PubMed Health⌕ Search

Biomedical subjects

W T Jenkins

Publications and source records attributed to W T Jenkins.

At least 37 records · Page 2Linked to original sources
PubMed1996

Subunit dissociations in natural and recombinant hemoglobins.

A precise and rapid procedure employing gel filtration on Superose-12 to measure the tetramer-dimer dissociation constants of some natural and recombinant hemoglobins in the oxy conformation is described. Natural sickle hemoglobin was chosen to verify the validity of the results by comparing the values with those reported using an independent method not based on gel filtration. Recombinant sickle hemoglobin, as well as a sickle double mutant with a substitution at the Val-6(beta) receptor site, had approximately the same dissociation constant as natural sickle hemoglobin. Of the two recombinant hemoglobins with amino acid replacements in the alpha 1 beta 2 subunit interface, one was found to be extensively dissociated and the other completely dissociated. In addition, the absence of an effect of the allosteric regulators DPG and IHP on the dissociation constant was demonstrated. Thus, a tetramer dissociation constant can now be determined readily and used together with other criteria for characterization of hemoglobins and their interaction with small regulatory molecules.

Allosteric Regulation↗
PubMed1996

Noninvasive imaging of the distribution in oxygen in tissue in vivo using near-infrared phosphors.

A newly developed water-soluble phosphor suitable for measuring oxygen pressure in the blood (Green 2W) was used for noninvasive, in vivo imaging of oxygen distribution in the vascular systems of mice. Oxygen quenches the phosphorescence of Green 2W, measured in the presence of 2% albumin, according to the Stern-volmer relationship. This oxygen-dependent quenching of phosphorescence has been used to obtain digital maps of the oxygen distribution in the tissue vasculature. EMT-6 mammary carcinoma tumors were grown by injecting 1 x 10(6) cells in 0.1-ml carrier into the subcutaneous space over the muscle on the hindquarter. When the tumors were approximately 8 mm in diameter, 300 micrograms of phosphorescence probe (Green 2W; absorption maximum 636 nm) was injected into the tail vein. The mice were immobilized with intraperotoneal Ketamine (133 mg/kg) and Xylazine (10 mg/kg) and illuminated with flashes (< 4-microseconds t1/2) of light of 630 +/- 12 nm. The emitted phosphorescence (790-nm maximum) was imaged an intensified CCD camera. Images were collected beginning at 30, 50, 80, 120, 180, 240, 420, and 2500 microseconds after the flash and used to calculate digital maps of the phosphorescence lifetimes and oxygen pressure. Both the illumination light and the phosphorescence were in the near-infrared region of the spectrum, where tissue has greatly decreased absorbance. The light therefore readily passed through the skin and centimeter thicknesses of tissue. The oxygen maps could be obtained by illuminating from the side of the mouse opposite the camera (and tumor). The tumors were readily observed as regions with oxygen pressures substantially below those of the surrounding tissue. Thus, phosphorescence measurements can noninvasively detect volumes of tissue with below-normal oxygen pressure in the presence of much larger volumes of tissue with normal oxygen pressures. In addition, tissue oxygen pressures can be monitored in real time, even through centimeter thicknesses of tissue.

Animals↗
PubMed1996

Biodistribution of the nitroimidazole EF5 (2-[2-nitro-1H-imidazol-1-yl]-N-(2,2,3,3,3-pentafluoropropyl) acetamide) in mice bearing subcutaneous EMT6 tumors.

The characteristic reduction and binding of nitroimidazoles to cellular macromolecules in the absence of oxygen allows their use for detection and characterization of hypoxia. The biodistribution of a new nitroimidazole, EF5 (2-[2-nitro-1H-imidazol-1-yl]-N-(2,2,3,3,3-pentafluoropropyl) acetamide), in mice bearing EMT6 tumors is described. Detection methods based on radioactivity and monoclonal antibody techniques are compared for liver and tumor. All nonexcretory tissues demonstrated similar levels of radioactivity at 0.5 hr postinjection of drug, demonstrating equivalent access of EF5 to all tissues. At 24 hr, when unbound drug has been cleared, the tissues with the highest binding are the liver, esophagus, bladder and tumor. Typically, liver tissue contains the highest level of radioactivity at this time. Examination of tumor and liver tissue by use of fluorescence microscopy and Cy3-bound monoclonal antibodies specific for EF5 adducts showed that the patterns of binding in tumor are considerably more heterogeneous than those of liver. Histograms of fluorescence intensity, with use of these antibodies, demonstrate average and maximal binding higher in tumors than in the liver. This divergence from the radioactivity data was determined to be unrelated to sampling error, differential antibody access or staining efficiency of liver vs. tumor tissue. A possible cause is the scavenging of radioactive drug metabolites by liver. The data presented herein suggest that EF5 is useful as a hypoxia detector and that monoclonal antibody detection methods can give detailed information on the distribution of EF5 binding. This technology may allow an accurate estimation of the oxygenation and/or nitroreductase levels in both tumor and normal tissues.

Animals↗
PubMed1995

Identification of hypoxia in cells and tissues of epigastric 9L rat glioma using EF5 [2-(2-nitro-1H-imidazol-1-yl)-N-(2,2,3,3,3-pentafluoropropyl) acetamide].

One of the most sensitive hypoxia detection methods is based on the observation that binding of nitroimidazoles to cellular macromolecules occurs as a result of hypoxia-dependent bioreduction by cellular nitroreductases. Nitroimidazole-binding techniques provide measurements of hypoxia to virtually any degree of spatial resolution and with a multiplicity of techniques. This paper demonstrates hypoxia imaging using in vivo EF5 binding with detection by a fluorochrome-conjugated monoclonal antibody. We investigated these techniques in the 9L glioma tumour, in part because the exact nature of the hypoxia in this tumour system is controversial. Our results demonstrate that following intravenous injection of EF5, binding and detection using a monoclonal antibody in 9L gliomas is specific and oxygen dependent. Detection of binding using fluorescence microscopy can be performed on frozen tissues; tissue sections can be counterstained with haematoxylin and eosin for light microscopic analysis. Alternatively, the distribution of hypoxia in a tumour can be inferred by examining individual tumour cells using flow cytometric techniques. Based upon the results presented herein, the radiation-resistant phenotype of 9L epigastric tumours grown in our laboratories can be associated with the presence of hypoxic cells.

Animals↗
PubMed1994

Yeast mitochondrial F1-ATPase--effects of metal ions.

The effects of a series of metal ions (M) on the velocity (V) of the reaction catalyzed by the yeast mitochondrial F1-ATPase were investigated in N-tris(hydroxymethyl)methyl-2-aminoethanesulfonic acid and Imidazole buffers (pH 7.45), with total concentrations of ATP ranging from 2 to 8 mM. The biphasic metal ion activation/inhibition effects, observed with a constant total amount of ATP, were found to be consistent with a simple empirical kinetic equation, V1/V = 1 + K1/[M] + [M]/K2, where V1, K1, and K2 are empirical parameters and [M] the concentrations of metal ions not complexed with the ATP. Three alternative kinetic equations, involving the concentrations of either ATP or its metal chelate, consistent with this empirical equation, were shown to be invalid since changing the total amount of ATP did not affect the empirical reaction parameters V1, K1, and K2. The fact that changing the total ATP concentration failed to change the empirical reaction parameters suggests that the inhibition by an excess of ATP, which is observed with a constant total metal ion concentration, is due to chelation of free metal ions that are required for the ATP hydrolysis. When the total amount of added ATP was in excess of that of added magnesium ions it was found that calcium, strontium, and aluminum ions, which liberate free magnesium from the Mg-ATP chelate by complexing the ATP, all activated. These activations apparently confirm the fact that the ATPase requires a free magnesium ion as a cofactor for the hydrolysis of ATP.

Adenosine Diphosphate↗
PubMed1994

Yeast mitochondrial F1-ATPase: the unusual kinetics with excess magnesium ions at low salt concentrations.

Studies with the yeast mitochondrial F1-ATPase showed that non-Michaelis-Menten kinetics occurred around pH 8 when there was an excess of magnesium over ATP and a low salt concentration. Increasing the salt concentration activated the enzyme and resulted in Michaelis-Menten kinetics. In contrast, increasing the salt concentration had no effect when ATP was in excess of magnesium. The data were interpreted to mean that magnesium ions inhibit by limiting the amount of free ATP and that the salt anions are able to overcome this effect by replacing the ATP. Mechanistically this is consistent with a ligand substitution reaction in which either free ATP or salt anions are required to displace a product from the enzyme.

Anions↗
PubMed1991

The pyruvate kinase-coupled assay for ATPases: a critical analysis.

The pyruvate kinase assay for ATPases suffers from major systematic errors due to the variable amounts of the inhibitory product, ADP, present as the rates change. A simple method is presented to estimate the true uninhibited rates and the ADP inhibition constant for the ATPase.

Adenosine Diphosphate↗
PubMed1989

Two kinetic methods to study the regulation of mammalian hexokinases.

Two new kinetic analyses for mammalian hexokinases are presented, which permit one to study the regulation of these enzymes by product inhibition. One method uses the pyruvate kinase-coupled assay and the other the glucose-6-phosphate dehydrogenase-coupled assay. Both methods give simple linear plots, which indicate that the magnesium-ATP complex overcomes the glucose 6-phosphate inhibition competitively, but by atypical kinetics. A new regulation coefficient (Kr) was defined and it was shown that, with both assay methods, the reciprocals of the slopes of the simple linear plots are proportional to Kr.[Mg.ATP].NADP, but not NAD, was found to be a powerful inhibitor of pig heart hexokinase.

Animals↗
PubMed1989

Thermodynamic bookkeeping: a reinvestigation of proton and dicarboxylic acid binding to aspartate aminotransferase.

A reinvestigation of the effects of pH and salt concentration on the proton and dicarboxylic acid dissociation constants of pig heart aspartate aminotransferase shows that both anions and cations were involved concomitantly, both as stoichiometric reactants and bioenergetically. Equations are presented which can be used experimentally, to determine the numbers of salt ions (their thermodynamic stoichiometries) involved in biochemical equilibria such as proton and ligand dissociations from macromolecules. These equations were used to reinvestigate the effects of salts on the chromophoric pKa of the enzyme prosthetic group, the interaction of the enzyme with dicarboxylic acids, and the overall equilibrium for the transamination half-reaction.

Aspartate Aminotransferases↗
PubMed1988

Guanine nucleotide regulation of the interconversion of the two-state hepatic glucagon receptor system of rat.

To investigate whether guanine nucleotides regulate interconversion of the two-state hepatic glucagon receptor we have utilized kinetic assays of glucagon binding to partially purified rat liver plasma membranes. Dissociation of glucagon at 30 degrees C exhibited biexponential character in either the absence or presence of GTP, indicating that the system previously seen in intact hepatocytes is independent of intracellular modulators. In each case the receptors underwent a time-dependent conversion from a low affinity to a high affinity state. However, GTP decreased the fraction of receptors in the high affinity state. The rank order for stabilizing the low affinity state was Gpp(NH)p greater than GTP greater than GDP much greater than GMP = no nucleotides. Data from competition binding assays with increasing concentrations of GTP allow calculation of equilibrium constants which are 3.32 nM for glucagon and receptor in the absence of GTP, 18.6 nM for glucagon and receptor in the presence of GTP, 1.55 microM for the association of receptor and GTP presumably linked to an N protein, and 8.86 microM for the association of the glucagon-receptor complex and GTP again presumably linked to an N protein, Glucagon binding to receptor is noncooperative in both the absence and presence of GTP, distinguishing this system from the beta-adrenergic system. With GTP, binding to the low affinity state is favored because of the relative affinities reported. Therefore, GTP regulates the activation by slowing the conversion of the receptor from a low affinity to high affinity form.

Animals↗
PubMed1988

Thermodynamic bookkeeping when nucleotides bind. Applications of the theory of linked functions.

The thermodynamic theory of linked functions was used to determine the numbers of modifier ions involved when nucleotides dissociate. Nucleotide dissociation constants, obtained spectrophotometrically using Dowex-1 resin as a model system, were plotted on log/log paper with respect to the modifier concentrations. The slopes of the lines represent the net number of modifier molecules/ions involved in the dissociation. Varying numbers of nucleotides are bound to the resin because the resin capacity is determined by the total number of charges bound. The nucleotides bind to the resin at comparable diffusion-limited rates, irrespective of how tightly they bind. When ATP binds at pH 6.8, 4 chlorides, 4 formates, 2 succinates or 1.4 citrates are displaced, indicating that the fully charged (ATP4-) nucleotide binds. By comparing ATP, ADP and AMP it was possible to evaluate the contributions of the adenosine moiety and each phosphate to the binding. Between pH 2 and 3, where ATP has two negative charges, ATP binds largely as the trianion, displacing 2.7 chlorides and 0.7 protons. In the presence of 4 mM magnesium, 0.58 magnesiums facilitate the dissociation by chelating 58% of the liberated ATP. Calcium behaved similarly to magnesium but aluminum, at pH 6.8, promoted the binding of ATP as an (A1.ATP)3- complex with the concomitant liberation of three chloride ions. These experimental thermodynamic stoichiometries were found to be independent of the concentrations of the other modifiers present. Thermodynamic linkage stoichiometries can be evaluated from log K vs. log (modifier) plots when a direct determination of modifier binding is impossible.(ABSTRACT TRUNCATED AT 250 WORDS)

Adenosine Triphosphate↗
PubMed1988

Factors affecting the rates at which nucleotides bind to Dowex 1 resin.

The relative rates at which adenine nucleotides bind to Dowex 1 anion-exchange resin are determined by diffusion through the immobile fluid film surrounding the resin particles and thus are unaffected by how tightly the nucleotides bind. By using a fine resin and stirring efficiently, the time needed for equilibration can be reduced from 4 h to less than 3 min.

Adenine Nucleotides↗
PubMed1988

Reactions of dysprosium with adenine nucleotides.

Dysprosium catalyzes a rapid hydrolysis of both ATP and ADP, at ambient temperatures, pH 7.0, where no hydroxide precipitates. The reactive complexes, at pH 6.7, were found to contain 2Dy:1ATP and 3Dy:2ADP. AMP forms an insoluble complex containing 1Dy:2AMP, which does not hydrolyze. ATP also forms a soluble 1Dy:1ATP complex, which does not react. Dysprosium only catalyzes the hydrolysis of ATP above pH 5.8, where it has been titrated to the hydroxide. At the optimum pH (pH 7) the stoichiometric composition is Dy2.ATP.(OH)2, indicating that the active complex is neutral, whereas at pH 5.8 the stoichiometric composition is Dy2.ATP.(OH)+, indicating an inactive cationic complex. The mechanism proposed for the hydrolysis is consistent with those proposed for other in vitro systems known to catalyze the hydrolysis of ATP.

Adenine Nucleotides↗
PubMed1987

Reactions of 3'-O-methylpyridoxal 5'-phosphate in aspartate aminotransferase.

The reaction of 3'-O-methylpyridoxal 5'-phosphate bound into the active site of aspartate aminotransferase with the substrate L-aspartate has been investigated. This methylated coenzyme is a very poor catalyst but it does function slowly to produce normal products of a transamination half-reaction. At pH 8.5 and above the characteristic absorption band of a quinonoid intermediate appears rapidly and becomes very intense when the aspartate concentration is raised to 2 M. At pH 6 the quinonoid band is not seen, but the conversion of the methylated coenzyme into 3'-O-methylpyridoxamine 5'-phosphate is about 7 times faster than at high pH with the pH dependence being determined by an apparent pKa of 8.1 at 30 degrees C. We suggest that the active site containing the methylated coenzyme carries a net charge 1 unit more positive than that of native enzyme. This causes a loss of some other proton from the active site and could leave the catalytic lysine-258 deprotonated in the quinonoid species. This may explain its inability to react rapidly. We have measured the spectral band shapes of the quinonoid species studied here and have compared it with that seen with native enzyme. Because of the close similarity we conclude that during normal transamination the proton bound to the imine nitrogen probably shifts onto the phenolic oxygen prior to or synchronously with the formation of the observed quinonoid species.

Aspartate Aminotransferases↗
PubMed1987

The induction of DNA-protein crosslinks in hypoxic cells and their possible contribution to cell lethality.

The induction of single-strand breaks (SSBs) in the DNA of Chinese hamster ovary cells by X rays under different irradiation conditions was measured by the alkaline elution technique. The oxygen enhancement ratio (OER) for SSB induction determined for cells irradiated in air versus irradiation of cells made hypoxic by metabolic depletion of O2 was 9.7. However, when proteinase K was included in the cell lysis solution the OER was reduced to 4.2. The proteinase affected the elution rate only of the cells irradiated under hypoxic conditions, suggesting that DNA-protein crosslinks (DPCs) are preferentially produced in hypoxic cells by radiation. The ability to repair these DPCs was compared in two cell lines: the wild-type AA8 line and an excision-repair-deficient mutant line, UV-41. The AA8 line removed about 80% of the DPCs induced by radiation under hypoxic conditions within a 24-h repair incubation. The UV-41 line, on the other hand, removed only about 20% of the DPCs in the same time. The OERs for cell survival of these two lines are 3.1 for AA8 but only 1.9 for UV-41, suggesting that the DPCs preferentially induced in the DNA of cells irradiated under hypoxic conditions may contribute to cell killing when the normal DNA-repair mechanisms are compromised.

Animals↗
PubMed1986

Catalysis of the dephosphorylation of an ATP-molybdate complex by calcium and magnesium ions.

Calcium and magnesium catalyze the dephosphorylation of a molybdate complex of adenosine triphosphate but not the corresponding molybdate complex of adenosine disphosphate. We conclude that catalysis of breaking the bond between the beta and gamma phosphates involves metal chelation of the alpha and beta phosphates. ATP hydrolysis with calcium was stimulated by phosphate, apparently because of formation of a calcium-phosphomolybdate complex. The reaction with magnesium, which does not form a comparable complex, was not affected by phosphate. Strontium, cadmium, and barium behaved like calcium. The reactions with transition metal cations showed autoinhibition.

Adenosine Diphosphate↗
PubMed1985

Quantitative analysis of intermediate phosphate-oxygen exchange during hydrolyses by ATPases.

A simple mathematical model is presented that predicts the paradoxical effect of substrate concentration on the extent of oxygen exchange into the phosphate produced during hydrolyses of nucleotide triphosphates by ATPases in labelled water. The same model also predicts the deviations from Michaelis-Menten kinetics observed at low salt concentrations.

Adenosine Triphosphatases↗