Characteristic properties of retinal oxidase (retinoic acid synthase) from rabbit hepatocytes.
Retinal oxidase (retinoic acid synthase) (EC 1.2.3.11) was purified electrophoretically, as a single protein band, from rabbit liver cytosol. The characteristic properties, enzymatic reaction mechanism, substrate specificity and kinetic parameters for retinals and molecular oxygen of the retinal oxidase were investigated. The Km values for all-trans-retinal of the retinal oxidase was the lowest than those for the other retinal derivatives. The retinal oxidase is a metalloflavoenzyme containing 2 FADs as the coenzyme, and 8 irons, 2 molybdenums, 2 disulfide bonds and 8 inorganic sulfurs. Its relative molecular mass was determined to be 270 kDa by gel filtration HPLC on a TSKgel G3000swXL column. Its minimum molecular mass was estimated to be 135 kDa by SDS-PAGE. The optical spectrum of the retinal oxidase showed absorption peaks at 275, 340 and 450 nm, and shoulders at 420 and 473 nm, in the oxidized form. The molecular extinction coefficients of the oxidase at selected wavelengths were determined. Circular dichroism spectra of the retinal oxidase were measured in the ultraviolet and visible regions. These spectra showed positive absorption in the visible region. The amino-acid composition was determined. The activity of the oxidase was not affected by any cofactors, such as NADP+, NAD+, NADPH and NADH, and it did not occur under anaerobic conditions. The oxidase was not inhibited by BOF-4272, a potent inhibitor of xanthine dehydrogenase, or rat anti-xanthine dehydrogenase IgG. Experiments on retinoic acid formation under 18O2 or H2(18)O demonstrated that the oxygen of water was incorporated into retinoic acid by the retinal oxidase, but not molecular oxygen.
Tsujita
M
M
Department of Biochemistry, Kagawa Medical School, Japan.
Tomita
S
S
Miura
S
S
Ichikawa
Y
Y
D000445:Aldehyde Oxidoreductases / Q000037:antagonists & inhibitors / Q000737:chemistry* / Q000302:isolation & purification; D000596:Amino Acids / Q000032:analysis; D000818:Animals; D003600:Cytosol / Q000201:enzymology; D007700:Kinetics; D008099:Liver / Q000201:enzymology*; D008297:Male; D008670:Metals / Q000032:analysis; D011817:Rabbits; D050697:Retinal Dehydrogenase; D012172:Retinaldehyde / Q000378:metabolism; D013455:Sulfur / Q000032:analysis; D013696:Temperature
D000596:Amino Acids; D008670:Metals; D013455:Sulfur; D000445:Aldehyde Oxidoreductases; D050697:Retinal Dehydrogenase; D012172:Retinaldehyde