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Biomedical subjects

K Ugurbil

Publications and source records attributed to K Ugurbil.

105 records · Page 6Linked to original sources
PubMed1981

31P NMR studies of intact anchorage-dependent mouse embryo fibroblasts.

31P NMR spectra of "normal" and x-ray-transformed anchorage-dependent mouse embryo fibroblasts (C3H/10T 1/2) at 145.7 MHz and 37 degrees C were obtained by using a solenoid coil probe with a sensitivity approximately 2.6 times higher than that of the standard Helmholtz coil probe. The cells were grown as monolayers on the surface of microcarrier beads. Results show that these cells have low levels of NMR-detectable ADP, are highly impermeable to protons, are capable of maintaining high ATP levels in the absence of respiration, and, when ATP generation is blocked, ultimately hydrolyze their existing ATP to a nonphosphorylated product and Pi. Only small differences were observed between the spectra of "normal" and transformed cells. However, the latter accumulated 2-deoxyglucose 6-phosphate at a faster rate.

Adenine Nucleotides↗
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

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

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

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

Nuclear magnetic resonance and chemical modification studies of bovine erythrocyte superoxide dismutase: evidence for zinc-promoted organization of the active site structure.

Nuclear magnetic resonance (NMR) spectroscopy of the exchangeable protons, tentatively assigned as histidine resonances, of bovine erythrocyte superoxide dismutase in H2O has been found to be a powerful method to study the active site of the enzyme. This technique has been employed in conjunction with chemical modification of the histidine residues using diethylpyrocarbonate (DEP) to show that zinc alone organizes the active site structure. All eight histidines per subunit of apoenzyme react with DEP. The accessibility of these residues to solvent is borne out by the broad, featureless NMR spectrum of the apoprotein. In the holoenzyme only His-19, which is exposed to solvent, can be modified with DEP. The reduced holoenzyme shows a well-resolved NMR spectrum compared with the oxidized form in which the lines are broadened by the paramagnetic copper ion. A spectrum very similar to that of the reduced enzyme is generated by addition of one zinc ion per subunit of apoprotein showing that zinc alone restores much of the native structure. This interpretation is supported by the fact that addition of up to 1 mol of zinc per subunit statistically reduces the number of histidine residues that can be modified by DEP until, at Zn: apoprotein ratios greater than or equal to 1, only His-19 reacts. The NMR spectrum of the apo plus 2 Zn2+ protein has additional structure that is briefly discussed.

Animals↗
PubMed1977

Studies of individual carbon sites of azurin from Pseudomonas aeruginosa by natural-abundance carbon-13 nuclear magnetic resonance spectroscopy.

The environments of the aromatic residues (and of the single arginine residue) of azurin from Pseudomonas aeruginosa are investigated by means of natural-abundance 13C Fourier transform NMR spectroscopy. In the case of the diamagnetic Cu(I) azurin, all 17 nonprotonated aromatic carbons (and Czota of Arg-79) yield narrow resonances. Furthermore, a single-carbon amide carbonyl resonance with an unusual chemical shift (peak chi) is observed. The pH dependence of chemical shifts is used to identify the resonances of Cgamma of titrating histidines, and of Cgamma and Czota of the two tyrosines. The resonances of Cgamma and Cdelta2 of the single tryptophan residue (and Czota of Arg-79) are also identified. The pKa values of the two tyrosines are different from each other and higher than typical values of "solvent-exposed" tyrosine residues. Two of the four histidine residues do not titrate (in the pH range 4 to 11). The resonance of Cgamma of one histidine exhibits a pH titration with fast proton exchange behavior and a pKa of 7.5 +/- 0.2. The direction of the titration shift indicates that the imidazole form of this histidine is the Ndelta1-H tautomer. The Cgamma resonance of the other titrating histidine exhibits slow exchange behavior with a pKa of about 7. The imidazole form of this histidine is the Nepsilon2-H tautomer. When going to the paramagnetic Cu(II) protein, only 11 of the 19 carbons mentioned above yield resonances that are narrow enough to be detected. Also, some of the observed resonances exhibit significant paramagnetic broadening. A comparison of spectra of fully reduced azurin, mixtures of reduced and oxidized azurin, and fully oxidized azurin yields the following information. (i) Peak chi arises from an amide group that probably is coordinated to the copper. (ii) The two nontitrating histidine residues are probably copper ligands, with Ndelta1 coordinated to the metal. (iii) The side chains of Arg-79 and the two tyrosine residues are not coordinated to the copper, and Trp-48 is probably not a ligand either. (iv) The gamma carbons of Trp-48, the tyrosine with the lower pKa, the titrating histidine with slow exchange behavior, and three or four of the six phenylalanine residues are sufficiently close to the copper to undergo significant paramagnetic broadening in the spectrum of oxidized azurin.

Azurin↗
PubMed1977

Tyrosine emission in the tryptophanless azurin from Pseudomonas fluorescens.

A strain of Pseudomonas fluorescens contains an azurin with no tryptophan and two tyrosines. This protein is interesting because it allows one to study both the structure of azurin and the emission of tyrosines in proteins. Comprehensive measurements were carried out including spectrophotometric and fluorimetric titration, fluorescence quantum yield, fluorescence polarization, and I- quenching. In the copper-containing protein, almost independent of the copper ion oxidation, the fluorescence quantum yield is approximately 60% of that of the apoprotein. The latter has the remarkable property that its quantum yield is even greater than free tyrosine. The two tyrosines in the metalloprotein have different pKa's, 10.75 and 12.78, but there is only one average pKa, 10.9 in the apoprotein. The polarization of the fluorescence at 310 nm (290-nm excitation) is 0.32 for the metalloproteins and 0.34 for the apoprotein. I- hardly quenches the fluorescence. The conclusion is that the two tyrosines are inaccesible to the solvent, located in nonpolar environments, larger than or equal to 20 A apart, and not adjacent to the disulfide bridge.

Azurin↗
PubMed1977

Study of the triplet state properties of tyrosines and tryptophan in azuring using optically detected magnetic resonance.

Optically detected magnetic resonance (ODMR) signals and phosphorescence spectra were seen of tyrosine in the P. aeruginosa and tryptophanless P. fluorescens azurins and of tryptophan in the former. This confirmed a conclusion from other experiments that the tryptophan of P. aeruginosa cannot effectively quench the singlet energy of both tyrosines. The ODMR signals were all very narrow, additional evidence that the chromophores are buried in the interior of the protein. Accurate values of the zero-field coupling constants D and E lead to a tentative correlation of D values with the red shift of the 0 leads to 0 peak of the phosphorescence spectrum. The environment of tryptophan in P. aeruginosa is the most hydrocarbon like of any tryptophan so far observed. The experiments raise a number of unanswered questions concerning rate processes. The intensities of the 2E transition of tyrosine and the phosphorescence of both tyrosine and tryptophan are substantially reduced when the copper is oxidized. Nevertheless the phsphorescence lifetimes are unaffected. A hole cannot be burned in the ODMR resonances. The homogeneously broadened lines may conceivably be a result of low-temperature proton tunnelling.

Azurin↗
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↗
PubMed1975

Intracellular bactericidal activity of leukocytes in whole blood for the diagnosis of chronic granulomatous disease of childhood.

A simplified method for the assay fo intracellular bactericidal activity of leukocytes in whole blood was developed. Providing that serum-resistant organisms are studied, the method can be used with gram-negative baccilli as well as with Staphylococcus aureus. The abnormalities characteristic of patients with chronic granulomatous disease of childhood and their mothers can be readily detected by this technique. By this means an abnormality in intracellular killing of Pseudomonas cepacia in a patient with chronic granulomatous disease and in his heterozygotic mother was found.

Benzenesulfonates↗
PubMedDate not supplied

Investigation of the initial dip in fMRI at 7 Tesla.

In agreement with optical imaging studies, previous fMRI studies have reported an initial decrease (i.e. the initial dip) in the BOLD response, which is believed to arise from an increase in oxygen consumption and to be mostly microvascular. To date, experimental studies of the initial dip in humans have been performed at fields up to 4 T, with relatively low spatial resolution. Because the sensitivity to microvascular contribution is increased at high magnetic fields, the present study investigated the initial dip at 7 T. In addition, to reduce the partial volume effect, the study is conducted at a high spatial resolution. The initial dip was detected in all subjects studied and was found to reside mostly in the gray matter. The relative amplitude of the early response was found to be 0.6, higher than that at 4 T (0.3) and 1.5 T (0.11). In addition, based on the assumption that the initial dip is a result of increased oxygen utilization, the fractional change in oxygen utilization was estimated to be 40% of that of the fractional change in cerebral blood flow. These results are in agreement with the notion that the initial dip arises from an increase in oxygen consumption.

Adult↗