[Femur head necrosis--value of so-called radiologic early signs for diagnosis and therapy].
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Cortisone↗
Biomedical subjects
C H Kang
Publications and source records attributed to C H Kang.
Kinetics and energetics of intramolecular electron transfer in yeast cytochrome c peroxidase.
The oxidation of ferric cytochrome c peroxidase by hydrogen peroxide yields a product, compound ES [Yonetani, T., Schleyer, H., Chance, B., & Ehrenberg, A. (1967) in Hemes and Hemoproteins (Chance, B., Estabrook, R. W., & Yonetani, T., Eds.) p 293, Academic Press, New York], containing an oxyferryl heme and a protein free radical [Dolphin, D., Forman, A., Borg, D. C., Fajer, J., & Felton, R. H. (1971) Proc. Natl. Acad. Sci. U.S.A. 68, 614-618]. The same oxidant takes the ferrous form of the enzyme to a stable Fe(IV) peroxidase [Ho, P. S., Hoffman, B. M., Kang, C. H., & Margoliash, E. (1983) J. Biol. Chem. 258, 4356-4363]. It is 1 equiv more highly oxidized than the ferric protein, contains the oxyferryl heme, but leaves the radical site unoxidized. Addition of sodium fluoride to Fe(IV) peroxidase gives a product with an optical spectrum similar to that of the fluoride complex of the ferric enzyme. However, reductive titration and electron paramagnetic resonance (EPR) data demonstrate that the oxidizing equivalent has not been lost but rather transferred to the radical site. The EPR spectrum for the radical species in the presence of Fe(III) heme is identical with that of compound ES, indicating that the unusual characteristics of the radical EPR signal do not result from coupling to the heme site. By stopped-flow measurements, the oxidizing equivalent transfer process between heme and radical site is first order, with a rate constant of 0.115 s-1 at room temperature, which is independent of either ligand or protein concentration.(ABSTRACT TRUNCATED AT 250 WORDS)
Cytochrome-c Peroxidase↗
Control of the transfer of oxidizing equivalents between heme iron and free radical site in yeast cytochrome c peroxidase.
A procedure has been developed for obtaining yeast cytochrome c peroxidase with the heme iron in the Fe(IV) state, without the concomitant formation of the protein free radical that occurs in the ES compound resulting from the oxidation of ferric peroxidase with hydrogen peroxide. In this procedure, ferrous peroxidase, prepared either by photochemical reduction or by trapping the dithionite-reduced enzyme with carbon monoxide, is oxidized with a stoichiometric amount of hydrogen peroxide. The resulting Fe(IV) enzyme oxidizes ferrocyanide monophasically, with a rate constant of 4 x 10(3) M-1 S-1. The optical spectrum of the free radical was obtained as the difference between the spectra of the ES and Fe(IV) compounds. EPR spectra of ES compound prepared with [16O]-and [17O]hydrogen peroxide are identical, demonstrating that no fragment of the oxidant is associated with the free radical. The heme in the Fe(IV) enzyme is stable and does not oxidize the free radical site either intra- or intermolecularly. On the other hand, previous results from the presteady state kinetics of reduction and reductive titrations of the ES compound with ferrocyanide imply that the heme and free radical sites exchange oxidizing equivalents, in particular that the radical site once reduced can be reoxidized, either intra- or intermolecularly, by the ferryl heme. To resolve these contradictions, we propose a catalytic mechanism for cytochrome c peroxidase in which the radical site can exist in two conformations having very different reduction potentials and in which a significant flow of oxidizing equivalents between heme and free radical sites occurs only (i) during the hydrogen peroxide oxidation of the resting Fe(III) enzyme to form compound ES and (ii) within the initial transient intermediate formed upon the one-electron reduction of this oxidized product.
Cytochrome-c Peroxidase↗
Electron paramagnetic and electron nuclear double resonance of the hydrogen peroxide compound of cytochrome c peroxidase.
We have collected electron paramagnetic resonance (EPR) and electron nuclear double resonance (ENDOR) spectra from the hydrogen peroxide compound of yeast cytochrome c peroxidase, termed ES, employing EPR microwave frequencies of 9.6 and 11.6 GHz. We have measured and analyzed the temperature dependence of the spin-lattice relaxation rate (1/T1) of the paramagnetic center of ES over the temperature range 1.9 to 4 K. In addition, an upper bound to exchange coupling between the ferryl heme and EPR-visible centers of ES has been calculated and expressions for the dipolar interaction between a ferryl heme and a free radical have been derived. These results all confirm that the EPR signal of ES is not associated with an aromatic amino acid radical, and in particular not with a tryptophanyl radical. This conclusion has led us to consider an explanation of the EPR signal in terms of a nucleophilically stabilized methionyl radical.
Binding Sites↗
Electron-nuclear double resonance of the hydrogen peroxide compound of cytochrome c peroxidase: identification of the free radical site with a methionyl cluster.
The results of electron-nuclear double resonance and electron paramagnetic resonance (EPR) studies on the hydrogen peroxide compound of yeast cytochrome c peroxidase are inconsistent with previous proposals for the source of the EPR signal in this compound, in particular with its identification with an aromatic amino acid radical such as would arise by oxidation of a tryptophanyl side chain. The present observations lead us to propose that the EPR signal is associated with a cluster containing at least one methionine and in which proximate side chains share the charge created by loss of one electron.
Binding Sites↗
Definitaion of cytochrome c binding domains by chemical modification. Reaction of carboxydinitrophenyl- and trinitrophenyl-cytochromes c with baker's yeast cytochrome c peroxidase.
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Binding Sites↗
Steady state kinetics and binding of eukaryotic cytochromes c with yeast cytochrome c peroxidase.
1. The steady state kinetics for the oxidation of ferrocytochrome c by yeast cytochrome c peroxidase are biphasic under most conditions. The same biphasic kinetics were observed for yeast iso-1, yeast iso-2, horse, tuna, and cicada cytochromes c. On changing ionic strength, buffer anions, and pH, the apparent Km values for the initial phase (Km1) varied relatively little while the corresponding apparent maximal velocities varied over a much larger range. 2. The highest apparent Vmax1 for horse cytochrome c is attained at relatively low pH (congruent to 6.0) and low ionic strength (congruent to 0.05), while maximal activity for the yeast protein is at higher pH (congruent to 7.0) and higher ionic strength (congruent to 0.2), with some variations depending on the nature of the buffering ions. 3. Direct binding studies showed that cytochrome c binds to two sites on the peroxidase, under conditions that give biphasic kinetics. Under those ionic conditions that yield monophasic kinetics, binding occurred at only one site. At the optimal buffer concentrations for both yeast and horse cytochromes c, the KD1 and KD2 values approximate the Km1 and Km2 values. At ionic strengths below optimal, binding becomes too strong and above optimal, too weak. 4. Under ionic conditions that are optimal and give monophasic kinetics with horse cytochrome c but are suboptimal for the yeast protein, yeast cytochrome c strongly inhibits the reaction of horse cytochrome c with peroxidase, uncompetitively at one site and competitively at a second site. The appearance of the second site under monophasic conditions is interpreted as an allosteric effect of the inhibitor binding to the first site. 5. The simplest model accounting for these observations postulates two kinetically active sites on each molecule of peroxidase, a high affinity and a low affinity site, that may correspond to the free radical and the heme iron (IV) of the oxidized enzyme, respectively. Both oxidizing equivalents may be discharged at either site. Furthermore, the enzyme appears to exist as an equilibrium mixture of a high ionic strength form, EH and a low ionic strength form, EL, the former reacting optimally with yeast cytochrome c, and the latter with horse cytochrome c.
Animals↗
Preparation of cytochrome c peroxidase from baker's yeast.
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Chromatography, DEAE-Cellulose↗
Do evolutionary changes in cytochrome c structure reflect functional adaptations?
Following the demonstration that the rate of evolutionary change in the amino acid sequences of cytochromes c of eukaryotic species was not constant either for a single line of phylogenetic descent during different evolutionary intervals or for separate lines of descent, the concept that neutral mutations account for the vast majority of the evolutionary variations could no longer be accepted. Previous studies had shown that all eukaryotic cytochromes c tested appeared to be functionally indistinguishable in their reaction with mitochondrial respiratory chain components. However, an examination of the kinetics at low ionic strength led to the discovery of a high affinity reaction of cytochrome c with cytochrome c oxidase that revealed large differences in activity between the cytochromes of the horse, baker's yeast and the protist Euglena. Observed Km values for this reaction of 10(-7) to 10(-8) M appear to represent actual dissociation constants, as demonstrated by direct binding studies of cytochrome c with purified cytochrome c oxidase. The high affinity reaction is sensitive to ionic strength and inhibited by ADP and ATP in the range of physiological concentrations, ATP being three times as effective as ADP. The possibility is discussed that this effect of ATP on cytochrome c binding to its oxidase could provide the basis of a mechanism for mitochondrial respiratory control. The demonstration of differences between cytochrome c of various species in this kinetic system opens the way to a systematic study of the possible evolutionary adaptations of cytochromes c to their oxidases.
Amino Acid Sequence↗
Separate intramolecular pathways for reduction and oxidation of cytochrome c in electron transport chain reactions.
The monoiodotyrosine 74, formyltryptophan 59, mononitrotyrosine 67, and carboxymethylmethionine 80 derivatives of horse cytochrome c are defective in their ability to accept electrons from the succinate-cytochrome c reductase system, while their reactions with purified cytochrome c oxidase are essentially those of the native protein. The 4-nitrobenzo-2-oxa-1,3-diazole derivative of lysine 13 of horse cytochrome c and the bis-phenylglyoxal derivative of arginine 13 of Candida krusei cytochrome c have the opposite properties, in that they are readily reduced by the succinate-cytochrome c reductase (EC 1.3.99.1) system but are defective in their capability of transferring electrons to cytochrome c oxidase (EC 1.9.3.1). We conclude that electrons from mitochondrial cytochrome c reductase are transmitted to ferricytochrome c by a different pathway than electrons from ferrocytochrome c to cytochrome c oxidase. The present results are compatible with the concept that the mechanism of reduction involves an aromatic ring channel comprising residues 74, 59, 67, and 80, leading from the "left back" part of the protein to the heme iron. On the other hand, since residue 13 is immediately above the edge of the heme that is at the "front surface" of the molecule, we suggest that the electron leaves ferrocytochrome c to cytochrome c oxidase by way of the edge of pyrrole ring II or the adjacent surface-located sulfur of cysteinyl residue 17, which is thioether bonded to the heme. On this basis, the sites of electron entry and exit in cytochrome c would appear to be some 110 degrees of arc away from each other along the surface of the protein, explaining several previously observed phenomena.
Animals↗
A sensitive technique for the detection of the alpha 7 neuronal nicotinic acetylcholine receptor antagonist, methyllycaconitine, in rat plasma and brain.
Methyllycaconitine (MLA) is the most potent and selective antagonist of the alpha-bungarotoxin sensitive neuronal nicotinic acetylcholine receptor (nAChR). In the present study, an accurate and reproducible technique for the extraction and analysis of MLA from rat plasma and brain is described. This study further sought to determine whether pharmacologically relevant concentrations of MLA could be achieved in brain following peripheral administration. The detection limits for MLA were 0.5 ng/ml for plasma samples and 1.0 ng/g for brain samples. The pharmacokinetic properties of MLA in rat are characterized by a short elimination half-life (19 min) following intravenous (i.v.) administration and poor bioavailability following oral (p.o.) administration. Remarkably, the elimination half-life is significantly longer following p.o. administration (408 min). To assess the extent to which MLA can penetrate into brain, brain and plasma levels of MLA were determined at different time points following intraperitoneal (i.p.) administration of a dose of MLA that produced no observable side effects. Maximal plasma and brain levels were 694 +/- 106 ng/ml and 32 +/- 3 ng/g, respectively. These concentrations are within a range previously reported to selectively block alpha 7 nAChR mediated responses in vitro. Peripherally administered MLA may therefore be a useful tool to further probe the central nervous system functions of the alpha 7 nAChR subunit in vivo.
Aconitine↗
Lung metastases manifesting as pulmonary infarction by mucin and tumor embolization: radiographic, high-resolution CT, and pathologic findings.
We present a case of lung metastasis manifesting as lung infarction by mucin and tumor embolization. The radiograph and high-resolution CT showed multiple focal consolidations with ground glass attenuation in subpleural areas of both lungs. Diagnosis was made by open lung biopsy, which revealed pulmonary infarction caused by intravascular adenocarcinoma with numerous mucus emboli in pulmonary arteries. Subpleurally located consolidations on high-resolution CT correlated well with the histologic findings of lung infarction by mucin and tumor emboli.
Humans↗