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Biomedical subjects

R G Shulman

Publications and source records attributed to R G Shulman.

At least 199 records · Page 11Linked to original sources
PubMed1979

Cellular applications of 31P and 13C nuclear magnetic resonance.

High-resolution nuclear magnetic resonance (NMR) studies of cells and purified mitochondria are discussed to show the kind of information that can be obtained in vivo. In suspensions of Escherichia coli both phosphorus-31 and carbon-13 NMR studies of glycolysis and bioenergetics are presented. In rat liver cells the pathways of gluconeogenesis from carbon-13-labeled glycerol are followed by carbon-13 NMR. In the intact liver cells cytosolic and mitochondrial pH's were separately measured by phosphorus-31 NMR. In purified mitochondria the internal and external concentrations of inorganic phosphate, adenosine diphosphate, and adenosine triphosphate were determined by phosphorus-31 NMR while the pH difference across the membrane was measured simultaneously.

Adenosine Diphosphate↗
PubMed1979

Effects of ethanol on alanine metabolism in perfused mouse liver studied by 13C NMR.

Time courses of 13C labeling from alanine and ethanol in perfused mouse livers have been followed by NMR. The enrichment at specific carbons of glucose, glutamate, glutamine, aspartate, acetate, acetoacetate, beta-hydroxybutyrate, and lactate has been measured. The specific labeling of glutamate in the presence of labeled alanine and labeled or unlabeled ethanol shows that, under these conditions, alanine enters the tricarboxylic acid cycle almost exclusively through pyruvate carboxylation, whereas ethanol is the exclusive source of acetyl-CoA. In the absence of ethanol, the alanine label flows through both paths. By comparing the scrambling of 13C between C3 and C2 of glutamate it is possible to estimate the mitochondrial fumarase activity; the C6-to-C5 ratios in glucose give the additional scrambling by cytosolic fumarase activity. In addition, the C6-to-C1 and C5-to-C2 ratios in glucose show that there is about 15% flux through the pentose cycle. Finally, the C4-to-C2 ratios in glutamine and glutamate are unequal at any time (the glutamine labels reflect the label distribution in glutamate measured 1 hr earlier), providing a method for studying flow through glutamine synthetase in situ.

Alanine↗
PubMed1979

13C nuclear magnetic resonance studies of anaerobic glycolysis in suspensions of yeast cells.

Anaerobic glycolysis in Saccharomyces cerevisiae has been studied by 13C NMR at 90.5 MHz. [1-13c]Glucose and [6-13C]glucose were fed to suspensions of yeast cells. Time courses for concentration changes of the starting material, of courses for concentration changes of the starting material, of the intermediate fructose 1,6-bisphosphate (Fru-P2), and of the end products, ethanol and glycerol, have been followed with 1-min time resolution. The glucose uptake was well fitted by a Michaelis-Menten model, assuming competition of alpha- and beta-glucose for the same site. The Km for the uptake was found to be 10 mM for beta-glucose and 5 mM for alpha-glucose. The concentration of Fru-P2 showed an initial oscillation before it reached a co,stant level. The 13C label, introduced only as [-13C]- or [6-13C]glucose, was observed in Fru-P2 in both the C1 and C6 positions, simultaneously. From the relative intensities of the C1 Fru-P2 and C6 Fru-P2 peaks in the presence of [1-13C]- and [6-13C]glucose, in vivo kinetic information was obtained about the aldolase-triosephosphate isomerase triangle. We found that under the conditions of these experiments the ratios of backward to forward velocities through aldolase and triosephosphate isomerase were 0.9 +/- 0.1 and 0.8 +/- 1, respectively, indicating they were close to equilibrium.

Anaerobiosis↗
PubMed1979

13C NMR studies of gluconeogenesis in rat liver cells: utilization of labeled glycerol by cells from euthyroid and hyperthyroid rats.

The gluconeogenic pathway from [2-13C]glycerol and [1,3-13C]glycerol has been followed in suspensions of isolated rat hepatocytes at 25 degrees C by 13C NMR at 90.5 MHz. The flow of label through the major pathway from glycerol to L-glycerol 3-phosphate and into glucose was followed in cells from control and triiodothyronine-treated rats. Treatment increased the rates of glucose formation and glycerol consumption 2-fold and decreased the alphaGP level to 40%. We calculate that approximately 60% of the flux is through the mitochondrial glycerol phosphate dehydrogenase in cells from triiodothyronine-treated rats, compared with approximately 15% in cells from the controls. Equal distribution of label between the trioses of glucose was obtained and, because the C3-C4 spin-spin coupling gives the distribution of labeled carbons in the same molecule, it was possible to measure the amount of triose from unlabeled fructose incorporated into the glucose labeled at carbons 1, 3, 4, and 6. About 10% of the hexoses had flowed through the pentose cycle and back into the hexose pathway in cells from fasted rats. From the distribution of label at glucose carbons not labeled via the major pathway and from the carbon spin-spin splitting patterns observed, we conclude that transketolase is reversible whereas transaldolase is essentially irreversible in the nonoxidative pentose branch.

Animals↗
PubMed1979

Adenine nucleotide storage and secretion in platelets as studied by 31P nuclear magnetic resonance.

Suspensions of human and pig blood platelets have been studied by 31P NMR at 145.7 MHz and by chemical and radiochemical determination of nucleotide levels. In both types of platelets the cytoplasmic nucleotide pool, which was prelabeled by incubation with [14C]adenine, was selectively reduced by addition of H2O2/NaN3 or 2-deoxyglucose/antimycin A. After the reduction of cytoplasmic ATP in human platelets, the 31P NMR spectra showed an almost complete loss of the nucleoside di- and triphosphate resonances at temperatures examined (4--50 degrees C), indicating that only the cytoplasmic nucleotides had been observed, with no detectable contributions from the granular ATP, ADP, and pyrophosphate. Slow tumbling of the granular nucleotides, possibly due to aggregation, is the probable explanation of their undetectability at 145.7 MHz. Similar experiments showed that in pig platelets, granular ATP and ADP were not detected by 31P NMR at 4 degrees C but were observed at higher temperatures, indicating that aggregation may be occurring at the lower temperatures. Upon thrombin stimulation of human platelets, the NMR spectra and the chemical and radioactivity analyses showed that the granular adenylates and pyrophosphate were secreted, and that cytoplasmic ATP levels were appreciably reduced.

Adenine Nucleotides↗
PubMed1978

Extended X-ray absorption fine structure determination of iron nitrogen distances in haemoglobin.

EXAFS spectra have been obtained of oxy and deoxy complexes of haemoglobin and of the 'picket fence' porphyrin, using synchrotron radiation as a source of X rays. The fluorescence data were Fourier filtered to obtain distances to the first shell and corrections applied to remove contributions from the axial ligands. In this way, the iron to porphinato nitrogen distances were determined to be 1.98 +/- 0.01 A for both oxygenated complexes and 2.055 +/- 0.01 A for both deoxy forms.

Hemoglobins↗
PubMed1978

On the measurement of pH in Escherichia coli by 31P nuclear magnetic resonance.

The 31P high resolution NMR spectra of concentrated suspensions of Escherichia coli cells have been measured at 145.8 MHz. The position of the orthophosphate resonance is used as a measure of internal and external pH. In accord with Paddan, Zilberstein and Rottenberg ((1976) Eur. J. Biochem. 63, 533--541) it is shown that when properly energized the internal pH is 7.5 +/- 0.1. By synchronizing the NMR data acquisition with 3-s bursts of O2 it is possible to measure the internal pH with a time resolution of about 1 s. It is shown that at 20 degrees C the pH remains constant for times longer than 15 s after the oxygen is discontinued and it decays in several minutes.

Escherichia coli↗
PubMed1978

Phosphate metabolites in lymphoid, Friend erythroleukemia, and HeLa cells observed by high-resolution 31P nuclear magnetic resonance.

High-resolution phosphorus-31 nuclear magnetic resonance spectra were observed for cell lines from mammalian sources: lymphoid cells, Friend erythroleukemia cells, and HeLa cells. Some phosphate metabolites were identified and their concentrations were found to vary among the different cell lines. ATP/ADP ratios could be measured in the living cells. The phospholipid metabolites phosphorylcholine and phosphorylethanolamine and their glyceryl esters were present in relatively high concentrations in the cells, but their ratios varied considerably. Also, a high concentration of an unidentified diphosphodiester compound was observed in the Friend erythroleukemia and HeLa cells. No appreciable pH gradient across the cell membrance was observed in these three mammalian cell lines, in contrast to the large pH gradient detected in microorganisms.

Adenosine Diphosphate↗
PubMed1978

High-resolution 31P nuclear magnetic resonance study of rat liver mitochondria.

Intact mitochondria were studied by high-resolution 31P nuclear magnetic resonance. Observable internal phosphate compounds included inorganic phosphate (Pi), ADP, and ATP. The internal pH was determined by the chemical shift of the internal Pi, the pK2 (6.7) of which was measured in uncoupled mitochondria. The observed equilibrium relation between the internal and the external Pi was consistent with the exchange equilibrium through the H2PO4-/OH- carrier. The internal ATP and ADP were essentially Mg2+ bound and their resonances were distinguishable from those of the external ATP and ADP by the chemical shift differences due to the Mg2+ concentration gradient and deltapH. Oxidative phosphorylation was followed by the separate resonances of Pi and adenine nucleotides both internal and external to mitochondria. From these resonances the internal and external phosphate potentials could be estimated.

Animals↗
PubMed1978

31P nuclear magnetic resonance studies of bioenergetics and glycolysis in anaerobic Escherichia coli cells.

31P nuclear magnetic resonance spectra of glycolyzing, anaerobic Escherichia coli cells and their perchloric acid extracts were obtained at 145.7 MHz. Time-dependent intracellular concentrations of nucleoside di- and triphosphates, Pi, and sugar phosphates were measured during glycolysis with 2-min resolution, while intracellular and extra-cellular pH values were monitored simultaneously. Upon glucose addition, anaerobic E. coli cells rapidly produce acids and develop a transmembrane pH gradient (delta pH). Glycolysis rates were calculated from the changes in the external pH. It was found that glycolysis rates are strongly dependent on internal pH, sharply decreasing when the pH drops below approximately 7.2. The ATPase inhibitor, dicyclohexylcarbodiimide (DCCD), prevented NTP hydrolysis and inhibited delta pH formation. The uncoupler, carbonyl cyanide p-triflouromethoxyphenyl hydrazone (FCCP), drastically reduced both the delta pH and the NTP level. When the cells were previously treated with DCCD, FCCP collapsed the delta pH while the NTP levels remained high. It is concluded that ATP produced by glycolysis is hydrolyzed by the membrane ATPase to generate a delta pH and that FCCP stimulates ATP hydrolysis by ATPase and collapses the proton gradient.

Anaerobiosis↗
PubMed1978

High-resolution 13C nuclear magnetic resonance studies of glucose metabolism in Escherichia coli.

High-resolution 13C nuclear magnetic resonance spectra of suspensions of Escherichia coli cells have been obtained at 90.5 MHz by using the Fourier transform mode. Anaerobic cells incubated with [I-13C]glucose show a time course of glycolysis in which the alpha and beta glucose anomers disappear at different rates, lactate, succinate, acetate, alanine, and valine accumulate as end products of glycolysis, and fructose bisphosphate appears as an intermediate. It is shown that fructose bisphosphate is labeled at C-1 and C-6 during [I-13C]-glucose catabolism. Upon oxygenation, glutamate appears with the 13C ENRICHMENT AT THE C-4, C-3, and C-2 positions, with the C-4 most intense. From the position of the 13C label we conclude that valine is formed by condensation of pyruvate and that carbon enters the tricarboxylic acid cycle mainly through acetyl CoA.

Anaerobiosis↗
PubMed1977

31P nuclear magnetic resonance studies of Ehrlich ascites tumor cells.

High-resolution 31P nuclear magnetic resonance spectra at 145.7 MHz are reported for intact Ehrlich ascites tumor cells and their perchloric acid extracts. In the extracts it was possible to assign resonances to fructose 1,6-bisphosphates, dihydroxyacetone phosphate, ATP, ADP, AMP, Pi, NAD+, phosphorylcholine, glycero-3-phosphorylcholine, glycero-3-phosphorylethanolamine, and glyceraldehyde 3-phosphate from their chemical shifts, pH behavior, and spin couplings. All but glyceraldehyde 3-phosphate were observed and assigned in the intact cells. It was possible to show that the hydrolysis of fructose 1,6-bisphosphate to dihydroxyacetone phosphate and glyceraldehyde 3-phosphate is in equilibrium, that the dihydroxyacetone phosphate leads to glyceraldehyde 3-phosphate reaction is not, and that in the intact cell without added oxygen or glucose the reaction 2ADP in equilibrium ATP + AMP is in equilibrium. From the known pH dependence of the Pi resonance it was possible to show that during aerobic or anerobic glycolysis the difference between intracellular and extracellular pH values was less than 0.2 pH units. Upon oxygenation the ATP concentration increased while the ADP concentration fell. Introducing deoxyglucose depleted the ATP and resulted in an AMP signal and one from deoxyglucose 6-phosphate, which is transported and phosphorylated but not catabolized.

Adenine Nucleotides↗
PubMed1977

31P nuclear magnetic resonance measurements of ATPase kinetics in aerobic Escherichia coli cells.

We have measured the in vivo unidirectional rates between the terminal phosphate of ATP and intracellular inorganic phosphate (PiIN) in aerobic suspensions of Escherichia coli cells using 31P nuclear magnetic resonance saturation transfer techniques. Typically, the measurements consisted of saturating the ATPgamma resonance and observing a 20 +/- 5% reduction in the intensity of the PiIN resonance. No saturation transfer was observed after incubation with 1 mM dicyclohexylcarbodiimide, an ATPase inhibitor. From the measured decrease in intensity of PiIN coming from saturation transfer, the apparent unimolecular rate constant of PiIN to ATP was calculated to be 0.6 +/- 0.15 sec-1.

Adenosine Triphosphatases↗
PubMed1977

High-resolution 31P nuclear magnetic resonance studies of metabolism in aerobic Escherichia coli cells.

31P nuclear magnetic resonance spectra at 145.7 MHZ were obtained of concentrated suspensions of E. coli cells. The position of the Pi resonance was used to determine the pH, and in most experiments it was possible to distinguish the intracellular (pHin) and extracellular (pHex) values. During respiration pHin approached 7.55, while pHex varied from 6.0 to 8.0. With succinate as a carbon source and in a N2 environment, pHin - pHex. Upon addition of glucose, pHin greater than pHex. In the presence of an ATPase (adenosinetriphosphatase; ATP phosphohydrolase; EC 3.6.1.3) inhibitor dicyclohexylcarbodiimide, pHin remained equal to pHex even in the presence of glucose. In other experiments, oxygenation brought pHin above pHex even in the presence of dicyclohexylcarbodiimide. These experiments are consistent with Mitchell's hypothesis that, first, delta pH can be created by the reversal of the ATPase reaction and, second, that protons are pumped outward during respiration. In addition to Pi, about 10 more resonances were resolved, several of which were assigned to different phosphate metabolites.

Adenosine Triphosphatases↗
PubMed1977

X-ray absorption studies of halide binding to carbonic anhydrase.

X-ray absorption measurements of bovine carbonic anhydrase B have been made at the the Stanford Synchrotron Radiation Project with a spectrometer operating in the fluorescence mode. Differences in absorption at and beyond the zinc K-edge near 9664 ev have been observed upon the addition of bromide or iodide. The additional absorption in k space, out to k approximately 7 A-1, obtained upon the addition of iodide has been compared with the absorption in this region of a ZnI2 sample. The similarities between these absorptions lead to the conclusion that the zinc-iodide distance in the protein is 2.65 +/-0.06 A; it is known to be 2.62 A in ZnI2. This shows that the iodide binds directly to the zinc in the protein.

Animals↗