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.     

D-glyceraldehyde-3-phosphate dehydrogenase purified from rabbit muscle contains phosphotyrosine.

Homogeneous preparations of D-glyceraldehyde-3-phosphate dehydrogenase purified from rabbit muscle were found to contain 0.2-0.7 moles of covalently bound phosphate per mole of the enzyme. With the use of anti-phosphotyrosine antibodies, evidence was obtained that the enzyme is phosphorylated at tyrosine residues.     

Purification and properties of d-4-deoxy-5-oxoglucarate hydro-lyase (decarboxylating)

1. An enzyme extracted from Pseudomonas acidovorans was purified and shown to catalyse the simultaneous dehydration and decarboxylation of d-4-deoxy-5-oxoglucarate. It is proposed to name the enzyme d-4-deoxy-5-oxoglucarate hydro-lyase (decarboxylating), trivial name ;deoxyoxoglucarate dehydratase'. 2. No added cofactors were required, and the enzyme was inactivated when incubated with its substrate in the presence of sodium borohydride. Under these conditions the substrate and enzyme appeared to be bound covalently. 3. The action of the enzyme is readily explained if it is assumed that d-4-deoxy-5-oxoglucarate forms a Schiff base with a lysine residue in the enzyme.     

Purification of the peroxisomal fatty acyl-CoA oxidase from rat liver

Fatty acyl-CoA oxidase, the rate limiting enzyme of the peroxisomal fatty acid oxidizing system, has been purified from rat liver to near homogeneity by a procedure involving affinity chromatography of its apoenzyme on flavin adenin dinucleotide-Sepharose. The oxidase presents an absolute requirement for the dinucleotide which is weakly bound to the apoenzyme (K_D, 0.6 μM). The highest specific activity obtained was 27 units/mg protein. The purified enzyme has two major polypeptides with apparent molecular weights of 45,000 and 22,000. These results suggest that the enzyme is a flavoprotein with non covalently bound flavin adenin dinucleotide composed of four subunits, two of 45,000 m.w. and two of 22,000 m.w.     

Influence of associated lipid on the properties of purified bovine erythrocyte acetylcholinesterase

Acetylcholinesterase has been isolated from bovine erythrocyte membranes by affinity chromatography using a m-trimethylammonium ligand. The purified enzyme had hydrophobic properties by the criterion of phase partitioning into Triton X-114. The activity of the hydrophobic enzyme was seen as a slow-moving band in nondenaturing polyacrylamide gels. After treatment with phosphatidylinositol-specific phospholipase C, another form of active enzyme was produced that migrated more rapidly toward the anode in these gels. This form of the enzyme partitioned into the aqueous phase in Triton X-114 phase separation experiments and was therefore hydrophilic. The hydrophobic form bound to concanavalin A in the absence of Triton X-100. As this binding was partially prevented by detergent, but not by alpha-methyl mannoside, D-glucose, or myo-inositol, it is in part hydrophobic. Erythrocyte cell membranes showed acetylcholinesterase activity present as a major form, which was hydrophobic by Triton X-114 phase separation and in nondenaturing gel electrophoresis moved at the same rate as the purified enzyme. In the membrane the enzyme was more thermostable than when purified in detergent. The hydrophobic enzyme isolated, therefore, represents a native form of the acetylcholinesterase present in the bovine erythrocyte cell membrane, but in isolation its stability becomes dependent on amphiphile concentration. Its hydrophobic properties and lectin binding are attributable to the association with the protein of a lipid with the characteristics of a phosphatidylinositol.     

The purification of formiminotransferase and cyclodeaminase by combination of affinity chromatography and isoelectric focusing

The twin enzyme glutamate-formiminotransferase and formiminotetrahydrofolate-cyclodeaminase were purified by subsequent ammonium sulphate fractionation, affinity chromatography with tetrahydrofolate covalently bound to Sepharose 4B and following isoelectric focusing. In the presence of formiminoglutamate the major part of the enzyme focused at pH 5.8 and was electrophoretically homogeneous. Another peak with the enzyme activity focusing at pH 4.5 was found in low amount but it was a heterogeneous protein mixture. The presence of formiminoglutamate in the course of affinity chromatography appeared to be necessary for achieving the purified enzyme.     

Isolation and characterization of methylmalonyl-CoA mutase from human placenta

Methylmalonyl-CoA mutase, one of two known cobalamin-dependent enzymes present in mammalian tissues, has been isolated from 2.5 kg of human placenta utilizing affinity chromatography on 5'-deoxyadenosylcobalamin-Sepharose as the major purification step. The enzyme gives a single band on polyacrylamide disc gel electrophoresis. The Mr of the enzyme is 145,000 and it has two subunits of Mr = 72,000. Amino acid analysis reveals major differences from other human cobalamin-binding proteins. Based on x-ray fluorescence, the enzyme has 2 mol of cobalamin bound/mol of enzyme. In contrast to purified cobalamin transport proteins, most of the cobalamin bound to the enzyme is not released by boiling at low pH in the presence of KCN, or dialysis against 7.5 M guanidine containing 0.2 M dithiothreitol, or both, suggesting the possibility that cobalamin may be covalently attached to the purified enzyme. Both precipitating antibodies and antibodies that inhibit enzyme activity have been raised in a chicken.     

Control of 5-aminolaevulinate synthetase activity in Rhodopseudomonas spheroides. Binding of pyridoxal phosphate to 5-aminolaevulinate synthetase.

1. Pyridoxal 5'-phosphate is a cofactor essential for the enzymic activity of aminolaevulinate synthetase from Rhodopseudomonas spheroides. It also aids activation of the low-activity enzyme by trisulphides such as cystine trisulphide, whereas inactivation of enzyme is facilitated by its absence. 2. The fluorescence spectrum of purified high-activity enzyme is that expected for a pyridoxal phosphate--Schiff base, but the firmly bound cofactor does not appear to be at the active centre. In dilute solutions of enzyme this grouping is inaccessible to nucleophiles such as glycine, hydroxylamine, borohydride and cyanide, at pH 7.4. 3. An active-centre Schiff base is formed between enzyne and added pyridoxal phosphate, which is accessible to nucleophiles. Concentrated solutions of this enzyme--Schiff base on treatment with glycine yield apo- and semi-apoenzyme, which can re-bind pyridoxal phosphate. 4. Two types of binding of pyridoxal phosphate are distinguishable in dilute solution of enzyme, but these become indistinguishable when concentrated solutions are treated with cofactor. A change occurs in the susceptibility towards borohydride of the fluorescence of the "structural" pyridoxal phosphate. 5. One or two molecules of cofactor are bound per subunit of mol. wt. 50 000 in semiapo- or holo-enzyme. The fluorescence of pyridoxamine phosphate covalently bound to enzyme also indicates one to two nmol of reducible Schiff base per 7000 units of activity in purified and partially purified samples of enzyme. 6. Cyanide does not convert high-activity into low-activity enzyme, but with the enzyme-pyridoxal phosphate complex it forms a yellow fluorescent derivative that is enzymically active.     

Covalent binding of folic acid to dimethylglycine dehydrogenase

Dimethylglycine dehydrogenase (EC 1.5.99.2) carries out the oxidative demethylation of dimethylglycine to sarcosine in liver mitochondria. In vivo, the enzyme uses tightly bound tetrahydropteroyl pentaglutamate (H₄PteGlu₅) as an acceptor of the one-carbon group generated during the reaction. The purified enzyme can use, but does not require, H₄PteGluB and under these conditions formaldehyde is the one-carbon unit produced. It is reported that folic acid may be covalently linked to dimethylglycine dehydrogenase in a specific and saturable manner so that only 1 mole of folic acid is bound per mole of enzyme. Covalently bound folic acid blocks the subsequent binding of H₄PteGlu, and does not inhibit the rate of dimethylglycine dehydrogenase activity in vitro.     

Purification and characterization of beta-1,6-glucanase of Streptomyces rochei application in the study of yeast cell wall proteins

A beta-1,6-glucanase was purified to apparent homogeneity from a commercial yeast digestive enzyme prepared from Streptomyces rochei by a series of column chromatographies. The molecular mass of the purified enzyme was 60 kDa by SDS-PAGE. The purified enzyme had an optimum pH range from 4.0 to 6.0 and was stable in the same pH range. The enzyme was stable under 50 degrees C but lost almost all activity at 60 degrees C. The enzyme was specific to beta-1,6-glucan and had little activity towards beta-1,3-glucan and beta-1,4-glucan. When the beta-1,6-glucan was hydrolyzed with the purified enzyme for 5 h, the reaction products contained 20% glucose, 36% gentiobiose, and 44% other oligosaccharides, suggesting that the enzyme is an endo-type glucanase. When the purified enzyme was used for the digestion of the cell wall of Saccharomyces cerevisiae, cell-wall proteins covalently bound to the cell-wall glucan were recovered as soluble forms, suggesting that this enzyme is useful for analysis of yeast-cell wall proteins.     

Purification of UDP-N-acetylglucosamine:glycoprotein N-acetylglucosamine-1-phosphotransferase from Acanthamoeba castellanii and identification of a subunit of the enzyme.

UDP-N-acetylglucosamine:glycoprotein N-acetylglucosamine-1-phosphotransferase (GlcNAc-phosphotransferase) from the soil amoeba Acanthamoeba castellanii has been purified over 100,000-fold by means of wheat germ agglutinin-Sepharose affinity chromatography, DEAE-cellulose chromatography, concanavalin A-Sepharose affinity chromatography, orange A-agarose dye chromatography, and gel filtration on Superose 6. The most purified enzyme has an estimated specific activity of at least 5 mumol of GlcNAc-phosphate transferred/min/mg of protein using alpha-methylmannoside as acceptor. The molecular weight of the native enzyme is approximately 250,000, as determined by gel filtration and glycerol gradients in H2O and D2O. A protein with an apparent M(r) of 97,000 in small scale preparations and its putative proteolytic fragment of 43,000 in large scale preparations co-purifies with the enzyme activity. This protein is covalently modified with GlcNAc-[32P]phosphate when the enzyme preparation is incubated with [beta-32P]UDP-GlcNAc in the absence of an acceptor substrate. The labeling of the 97(43)-kDa protein requires active enzyme and is completely inhibited by the addition of the acceptor substrate alpha-methylmannoside. The GlcNAc-[32P]phosphate transferred to the protein is not bound to serine, threonine, tyrosine, or mannose residues. The 97(43)-kDa protein with covalently bound GlcNAc-P does not serve as a kinetically competent enzyme-substrate intermediate. However, preincubation of GlcNAc-phosphotransferase with UDP-GlcNAc does result in a decrease in the Vmax of the enzyme in subsequent assays. Taken together, these data are consistent with the 97(43)-kDa protein being a subunit of GlcNAc-phosphotransferase.     

A myo-inositol D-3 hydroxykinase activity in Dictyostelium.

A soluble ATP-dependent enzyme which phosphorylates myo-inositol has been characterized in Dictyostelium. The myo-inositol kinase activity was partially purified from amoebae by chromatography on DEAE-Sepharose and phenyl-Sepharose columns. The product of both the partially purified activity and of a crude cytosolic fraction was myo-inositol 3-phosphate. The partially purified preparations of myo-inositol kinase (a) possessed a Km for myo-inositol of 120 microM (in the presence of 5 mM-ATP) and for ATP of 125 microM (in the presence of 1 microM-myo-inositol), (b) did not recognize allo-, epi-, muco-, neo-, scyllo-, 1 D-chiro or 1 L-chiro-inositol as substrates, (c) were competitively inhibited by three naturally occurring analogues of myo-inositol: 1 L-chiro-inositol (Ki 49.5 +/- 0.7 microM: the structural equivalent of myo-inositol, except that the D-3 hydroxy moiety is axial), D-3-deoxy-myo-inositol [Ki 103 +/- 1 microM: (-)-viburnitol], and sequoyitol (Ki 271 +/- 7 microM; unlike 1 L-chiro-inositol and D-3-deoxy-myo-inositol, this was a substrate for the kinase), and finally (d) were apparently non-competitively inhibited by myo-inositol 3-phosphate. The product of myo-inositol kinase could be detected in intact amoebae and was a substrate for the first in a series of inositol polyphosphate kinases present in Dictyostelium which ultimately yield myo-inositol hexakisphosphate. The activity of myo-inositol D-3-hydroxykinase in Dictyostelium lysates showed evidence of developmental regulation.     

Purification and characterization of peroxisomal L-pipecolic acid oxidase from monkey liver

L-Pipecolic acid oxidase has been purified to near homogeneity from Rhesus monkey liver. The protein, a yellow monomer, has a molecular weight of 46,000 by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and a pI of 8.9. It contains a covalently bound flavin with absorption maxima at 457 and 383 nm and a shoulder at 480 nm. The purified enzyme is most reactive toward L-pipecolic acid, with lesser reactivities toward L-proline and sarcosine. The enzyme has no significant reactivity toward the D-enantiomer of pipecolic acid or toward any other amino acid tested. Benzoic acid is a competitive inhibitor of the enzyme with a Ki of 750 microM. The Km of the purified enzyme is 3.7 mM for L-pipecolic acid. With less purified preparations, the reaction product is alpha-aminodipic acid. The purified enzyme, however, produces an intermediate which reacts with ortho-aminobenzaldehyde to form an alpha-aminoadipic acid semialdehyde adduct. Thus, the formation of alpha-aminoadipic acid requires at least two enzymes.     

Covalent linking of poly(ethyleneglycol)-bound NAD with Thermus thermophilus malate dehydrogenase. NAD(H)-regeneration unit for a coupled second-enzyme reaction

Poly(ethyleneglycol)-bound NAD (PEG-NAD) was covalently linked to Thermus thermophilus malate dehydrogenase with a bifunctional reagent, 3,3'-(1,6-dioxo-1,6-hexanediyl)bis-2-thiazolidinethione. The covalently linked malate-dehydrogenase--PEG--NAD complex (MDH-PEG-NAD) was purified by DEAE-Sephadex column chromatography to remove unbound PEG-NAD, and fractionated by blue-Sepharose column chromatography into four preparations: MDH-PEG-NAD I, MDH-PEG-NAD II, MDH-PEG-NAD III and MDH-PEG-NAD IV. The average numbers of NAD moieties covalently bound per subunit of MDH-PEG-NAD I, MDH-PEG-NAD II, MDH-PEG-NAD III and MDH-PEG-NAD IV were 1.2, 1.2, 0.8 and 0.5, respectively, and the values were confirmed by sodium dodecyl sulfate/polyacrylamide gel electrophoresis. 60-80% bound NAD moiety of these preparations of MDH-PEG-NAD was reduced by the enzyme moiety in the presence of L-malate, and the specific activity of the enzyme moiety of the preparations was more than 80% that of the native enzyme. MDH-PEG-NAD I has the following properties. The Km value for exogenous NAD is three times that of the native enzyme. The coenzyme activity of its NAD moiety is 20-40% that of native NAD for alcohol and lactate dehydrogenases. The complex catalyzes the oxidation of L-malate in the presence of the redox system of 5-ethylphenazinium ethyl sulfate and a tetrazolium salt with a rate constant of 0.11 s-1. The coenzyme moiety of the complex can also be recycled by coupled reactions of the active site of the same complex and alcohol dehydrogenase. These results indicate that MDH-PEG-NAD works as an NAD(H)-regeneration unit for coupled reactions.     

Purification and properties of aldehyde dehydrogenase from Saccharomyces cerevisiae.

A procedure for the purification of aldehyde dehydrogenase from bakers' yeast (Saccharomyces cerevisiae) is reported. Treatment with acid, heat and organic solvents was avoided and chromatographic and filtration techniques in the presence of phenylmethylsulfonylfluoride were mainly used. An affinity chromatography step using the reactive dye Cibacron blue F3G-A, which was covalently bound to Sepharose 4B, was found to be essential. The enzyme was bound to and then released from the dye. The purified enzyme was shown to be homogeneous by gel filtration, disc electrophoresis and SDS electrophoresis. The molecular weight of the purified enzyme determined by gel filtration was 170,000, which agreed with that of the enzyme in the crude extract. The enzyme was composed of subunits of a molecular weight of 57,000. The specific activity of the enzyme was 20 units per mg of protein under the standard assay conditions. The substrate specificity, the relative maximal velocity, the michaelis constants, the pH optimum, the stability and the activation energy of the enzyme are reported.     

Immobilization of Chloroperoxidase on Aminopropyl-Glass

Chloroperoxidase (CPO) purified from Caldariomyces fumago CMI 89362 was covalently bound to aminopropyl-glass by using a modification of an established method. Acid-washed glass was derivatized by using aminopropyltriethoxysilane, and the enzyme was ionically bound at low ionic strength. Further treatment with glutaraldehyde covalently linked the enzyme to the glass beads in an active form. No elution of bound activity from glass beads could be detected with a variety of washings. The loading of enzyme protein to the glass beads was highest, 100 mg of CPO per g of glass, at high reaction ratios of CPO to glass, but the specific activity of the immobilized enzyme was highest, 36% of theoretical, at low enzyme-to-carrier ratios. No differences in the properties of the soluble and immobilized enzymes could be detected by a number of criteria: their pH-activity and pH-stability profiles were similar, as were their thermal stabilities. After five uses, the immobilized enzyme retained full activity between pH 6.0 and 6.7.     

Mammalian dihydroorotate dehydrogenase: physical and catalytic properties of the primary enzyme.

A number of physical and catalytic properties of purified dihydroorotate dehydrogenase from rat liver mitochondria are described. The only potentially reducible cofactor detected was iron. The enzyme was also found to contain zinc. The primary enzyme does not contain FAD, FMN, covalently bound flavin, ubiquinone, or labile sulfide. Certain metal chelators were shown to behave as noncompetitive inhibitors of dihydroorotate oxidation and as competitive inhibitors of the reduction of phenazine methosulfate. The purified preparation can use oxygen as sole electron acceptor, although the reaction rate is relatively slow. The activity of the purified enzyme differs from that of the membrane bound form in a number of ways: the pH maximum is apparently shifted, the effect of thenoyltrifluoroacetone and its K_i are markedly changed and the mode of electron transfer to dichlorophenolindophenol is altered. Therefore, only tentative extrapolations to the membrane system regarding activities such as Superoxide production can be made.     

Enzyme mechanism and catalytic property of beta propeller phytase

BACKGROUND: Phytases hydrolyze phytic acid (myo-inositol-hexakisphosphate) to less-phosphorylated myo-inositol derivatives and inorganic phosphate. Phytases are used in animal feed to reduce phosphate pollution in the environment. Recently, a thermostable, calcium-dependent Bacillus phytase was identified that represents the first example of the beta propeller fold exhibiting phosphatase activity. We sought to delineate the catalytic mechanism and property of this enzyme. RESULTS: The crystal structure of the enzyme in complex with inorganic phosphate reveals that two phosphates and four calcium ions are tightly bound at the active site. Mutation of the residues involved in the calcium chelation results in severe defects in the enzyme's activity. One phosphate ion, chelating all of the four calcium ions, is close to a water molecule bridging two of the bound calcium ions. Fluoride ion, which is expected to replace this water molecule, is an uncompetitive inhibitor of the enzyme. The enzyme is able to hydrolyze any of the six phosphate groups of phytate. CONCLUSIONS: The enzyme reaction is likely to proceed through a direct attack of the metal-bridging water molecule on the phosphorous atom of a substrate and the subsequent stabilization of the pentavalent transition state by the bound calcium ions. The enzyme has two phosphate binding sites, the "cleavage site", which is responsible for the hydrolysis of a substrate, and the "affinity site", which increases the binding affinity for substrates containing adjacent phosphate groups. The existence of the two nonequivalent phosphate binding sites explains the puzzling formation of the alternately dephosphorylated myo-inositol triphosphates from phytate and the hydrolysis of myo-inositol monophosphates.     

Extraction of acid alpha-glucosidase from human spleen]

An efficient method for isolation of acid alpha-glucosidase from human spleen is developed. The method involves chromatography of the enzyme on rho-aminophenyl-alpha-D-glucopyranoside covalently bound to CH-Sepharose 4B, with subsequent gel filtration on Sephadex G-200. The enzyme was homogeneous by the polyacrylamide gel electrophoresis data; it was purified about 1500-fold, as compared with the crude extract (the total yield 12.5%). Besides acid alpha-glucosidase, the preparations of alpha-L-fucosidase, alpha-D-galactosidase and beta-N-acetylglucosaminidase were isolated and purified 200-, 130- and 280-fold, respectively. The nature of interaction between acid alpha-glucosidase and immobilized rho-aminophenyl-alpha-glucopyranoside is discussed.     

Isolation and characterization of rat kidney alanine aminopeptidase

Rat alanine aminopeptidase was purified from kidney by isolation of the brush border membrane with CaCl2 followed by differential centrifugation and tryptic proteolysis. It is a glycoprotein with a molecular weight of approximately 210,000 daltons comprising two 110,000-dalton subunits and has an amino acid composition similar to that of the human enzyme. Two zinc atoms are covalently bound to each protein subunit.