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 characterization of mandelonitrile lyase from Prunus lyonii

The enzyme mandelonitrile lyase (EC 4.1.2.10) which catalyzes the decomposition of the cyanohydrin of benzaldehyde has been isolated and purified to homogeneity from mature seeds of the California cherry (Prunus lyonii). The purification procedure involved chromatography on DEAE-cellulose and Con-A-Sepharose with a final recovery of 60% of enzyme activity. Purification of only 4.3-fold yielded a nearly homogeneous preparation. The absorption spectrum of this protein shows maxima at 278, 389, and 463 nm, indicative of its flavoprotein character. The native molecular weight for the lyase was found to be 50,000. The subunit molecular weight of 59,000 was estimated by gel electrophoresis in the presence of sodium dodecylsulfate. The isoelectric point was estimated to be 4.75. The enzyme has a narrow pH optimum around 5.5 and is highly stable at 4 degrees C.     

The purification and characterization of glutaryl-coenzyme A dehydrogenase from porcine and human liver

Glutaryl-CoA dehydrogenase, a multifunctional enzyme responsible for dehydrogenation and decarboxylation of glutaryl-CoA to crotonyl-CoA, has been purified 1,680-fold from porcine liver mitochondria. The purified porcine enzyme has a subunit molecular weight of 47,800 and a native molecular weight of 190,500. Porcine glutaryl-CoA dehydrogenase catalyzed the conversion of [1,5-14C]glutaryl-CoA to [14C] crotonyl-CoA and 14CO2 in a 1:1:1 ratio. The porcine enzyme has Km values for electron transfer flavoprotein and glutaryl-CoA of 1.1 and 3.3 microM, respectively, and turnover numbers of 860 mol of electron transfer flavoprotein/min/mol of glutaryl-CoA dehydrogenase and 327 mol of glutaryl-CoA/min/mol of glutaryl-CoA dehydrogenase. Human glutaryl-CoA dehydrogenase has been purified 1,278-fold from human liver mitochondria. The purified human enzyme has a subunit molecular weight of 58,800 and a native molecular weight of 256,000. Human glutaryl-CoA dehydrogenase showed a reaction of only partial identity when compared to porcine glutaryl-CoA dehydrogenase by Ouchterlony double immunodiffusion analysis using antiserum raised against and monospecific for porcine glutaryl-CoA dehydrogenase.     

Purification, properties, and oligomeric structure of glutathione reductase from the cyanobacterium Spirulina maxima

Glutathione reductase [NAD(P)H:GSSG oxidoreductase EC 1.6.4.2] from cyanobacterium Spirulina maxima was purified 1300-fold to homogeneity by a simple three-step procedure involving ammonium sulfate fractionation, ion exchange chromatography on DEAE-cellulose, and affinity chromatography on 2',5'-ADP-Sepharose 4B. Optimum pH was 7.0 and enzymatic activity was notably increased when the phosphate ion concentration was increased. The enzyme gave an absorption spectrum that was typical for a flavoprotein in that it had three peaks with maximal absorbance at 271, 370, and 460 nm and a E1%271 of 23.3 Km values were 120 +/- 12 microM and 3.5 +/- 0.9 microM for GSSG and NADPH, respectively. Mixed disulfide of CoA and GSH was also reduced by the enzyme under assay conditions, but the enzyme had a very low affinity (Km 3.3 mM) for this substrate. The enzyme was specific for NADPH. The isoelectric point of the native enzyme at 4 degrees C was 4.35 and the amino acid composition was very similar to that previously reported from other sources. The molecular weight of a subunit under denaturing conditions was 47,000 +/- 1200. Analyses of pure enzyme by a variety of techniques for molecular weight determination revealed that, at pH 7.0, the enzyme existed predominantly as a tetrameric species in equilibrium with a minor dimer fraction. Dissociation into dimers was achieved at alkaline pH (9.5) or in 6 M urea. However, the equilibrium at neutral pH was not altered by NADPH or by disulfide reducing reagents. The Mr and S20,w of the oligomeric enzyme were estimated to be 177,000 +/- 14,000 and 8.49 +/- 0.5; for the dimer, 99,800 +/- 7000 and 5.96 +/- 0.4, respectively. Low concentrations of urea increased the enzymatic activity, but this increase was not due to changes in the proportions of both forms.     

Purification and some properties of component A of the 4-chlorophenylacetate 3,4-dioxygenase from Pseudomonas species strain CBS

Pseudomonas sp. strain CBS 3 possesses a two-component enzyme system which converts 4-chlorophenylacetate to 3,4-dihydroxyphenylacetate by the incorporation of 2 atoms of molecular oxygen. Component A of this enzyme system was purified to homogeneity by a 5-step procedure. After the last purification step the enzyme was homogeneous in analytical and sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The molecular weight of the native protein was determined to be 140,000 by Sephadex G-200 and 144,000 by analytical ultracentrifugation. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis showed that component A consists of three identical subunits with a molecular weight determined to range between 46,000 and 52,000. The isoelectric point was estimated to be 5.0. Component A shows an intensive red-brown color, and in the oxidized state it exhibits a visible absorption spectrum with a maximum at 458 nm and a shoulder at 560 nm. By reduction with sodium dithionite a new peak with a maximum at 518-520 nm is observed. The enzyme contains iron (1.6-1.8 mol/subunit) and acid-labile sulfide (1.6-1.9 mol/subunit) which suggests that component A is an iron-sulfur protein.     

Purification and characterization of 3-ketosteroid-delta 1-dehydrogenase from Nocardia corallina

The inducible 3-ketosteroid-delta 1-dehydrogenase of Nocardia corallina which catalyzes the introduction of a double bond into the position of carbon 1 and 2 of ring A of 3-ketosteroid has been obtained in four steps with a 50% yield and 360-fold purification. The enzyme is homogeneous as judged by SDS-gel electrophoresis and is a monomeric protein with a molecular weight of 60,500. The isoelectric point of the enzyme is about 3.1. The enzyme contains 1 mol of flavin adenine dinucleotide per mol of protein, and has a typical flavoprotein absorption spectrum with maxima of 458, 362 and 268 nm. The enzyme is very stable in the absence of added cofactors, and catalyzes the dehydrogenation of delta 4-3-ketosteroids in the presence of phenazine methosulfate, which acts as an excellent electron acceptor. Potassium ferricyanide and cytochrome c did not act as electron acceptors. The delta 1-dehydrogenation was also stimulated by molecular oxygen with stoichiometric production of hydrogen peroxide and delta 1,4-3-ketosteroid. The optimum pH is 10 for dehydrogenation using phenazine methosulfate, and is between 8.5 and 10 for the oxidase reaction. The enzyme oxidizes a wide variety of 3-ketosteroids, but not 3 beta-hydroxysteroids. 3-Ketosteroids having an 11 alpha- or 11 beta-hydroxyl group were oxidized at slow rates. The purified enzyme catalyzes efficiently aromatization of the A-ring of 19-nortestosterone and 19-norandrostenedione to produce estradiol and estrone. 19-Hydroxytestosterone, 19-hydroxyandrostenedion and 19-oxotestosterone were converted to the respective phenolic steroids with cleavage of the C10 side-chain. Activities of 3-ketosteroid-delta 4-dehydrogenase, delta 5-3-ketosteroid-4,5-isomerase, 3 beta-hydroxysteroid dehydrogenase and 17 beta-hydroxysteroid dehydrogenase were not observed in the purified preparations. Properties of this novel flavoprotein enzyme are discussed.     

Purification and characterization of NADH oxidase from a strain of Leuconostoc mesenteroides

An NADH oxidase was purified to homogeneity from Leuconostoc mesenteroides with a specific activity 100-fold higher than that of the crude extract. The purified NADH oxidase was an acidic protein having an S0 20,W of 5.49S and a molecular weight of 104,000, consisting of a dimer with 53,000 subunit size. The enzyme could use O2, dichlorophenolindophenol and methylene blue as oxidants, but not H2O2, cytochrome c, or ferricyanide. The physiological substrate was beta-NADH (Km = 0.12 mM) with O2 as the oxidant, probably forming H2O, rather than H2O2. Activity toward alpha-NADH was observed (Km = 0.14 mM), but the maximum velocity was 3 orders of magnitude lower than that with beta-NADH. alpha-NADPH and beta-NADPH were inert for the reaction. The enzyme showed a flavoprotein absorption spectrum with maxima at 273, 379, and 450 nm with a shoulder at 465 nm: the absorption at 450-465 nm disappeared on adding excess NADH or hydrosulfite. One mol of the holoenzyme contained approximately 2 mol of FAD. The apoenzyme was obtained by treatment with EDTA-KBr solution and could be reconstituted partially by adding FAD, but not riboflavin or FMN. The maximum activity of the reaction was observed at pH 6.5 in a temperature range of 35-45 degrees C. The activation energy was estimated to be 3.77 kcal/mol. The enzyme was inhibited by SH reagents, quinacrine, quinine, and Cu2+, but not by EDTA. Adenine and its nucleoside 5'-di- and triphosphates showed competitive inhibitions, while various metabolites, such as H2O2, FDP, acetyl phosphate, lactate, ethanol, and acetate, did not affect the reaction.     

D-aspartate oxidase from beef kidney. Purification and properties

The flavoprotein D-aspartate oxidase (EC 1.4.3.1) has been purified to homogeneity from beef kidney cortex. The protein is a monomer with a molecular weight of 39,000 containing 1 molecule of flavin. The enzyme as isolated is a mixture of a major active form containing FAD and a minor inactive form containing 6-hydroxy-flavin adenine dinucleotide (6-OH-FAD). The absorption and fluorescence spectral properties of the two forms have been studied separately after reconstitution of the apoprotein with FAD or 6-OH-FAD, respectively. FAD-reconstituted D-aspartate oxidase has flavin fluorescence, shows characteristic spectral perturbation upon binding of the competitive inhibitor tartaric acid, is promptly reduced by D-aspartic acid under anaerobiosis, reacts with sulfite to form a reversible covalent adduct, stabilizes the red anionic form of the flavin semiquinone upon photoreduction, and yields the 3,4-dihydro-FAD-form after reduction with borohydride. A Kd of 5 X 10(-8) M was calculated for the binding of FAD to the apoprotein. 6-OH-FAD-reconstituted D-aspartate oxidase has no flavin fluorescence, shows no spectral perturbation in the presence of tartaric acid, is not reduced by D-aspartic acid under anaerobiosis, does not stabilize any semiquinone upon photoreduction, and does not yield the 3,4-dihydro-form of the coenzyme when reduced with borohydride; the enzyme stabilizes the p-quinoid anionic form of 6-OH-FAD and lowers its pKa more than two pH units below the value observed for the free flavin. The general properties of the enzyme thus resemble those of the dehydrogenase/oxidase class of flavoprotein, particularly those of the amino acid oxidases.     

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.     

Rabbit liver glutathione reductase. Purification and properties

Hepatic glutathione reductase can be obtained in relatively good amounts from rabbit by a procedure which is fairly simple and sufficiently rapid. The purified flavoprotein shows absorbance ratios at 274, 379, and 463 nm of 8.2:0.92:1.0, respectively; the FAD fluorescence is nearly completely quenched by the protein. Gradient ultracentrifugation and sodium dodecyl sulfate gel electrophoresis indicate that the enzyme is a dimer, consisting of subunits of about 56,000 molecular weight; flavin content suggests one FAD per chain. Gel filtration under a variety of conditions, on the other hand, yields a molecular weight in the range 56,000–67,000. It is proposed that rabbit liver glutathione reductase can be active also as monomer. Kinetic parameters of the enzyme have been determined under optimal conditions. The rabbit liver glutathione reductase is, at physiological pH, absolutely specific for NADPH.     

Membrane-bound D-gluconate dehydrogenase from Pseudomonas aeruginosa. Purification and structure of cytochrome-binding form.

A membrane-bound D-gluconate dehydrogenase [EC 1.1.99.3] was solubilized from membranes of Pseudomonas aeruginosa and purified to a homogeneous state with the aid of detergents. The solubilized enzyme was a monomer in the presence of at least 0.1% Triton X-100, having a molecular weight of 138,000 on polyacrylamide gel electrophoresis or 124,000--131,000 on sucrose density gradient centrifugation. In the absence of Triton X-100, the enzyme became dimeric, having a molecular weight of 240,000--260,000 on sucrose density gradient centrifugation. Removal of Triton X-100 caused a decrease in enzyme activity. Enzyme activity was stimulated by addition of phospholipid, particularly cardiolipin, in the presence of Triton X-100. The enzyme had a cytochrome c1, c-554(551), which might be a diheme cytochrome, and it also contained a covalently bound flavin but not ubiquinone. In the presence of sodium dodecyl sulfate, the enzyme was dissociated into three components with molecular weights of 66,000, 50,000, and 22,000. The components of 66,000 and 50,000 daltons corresponded to a flavoprotein and cytochrome c1, respectively, but that of 22,000 dalton remained unclear as to its function.     

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.     

Properties of anthranilate hydroxylase (deaminating), a flavoprotein from Trichosporon cutaneum

Anthranilate hydroxylase was purified from the yeast Trichosporon cutaneum. This enzyme is a simple flavoprotein which apparently does not require any additional cofactor for the conversion of anthranilate to 2,3-dihydroxybenzoate. Anthranilate hydroxylase has Mr of approximately 95,000, with subunit Mr of 50,000; it contains 2 mol of FAD/mol of enzyme. A number of compounds in addition to anthranilate serve as substrates, or effectors, for this enzyme. Oxygen-labeling experiments show that the oxygen atom at the 3-position of the product, 2,3-dihydroxybenzoate, originates from O2, while that at the 2-position is derived from H2O. A mechanism is proposed involving imine formation and hydrolysis during the reaction with the flavin hydroperoxide formed from reduced enzyme flavin and molecular oxygen. This proposal is in accord with the mechanism postulated for other flavoprotein aromatic hydroxylases.     

A cytosolic FAD-containing enzyme catalyzing cytochrome c reduction in Trypanosoma cruzi. I. Purification and some properties

A cytosolic flavoprotein enzyme for the protozoan, Trypanosoma cruzi, has been purified essentially to homogeneity by DEAE-cellulose and 2',5'-ADP-agarose column chromatography. The native enzyme has a molecular weight of 100,000 +/- 6,000, is composed of two identical subunits of molecular weight 52,000 +/- 1,000, and contains FAD in the ratio of 1 mol of FAD per mol of enzyme subunit. The enzyme is NADPH-dependent and is capable of reducing cytochrome c, ferricyanide, 2,6-dichloroindophenol, and menadione, but not adrenalin. It does not hydroxylate either sodium salicylate or sodium p-hydroxybenzoate, but N-methylaniline and N,N-dimethylaminobenzaldehyde-supported oxidation of NADPH has been demonstrated. Plots of initial velocity against NADPH concentration give hyperbolic curves with Km values of 6.289 X 10(-5) M. The enzyme is clearly different from the microsomal NADPH-cytochrome c reductase in its intracellular distribution, molecular weight, dimeric nature, presence of only FAD, and activity against secondary and tertiary aromatic amines.     

Isolation and some properties of NAD+ reductase of the green photosynthetic bacterium Prosthecochloris aestuarii.

NAD+ reductase of the green photosynthetic bacterium Prosthecochloris aestuarii was isolated and purified by ammonium sulfate fractionation, DEAE-cellulose column chromatography, and Sephadex G-200 gel filtration. This enzyme is an FAD-containing flavoprotein and has absorption maxima at 485 (shoulder0 452, 411, and 385 nm (the 411 nm band is due to cytochrome). The molecular weight of the enzyme as determined by gel filtration using Sephadex G-200 is 119,000. The enzyme catalyzes the reduction of NAD+ and NADP+ by photoreduced spinach ferredoxin or reduced benzyl viologen...     

Assay, purification and properties of mammalian d-2-hydroxy acid dehydrogenase

1. A new method is described for the measurement of d-2-hydroxy acid dehydrogenase in samples of animal tissues. 2. The distribution of the enzyme in a number of animals was determined. Of the animal tissues tested, the most active source of the enzyme was found to be rabbit kidney cortex. 3. The enzyme was purified from rabbit kidney to a stage at which it appears to be homogeneous in the analytical ultracentrifuge and on polyacrylamide-gel electrophoresis. 4. The molecular weight was estimated by gel filtration to be approx. 102000; combination of gelfiltration data and the sedimentation coefficient gave a value of 95000. 5. The purified enzyme has a spectrum typical of a flavoprotein. The change induced in the spectrum on addition of d-malate or d-lactate suggests the formation of a flavin semiquinone. 6. Flavin can be removed by treatment with acid ammonium sulphate, and activity can be restored to the inactive apoenzyme by addition of FAD, but not of FMN or riboflavin. 7. Studies of acceptor specificity showed that the enzyme has a relatively weak d-2-hydroxy acid oxidase activity.     

Purification and Properties of Cystathionine [gamma]-Synthase from Wheat (Triticum aestivum L.)

Cysthathionine [gamma]-synthase (CS), an enzyme involved in methionine biosynthesis, was purified from an acetone powder prepared from wheat (Triticum aestivum L.). After several chromatographic steps and radiolabeling of the partially purified enzyme with sodium cyanoboro[3H]hydride, a single polypeptide with a molecular weight of 34,500 was isolated by sodium dodecyl sulfate-high performance electrophoresis chromatography. Since the molecular weight of the native enzyme was 155,000, CS apparently consists of four identical subunits. The pyridoxal 5[prime]-phosphate-dependent forward reaction has a pH optimum of 7.5 and follows a hybrid ping-pong mechanism with Km values of 3.6 mM and 0.5 mM for L-homoserine phosphate and L-cysteine, respectively. L-Cysteine methyl ester, thioglycolate methyl ester, and sodium sulfide were also utilized as thiol substrates. The latter observation suggests that CS and phosphohomoserine sulfhydrase might be a single enzyme. CS does not seem to be a regulatory enzyme but was irreversibly inhibited by DL-propargylglycine (Ki = 45 [mu]M, Kinact = 0.16 min-1). Furthermore, the homoserine phosphate analogs 4-(phosphonomethyl)-pyridine-2-carboxylic acid, Z-3-(2-phosphonoethen-1-yl)pyridine-2-carboxylic acid, and DL-E-2-amino-5-phosphono-3-pentenoic acid acted as reversible competitive inhibitors with Ki values of 45, 40, and 1.1 [mu]M, respectively.     

Solubilization, purification and characterization of D-alanine dehydrogenase from Pseudomonas aeruginosa and effects of solubilization on its properties

D-alanine dehydrogenase, an inducible, membrane associated enzyme of Pseudomonas aeruginosa was solubilized from envelope preparations by treatment with Triton X-100 and purified 31-fold in the presence of 0.05% Triton X-100 to 60% homogeneity. Gel electrophoresis indicated the presence of a single subunit of approximately 49,000 molecular weight. The enzyme contained FAD, and absorption spectra were typical of an iron-sulfur flavoprotein. Solubilization produced significant changes in some properties of the enzyme: solubilized enzyme showed increased affinity for D-alanine; a broader substrate specificity; and increased temperature sensitivity, compared with the membrane associated form.     

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.     

Presence of a flavoprotein in O2-evolving Photosystem II preparations from the cyanobacterium Anacystis nidulans

A highly active O₂-evolving Photosystem II complex which was greatly depleted of phycobiliproteins was isolated from the cyanobacterium Anacystis nidulans. This complex contained the flavoprotein with l-amino acid oxidase activity which we have previously shown to be present in thylakoid preparations of this cyanobacterium (Pistorius, E.K. and Voss, H. (1982) Eur. J. Biochem. 126, 203–209). One of the most prominent polypeptides in this O₂-evolving Photosystem II complex had a molecular weight of 49 kDa. This polypeptide co-chromatographed on SDS-polyacrylamide gels with the purified l-amino acid oxidase which consists of two subunits of 49 kDa. The antagonistic effect of CaCl₂ on the two examined reactions could also be demonstrated with this O₂-evolving Photosystem II complex: CaCl₂ stimulated photosynthetic O₂ evolution, but inhibited the l-amino acid oxidase activity. Both reactions were inhibited by o-phenanthroline. These results further support a functional relationship between the flavoprotein with l-amino acid oxidase activity and Photosystem II activities in A. nidulans. However, we only found 1 mol FAD per 350–650 mol chlorophyll, although 1 gatom Mn per 5–10 mol chlorophyll was present. When we assume a photosynthetic unit of about 40 chlorophylls, then in most preparations the FAD values were more than a factor of 10 too low. Results which we obtained with the purified l-amino acid oxidase showed that the FAD values were in most enzyme samples lower than the theoretically expected value of 2 mol FAD per mol enzyme. Moreover, in some cases the absorption spectrum of the enzyme showed substantial deviations from the spectrum of oxidized FAD. These experiments indicated that the flavin in the enzyme could partly exist in a form which was different from ‘authentic oxidized FAD’. We do not yet know the chemical nature of this ‘modified flavin’.