Studies on cytochrome c oxidase activity of the cytochrome c1aa3 complex from Thermus thermophilus.
(57/79)
Cytochrome oxidase from T. thermophilus is isolated as a noncovalent complex of cytochromes c1 and aa3 in which the four redox components of aa3 appear to be associated with a single approximately 55,000-D subunit while the heme C is associated with a approximately 33,000-D peptide (Yoshida, T., Lorence, R. M., Choc, M. G., Tarr, G. E., Findling, K. L., and Fee, J. A. (1983) J. Biol. Chem. 258, 112-123). We have examined the steady state transfer of electrons from ascorbate to oxygen by cytochrome c1aa3 as mediated by horse heart, Candida krusei, and T. thermophilus (c552) cytochromes c as well as tetramethylphenylenediamine (TMPD). These mediators exhibit simple Michaelis-Menten kinetic behavior yielding Vmax and KM values characteristic of the experimental conditions. Three classes of kinetic behavior were observed and are qualitatively discussed in terms of a reaction scheme. The data show that tetramethylphenyldiamine and cytochromes c react with the enzyme at independent sites; it is suggested that cytochrome c1 may efficiently transfer electrons to cytochrome aa3. When incorporated into phospholipid vesicles, the highly purified cytochrome c1aa3 was found to translocate one proton into the exterior medium for each molecule of cytochrome c552 oxidized. The combined results suggest that this bacterial enzyme functions in a manner generally identical with the more complex eucaryotic enzyme. (+info)
Pulse-radiolysis studies on the interaction of one-electron-reduced species with ascorbate oxidase in aqueous solution.
(58/79)
The interaction of e-aq., CO2-. and one-electron reduced nitroaromatics (RNO2-.) with ascorbate oxidase (AAO) was studied in aqueous solution at pH 6.0 and 7.5 by using the technique of pulse radiolysis. From observations at 330, 410 and 610 nm, interaction of e-aq. and CO2-. with AAO results in non-specific reduction of the protein followed by reduction of Type 1 Cu in a rate-determining intramolecular step. Only a few per cent of the reducing equivalents ultimately results in reduction of Type 1 Cu. With large excesses of reducing equivalents (e-aq. and CO2-.) with respect to the copper concentration, the amount of Type 1 copper reduced never exceeds 50% of the total amount of Type 1 copper after a single radiation pulse. With less-powerful reducing agents, e.g. RNO2-. reduction of Type 1 Cu occurs via a bimolecular step, and there is no evidence for formation of radicals on protein residues. From observations at 330 nm it is evident that Type 2 and/or Type 3 Cu may also be reduced along with Type 1 Cu. Almost stoichiometric reduction of AAO by RNO2-. was observed, e.g. the protein accepts 6-7 reducing equivalents. It is inferred that the various types of redox couples Cu2+/Cu+ are in equilibrium and that intramolecular electron transfer between the different types of Cu is not rate-determining when using RNO2-. as reducing agent. (+info)
Characterization of ascorbic acid transport by adrenomedullary chromaffin cells. Evidence for Na+-dependent co-transport.
(59/79)
Ascorbic acid transport by bovine adrenomedullary chromaffin cells in primary cultures has been characterized. Ascorbic acid uptake can be measured by either high performance liquid chromatography with electrochemical detection or radiometric techniques with L-[1-14C]ascorbic acid. The transport system is temperature- and energy-dependent and exhibits Michaelis-Menten kinetics with an apparent Km of 29 microM when the external Na+ concentration is 150 mM. Uptake of ascorbate by chromaffin cells is ouabain-sensitive and dependent on the presence of external Na+. Ascorbate transport by chromaffin cells is, thus, an active process driven by the Na+ electrochemical gradient. The kinetics of this co-transport system fits an "affinity type" model where binding of Na+ to the carrier increases the affinity to ascorbate and vice versa. Thus, the data suggest that binding of either Na+ or ascorbate induces a conformational change in the transporter, which results in a change in the association constant for the second ligand while the mobility of the carrier remains unchanged. Cellular uptake of ascorbate into adrenomedullary chromaffin cells appears to be followed by its distribution into several subcellular compartments. One subcellular compartment for concentration of ascorbate is the chromaffin vesicle where it accumulates at a relatively slow rate. The interrelationships between ascorbate transport and other aspects of ascorbate metabolism and chromaffin vesicle function and dopamine beta-hydroxylation are also considered. (+info)
Immunochemical evidence for phytochrome regulation of the specific activity of ascorbate oxidase in mustard seedlings.
(60/79)
A monospecific antiserum to a Cucurbita ascorbate oxidase was shown to cross-react with the mustard enzyme. Purified antiserum was used in rocket immunoelectrophoresis plates to measure the amount of ascorbate oxidase protein in mustard cotyledon and hypocotyl extracts. On transfer from darkness to far-red light and activity of the enzyme in expanding cotyledons increased but the amount of enzyme protein remained the same. Unimbibed mustard seeds were shown to contain the same amount of antigenic protein as expanding cotyledons although in the seeds the enzyme was inactive. In hypocotyls continuous far-red light treatment also led to an increase in ascorbate oxidase activity; in this case there was also an increase in enzyme protein although this was relatively small. The results are discussed in relation to the mechanism by which phytochrome may control the activity of this enzyme. (+info)
Specific spectrophotometry of ascorbic acid in serum or plasma by use of ascorbate oxidase.
(61/79)
We describe a specific enzymatic spectrophotometric method for ascorbic acid in serum or plasma. Samples are analyzed indirectly by measuring the absorbance at 593 nm of a reaction product, a complex of ferrous ion and 2,4,6-tris(2-pyridyl)-s-triazine (Fe2+-TPTZ). This product is formed by reduction of the corresponding ferric ion complex (Fe3+-TPTZ), which is nonspecifically reduced by various biological reducing agents under acidic conditions. Ascorbic acid is specifically quantified by pretreating one of a pair of replicate samples with ascorbate oxidase (EC 1.10.3.3), to oxidize the ascorbic acid, then reacting both samples with Fe3+-TPTZ and measuring the difference between the absorbances at 593 nm of the treated and untreated samples. This difference is linearly related to ascorbic acid concentrations from 10 to 100 mg/L. Ten repeat determinations of a serum pool with added ascorbic acid yielded a CV of 2.8% and a mean of 47.2 mg/L. The correlation (r) between the proposed method and the dinitrophenylhydrazine method was 0.93 for 32 samples analyzed by both methods. The present method is specific for ascorbic acid and requires no deproteinization. (+info)
Spectrophotometric determination of ascorbic acid and dehydroascorbic acid.
(62/79)
We present a method for measuring ascorbic acid in methanol/trichloroacetic acid extracts prepared from human plasma after enzymatic oxidation of ascorbic acid to dehydroascorbic acid by ascorbate oxidase. Samples were assayed by spectrophotometrically monitoring the kinetics of the concentration-dependent absorbance changes of dehydroascorbic acid with phosphate-citrate-methanol buffers. Ascorbic acid was determined as the difference between dehydroascorbic acid and total ascorbic acid content. The detection limit was < 0.5 mumol/L. The calibration curve was linear (r > 0.995) over the range 0-1000 mumol/L. Analytical recovery of ascorbic acid added to plasma was 93-105%. The between-day variance was < 7%. Comparison of the spectrophotometric determination (y) with a chromatographic procedure (x) gave y = 1.02x - 0.653 (Sylx = 3.61) over the range of physiologically relevant concentrations. Total analysis time is < 10 min per sample and allows the simultaneous analysis of multiple samples. (+info)
Stability of Japanese-lacquer-tree (Rhus vernicifera) laccase to thermal and chemical denaturation: comparison with ascorbate oxidase.
(63/79)
The thermal denaturation of laccase from the Japanese lacquer tree (Rhus vernicifera) was studied by differential scanning calorimetry. The endotherms of holo-laccase, type 2-Cu-depleted laccase and apo-laccase were deconvoluted into two independent two-state transitions, providing evidence for a domain structure of the protein. The correlation of the two transitions with the bleaching of copper optical bands and the decrease of the transitions' enthalpy on Cu removal show that the process involves the denaturation of Cu sites. No detectable unfolding of secondary structure was observed, since the thermal transitions, characterized by low overall specific enthalpy, did not modify either the laccase c.d. spectra in the beta-fold region or the maximum wavelength of the fluorescence emission. On chemical denaturation, however, the emission was red-shifted by about 20 nm. The laccase behaviour is substantially different from that of stellacyanin, a protein containing a single blue Cu ion, in which the thermal transition had higher specific enthalpy and induced a large change of the c.d. spectrum in the beta-fold region. The laccase denaturation behaviour is similar to that of ascorbate oxidase from zucchini (courgette; Cucurbita pepo) [Savini, D'Alessio, Giartosio, Morpurgo and Avigliano (1990) Eur. J. Biochem. 190, 491-495], suggesting a structural analogy. In both proteins heating may cause a change of tertiary structure through modifications of Cu co-ordination with loosening of the bonds between the structural domains at the interface of which the trinuclear Cu cluster is located. (+info)
Characterization of ascorbate oxidase from Acremonium sp. HI-25.
(64/79)
The ascorbate oxidase obtained from a microorganism, Acremonium sp. HI-25 (molecular weight, 80 kDa; monomeric protein), was studied with respect to atomic absorption, EPR, absorption spectra, circular dichroism (CD) spectra, and steady-state kinetics. The enzyme was found to be a multicopper protein, containing four copper atoms of three kinds, types 1, 2, and 3 copper, in the ratio of 1:1:2. The EPR parameters of the type 1 and 2 copper atoms in the ascorbate oxidase are very similar to those in the case of the ascorbate oxidase obtained from cucumber, which is a dimeric protein. The apparent Km and kcat values for ascorbic acid of the ascorbate oxidase from Acremonium sp. HI-25 are almost the same as those of the monomeric unit of the ascorbate oxidase from cucumber. (+info)