo. A three-site exchange model is used to investigate the effects of Pi-ATP exchange on saturation transfer measurements of the reverse flux in the creatine kinase reaction. A discrepancy in the measured and forward and reverse fluxes in this reaction has been attributed previously to the participation of the gamma-phosphate of ATP in other exchange reactions.
Adenosine Triphosphate↗
NMR studies of kinetics in cells and tissues.
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Animals↗
A 1H-NMR study of the activity expressed by lactate dehydrogenase in the human erythrocyte.
The activity expressed by lactate dehydrogenase in the human erythrocyte has been compared with the activity displayed by the isolated enzyme in vitro. Enzyme activity was measured by using 1H spin-echo NMR to measure non-invasively the velocity of hydrogen label exchange between the C-2 positions of two methyl-labelled lactate species. This method has significant advantages over a method which has been described previously. The exchange velocity observed in the cell was much lower than that expected based on a comparison with measurements of the exchange velocity in vitro under conditions thought to simulate the intracellular environment. Measurements of enzyme inhibition in cell extracts suggest that the low intracellular activity is due to inhibition of the enzyme by low-molecular-mass compounds present in the cell.
Erythrocytes↗
Measurements of exchange in the reaction catalysed by creatine kinase using 14C and 15N isotope labels and the NMR technique of saturation transfer.
31P-NMR measurements of saturation transfer have been used to measure exchange between the gamma-phosphate of ATP and phosphocreatine and between the beta-phosphate of ATP and the beta-phosphate of ADP in the reaction catalysed by creatine kinase in vitro. The calculated exchange fluxes have been compared with measurements of 15N label exchange between creatine and phosphocreatine and 14C label exchange between ATP and ADP. At pH 8.0 the fluxes between phosphocreatine and the gamma-phosphate of ATP and between the beta-phosphates of ATP and ADP, measured by saturation transfer, were the same and equal, within experimental error, to the fluxes between creatine and phosphocreatine, measured by 15N label exchange, and between ADP and ATP, measured by 14C label exchange. At pH 7.0 the flux between phosphocreatine and the gamma-phosphate of ATP, measured by saturation transfer, was equal, within experimental error, to the flux between creatine and phosphocreatine, measured by 15N label exchange. However, at low ADP concentrations (less than 0.2 mM), the flux between ATP and ADP measured by saturation transfer was significantly less than that between phosphocreatine and ATP and, more importantly, less than the ADP-ATP exchange flux measured by 14C label exchange. The saturation transfer and isotope exchange measurements at pH 7.0 have shown that it is valid to equate saturation transfer measurements of exchange between phosphocreatine and ATP in vivo with the potential for net chemical flux through the reaction. The observed discrepancy at pH 7.0 between the 14C and saturation transfer measurements of ATP----ADP exchange can be explained if there is significant loss of saturation in an intermediate in the exchange reaction. Under these conditions analysis of the exchange according to two-site exchange model is invalid. In magnetisation transfer measurements of exchange in other enzyme catalysed reactions, the possible presence of a kinetically significant intermediate and therefore the validity of data analysis using a two-site exchange model should be considered.
Adenosine Diphosphate↗
A multinuclear NMR study of 2,3-bisphosphoglycerate metabolism in the human erythrocyte.
Resonances from 13C, 31P and 1H have been detected simultaneously in suspensions of human erythrocytes using a modified NMR spectrometer equipped with a probe tuned to four different frequencies. The utility of multinuclear NMR in the study of cellular metabolism is demonstrated with an investigation of 13C label flux through the 2,3-bisphosphoglycerate bypass in human erythrocytes. In a single experiment, the respective contributions of this bypass and the pentose-phosphate shunt were found to be 27 and 10% of the total glycolytic rate.
2,3-Diphosphoglycerate↗
The inhibition of erythrocyte glyceraldehyde-3-phosphate dehydrogenase. In situ PMR studies.
The kinetics of inhibition of human erythrocyte glyceraldehyde-3-phosphate dehydrogenase by iodoacetate were studied in the intact cell and in vitro. The kinetics were determined using 1H-NMR to follow solvent exchange of 1H and 2H at the C-2 position of lactate. The exchange occurs via a series of enzyme-catalysed reactions, including that catalysed by glyceraldehyde-3-phosphate dehydrogenase. A direct assay with quenching of the inhibition was also used to check the results. Iodoacetate was shown to act as an active site-directed inhibitor of the dehydrogenase. The enzyme inhibition patterns, which are characterised by a binding step and a kinetic step, are similar in situ and in vitro. Membrane binding, however, was found to alter the inhibition pattern for the enzyme in vitro.
Deuterium↗
A comparison of 31P-NMR saturation transfer and isotope-exchange measurements of creatine kinase kinetics in vitro.
31P-NMR measurements of saturation transfer have been used to measure the exchange of phosphate between phosphocreatine and the gamma-phosphate of ATP in the reaction catalysed by creatine kinase in vitro. The similarity of the calculated exchange flux with the flux estimated from an isotope-exchange experiment, in which exchange of 15N label between creatine and phosphocreatine was measured, showed that the two-site-exchange model, normally used in the analysis of saturation-transfer data, is valid in this case. 15N label exchange was monitored using a heteronuclear 31P/15N spin-echo NMR experiment in which the incorporation of 15N label into phosphocreatine was detected by following the phase modulation of the spin-spin coupled 31P resonance. The isotope-exchange experiment should prove to be useful in studies of creatine kinase in systems where the low concentration of the enzyme precludes saturation-transfer measurements, for example in muscle mitochondria preparations.
Creatine Kinase↗
Proton NMR measurements of hydrogen exchange at the C-3 position of 3-hydroxybutyrate in suspensions of rat liver mitochondria.
Rat liver mitochondria suspended in buffer made with 2H2O catalyse exchange C-3 proton of added D-3-hydroxybutyrate with solvent deuterons. The kinetics of this process can be followed using spin-echo proton NMR. The observed isotope exchange velocity is sensitive to the activity of D-3-hydroxybutyrate dehydrogenase, which is an integral protein of the inner mitochondrial membrane. A method is described which can be used to obtain the specific isotope exchange velocity of the enzyme in the intact mitochondrion.
3-Hydroxybutyric Acid↗
Centrifugal analysis of undiluted packed human erythrocyte lysates. Studies of the association of glyceraldehyde-phosphate dehydrogenase with the membrane fraction.
Concentrated human erythrocyte lysates (greater than 99% initial haematocrit) were subjected to high centrifugal fields. This caused the membrane fraction to separate from the cytoplasmic portion, due to the lower density of the former. Enzyme distribution data indicated that glyceraldehyde-phosphate dehydrogenase was predominantly in the cytoplasmic fraction.
Cell Fractionation↗
Observation of carbon labelling in cell metabolites using proton spin echo NMR.
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Alanine↗