Agric. Biol. Chem ., 44 (10), 2329•`2335 , 1980 2329•@ Molecular and Enzymatic Properties of Pyridoxamine (Pyridoxine) •@5 '-Phosphate Oxidas•@ from Baker's Yeast •@ Haruh ito TSUGE, Kenji OZEKI and Kazuj i OHASHI •@ Department of Agricu ltural Chemi stry , Gifu University, •@ Kagamigaha ra, Gifu 504 , Japan •@ Receiv ed March 17 , 1980 •@ Pyridoxamine (pyridoxine) 5'-phosphate oxidase purified from baker's yeast was found to h ave a molecular weight of ca, 55 ,000 daltons based on polyacrylamide gel electrophoresis. The size of the enzyme subunit was analyzed by gel electrophoresis in the presence of sodium dodecylsulfate . Thi s showed that the enzyme was composed of two nonidentical subunits with a molecular weight of 27,000 and 25,000 daltons . Fluorescence titration of the apoenzyme with FMN suggested that the holoenzyme contained one mol of FMN per mol of the enzyme . The Km value of FMN for apoenzyme was calculated to be ca . 16 nM on both activities of pyridoxamine 5'-phosphate oxidase and pyridoxine 5'-phosphate oxidase . Chemical Co. PNP was prepared by reduction of PLP with NaBH4 and purified by•@recrystallization.4) Commercially available FMN was purified according to the method of Moffatt and Khorana.7) Pooled FMN fraction was checked by thin-layer chromatography as giving one spot by two solvent systems8) (I: 5% Na2HPO4.12H2O, and ‡U: n-butanol-acetic acid-water (12:3:5)) prior to use. PMP(PNP) oxidase was purified from dried baker's yeast as previously described.2) Enzyme preparation of eluate on calcium phosphate gel column, whose specific activity was more th an 25 ƒÊmol PLP formed/hr/mg protein as meas ured with the PLP formation and PMP as standard substrate, was extensively used. •@ Refer ence protein s with known molecular weight were purchased from Boehringer Mannheim GmbH (oval bumin and aldolase from rabbit muscle), and from Sigma Chemical Co. (cytochrome c from horse heart; chymo trypsinogen A, Type ‡U; triosephosphate isomerase from rabbit muscle, Type X;9) bovine serum albumin, Frac tion V powder and hexokinase from yeast, Type F-30010)). Enzyme assay. All experiments were carried out under reduced light to minimize photo-decompositi on of sub strate and coenzyme. PMP and PNP oxidase activities were assayed as described elsewhere.11) Preparation of apoenzyme. Electrophoretically pure enzyme was dialyzed against 200 ml of 0.2M potassium acetate buffer (pH 4.0, 5.0 and 6.0) containing 2M KBr and 0.1 mM EDTA for appropriate periods at 5•Ž, then against 200 ml of 0.2M•@ potassium phosphate buffer (pH 7.0) for desired periods at 5•Ž in the dark. Both buffer solutions were changed every 6 hr. The amount of active apoenzyme so obtained was estimated from the assays in •@n the previous paper,1,2) we described a purification method and some properties of pyridoxamine (pyridoxine) 5'-phosphate oxi dase [PMP(PNP) oxidase*; EC 1.4.3.5] from dried baker's yeast. Yeast enzyme is a typical flavoprotein similar to the enzymes from Alcaligenes faecalis3) and rabbit liver, 4) but the enzyme differed from these enzymes in that it oxidizes PMP much faster than PNP.2) Considerable differences in molecular weight of the enzymes from these sources have been reported. The enzyme from A . faecalis is 38,000 with on e FMN, probably in a single subunit,5) and that from rabbit liver is established as 54,000, being composed of possibly two identical subunits and of one FMN.4,6) •@ The prese nt invest igation aimed to establish the physico-chemical properties of the purified yeast enzyme, and a subunit structure was revealed .•@ EXPERIMENTAL •@ Materials. PMP HCl was purc hased from Sigma * Abbreviations: PMP , pyridoxamine 5'-phosphate; PNP, pyridoxine 5'-phosphate; PLP, pyridoxal 5'-phos phate; SDS, sodium dodecylsulfate.
