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K M Brindle

Publications and source records attributed to K M Brindle.

At least 37 records · Page 2Linked to original sources
PubMed1999

Mitochondrial proton leak and the uncoupling proteins.

An energetically significant leak of protons occurs across the mitochondrial inner membranes of eukaryotic cells. This seemingly wasteful proton leak accounts for at least 20% of the standard metabolic rate of a rat. There is evidence that it makes a similar contribution to standard metabolic rate in a lizard. Proton conductance of the mitochondrial inner membrane can be considered as having two components: a basal component present in all mitochondria, and an augmentative component, which may occur in tissues of mammals and perhaps of some other animals. The uncoupling protein of brown adipose tissue, UCP1, is a clear example of such an augmentative component. The newly discovered UCP1 homologs, UCP2, UCP3, and brain mitochondrial carrier protein 1 (BMCP1) may participate in the augmentative component of proton leak. However, they do not appear to catalyze the basal leak, as this is observed in mitochondria from cells which apparently lack these proteins. Whereas UCP1 plays an important role in thermogenesis, the evidence that UCP2 and UCP3 do likewise remains equivocal.

Adipose Tissue, Brown↗
PubMed1999

The significance and mechanism of mitochondrial proton conductance.

There is a futile cycle of pump and leak of protons across the mitochondrial inner membrane. The contribution of the proton cycle to standard metabolic rate is significant, particularly in skeletal muscle, and it accounts for 20% or more of the resting respiration of a rat. The mechanism of the proton leak is uncertain: basal proton conductance is not a simple biophysical leak across the unmodified phospholipid bilayer. Equally, the evidence that it is catalysed by homologues of the brown adipose uncoupling protein, UCP1, is weak. The yeast genome contains no clear UCP homologue but yeast mitochondria have normal basal proton conductance. UCP1 catalyses a regulated inducible proton conductance in brown adipose tissue and the possibility remains open that UCP2 and UCP3 have a similar role in other tissues, although this has yet to be demonstrated.

Animals↗
PubMed1998

Induction of apoptosis in two mammalian cell lines results in increased levels of fructose-1,6-bisphosphate and CDP-choline as determined by 31P MRS.

Programmed cell death or apoptosis was induced in human promyelocytic leukemia (HL-60) and Chinese hamster ovary (CHO-K1) cells using several cytotoxic drugs that have different modes of action, including camptothecin, ceramide, chelerythrine, etoposide, farnesol, geranyl geraniol, and hexadecylphosphocholine. The consequent changes in cellular metabolism were monitored using 31P MRS measurements on intact cells and cell extracts. Cells undergoing programmed cell death exhibited characteristic changes in the levels of glycolytic and phospholipid metabolites. The most significant changes were increases in the concentration of the glycolytic intermediate, fructose-1,6-bisphosphate and in the concentration of CDP-choline, which is an intermediate in phosphatidylcholine biosynthesis. In HL-60 cells, the increase in fructose-1,6-bisphosphate levels could be explained by depletion of cellular NAD(H) levels. All of the agents used to induce apoptosis caused the accumulation of CDP-choline. Since the resonances of this compound occur in a relatively well resolved region of tissue spectra, it could provide a marker for apoptosis that would allow the noninvasive detection of the process in vivo using 31P MRS measurements.

Animals↗
PubMed1998

Magnetic resonance imaging and spectroscopy of combretastatin A4 prodrug-induced disruption of tumour perfusion and energetic status.

The effects of combretastatin A4 prodrug on perfusion and the levels of 31P metabolites in an implanted murine tumour were investigated for 3 h after drug treatment using nuclear magnetic resonance imaging (MRI) and spectroscopy (MRS). The area of regions of low signal intensity in spin-echo images of tumours increased slightly after treatment with the drug. These regions of low signal intensity corresponded to necrosis seen in histological sections, whereas the expanding regions surrounding them corresponded to haemorrhage. Tumour perfusion was assessed before and 160 min after drug treatment using dynamic MRI measurements of gadolinium diethylenetriaminepentaacetate (GdDTPA) uptake and washout. Perfusion decreased significantly in central regions of the tumour after treatment. This was attributed to disruption of the vasculature and was consistent with the haemorrhage seen in histological sections. The mean apparent diffusion coefficient of water within the tumour did not change, indicating that there was no expansion of necrotic regions during the 3 h after drug treatment. Localized 31P-MRS showed that there was decline in cellular energy status in the tumour after treatment with the drug. The concentrations of nucleoside triphosphates within the tumour fell, the inorganic phosphate concentration increased and there was a significant decrease in tumour pH for 80 min after drug treatment. The rapid, selective and extensive damage caused to these tumours by combretastatin A4 prodrug has highlighted the potential of the agent as a novel cancer chemotherapeutic agent. We have shown that the response of tumours to treatment with the drug may be monitored non-invasively using MRI and MRS experiments that are appropriate for use in a clinical setting.

Animals↗
PubMed1997

19F NMR measurements of the rotational mobility of proteins in vivo.

Three glycolytic enzymes, hexokinase, phosphoglycerate kinase, and pyruvate kinase, were fluorine labeled in the yeast Saccharomyces cerevisiae by biosynthetic incorporation of 5-fluorotryptophan. 19F NMR longitudinal relaxation time measurements on the labeled enzymes were used to assess their rotational mobility in the intact cell. Comparison with the results obtained from relaxation time measurements of the purified enzymes in vitro and from theoretical calculations showed that two of the labeled enzymes, phosphoglycerate kinase and hexokinase, were tumbling in a cytoplasm that had a viscosity approximately twice that of water. There were no detectable signals from pyruvate kinase in vivo, although it could be detected in diluted cell extracts, indicating that there was some degree of motional restriction of the enzyme in the intact cell.

Fluorine↗
PubMed1996

31P NMR magnetization transfer study of the control of ATP turnover in Saccharomyces cerevisiae.

31P NMR magnetization transfer measurements have been used to measure the steady state flux between Pi and ATP in yeast cells genetically modified to overexpress an adenine nucleotide translocase isoform. An increase in Pi -> ATP flux and apparent ratio of moles of ATP synthesized/atoms of oxygen consumed (P:O ratio), when these cells were incubated with glucose, demonstrated that the reactions catalyzed by the translocase and F1F0 ATP synthase were readily reversible in vivo. However, when the same cells were incubated with ethanol alone, translocase overexpression had no effect on the measured Pi -> ATP flux or apparent P:O ratio, suggesting that the synthase was now operating irreversibly. This change was accompanied by an increase in the intracellular ADP concentration. These observations are consistent with a model proposed for the kinetic control of mitochondrial ATP synthesis, which was based on isotope exchange measurements with isolated mammalian mitochondria [LaNoue, K. F., Jeffries, F. M. H. & Radda, G. K. (1986) Biochemistry 25, 7667-7675].

Adenosine Triphosphate↗
PubMed1996

Analysis of metabolic control in vivo using molecular genetics.

The aim of this brief review has been to illustrate the enormous power of molecular genetic techniques for testing out our, sometimes old, models of metabolic control. The technology allows us to ask some very simple but direct questions about the importance of specific enzymes in the control of flux. The answers, however, may not always be straightforward in that the absence of a change in pathway flux does not necessarily indicate that the enzyme is unimportant in flux control. Rather it would seem, from the relatively few studies that have been done, that the changes in the biological system which occur in response to a change in the concentration of a specific enzyme could tell us a lot about the role of that enzyme in metabolism and how it is controlled. I have restricted my discussion to studies in which relatively large numbers of cells have been modified genetically and subsequently characterized. This ignores a large and growing area in which micro-injection techniques are being used to introduce plasmid DNA or proteins into individual cells. The resultant phenotypes are then characterized at the single cell level using very sensitive optical techniques, such as fluorescence and bioluminescence. The reader is referred to a recent article for an example of this type of approach.

Animals↗
PubMed1996

31P NMR measurements of the effects of unsaturated fatty acids on cellular phospholipid metabolism.

31P NMR measurements on extracts prepared from a variety of cultured mammalian cell lines and primary rat hepatocytes have shown changes in the levels of several phospholipid metabolites after incubation of cells with unsaturated fatty acids. These data suggest a possible link between the accumulation of neutral lipid and the changes in phospholipid metabolite concentrations that have been observed in some tumor cells and other rapidly growing tissues such as the regenerating liver and mitogen-stimulated lymphocytes.

Animals↗
PubMed1995

Differential localization of the mRNA of the M and B isoforms of creatine kinase in myoblasts.

Creatine kinase (CK) plays an important role in buffering ATP and ADP levels in tissues which have intermittently high and fluctuating energy demands, such as skeletal muscle. This buffering function has a spatial, as well as a temporal aspect, which is dependent on the localization of different enzyme isoforms within the cell. We show here, by in situ hybridization, that the mRNAs for the cytoplasmic isoforms of CK are differentially localized in a mouse myoblast cell line (C2C12). The mRNA for the M form is localized at the cell periphery, while that for the B form is localized in the perinuclear region. Deletion of segments of the 3' untranslated regions of these mRNAs or swapping of these segments between the mRNAs for the two isoforms demonstrated that localization signals lie within these regions. Localization appears to be tissue-specific, since both the M and B mRNAs were distributed uniformly over the cytoplasm in a non-muscle cell line. These results, in conjunction with other studies which have shown that mRNA localization can lead to co-localization of the encoded protein, suggest that the localization of the mRNAs for the cytoplasmic isoforms of CK may be involved in the localization of the enzymes themselves.

Animals↗
PubMed1993

Estimation of the intracellular free ADP concentration by 19F NMR studies of fluorine-labeled yeast phosphoglycerate kinase in vivo.

Yeast phosphoglycerate kinase was selectively fluorine-labeled in vivo by inducing enzyme synthesis in stationary phase cells in the presence of 5-fluorotryptophan. Inducible expression was obtained using a galactose-inducible expression vector containing the yeast phosphoglycerate kinase coding sequence. 19F NMR measurements on intact cells showed two resolved resonances, from the two tryptophan residues in the protein, which underwent reversible changes in chemical shift under different metabolic conditions. Measurements in vitro showed that the difference in the chemical shifts of these two resonances was dependent on the adenine nucleotide concentration, in particular the MgADP concentration. A comparison of the spectra obtained in vitro with those obtained from the intact cell indicated that in glucose-fed cells the cytosolic free MgADP concentration was less than 50 microM, which is significantly lower than the concentrations measured in whole-cell extracts.

Adenine Nucleotides↗
PubMed1993

A 1H/15N n.m.r. study of nitrogen metabolism in cultured mammalian cells.

1. Heteronuclear 1H/15N n.m.r. experiments are described in which 15N labelling of cellular metabolites is detected via their proton resonances. 2. These n.m.r. experiments have been used to monitor label redistribution amongst extracellular metabolites in cultures of mammalian cells incubated with L-[2-15N]glutamine, L-[5-15N]glutamine and 15NH4Cl. Label redistribution was monitored in two HeLa cell lines and in two CHO cell lines which showed a range of extractable activities of glutamate dehydrogenase, glutaminase and glutamine synthetase. 3. In cells incubated with L-[2-15N]glutamine the 15N label was subsequently found in a number of metabolites including alanine, aspartate, glycine and pyrrolidone-5-carboxylic acid. There was no detectable production of 15NH4+, showing that most of the glutamate formed in the reaction catalysed by glutaminase was subsequently transaminated rather than oxidatively deaminated by glutamate dehydrogenase. 4. Incubation of cells with L-[5-15N]glutamine showed that the ammonia in the cultures was derived predominantly from the amide group of glutamine. 5. The rate of formation of L-[5-15N]glutamine in cells incubated with 15NH4Cl was used to estimate glutamine synthetase flux in vivo. Flux in this reaction was only observable in the two CHO cell lines which express relatively high levels of the enzyme.

Ammonia↗
PubMed1993

Enzymology in vivo using NMR and molecular genetics.

Models of metabolic flux regulation are frequently based on an extrapolation of the kinetic properties of enzymes measured in vitro to the intact cell. Such an extrapolation assumes a detailed knowledge of the intracellular environment of these enzymes in terms of their free substrate and effector concentrations and possible interactions with other cellular macromolecules, which may modify their kinetic properties. There is a considerable incentive, therefore, to study the properties of enzymes directly in vivo. We have been using non-invasive NMR techniques, in conjunction with molecular genetic manipulation of enzyme levels, to study the kinetic properties of individual enzymes in vivo. We have also developed a novel labelling strategy which has allowed us to monitor, by NMR, the ligand binding properties and mobilities of enzymes in the intact cell. This technique may also allow us to measure the diffusion coefficients of these proteins in the cell. These studies should give new insight into the properties of enzymes in vivo.

Animals↗
PubMed1993

The appearance of neutral lipid signals in the 1H NMR spectra of a myeloma cell line correlates with the induced formation of cytoplasmic lipid droplets.

The appearance of high resolution neutral lipid signals in the 1H NMR spectra of myeloma cells grown in the presence of oleate was shown to correlate with the appearance of cytoplasmic lipid droplets observable by electron microscopy. The spin-spin relaxation times of these lipid signals were similar to those measured previously for lipid resonances in other cell types. These data suggest that cytoplasmic lipid droplets could make a significant contribution to the neutral lipid signals observed in the 1H NMR spectra of some cells.

Animals↗
PubMed1992

Endoplasmic reticulum: the major contributor to the PDE peak in hepatic 31P-NMR spectra at low magnetic field strengths.

31P-NMR spectra of liver in vivo, subcellular fractions and model systems were acquired in order to characterise further the hepatic phosphodiester peak seen at low magnetic field strengths previously shown to be predominantly due to phospholipid bilayers. The data obtained in this study in vitro suggested that the phospholipid membranes of the endoplasmic reticulum provide the dominant contribution to this phosphodiester peak. Support for this hypothesis was provided by experiments on rats. Phenobarbitone, which is known to induce proliferation of the endoplasmic reticulum produced a considerable increase in intensity of the phosphodiester peak in liver spectra in vivo.

Animals↗
PubMed1992

Effects of overexpression of phosphofructokinase on glycolysis in the yeast Saccharomyces cerevisiae.

The influence of 6-phosphofructo-1-kinase on glycolytic flux in the yeast Saccharomyces cerevisiae was assessed by measuring the effects of enzyme overexpression on glucose consumption, ethanol production, and glycolytic intermediate levels under aerobic and anaerobic conditions. Enzyme overexpression had no effect on glycolytic flux under anaerobic conditions, but under aerobic conditions, it increased glycolytic flux up to the anaerobic level. The Pasteur effect was thus abolished in these cells. The increased glycolytic flux was accompanied by a compensatory decrease in flux in oxidative phosphorylation. The concentrations of the enzyme substrates showed only small or insignificant changes. These data imply that the enzyme has a low flux control coefficient for glycolysis. However, in cells overexpressing the enzyme, there was a compensatory decrease in 6-phosphofructo-2-kinase activity which was accompanied by a corresponding decrease in fructose 2,6-bisphosphate concentration. Measurements in vitro showed that the decrease in the concentration of this positive allosteric effector of 6-phosphofructo-1-kinase could significantly lower its specific activity in the cell and that this could compensate for the increased enzyme concentration in the overproducer.

Anaerobiosis↗
PubMed1992

Changes in phosphatidylethanolamine metabolism in regenerating rat liver as measured by 31P-NMR.

31P-NMR spectra of regenerating rat liver in vivo show increases in resonance intensities in the phosphomonoester (PME) region and decreases in the phosphodiester (PDE) region as early as 12 h post partial hepatectomy, which return to normal by 8 days. The compounds primarily responsible for these changes have been identified in perchloric acid extracts as the phosphomonoester phosphoethanolamine and the phosphodiester glycerophosphoethanolamine (GPE), indicating altered phosphatidylethanolamine metabolism. A corresponding increase in diacylglycerol (DAG) levels during regeneration indicates a possible role for a phosphatidylethanolamine-specific phospholipase C in cellular proliferation. These results suggest that changes in phospholipid metabolites previously associated with neoplastic tissue can also be induced by normal tissue undergoing rapid cellular proliferation. The spectral changes observed in the regenerating rat liver are similar to changes seen in spectra from the livers of human patients in several disease states, indicating that 31P-NMR may allow non-invasive study of cell turnover in liver disease.

Adenosine Triphosphate↗