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C J Kay

Publications and source records attributed to C J Kay.

At least 37 records · Page 2Linked to original sources
PubMed1989

EPR and kinetic analysis of the interaction of halides and phosphate with nitrate reductase.

Electron paramagnetic resonance spectra obtained during turnover of the Mo center of NADH:nitrate reductase at pH 8 were comprised of two Mo(V) species, signal A (g1 = 1.996, g2 = 1.969, g3 = 1.967, A1H = 1.25 mT, A2H = 1.18 mT, and A3H = 1.63 mT) and signal B (g1 = 1.996, g2 = 1.969, and g3 = 1.967), the former exhibiting superhyperfine interaction due to strong coupling with a single, exchangeable proton. Binding of halides and nitrite to the Mo center increased the proportion of signal A whereas phosphate had no effect on the EPR line shape. Halides decreased and phosphate increased the rates of enzyme activities involving the Mo center (NADH:nitrate reductase and reduced methyl viologen:nitrate reductase), but neither had any effect on activities involving FAD (NADH:ferricyanide reductase) or heme (NADH:cytochrome c reductase), indicating specific binding of halides to the Mo center. Halides were found to be weak, mixed competitive-noncompetitive inhibitors (Cl- KI = 39 mM, mu = 0.2 M, pH 8) of nitrate reductase forming a catalytically inactive ternary halide-nitrate-enzyme complex. Inhibition patterns changed from nearly noncompetitive (F-) to nearly competitive (I-). The weakening of nitrate binding due to halide binding correlated with increased halide electronegativity rather than ionic radius. In contrast, phosphate (Kd = 7.4 mM, mu = 0.2 M, pH 8) and arsenate were determined to be nonessential activators, characterized by a constant value of (Vmax/Km)app, increasing nitrate reductase activity by weakening nitrate binding without affecting the stability of the transition state. Phosphate had no effect on product inhibition by nitrite (KI = 0.33 mM) or the oxidation-reduction midpoint potentials of the Mo center.

Arsenates↗
PubMed1989

Chloride inhibition of spinach nitrate reductase.

Initial rate studies of spinach (Spinacia oleracea L.) nitrate reductase showed that NADH:nitrate reductase activity was ionic strength dependent with elevated ionic concentration resulting in inhibition. In contrast, NADH:ferricyanide reductase was markedly less ionic strength dependent. At pH 7.0, NADH:nitrate reductase activity exhibited changes in the V(max) and K(m) for NO(3) (-) yielding V(max) values of 6.1 and 4.1 micromoles NADH per minute per nanomoles heme and K(m) values of 13 and 18 micromolar at ionic strengths of 50 and 200 millimolar, respectively. Control experiments in phosphate buffer (5 millimolar) yielded a single K(m) of 93 micromolar. Chloride ions decreased both NADH:nitrate reductase and reduced methyl viologen:nitrate reductase activities, suggesting involvement of the Mo center. Chloride was determined to act as a linear, mixed-type inhibitor with a K(i) of 15 millimolar for binding to the native enzyme and 176 millimolar for binding to the enzyme-NO(3) (-) complex. Binding of Cl(-) to the enzyme-NO(3) (-) complex resulted in an inactive E-S-I complex. Electron paramagnetic resonance spectra showed that chloride altered the observed Mo(V) lineshape, confirming Mo as the site of interaction of chloride with nitrate reductase.

Journal Article↗
PubMed1988

Circular dichroism and potentiometry of FAD, heme and Mo-pterin prosthetic groups of assimilatory nitrate reductase.

Oxidation-reduction midpoint potentials for flavin, heme, and molybdenum-pterin prosthetic groups of assimilatory nitrate reductase (NR) from Chlorella vulgaris were measured at room temperature by using CD and EPR potentiometry. The CD changes accompanying reduction of each prosthetic group were determined by using enzyme fragments containing either FAD or heme and molybdenum prosthetic groups, obtained by limited proteolysis, and by poising the enzyme at various redox potentials in the presence of dye mediators. Limited proteolysis did not appear to alter the environment of the prosthetic groups, as judged by their CD spectra. Also, CD potentiometric titration of FAD in intact NR (Em' = -272 mV, n = 2) gave a similar value (Em' = -286 mV) to the FAD of the flavin-containing proteolytic domain, determined by visible spectroscopy. Less than 1% of the flavin semiquinone was detected by EPR spectroscopy, indicating that Em' (FAD/FAD.-) may be more than 200 mV lower than Em' (FAD.-/FADH-). Reduction of heme resulted in splitting of both Soret and alpha CD bands into couplets. The heme Em' was -162 mV (n = 1) determined by both CD and visible spectroscopy. Reduction of Mo-pterin was followed by CD at 333 nm, and Mo(V) was monitored by room temperature EPR spectroscopy. Most of the change in the Mo-pterin CD spectrum was due to the Mo(VI)/Mo(V) transition. The Em' values determined for Mo(VI)/Mo(V) were +26 mV by CD and +16 mV by EPR, whereas Mo(V)/Mo(IV) values were -40 mV by CD and -26 mV by EPR.(ABSTRACT TRUNCATED AT 250 WORDS)

Circular Dichroism↗
PubMed1987

Radiation inactivation analysis of assimilatory NADH:nitrate reductase. Apparent functional sizes of partial activities associated with intact and proteolytically modified enzyme.

Recently we demonstrated that target sizes for the partial activities of nitrate reductase were considerably smaller than the 100-kDa subunit which corresponded to the target size of the full (physiologic) activity NADH:nitrate reductase. These results suggested that the partial activities resided on functionally independent domains and that radiation inactivation may be due to localized rather than extensive damage to protein structure. The present study extends these observations and addresses several associated questions. Monophasic plots were observed over a wide range of radiation doses, suggesting a single activity component in each case. No apparent differences were observed over a 10-fold range of concentration for each substrate, suggesting that the observed slopes were not due to marked changes in Km values. Apparent target sizes estimated for partial activities associated with native enzyme and with limited proteolysis products of native enzyme suggested that the functional size obtained by radiation inactivation analysis is independent of the size of the polypeptide chain. The presence of free radical scavengers during irradiation reduced the apparent target size of both the physiologic and partial activities by an amount ranging from 24 to 43%, suggesting that a free radical mechanism is at least partially responsible for the inactivation. Immunoblot analysis of nitrate reductase irradiated in the presence of free radical scavengers revealed formation of distinct bands at 90, 75, and 40 kDa with increasing doses of irradiation rather than complete destruction of the polypeptide chain.

Chlorella↗
PubMed1986

Assimilatory nitrate reductase from Chlorella. Effect of ionic strength and pH on catalytic activity.

Initial velocity studies of Chlorella nitrate reductase showed that increased ionic strength stimulated NADH:nitrate reductase activity by increasing both Vmax and Km for nitrate. Examination of the effect of ionic strength on the various partial activities of nitrate reductase revealed that while NADH:ferricyanide and reduced methyl viologen:nitrate reductase activities were unaffected by ionic strength, NADH:cytochrome c and reduced flavin:nitrate reductase activities were inhibited and stimulated by increased ionic strength, respectively. Comparison of the rates for the partial activities indicated electron transfer from heme to molybdenum to be the rate-limiting step in enzyme turnover. The pH optimum for NADH:nitrate reductase activity was found to be 7.9 while values for the partial activities ranged from 5.5 to 8.1. Phosphate was found to stimulate both NADH:nitrate and reduced methyl viologen:nitrate reductase activities indicating the molybdenum center as the site of interaction.

Chlorella↗
PubMed1986

Chlortetracycline and the transmembrane potential of the inner membrane of plant mitochondria.

The oxidation of NADH or succinate by Jerusalem-artichoke (Helianthus tuberosus L.) mitochondria in the presence of chlortetracycline induced an increase in chlortetracycline fluorescence. Any treatment that prevented the formation of a transmembrane potential (as monitored by changes in safranine absorbance, A511-A533), e.g. uncoupling with carbonyl cyanide p-trifluoromethoxyphenylhydrazone, inhibition of dehydrogenase activity or electron transport, anaerobiosis or depletion of substrate, prevented the increase in chlortetracycline fluorescence or caused it to disappear. Changes in chlortetracycline fluorescence were always slower than changes in the safranine absorbance. The increase in chlortetracycline fluorescence caused by succinate oxidation had an excitation maximum at 393 nm, indicating that a Ca2+-chlortetracycline complex was involved. The increase in fluorescence was observed even in the presence of EDTA, which removes all external bivalent cations, indicating that internal Ca2+ is mobilized. Although NADH and succinate oxidations gave the same membrane potential and qualitatively had the same effect on chlortetracycline fluorescence, NADH oxidation caused a much larger (over 3-fold) increase in chlortetracycline fluorescence than did succinate oxidation. It is possible that this is connected with the Ca2+-dependence of NADH oxidation. In the presence of 2 mM external Ca2+, chlortetracycline collapsed the transmembrane potential and uncoupled succinate and duroquinone oxidation.

Calcium↗
PubMed1986

Thermodynamic properties of the heme prosthetic group in assimilatory nitrate reductase.

The oxidation-reduction midpoint potential for the heme prosthetic group present in assimilatory nitrate reductase from Chlorella vulgaris has been determined by optical potentiometric titrations in the presence of dye mediators. At pH 7, the midpoint potential was determined to be -160 mV and corresponds to a reversible n = 1 redox process. The midpoint potential was unaltered by the use of NADH as reductant, unaffected by the presence of NAD+, cytochrome c, phosphate, cyanide, or alkaline pH. In addition, the redox potential of the heme was independent of modifications to the enzyme such as substitution of the molybdenum center with tungsten, or cleavage and separation of the enzyme into its flavin and heme/molybdenum domains. In contrast, the midpoint potential increased on decreasing the pH yielding a pH dependence of approximately 20 mV/pH unit within the range 5.5 to 7, suggesting the presence of a single, redox-associated, ionizable functional group on the protein with pKox = 5.8 and pKred = 6.1. At pH 7 and within the range 12 to 38 degrees C, the midpoint potential of the heme decreased by approximately 1 mV/degree. Values for delta S0 and delta H0 were calculated to be -25.6 e.u. and -4.0 kcal/mol.

Chlorella↗
PubMed1985

Solubilization of the alternative oxidase of cuckoo-pint (Arum maculatum) mitochondria. Stimulation by high concentrations of ions and effects of specific inhibitors.

Selective solubilization of cyanide- and antimycin-insensitive duroquinol oxidase activity from cuckoo-pint (Arum maculatum) mitochondria was achieved using taurocholate. Inhibitor-sensitivities and water-forming DQH2 (tetramethyl-p-hydroquinone, reduced form): O2 stoichiometry were the same for the alternative oxidase of intact Arum mitochondria. Cyanide-insensitive oxidation of DQH2 by intact and solubilized mitochondria was stimulated by up to four-fold by high concentrations of anions high in the Hofmeister series, such as phosphate, sulphate or citrate. Optimal (0.7 M) sodium citrate increased Vmax. for DQH2 oxidation by the solubilized preparation from 450 to 2400 nmol of O2 X min-1 X mg of protein-1 and decreased the apparent Km for DQH2 from 0.53 to 0.38 mM. Inhibition of solubilized DQH2 oxidase activity by CLAM (m-chlorobenzhydroxamic acid) and SHAM (salicylhydroxamic acid) was mixed competitive/non-competitive, with apparent inhibition constants for CLAM of 25 microM (Ki) and 81 microM (KI) and for SHAM of 53 microM (Ki) and 490 microM (KI). Propyl gallate and UHDBT were non-competitive inhibitors with respect to DQH2 (apparent Ki = 0.3 microM and 12 nM respectively). Low concentrations of C18 fatty acids selectively inhibited cyanide-insensitive oxidation by intact and solubilized mitochondria, and inhibition was reversed by 1% (w/v) bovine serum albumin. Inhibition was competitive with DQH2, suggesting that fatty acids interfere reversably with the binding of DQH2 to the oxidase. These results tend to support the view that quinol oxidation by the alternative pathway of Arum maculatum mitochondria is catalysed by a quinol oxidase protein, rather than by a non-enzymic mechanism involving fatty acid peroxidative reaction. [Rustin, Dupont & Lance (1983) Trends Biochem. Sci. 8, 155-157; (1983) Arch. Biochem. Biophys. 225, 630-639].

Citrates↗
PubMed1984

Electrostatic screening stimulates rate-limiting steps in mitochondrial electron transport.

The effect of electrostatic screening of fixed negative charges on uncoupled mitochondrial electron transport was investigated with substrates with different charge and different sites of donation of electrons to the electron-transport chain of Jerusalem-artichoke (Helianthus tuberosus L.) mitochondria. Duroquinol (neutral substrate) was oxidized with a pH optimum of 7.6-7.8. The addition of cations caused a doubling of Vmax. (order of efficiency C3+ greater than C2+ greater than C+) through electrostatic screening, whereas the Km was unaffected. Screening stimulated (by 150%) the Vmax. for the oxidation of reduced cytochrome c (positive substrate; to O2), but in this case the Km doubled. The Vmax. of the oxidation of exogenous NADH (negative substrate) was also stimulated by screening when the acceptor was O2, but unaffected when duroquinone was the acceptor. In both cases, the Km for NADH was considerably decreased. The effect of screening on the Km for the different substrates can be explained by the changes in the effective concentration of substrate near the active site due to the lowering in the size of the surface potential. The effect of screening on the Vmax. of the different partial processes indicates that increasing the salt concentration of the medium enhances the maximal activity of cytochrome c oxidase. However, the results also point at the existence of other rate-limiting steps, which are affected by screening and may involve ubiquinone, in electron transport in plant mitochondria.

Cell Membrane↗
PubMed1984

Soft-tissue CT changes in pelvic venous thrombosis.

The computed tomographic (CT) changes in the soft tissues of the pelvis are presented in three patients with pelvic venous thrombosis. Pelvic venous thrombosis may lead to swelling of surrounding muscles and may present as a pelvic mass. This mass can simulate a tumor clinically, radiographically, and sonographically, but its origin and cause can be demonstrated by CT. Proper understanding of such changes may prevent an erroneous diagnosis of enlarged pelvic lymph nodes or an inflammatory or neoplastic mass.

Adult↗
PubMed1982

Primary amyloidoma of the spine.

A rare case of primary solitary amyloidoma involving the dorsal spine is reported. Destruction of the fourth thoracic vertebra and extension of the soft tissue mass around the vertebra were visualized on linear tomography and computed tomography. Diagnosis was made on the basis of histology and histochemical reactions.

Aged↗
PubMed1982

Sonography of splenic abscess.

Gray-scale sonographic examination was performed in seven cases of splenic and parasplenic abscess. The splenic abscesses appeared as irregular, poorly defined anechoic masses with varying internal echogenicity and acoustical transmission. Increased echogenicity and distal acoustical shadowing due to gas within the abscess were also noted. Gray-scale sonography was instrumental in the diagnosis of splenic abscess in this group of patients whose symptomatology was of uncertain etiology.

Abscess↗
PubMed1981

Spectral opponency of on-type ganglion cells and the blue preference of Rana pipiens.

Spectrally opponent processes of ON-type retinal ganglion cells and the blue preference behavior were identified by parallel physiological and behavioral experiments in Rana pipiens. Spectral opponency of retinal ON-units was measured by recording from optic nerve terminals in the anterior thalamus, while the retina was stimulated by combinations of monochromatic stimuli. Spectral opponency of the blue preference was determined in a Y-maze, using similar combinations of monochromatic stimuli. The opponent processes of the ON-units and blue preference are similar in the spectral ranges of excitatory and inhibitory effects. In both cases the spectral opponency can be described as short wavelength excitation and long wavelength inhibition. The data suggest that the short wavelength excitation is based, at least in part, on the green rod (P432) channel, while the long wavelength inhibition is caused, at least in part, by stimulation of the principal and/or single cone (P580) channel. A model is presented to show how receptor interactions may encode this spectrally opponent process. The results support the hypothesis that the blue preference is dependent on information supplied to the anterior thalamus by ON-type retinal ganglion cells.

Animals↗
PubMed1980

Evaluation of solitary simple renal cysts in children.

Because there is a proportionately higher incidence of solid renal neoplasm in children than in adults early operative intervention is more common in these cases. For this reason the most important consideration when evaluating children is to exclude the presence of a malignant neoplasms. The clinical features, radiologic manifestations and management of solitary simple renal cysts in 3 children are discussed. We suggest that the increased accuracy and availability of newer imaging techniques now permit this entity to be managed in a fashion similar to its more common adult counterpart. Surgical exploration should be reserved for those cases in which there is doubt as to the benign nature of the cyst.

Child↗
PubMed1980

Gray-scale ultrasonography in the evaluation of renal trauma.

Static and real-time gray-scale ultrasound examinations were performed on seven patients who had minor, major, and catastrophic renal trauma. The sonographic findings in avulsion of the renal pedicle, fractured kidney, ruptured renal cyst, perinephric and subcapsular hematomas, and pelvic blood clots are presented. Ultrasonography provided a valuable adjunct to conventional urography and radionuclide studies by adding information on the extent of renal and retroperitoneal injury.

Adolescent↗
PubMed1979

Ultrasonic characteristics of chronic atrophic pyelonephritis.

Six cases of chronic atrophic pyelonephritis were studied with gray scale ultrasound. The findings are analogous to those seen pathologically, namely a focal or multifocal process with loss of renal parenchyma, retraction of one or more calyces, decrease in renal size, and increased echoes from fibrosis. In the proper clinical setting, the diagnosis of chronic atrophic pyelonephritis can be made using ultrasound technique.

Adolescent↗