Toshiaki Miki

Variety of Nucleotide Sugar Transporters with Respect to the Interaction with Nucleoside Mono- and Diphosphates

Nucleotide sugar transporters have long been assumed to be antiporters that exclusively use nucleoside monophosphates as antiport substrates. Here we present evidence indicating that two other types of nucleotide sugar transporters exist that differ in their antiport substrate specificity. Biochemical studies using microsomes derived from Saccharomyces cerevisiae cells expressing either human (h) UGTrel7 or the Drosophila (d) FRC (Fringe connection) transporter revealed that (i) efflux of preloaded UDP-glucuronic acid from the yeast microsomes expressing hUGTrel7 was strongly enhanced by UDP-GlcNAc added in the external medium, but not by UMP or UDP, suggesting that hUGTrel7 may be described as a UDP-sugar/UDP-sugar antiporter, and (ii) addition of UDP-sugars, UDP, or UMP in the external medium stimulated the efflux of preloaded UDP-GlcNAc from the yeast microsomes expressing dFRC to a comparable extent, suggesting that UDP, as well as UMP, may serve as an antiport substrate of dFRC. Antiport of UDP-sugars with these specific substrates was reproduced and definitively confirmed using proteoliposomes reconstituted from solubilized and purified transporters. Possible physiological implications of these observations are discussed.

Oxidation Process of Bovine Heart Ubiquinol-Cytochrome c Reductase as Studied by Stopped-flow Rapid-scan Spectrophotometry and Simulations Based on the Mechanistic Q Cycle Model

Stopped-flow rapid-scan spectrophotometry was employed to study complicated oxidation processes of ubiquinol-cytochrome c reductase (QCR) that was purified from bovine heart mitochondria and maximally contained 0.36 mol of ubiquinone-10/mol of heme c 1. When fully reduced QCR was allowed to react with dioxygen in the presence of cytochrome c plus cytochrome c oxidase, the oxidation of b-type hemes accompanied an initial lag, apparently low potential heme b L was oxidized first, followed by high potential heme b H. Antimycin A inhibited the oxidation of both b-type hemes. The oxidation of heme c 1 was triphasic and became biphasic in the presence of antimycin A. On the other hand, starting from partially reduced QCR that was poised at a higher redox potential with succinate and succinate-cytochrome creductase, the b-type hemes were oxidized immediately without a lag. When the ubiquinone content in QCR was as low as 0.1 mol/mol heme c 1 the oxidation of theb-type hemes was almost suppressed. As the Q-deficient QCR was supplemented with ubiquinol-2, the rapid oxidation ofb-type hemes was restored to some extent. These results indicate that a limited amount of ubiquinone-10 found in purified preparations of QCR is obligatory for electron transfer from theb-type hemes to iron-sulfur protein (ISP) and hemec 1. The characteristic oxidation profiles of hemeb L, heme b H, and hemec 1 were simulated successfully based on a mechanistic Q cycle model. According to the simulations the two-electron oxidation of ubiquinol-10 via the ISP and hemec 1 pathway, which is more favorable thermodynamically than the bifurcation of electron flow into both ISP and heme b L, does really occur as long as hemeb L is in the reduced state and provides ubiquinone-10 at center i. Mechanistically this process takes time, thus explaining the initial lag in the oxidation of theb-type hemes. With the partially reduced QCR, inherent ubisemiquinone at center i immediately oxidizes reduced heme b H thus eliminating the lag. The mechanistic Q cycle model consists of 56 reaction species, which are interconnected by the reaction paths specified with microscopic rate constants. The simulations further indicate that the rate constants for electron transfer between the redox centers can be from 105 to 103 s−1 and are rarely rate-limiting. On the other hand, a shuttle of ubiquinone or ubiquinol between center o and center i and the oxidation of heme c 1 can be rate-limiting. The interplay of the microscopic rate constants determines the actual reaction pathway that is shown schematically by the “reaction map.” Most significantly, the simulations support the consecutive oxidation of ubiquinol in center o as long as both hemeb L and heme b H are in the reduced state. Only when heme b L is oxidized and ISP is reduced can SQ o donate an electron to hemeb L. Thus, we propose that a kinetic control mechanism, or “a kinetic switch,” is significant for the bifurcation of electron flow.

The reaction of horseradish peroxidase with hydroperoxides derived from Triton X-100

All of the commercially available Triton X-100 examined gave Compound I upon reaction with horseradish peroxidase, followed by its gradual transition into Compound II. Titration of horseradish peroxidase with Triton X-100 to form Compound I indicated that 1% (v/v) aqueous solutions of the detergent contained 0.4 to 3.2 microM equivalent peroxide but iodometric titration revealed 1.1 to 5.0 microM peroxide, suggesting the occurrence of different types of peroxides, reactive and unreactive with the peroxidase. The rate constant for Compound I formation was 1.5 X 10(7) M-1 S-1 at pH 7.4 at 25 degrees C, and for conversion into Compound II apparent first-order rate constants were 5.2 X 10(-3) to 1.7 X 10(-2) S-1. These results indicate that the Triton peroxides are as highly reactive as hydrogen peroxide. The amount of Triton peroxides increased as aqueous solutions of the detergent were allowed to stand, but the peroxides were destroyed by treatment with sodium borohydride. Although freshly prepared aqueous solutions of sodium cholate, sodium dodecyl sulfate, Tween 20 (polyoxyethylene sorbitan monolaurate), and Emasol 1130 (an equivalent of Tween 20) did not contain any detectable amount of peroxide, aged solutions of sodium dodecyl sulfate and Emasol 1130 contained peroxides. These observations suggest the need for appropriate precautions when biologically active substances vulnerable to attack by peroxides are incubated with Triton X-100 either for their solubilization from biomembranes or for other processing.

KINETIC CHARACTERIZATION OF THE REDOX COMPONENTS IN SOLUBILIZED MEMBRANES FROM PORCINE NEUTROPHILS: REDUCTION WITH DITHIONITE AND PHOTOEXCITED NAD(P)H

Abstract— Cytochrome b558 in solubilized membranes prepared from porcine neutrophils was reduced by dithionite with a second‐order rate constant of 2.5 times 106 M‐1 s‐1 at pH 7.4 and 20°C accompanied by spectral changes with peaks at 428 nm and 560 nm and isosbestic points at 420 and 441 nm. When an anaerobic mixture of solubilized membranes and NAD(P)H was exposed to a white light, cytochrome b558 was reduced biphasically but with almost the same spectral profiles as in the dithionite reduction. Thus, participation of redox component(s) of unknown nature in the photochemical reduction was suggested.

Cytochrome C Peroxidase Activity of Cytochrome Oxidase and its Coupling to Proton Pumping

Coupled with reduction of molecular oxygen, cytochrome oxidase of mammalian as well as bacterial origin translocates protons from one side of the energy transducing membrane to the other developing an electrochemical potential gradient across the membrane to drive H+-ATP synthase (1-8). The proton pump capacity has been demonstrated with phospholipid vesicles reconstituted with purified cytochrome oxidase preparation. (9-28). These vesicles are very useful for studying the coupling mechanism between the redox reactions and the proton translocation, but no systematic studies have been done successfully to solve this problem. In pursuing this mechanism and identifying the elementary step(s) for the coupling, it would be advantageous experimentally if the oxygen reduction can be resolved into partial reactions.