Purification and properties of cyclopentanone oxygenase of Pseudomonas NCIB 9872.

1. Cyclopentanone oxygenase from Pseudomonas NCIB 9872 has been purified some 40-fold. It gives a single peak in the ultracentrifuge and a single major protein band on polyacrylamide gels contaminated with about 5% of a slower migrating impurity. Flavin dissociates from the protein during electrophoresis. 2. The enzyme has a molecular weight of about 200000 and is a homopolymeric assemblage of either three of four subunits of molecular weight 54000-58000. 3. The prosthetic group is FAD and values of about 2.5 are typically obtained for the number of moles bound to each mole of holoenzyme. Some FAD probably dissociates during purification and it seems likely that each subunit binds one FAD in the undamaged protein.     

Rapid purification of 2,4-dichlorophenol hydroxylase by biospecific desorption from 10-carboxydecylamino-sepharose

10-Carboxydecylamino-Sepharose, which bears a mixture of ionic and aliphatic substituent groups, adsorbs 2,4-dichlorophenol hydroxylase from Acinetobacter in a non-biospecific manner. The enzyme has been specifically desorbed by its substrate, 2,4-dichlorophenol, giving a 42-fold purification (to greater than 90% purity) in a single step. The enzyme contained 3.1 moles of FAD per mole and displayed a catalytic constant of 14.7 s(-1). Mixed-function adsorbents probably have wide applicability for biospecific desorption of proteins. The present report indicates that they may be useful in the purification of aromatic hydroxylases bearing flavin prosthetic groups that readily dissociate in conventional purification procedures employing conditions of high ionic strength.     

Identification of pyruvate in S-adenosylmethionine decarboxylase from rat liver

S-Adenosylmethionine decarboxylase has been purified to homogeneity (26,000-fold) from rat liver. The enzyme has a molecular weight of 155,000 and a subunit molecular weight of 42,000. One mole of covalently bound pyruvate was found to be present per mole of enzyme subunit. This is the first mammalian enzyme found to contain covalently linked pyruvate.     

Purification and properties of Escherichia coli 4'-phosphopantothenoylcysteine decarboxylase: presence of covalently bound pyruvate

4'-Phosphopantothenoylcysteine decarboxylase was purified 900-fold from Escherichia coli B with an overall yield of 6%. The enzyme migrates as a single band with a molecular weight of 35,000 +/- 3000 in 10% polyacrylamide gel electrophoresis under denaturing conditions. The native enzyme has an apparent molecular weight of 146,000 +/- 9000 as determined by a gel exclusion column. At pH 7.6 and 25 degrees C, Km = 0.9 mM and Vmax = 600 nmol/(min X mg of protein). The pH optimum for Vmax is between 7.5 and 7.7. Hydroxylamine, phenylhydrazine, potassium cyanide, and sodium borohydride as well as pyridoxal phosphate and pyridoxal inactivated the enzyme. The enzyme contains covalently bound pyruvate as suggested by the isolation of [3H]lactate and pyruvate from [3H]NaBH4-reduced enzyme and native enzyme, respectively. One mole of [3H]lactate was isolated per 39,000 g of [3H]NaBH4-reduced and completely inactivated enzyme, and 1 mol of pyruvate was isolated per 31,000 +/- 4000 g of native enzyme. Mild base treatment released lactate and pyruvate from the reduced and the native enzymes, respectively, suggesting the pyruvate is attached to the enzyme by an ester bond. These findings are in accord with similar results obtained with the horse liver enzyme (R. Scandurra, personal communication). The presence of covalently bound pyruvate in the bacterial and mammalian enzymes suggests that pyruvate plays a major role in the mechanism of action.     

Purification and properties of methylmalonyl coenzyme A mutase from human liver

Methylmalonyl coenzyme A (CoA) mutase has been purified to apparent homogeneity from human liver by a procedure involving column chromatography on DEAE-cellulose, Matrex-Gel Blue A, hydroxylapatite, and Sephadex G-150. The overall purification achieved is 500- to 600-fold, yield 3–5%. Electrophoresis of the native purified protein on nondenaturing polyacrylamide gels shows a single diffuse band coincident with the enzyme activity; dodecyl sulfate/polyacrylamide gels show a single protein band with an apparent molecular weight of 77,500. The native protein has a molecular weight of approximately 150,000 by Sephadex G-150 chromatography, suggesting that it is composed of two identical subunits. The activity of the purified enzyme is stimulated only slightly (10–20%) by the addition of its cofactor, adenosylcobalamin, indicating that the purified enzyme is largely saturated with coenzyme. The spectrum of the enzyme is consistent with the presence of about 1 mole of adenosylcobalamin per mole of subunit. The enzyme displays complex kinetics with respect to dl-methylmalonyl CoA; substrate inhibition by l-methylmalonyl CoA appears to occur. The enzyme activity is stimulated by polyvalent anions (PO₄^3? > SO₄^2? > Cl^?); monovalent cations are without effect, but high concentrations of divalent cations are inhibitory. The enzyme activity is insensitive to N-ethylmaleimide, is rapidly destroyed at temperatures > 50 °C, and shows a broad pH optimum around pH 7.5.     

Purification and characterization of the bifunctional thymidylate synthetase-dihydrofolate reductase from methotrexate-resistant Leishmania tropica

Thymidylate synthetase (TS) and dihydrofolate reductase (DHFR) in Leishmania tropica exist as a bifunctional protein. By use of a methotrexate-resistant strain, which overproduces the bifunctional enzyme, the protein was purified 80-fold to apparent homogeneity in two steps. The native protein has an apparent molecular weight of 110 000 and consists of two subunits with identical size and charge. Available data indicate that each of the subunits possesses TS and DHFR. The TS of the bifunctional protein forms a covalent 5-fluoro-2'-deoxyuridylate (FdUMP)-(+/-)-5,10-methylenetetrahydrofolate-enzyme complex in which 2 mol of FdUMP is bound per mole of enzyme. In contrast, titration of DHFR with methotrexate indicated that only 1 mol of the inhibitor is bound per mole of dimeric enzyme. Both TS and DHFR activities of the bifunctional enzyme were inactivated by the sulfhydryl reagent N-ethylmaleimide. Substrates of the individual enzymes afforded protection against inactivation, indicating that each enzyme requires at least one cysteine for catalytic activity. Kinetic evidence indicates that most, if not all, of the 7,8-dihydrofolate produced by TS is channeled to DHFR faster than it is released into the medium. Although the mechanism of channeling is unknown, the possibility that the two enzymes share a common folate binding site has been ruled out.     

Purification and some properties of cholesterol oxidase from Schizophyllum commune with covalently bound flavin.

Cholesterol oxidase [EC 1.1.3.6] from Schizophyllum commune was purified by an affinity chromatography using 3-O-succinylcholesterol-ethylenediamine (3-cholesteryl-3-[2-aminoethylamido]propionate) Sepharose gels. The resulting preparation was homogeneous as judged by sodium dodecyl sulfate (SDS) polyacrylamide gel electrophoresis. The molecular weight of the enzyme was estimated to be 53,000 by SDS-gel electrophoresis and 46,000 by sedimentation equilibrium. The enzyme contained 483 amino acid residues as calculated on the basis of the molecular weight of 53,000. The enzyme consumed 60 mumol of O2/min per mg of protein with 1.3 mM cholesterol at 37 degrees C. The enzyme showed the highest activity with cholesterol; 3 beta-hydroxysteroids, such as dehydroepiandrosterone, pregnenolone, and lanosterol, were also oxidized at slower rates. Ergosterol was not oxidized by the enzyme. The Km for cholesterol was 0.33 mM and the optimal pH was 5.0. The enzyme is a flavoprotein which shows a visible absorption spectrum having peaks at 353 nm and 455 nm in 0.1 M acetate buffer, pH 4.0. The spectrum was characterized by the hypsochromic shift of the second absorption peak of the bound flavin. The bound flavin was reduced on anaerobic addition of a model substrate, dehydroepiandrosterone. Neither acid not heat treatment released the flavin coenzyme from the enzyme protein. The flavin of the enzyme could be easily released from the enzyme protein in acid-soluble form as flavin peptides when the enzyme protein was digested with trypsin plus chymotrypsin. The mobilities of the aminoacyl flavin after hydrolysis of the flavin peptides on thin layer chromatography and high voltage electrophoresis differed from those of free FAD, FMN, and riboflavin. A pKa value of 5.1 was obtained from pH-dependent fluorescence quenching process of the aminoacyl flavin. AMP was detected by hydrolysis of the flavin peptides with nucleotide pyrophosphatase. The results indicate strongly that cholesterol oxidase from Schizophyllum commune contains FAD as the prothetic group, which is covalently linked to the enzyme protein. The properties of the bound FAD were comparable to those of N (1)-histidyl FAD.     

Purification and properties of dihydropterin oxidase from Drosophila melanogaster

The enzyme dihydropterin oxidase has been purified to apparent homogeneity from the fruit fly Drosophila melanogaster. This enzyme uses a variety of 2-amino-4-oxo-7,8-dihydropteridine compounds as substrates, including 2-amino-4-oxo-7,8-dihydropteridine (called dihydropterin), Km = 0.11 microM; 6-lactoyl-7,8-dihydropterin, Km = 1.80 microM; and 7,8-dihydrobiopterin, Km = 1.25 microM. The products in each case are the corresponding fully oxidized compounds 2-amino-4-oxopteridine, oxidized 6-lactoyl-7,8-dihydropterin, and 6-L-erythro-dihydroxypropylpterin, respectively. During the reaction, 1 mol of molecular oxygen is consumed per mole of substrate oxidized, and hydrogen peroxide is produced. The molecular weight of the enzyme is approximately 51,500. The enzyme apparently contains two polypeptide chains of identical molecular weight. The prosthetic group of the enzyme has been identified as FAD. From the determination of the occurrence of the enzyme in the various stages of the life cycle of D. melanogaster and from other considerations, the tentative conclusion is reached that the physiological role of dihydropterin oxidase is to convert dihydropterin to 2-amino-4-oxopteridine, a reaction that is believed to be essential in the formation of 2-amino-4-oxo-7-hydroxypterin in D. melanogaster.     

Regulation, purification, and properties of xanthine dehydrogenase in Neurospora crassa.

Xanthine dehydrogenase (EC 1.2.1.37) is the first enzyme in the degradative pathway by which fungi convert purines to ammonia. In vivo, the activity is induced 6-fold by growth in uric acid. Hypoxanthine, xanthine, adenine, or guanine also induce enzyme activity but to a lesser degree. Immunoelectrophoresis using monospecific antibodies prepared against Neurospora crassa xanthine dehydrogenase shows that the induced increase in enzyme activity results from increased numbers of xanthine dehydrogenase molecules, presumably arising from de novo enzyme synthesis. Xanthine dehydrogenase has been purified to homogeneity by conventional methods followed by immunoabsorption to monospecific antibodies coupled to Sepharose 6B. Electrophoresis of purified xanthine dehydrogenase reveals a single protein band which also exhibits enzyme activity. The average specific activity of purified enzyme is 140 nmol of isoxanthopterine produced/min/mg. Xanthine dehydrogenase activity is substrate-inhibited by xanthine (0.14 mM), hypoxanthine (0.3 mM), and pterine (10 micron), is only slightly affected by metal binding agents such as KCN (6 mM), but is strongly inhibited by sulfhydryl reagents such as p-hydroxymercuribenzoate (2 micron). The molecular weight of xanthine dehydrogenase is 357,000 as calculated from a sedimentation coefficient of 11.8 S and a Stokes radius of 6.37 nm. Sodium dodecyl sulfate-gel electrophoresis of the enzyme reveals a single protein band having a molecular weight of 155,000. So the xanthine dehydrogenase protein appears to be a dimer. In contrast to xanthine dehydrogenases from animal sources which typically possess as prosthetic groups 2 FAD molecules, 2 molybdenum atoms, 8 atoms of iron, and 8 acid-labile sulfides, the Neurospora enzyme contains 2 FAD molecules, 1 molybdenum atom, 12 atoms of iron, and 14 eq of labile sulfide/molecule. The absorption spectrum of the enzyme shows maxima between 400 and 500 nm typical of a non-heme iron-containing flavoprotein.     

Purification and properties of pyrazon dioxygenase from pyrazon-degrading bacteria.

Chromatography on DEAE-cellulose and gel filtration on Sephadex revealed that pyrazon dioxygenase from pyrazon-degrading bacteria consists of three different enzyme components. No component alone oxidizes the phenyl moiety of pyrazon, only when the three components are combined can oxidation be detected. Following electron paramagnetic resonance and ultraviolet measurements the protein nature of the three components was determined: component A1 (molecular weight about 180000,red-brown in colour) is an iron-sulphur protein. The existence of approximately two moles of iron and two moles of inorganic sulphur per mole of protein was demonstrated. This enzyme component was purified to homogeneity in disc electrophoresis. Component A2 is a yellow protein of a molecular weight of about 67000. FAD was shown to be the prosthetic group of this protein. Component B (molecular weight about 12000, brown in colour) is a protein of the ferredoxin type, which was purified to homogeneity, as demonstrated by disc electrophoresis. A hypothetical scheme for the cooperation of the three components is proposed: component A2 accepts as cosubstrate NADH and functions as a ferredoxin reductase. The ferredoxin, component B, has the function of an electron carrier. The conversion of the substrates is effected by component A1, the terminal dioxygenase.     

Purification and properties of maize polyamine oxidase: a flavoprotein

Polyamine oxidase was purified from maize shoots to homogeneityby the criteria of polyacrylamide gel electrophoresis and ultracentrifugation.The purified yellow enzyme showed absorption maxima at 278,380 and 460 nm. The molecular weight estimated by gel filtrationwas about 65,000 and the sedimentation coefficient was 5.95S. Sodium dodecylsulfate gel electrophoresis yielded a singleband at a molecular weight of 65,000. The enzyme contained 1mole of FAD per mole of enzyme. Amino acid composition and kineticproperties of the enzyme are presented. (Received April 30, 1980; )     

Purification and properties of NADH-ferredoxinNAP reductase, a component of naphthalene dioxygenase from Pseudomonas sp. strain NCIB 9816.

Cells of Pseudomonas sp. strain NCIB 9816, after growth with naphthalene or salicylate, contain a multicomponent enzyme system that oxidizes naphthalene to cis-(1R,2S)-dihydroxy-1,2-dihydronaphthalene. We purified one of these components to homogeneity and found it to be an iron-sulfur flavoprotein that loses the flavin cofactor during purification. Dialysis against flavin adenine dinucleotide (FAD) showed that the enzyme bound 1 mol of FAD per mol of enzyme protein. The enzyme consisted of a single polypeptide with an apparent molecular weight of 36,300. The purified protein contained 1.8 g-atoms of iron and 2.0 g-atoms of acid-labile sulfur and showed absorption maxima at 278, 340, 420, and 460 nm, with a broad shoulder at 540 nm. The purified enzyme catalyzed the reduction of cytochrome c, dichlorophenolindophenol, Nitro Blue Tetrazolium, and ferricyanide. These activities were enhanced in the presence of added FAD. The ability of the enzyme to catalyze the reduction of the ferredoxin involved in naphthalene reduction and other electron acceptors indicates that it functions as an NAD(P)H-oxidoreductase in the naphthalene dioxygenase system. The results suggest that naphthalene dioxygenase requires two proteins with three redox groups to transfer electrons from NADH to the terminal oxygenase.     

Rat liver cytosolic azoreductase. Purification and characterization.

An azoreductase has been purified to apparent homogeneity from the hepatic 105,000 x g supernatant fraction of 3-methylcholanthrene-treated rats. In the presence of sodium dodecyl sulfate, the purified enzyme preparation electrophoreses on polyacrylamide gels as a single protein band with a molecular weight of 30,000. In the absence of detergent, chromatography of the azoreductase on Sephadex G-100 gives a molecular weight of about 52,000 suggesting that the native enzyme may exist as a dimer. The purified azoreductase has a typical flavoprotein absorption spectrum and contains 2 mol of FAD/mol of enzyme. The enzyme catalyzes the reductive fission of methyl red (2'-carboxy-4-N,N-dimethylaminoazobenzene) and a structure-activity study indicates that the 2'-carboxyl group of methyl red is essential for catalysis since other structurally related analogs are totally inactive.     

Pig liver monoamine oxidase: Studies on the subunit structure

The molecular weight of pig liver MAO has previously been shown to be about 115,000 with 1 mole of covalently bound FAD per mole of enzyme. Gel filtration of purified enzyme on Sepharose 4B in 6 m guanidine and 0.1 m mercaptoethanol (MCE) and analytical ultracentrifugation in 0.1% sodium dodecyl sulfate (SDS) and 0.1% MCE yielded molecular weights of 55,000 and 63,000, respectively. By polyacrylamide electrophoresis in 0.1% SDS + MCE one band of 60,000 MW appeared. These results seem to imply that the enzyme is composed of two subunits of which one carries the active site. If MCE was omitted during the gel electrophoresis two equally large bands of about 60,000 MW were formed. By using enzyme inhibited by [¹⁴C]pargyline, a MAO-inhibitor blocking the active site of the enzyme in a 1:1 molar ratio, it was found, however, that both bands contained pargyline. Furthermore, amino acid analyses yielded the same amino acid composition of the two bands. The results are interpreted that the enzyme is composed of two subunits of identical molecular size (about 60,000) of which only one contains the active site and that the enzyme preparation contained two forms of the enzyme presumably differing in the number of disulfide bonds.     

Purification and properties of L-alpha-glycerophosphate oxidase from Streptococcus faecium ATCC 12755.

A procedure was developed to purify the Streptococcus faecium ATCC 12755 L-alpha-glycerophosphate oxidase. The molecular weight of the purified enzyme was 131,000 and the subunit molecular weight was 72,000. Two moles of FAD were bound/mol of enzyme. Apo-L-alpha-glycerophosphate oxidase displayed physical properties similar to the holoenzyme as judged by electrophoresis in 10% buffer gels at pH 8.5 and by centrifugation in a 5 to 20% linear sucrose gradient. The apoenzyme was completely reactivated by incubation with FAD. L-alpha-Glycerophosphate oxidase was specific for L-alpha-glycerophosphate when compared with several other pohsphorylated glycerol and sugar derivatives. Oxygen was the preferred electron acceptor. At 10 mM DL-alpha-glycerophosphate (below the Km of 26 mM for L-alpha-glycerophosphate), activity was increased from 2.6- to 10-fold by increasing the buffer concentration from 0.01 to 0.1 m. This buffer effect was observed with potassium phosphate and other anionic buffers. In 0.001 m potassium phosphate buffer, pH 7.0, activity was increased by several divalent metal ions, including 10 mM CaCl2 (7.7-fold activation) and 10 mM MgCl, (6.8-fold activation). Fructose 6-phosphate and fructose1-phosphate were inhibitors of the L-alpha-glycerophosphate oxidase.     

Human FAD synthase (isoform 2): a component of the machinery that delivers FAD to apo-flavoproteins

A soluble form of human FAD synthase (isoform 2; hFADS2) was produced and purified to homogeneity as a recombinant His-tagged protein. The enzyme binds 1 mole of the FAD product very tightly, although noncovalently. Complete release of FAD from the 'as isolated' protein requires extensive denaturation. A 75 : 25 mixture of apo/holoprotein could be prepared by treatment with mild chaotropes, allowing estimatation of the contribution made by bound FAD to the protein stability and evaluatation of whether structural rearrangements may be required for FAD release. Under turnover conditions, the enzyme catalyzes FAD assembly from ATP and FMN and, at a much lower rate, the pyrophosphorolytic hydrolysis of FAD. Several mechanistic features of both reactions were investigated in detail, along with their dependence on environmental conditions (pH, temperature, dependence on metals). Our data indicate that FAD release may represent the rate-limiting step of the whole catalytic cycle and that the process leading to FAD synthesis, and delivery to client apoproteins may be tightly controlled.     

A new oxygenase, 2-nitropropane dioxygenase of Hansenula mrakii. Enzymologic and spectrophotometric properties.

2-Nitropropane dioxygenase, purified to homogeneity from Hansenula mrakii (IFO 0895), has a molecular weight of approximately 62,000 and consists of two subunits nonidentical in molecular weight (39,000 and 25,000). Stoichiometrical studies and the results obtained with 18O2 showed that 2 atoms of molecular oxygen are incorporated into 2 molecules of acetone formed from 2-nitropropane. In addition to 2-nitropropane, nitroethane, 3-nitro-2-pentanol, and 1-nitropropane are oxidatively dentrified. The enzyme, which exhibits absorption maxima at 274, 370, 415, and 440 nm and a shoulder at 470 nm, contains 1 mol of FAD and 1 g atom of non-heme iron per mol of enzyme. The enzyme-bound FAD is reduced by 2-nitropropane under anaerogic conditions, but the enzyme-bound Fe3+ is not affected. The introduction of oxygen to the reduced form of enzyme causes reoxidation of the enzyme. The bound FAD and Fe3+ are reduced by the addition of nitromethane, which is not a substrate, under anaerobic conditions. The aerobic dialysis of the enzyme treated with nitromethane causes reoxidation of only the Fe2+. Sodium dithionite also reduces both the enzyme-bound FAD and Fe3+ under anaerobic conditions. When the enzyme is dialyzed against 10 mM potassium phosphate buffer (pH 7.0) immediately after reduction by dithionite, the absorption spectrum similar to that of the native enzyme appeared with concomitant restoration of approximately 80% of the activity. The enzyme activity is significantly inhibited by pyrocatechol-3,5-disulfonate disodium salt, 8-hydroxyquinoline, reducing agents such as 2-mercaptoethanol, and HgCl2. The Michaelis constants are as follows: 2-nitropropane (2.13 X 10(-2) M), nitroethane (2.43 X 10(-2) M), 3-nitro-2-pentanol (6.8 X 10(-3) M), 1-nitropropane (2.56 X 10(-2) M), and oxygen (3.03 X 10(-4) M, with 2-nitropropane).     

The mercuric and organomercurial detoxifying enzymes from a plasmid-bearing strain of Escherichia coli.

Two separate enzymes, which determine resistance to inorganic mercury and organomercurials, have been purified from the plasmid-bearing Escherichia coli strain J53-1(R831). The mercuric reductase that reduces Hg2+ to volatile Hg0 was purified about 240-fold from the 160,000 X g supernatant of French press disrupted cells. This enzyme contains bound FAD, requires NADPH as an electron donor, and requires the presence of a sulfhydryl compound for activity. The reductase has a Km of 13 micron HgCl2, a pH optimum of 7.5 in 50 mM sodium phosphate buffer, an isoelectric point of 5.3, a Stokes radius of 50 A, and a molecular weight of about 180,000. The subunit molecular weight, determined by gel electrophoresis in the presence of sodium dodecyl sulfate, is about 63,000 +/- 2,000. These results suggest that the native enzyme is composed of three identical subunits. The organomercurial hydrolase, which breaks the mercury-carbon bond in compounds such as methylmercuric chloride, phenylmercuric acetate, and ethylmercuric chloride, was purified about 38-fold over the starting material. This enzyme has a Km of 0.56 micron for ethylmercuric chloride, a Km of 7.7 micron for methylmercuric chloride, and two Km values of 0.24 micron and over 200 micron for phenylmercuric acetate. The hydrolase has an isoelectric point of 5.5, requires the presence of EDTA and a sulfhydryl compound for activity, has a Stokes radius of 24 A, and has a molecular weight of about 43,000 +/- 4,000.     

Steroid-transforming enzymes from microorganisms. X. Enrichment of a 4-en-3-oxosteroid-5 alpha-reductase from Mycobacterium smegmatis as well as separation and enrichment of the apoenzyme by means of affinity chromatography

The 4-en-3-oxosteroid-5 alpha-reductase from Mycobacterium smegmatis was bound biospecifically on the affinant containing an immobilized testosterone ligand. The enzyme obtained by elution with ethylene glycol and urea in a 32 fold purity has a S. A. of 8.73 X 10(-3) microM androstenedione min-1 mg-1. The coenzyme (FAD) could be separated from the immobilized enzyme substrate complex on the affinity matrix, in the presence of (NH4)2SO4 at pH 3.0. After elution of the apoenzyme 97% of the initial enzyme activity was obtained by incubation with FAD. The reactivated enzyme results in a 40-fold enrichment.     

Complete purification and general characterization of FAD synthetase from rat liver

Flavin adenine dinucleotide synthetase (ATP:FMN adenylyltransferase, EC 2.7.7.2) was purified about 10,000-fold from the high-speed supernatant of rat liver by a sequence of ammonium sulfate fractionation and column chromatographies on DEAE-Sephadex (A-50), chromatofocusing, FMN-agarose affinity, and Sephadex G-200. The specific activity of the purified enzyme was 133 units (nanomoles of FAD formed per min at 37 degrees C)/mg of protein. This preparation was free from contaminating FAD pyrophosphatase. The apparent molecular weight was estimated to be 97,000 by gel filtration on Sephadex G-200. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis revealed an apparent subunit molecular weight of 53,000. Hence, the enzyme is a dimer of approximately 100,000. The enzyme was found most active at pH 7.1, requires Mg2+, and is essentially irreversible in the direction of FAD formation. Kinetic analysis gave Km values of 9.6 microM for FMN and 53 microM for ATP.