坂根 郁夫

NADPH-cytochrome c reductase and cytochrome b559 were purified from the membrane fraction of phorbol myristate acetate-stimulated porcine polymorphonuclear leukocytes. The highly purified reductase oxidized NADPH and generated superoxide when combined with partially purified cytochrome b559 in the presence of phosphatidylcholine. In the same system, but under the anaerobic condition, the reductase was found to reduce cytochrome b559. © 1987 Academic Press, Inc.

The membrane fraction of Guinea pig polymorphonuclear leukocytes stimulated with phorbol myristate acetate exhibits the respiratory burst NADPH oxidase activity. This activity is markedly unstable at 37°C, disappearing with a half-life of 11.0 min. When the membrane fraction was pretreated with 0.1% glutaraldehyde, the NADPH oxidase was found to become more stable its half-life increased about sixfold without any enhancement of the initial activity. The glutaraldehyde treatment of the membrane fraction also protected the NADPH oxidase against inactivation with 0.1-0.2% Triton X-100. These stabilizing effects of glutaraldehyde on the NADPH oxidase seem to be due to its protein cross-linking ability, since its monovalent analogue, butyraldehyde, did not show any effect on the NADPH oxidase activity. In fact, sodium dodecyl sulfate-polyacrylamide gel electrophoresis revealed that glutaraldehyde cross-linked many proteins constituting the membrane. © 1987 The Journal of Biochemistry.

A membrane-bound NADPH-cytochrome c reductase, which is capable of forming the superoxide anion (O2 -) in the presence of menadione, was highly purified from membrane fractions of disrupted Guinea pig polymorphonuclear leukocytes by solubilization with 0.2% Triton X-100 and chromatographies on Sephacryl S-300 and 2',5'-ADP-agarose. The overall purification from the membrane fraction was over 110-fold, with a yield of about 6%. The purified preparation did not contain two other pyridine nucleotide-oxidizing enzymes: NADH- and NAD(P)H-oxidizing enzymes (J. Biochem. 94, 931-936, 1983).Besides cytochrome c, the purified enzyme was able to reduce menadione, Nitroblue tetrazolium (NBT) and 2,6-dichlorophenolindophenol. The reduction of menadione alone resulted in the formation of O2 -. The purified enzyme preparation contained FAD. When assayed by measuring O2 - generation in the presence of menadione, the enzyme showed an optimum pH at 7.0-7.4, and Km values for NADPH, NADH, and menadione were 25, 230, and 5.3 μM, respectively.The enzyme activity was not inhibited by NaN3 or dicumarol, but was by N-ethylmaleimide, EDTA, and quercetin; these inhibition profiles agree with those observed for the NADPH oxidase in the membrane fraction of phorbol-myristate acetate-stimulated leukocytes. Furthermore, when compared by means of the NBT-staining method combined with disc gel electrophoresis, the purified enzyme was electrophoretically indistinguishable from the NADPH-NBT reductase in the plasma membrane as well as phagosomes of the leukocytes. These results suggest that the purified NADPH-cytochrome c reductase is the putative flavoprotein of the NADPH oxidase system responsible for the respiratory burst. © 1984 Oxford University Press.

Pyridine nucleotide-oxidizing enzymes in guinea pig polymorphonuclear leukocytes were separated by Sephacryl S-300 gel filtration of the sonicated cells in the presence of 0.2% Triton X-100. Two peaks of NADPH-dependent cytochrome c reductase activities with apparent molecular weights of 400,000 and 120,000 were detected. The replacement of NADPH by NADH, on the other hand, revealed two NADH-dependent cytochrome c reductases with apparent molecular weights of 300,000 and 120,000. The addition of 40 μM menadione to assay mixtures considerably enhanced all the cytochrome c-reducing activities, and the enhancement was accompanied by the formation of superoxide anion (O2 -). Analysis of the subcellular localizations of these enzymes by fractional centrifugation demonstrated that the NADPH-dependent enzyme (400,000 daltons) was membrane-bound in nature, and that the NADH-dependent enzyme (300,000 daltons) and the NADPH- and NADH-dependent enzyme (120,000 daltons) existed in the cytosol of leukocytes. Thus, the leukocytes contained at least three types of menadione-dependent, O2 --forming enzymes: a membrane-bound NADPH-oxidizing enzyme, and soluble NADH-oxidizing and NAD(P)H-oxidizing enzymes.