Presence of Escherichia coli of a deaminase and a reductase involved in biosynthesis of riboflavin.

Two enzymes have been partially purified from extracts of Escherchia coli B which together catalyze the conversion of the product of the action of GTP cyclohydrolase II, 2,5-diamino-6-oxy-4-(5'-phosphoribosylamine)pyrimidine, to 5-amino-2,6-dioxy-4-(5'-phosphoribitylamine)pyrimidine. These two compounds are currently thought to be intermediates in the biosynthesis of riboflavin. The enzymatic conversion occurs in two steps. The product of the action of GTP cyclohydrolase II first undergoes hydrolytic deamination at carbon 2 of the ring, followed by reduction of the ribosylamino group to a ribitylamino group. The enzyme which catalyzes the first step, herein called the "deaminase," has been purified 200-fold. The activity was assayed by detecting the conversion of the product of the reaction catalyzed by GTP cyclohydrolase II to a compound which reacts with butanedione to form 6,7-dimethyllumazine. The enzyme has a molecular weight of approximately 80,000 and a pH optimum of 9.1. The dephosphorylated form of the substrate is not deaminated in the presence of the enzyme. The assay for the enzyme which catalyzes the second step, referred to here as the "reductase," involves the detection of the conversion of the product of the deaminase-catalyzed reaction to a compound which, after treatment with alkaline phosphatase, reacts with butanedione to form 6,7-dimethyl-8-ribityllumazine. The reductase has a molecular weight of approximately 40,000 and a pH optimum of 7.5. Like the deaminase, the reductase does not act on the dephosphorylated form of its substrate. Reduced nicotinamide adenine dinucleotide phosphate is required as a cofactor; reduced nicotinamide adenine dinucleotide can be used about 30% as well as the phosphate form. The activity of neither enzyme is inhibited by riboflavin, FMN, or flavine adenine dinucleotide.     

Purification and properties of 2-furoyl-coenzyme A hydroxylase from Pseudomonas putida F2

1. 2-Furoyl-CoA hydroxylase of Pseudomonas putida F2 has been purified 60-fold by a combination of (NH(4))(2)SO(4) fractionation, DEAE-cellulose chromatography and agarose chromatography. 2. The purified enzyme catalyses the formation of 5-hydroxy-2-furoyl-CoA, which tautomerizes to form 5-oxo-Delta(2)-dihydro-2-furoyl-CoA. 3. The enzyme has a requirement for an electron acceptor that can be satisfied by a membrane preparation from 2-furoate-grown Ps. putida F2 or by artificial electron acceptors, and so presumably the incorporated oxygen atom is derived from water rather than molecular oxygen. 4. The enzyme is a large protein with a molecular weight of 3.27x10(6) and is disrupted to form inactive subunits in the presence of 0.2% (w/v) sodium dodecyl sulphate. It has a pH optimum of 8.5-9.5, a K(m) for 2-furoyl-CoA of 20.2mum and an absorption spectrum with a trough at 265nm and a single peak at 273nm. No absorption peaks are detectable in the visible region of the spectrum. 5. The enzyme is resistant to the effects of a wide range of potential inhibitors, but is inhibited by the copper-chelating agents bathocuproin and cuprizone, though not by sodium diethyldithiocarbamate. 6. Flavins are absent and the iron content does not show a sustained increase during purification. The copper content of the protein increases in close correlation with the increase in specific activity during purification. 7. A catalytic sequence for the hydroxylation of 2-furoyl-CoA by a copper protein is proposed.     

Plasmodium falciparum: properties of an alpha-like DNA polymerase, the key enzyme in DNA synthesis

An alpha-like DNA polymerase has been identified and characterized in the extracts from the malarial parasite Plasmodium falciparum. The enzyme is sensitive to the specific inhibitors of alpha-DNA polymerase, N-ethylmaleimide and aphidicolin, and is cell-cycle specific. High activity has been found in the schizont, is lower in trophozoites, and has only negligible activity in the ring form. The enzyme has a molecular weight of about Mr 100,000-103,000 estimated by detecting activity in SDS-polyacrylamide electrophoresis and by Bio-Gel filtration. Another active band of a molecular Mr 68,000 was detected by SDS electrophoresis when the enzyme was stored for 2 months at -20 degrees C. The catalytic activity of parasite enzyme was optimal between pH 8 and pH 9. The apparent Michaelis constant for dTTP was 4.3 microM.     

Purification of inactivated photoresponsive nitrile hydratase

Photoresponsive nitrile hydratase from Rhodococcus sp. N-771 was purified in its inactivated form. The enzyme had a molecular weight of approximately 60 kDa and consisted of 2 subunits each having molecular weight of 27.5 and 28 kDa. The enzyme also contained 2 iron atoms/enzyme as a cofactor. The enzyme was more stable in its inactivated form, rather than the activated during storage in the dark. The enzyme was most stable in the temperature region of 0-35 degrees C, and lost its activity above 40 degrees C. The enzyme was most stable in the pH region of 6-8. The optimum temperature and pH for the enzyme activity was 30 degrees C and 7.8, respectively. The enzyme showed wide substrate specificity, and most of the metal ions did not affect enzyme activity significantly. The absorption spectrum revealed the presence of some cofactor which changed form after photoirradiation.     

Isolation of the hexameric form of purine nucleoside phosphorylase from E. coli. Comparative study of trimeric and hexameric forms of the enzyme

The presence of two forms (high and low molecular weight ones) of purine nucleoside phosphorylase II (purine nucleoside: orthophosphate ribosyltransferase, EC 2.4.2.1) was demonstrated. The high molecular weight form of the enzyme was purified, and the properties of both forms were compared. The enzyme forms were shown to differ in their quaternary structure (trimeric and hexameric), molecular weight of the native enzyme and its subunits (85,000 and 28,000 for the trimer, 150,000 and 25,000 for the hexamer, respectively) as well as substrate specificity (the trimer is specific for all major purine nucleosides, while the hexamer does not cleave adenine nucleosides). Adenosine is a competitive inhibitor of the hexameric form with respect to deoxyguanosine (Ki = 1.16 X 10(-3) M); the Km value for deoxyguanosine is 9.85 X 10(-5) M. The isoelectric point for the both forms of the enzyme in the presence of 9 M urea is about 5.5. Both forms have a pH optimum of phosphorolytic activity between 6.5 and 7.0.     

Purification and characterization of avian liver mevalonate-5-pyrophosphate decarboxylase

Mevalonate-5-pyrophosphate decarboxylase [ATP:5-diphosphomevalonate carboxy-lyase (dehydrating), EC 4.1.1.33] has been purified 5800 times from chicken liver and obtained in a stable and highly purified form. The protein is a dimer of molecular weight 85400 +/- 1941, and its subunits were not resolved by gel electrophoresis in denaturing conditions. The purified enzyme does not require the presence of SH-containing reagents for either activity or stability. The enzyme shows a high specificity for adenosine 5'-triphosphate (ATP) and requires for activity a divalent metal cation, Mg2+ being most effective. The optimum pH for the enzyme ranges from 4.0 to 6.5. Inhibitory effects for the enzyme activity were detected by citrate, phthalate, and phosphate. The isoelectric point, as determined by column chromatofocusing, is 4.8. The kinetics are hyperbolic for both substrates, showing a sequential mechanism; true Km values of 0.0141 mM and 0.504 mM have been obtained for mevalonate-5-pyrophosphate and ATP, respectively.     

The purification and properties of a ribonucleoenzyme, o-diphenol oxidase, from potatoes

1. o-Diphenol oxidase was isolated from potato tubers by a new approach that avoids the browning due to autoxidation. 2. There are at least three forms of the enzyme, of different molecular weights. The major form, of highest molecular weight, was separated from the others in good yield and with high specific activity by gel filtration through Bio-Gel P-300. 3. The major form is homogeneous by disc electrophoresis but regenerates small amounts of the species of lower molecular weight, as shown by rechromatography on Bio-Gel P-300. 4. There is an equal amount of RNA and protein by weight in the fully active enzyme. The RNA cannot be removed without loss of activity, and is not attacked by ribonuclease. 5. The pH optimum of the enzyme is at pH5.0 when assayed with 4-methylcatechol as substrate. It is ten times more active with this substrate than with chlorogenic acid or catechol. The enzyme is fully active in 4m-urea. 6. A minimal molecular weight of 36000 is indicated by copper content and amino acid analysis of the protein component of the enzyme. 7. The protein contains five half-cystinyl residues per 36000 daltons, a value similar to that found in o-diphenol oxidase from mushrooms. It also contains tyrosine residues although, when pure, it does not turn brown by autoxidation.     

Multiple molecular forms of glia maturation factor.

Glia maturation factor from the pig brain can be detected in two molecular forms: the high molecular weight form which is 200 000 dalton in size and the low molecular weight form which is 40 000 dalton in size, as determined by Sephadex gel filtration. The former accounts for 85% of the total biological activity extracted at physiologic pH. The proportion of the low molecular weight form increases following freeze-thawing and ion-exchange chromatography. In addition to the morphological effects, both forms possess mitogenic activity but no esteropeptidase activity. Both forms show similar enzyme susceptibility, being inactivated by papain, ficin and pronase but resistant to subtilisin, thermolysin and trypsin. The high molecular weight form is more resistant to denaturation by low pH, heating and urea than the low molecular weight form. The high molecular weight factor has an isoelectric point of 4.27 whereas the low molecular weight factor has one of 5.04.     

Fasciola hepatica: A proteolytic digestive enzyme

A proteolytic enzyme in the gut exudate of the common liver fluke has been purified. The enzyme is specific for globin with a pH optimum of 3.9–4.0 and breaks the protein down to peptides and a small percentage of free amino acids. Collagenase activity at pH 7.5 copurifies with the main globinolytic enzyme. The enzyme has a molecular weight of 12,000 and does not appear to be antigenic in fluke-infected animals.     

Guanylate cyclase in Escherichia coli. Purification and properties.

Guanylate cyclase has been purified from extracts of Escherichia coli. After a 1000-fold purification, the enzyme contains only minor contaminants as judged by disc gel electrophoresis. The Km for GTP is approximately 7 times 10(-5) M and the optimal pH is 8.0. More activity is observed with Mn2+ than with Mg2+, and maximal activity is observed at 0.14 mM Mn2+ and 1.4 mM Mg2+. Based on its behavior on Sephadex G-100, the molecular weight of E. coli guanylate cyclase is about 30,000. Disc gel electrophoretic analysis indicates that the enzyme consists of a single polypeptide chain. Guanylate cyclase does not form 3':5'-AMP from ATP, and therefore, is distinct from adenylate cyclase.     

Reversible activation of human neutrophil calpain promoted by interaction with plasma membranes

Human neutrophil calpain is a monomer of 85 kDa molecular weight. The proteinase shows an absolute requirement for Ca2+ with maximal catalytic activity at 0.1-0.2 mM Ca2+ and negligible activity at 1-5 microM Ca2+. At this concentration of Ca2+ neutrophil calpain becomes active and reaches 65% of its maximal catalytic activity following interaction with plasma membranes. The activation is fully reversible since the enzyme returns to its native, high Ca2+ requiring form following removal of the membranes. Membrane phospholipids appear to be the physiological compounds responsible for the promotion of such reversible activation. Unlike other Ca2+ dependent proteinases, neutrophil calpain does not undergo conversion to a low Ca2+ requiring form by limited autoproteolysis.     

Low and high pH form of cadmium carbonic anhydrase determined by nuclear quadrupole interaction.

The pH dependence of the nuclear quadrupole interaction between the excited 247-keV state in 111Cd bound to the active site in human carbonic anhydrase B and the nearest protein surroundings has been studied by means of the nuclear spectroscopic technique of perturbed angular correlation of gamma rays. The enzyme has been studied in the pH region 5.6-11.0 at 22 and -196 degrees C. The results show that the Cd enzyme changes from one form at low pH to another form at high pH both at 22 and -196 degrees C. The pK of the transition is 8.9 +/- 0.2 at -196 degrees C and close to 9 at 22 degrees C. Parallel to this transformation, the esterase activity of the Cd enzyme for the hydration of p-nitrophenyl acetate exhibits a pH dependency with a pH of 9.1 +/- 0.2. The sulfonamide inhibitor acetazolamide completely inhibits this activity of the Cd enzyme. The quadrupole interaction parameters for the Cd enzyme are not significantly different at -196 degrees C from those obtained at 22 degrees C. A measurement at 0 degrees C pH 5.7 shows, however, a form different from those at 22 degrees C pH 5.6 and -196 degrees C pH 5.7. The change in the quadrupole interaction with pH is, in a simple model, consistent with an ionization of a metal-bound water molecule.     

Carbamoyl-phosphate synthase in Phycomyces blakesleeanus

A carbamoyl-phosphate synthase has been purified from mycelia of Phycomyces blakesleeanus NRRL 1555 (-). The molecular weight of the enzyme was estimated to be 188,000 by gel filtration. Polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate showed that the enzyme consists of two unequal subunits with molecular weights of 130,000 and 55,000. The purified enzyme has been shown to be highly unstable. The carbamoyl-phosphate synthase from Phycomyces uses ammonia and not L-glutamine as a primary N donor and does not require activation by N-acetyl-L-glutamate, but it does require free Mg2+ for maximal activity. Kinetic studies showed a hyperbolic behavior with respect to ammonia (Km 6.34 mM), bicarbonate (Km 10.5 mM) and ATP.2 Mg2+ (Km 0.93 mM). The optimum pH of the enzyme activity was 7.4-7.8. The Phycomyces carbamoyl-phosphate synthase showed a transition temperature at 38.5 degrees C. It was completely indifferent to ornithine, cysteine, glycine, IMP, dithiothreitol, glycerol, UMP, UDP and UTP. The enzyme was inhibited by reaction with 5 mM N-ethylmaleimide.     

Characterization of alpha-Galactosidase from Cucumber Leaves

Two forms of alpha-galactosidase (alpha-d-galactoside galactohydrolase, E.C. 3.2.1.22) which differed in molecular weight were resolved from Cucumis sativus L. leaves. The enzymes were partially purified using ammonium sulfate fractionation, Sephadex gel filtration, and diethylaminoethyl-Sephadex chromatography. The molecular weights of the two forms, by gel filtration, were 50,000 and 25,000. The 50,000-dalton form comprised approximately 84% of the total alpha-galactosidase activity in crude extracts from mature leaves and was purified 132-fold. The partially purified 25,000-molecular weight form rapidly lost activity unless stabilized with 0.2% albumin and accounted for 16% of the total alpha-galactosidase activity in the crude extract. The smaller molecular weight form was not found in older leaves.The two forms were similar in several ways including their pH optima which were 5.2 and 5.5 for the 50,000- and 25,000-dalton form, respectively, and activation energies, which were 15.4 and 18.9 kilocalories per mole for the larger and smaller forms. Both enzymes were inhibited by galactose as well as by excess concentrations of p-nitrophenyl-alpha-d-galactoside sub-strate. K(m) values with this substrate and with raffinose and melibiose were different for each substrate, but similar for both forms of the enzyme. With stachyose, K(m) values were 10 and 30 millimolar for the 50,000- and 25,000- molecular weight forms, respectively.     

Purification and characterization of glucose oxidase from ligninolytic cultures of Phanerochaete chrysosporium.

Glucose oxidase, an important source of hydrogen peroxide in lignin-degrading cultures of Phanerochaete chrysosporium, was purified to electrophoretic homogeneity by a combination of ion-exchange and molecular sieve chromatography. The enzyme is a flavoprotein with an apparent native molecular weight of 180,000 and a denatured molecular weight of 80,000. This enzyme does not appear to be a glycoprotein. It gives optimal activity with D-glucose, which is stoichiometrically oxidized to D-gluconate. The enzyme has a relatively broad pH optimum of 4 to 5. It is inhibited by Ag+ (10 mM) and o-phthalate (100 mM), but not by Cu2+, NaF, or KCN (each 10 mM).     

Characterization of invertase activity from cariogenic Streptococcus mutans

Invertase activity from Streptococcus mutans GS-5 has been partially purified and shown to possess beta-fructofuranosidase specificity. The enzyme has a broad pH optimum between pH 5.5 and 7.5 and exhibits maximal activity at 37 C. Fructose, but not the glucose analogue alpha-methyl-d-glucoside, acts as a competitive inhibitor of the enzyme. None of the common glycolytic intermediates or adenine nucleotides had any significant effect on enzyme activity. A molecular weight of approximately 47,000 was estimated for the enzyme. The enzyme does not appear to be catabolically repressed by glucose nor inducible by sucrose. Higher specific activities of the enzyme are observed in fructose or glucose-grown cells compared to sucrose-grown cells. These results are discussed in terms of the regulation of invertase activity in vivo.     

Enzymes hydrolyzing ApppA and/or AppppA in higher plants. Purification and some properties of diadenosine triphosphatase, diadenosine tetraphosphatase, and phosphodiesterase from yellow lupin (Lupinus luteus) seeds

Three distinct enzymes hydrolyzing either ApppA or AppppA, or both, were separated and purified from yellow lupin seed extracts. Two of the enzymes were purified to homogeneity. These enzymes differ greatly in their catalytic and physical properties. One hydrolase, with a native molecular weight of 41,000, exhibits broad pH (from 5-8) optimum for activity, requires Mg2+ for activity, is inhibited by zinc ions (I0.5 = 25 microM) and hydrolyses ApppA (V = 1), ApppC (V = 0.38), ApppG (V = 0.2), and ribose(5')pppA (V = 0.2). The enzyme exhibits much lower activity with AppppA (V = 0.1), and ApppppA, AppppppA, ppppA, and ATP are hydrolyzed 25- to 100-fold slower then ApppA. ADP was always one of the products of the reactions catalyzed by the enzyme. AppA, NAD, NADP, FAD, cAMP, and p-nitrophenyl-thymidine 5'-phosphate were not hydrolyzed by the enzyme. The enzyme is diadenosine 5',5"'-P1, P3-triphosphatase. The second hydrolase, composed of one polypeptide chain of a molecular weight 18,000-18,500, exhibits optimal activity in the pH range from 7.5-9, requires Mg2+ for activity, is inhibited by calcium ions (I0.5 for calcium depends on the concentration of Mg2+ and is 35-180 microM in the presence of 0.5-10 mM Mg2+, respectively), and hydrolyzes AppppA (V = 1, Km = 1 microM), ApppppA (V = 0.42, Km = 1.8 microM), AppppppA (V = 0.34), AppppU (V = 0.73), AppppC (V = 0.67), AppppG (V = 0.27), and ppppA. ATP was always one of the products of the reactions catalyzed by the enzyme. Dinucleoside di- and triphosphates, ATP, cAMP, and p-nitrophenylthymidine 5'-phosphate were not hydrolyzed by the enzyme. This enzyme is diadenosine 5',5"'-P1,P4-tetraphosphatase (EC 3.6.1.17). The third hydrolase, composed of one polypeptide chain of a molecular weight of 56,000, exhibits maximal activity at pH 9-10.5, does not require Mg2+ ions for activity, is inhibited neither by divalent cations (Mg2+, Ca2+, Zn2+, Co2+, Mn2+, or Ni2+) nor by EDTA, and uses as substrates all compounds which are substrates for the diadenosine 5',5"'-P1,P3-triphosphatase and diadenosine 5',5"'-P1,P4-tetraphosphatase. In addition, the enzyme hydrolyzes p-nitrophenyl-thymidine 5'-phosphate, p-nitrophenylthymidine 3'-phosphate, bis-p-nitrophenylphosphate, ADP, AppA, NAD, NADP, and FAD, but not cAMP. With the exception of p-nitrophenylphosphate derivatives all other substrates of the enzyme yield AMP as one of the products of hydrolysis. This enzyme has a specificity similar to that of phosphodiesterases (EC 3.1.4.1) from other sources. With the lupin phosphodiesterase, ApppA (V = 1, Km = 2.2 microM) and AppppA (V = 1, Km = 2.0 microM) are better substrates than NAD (V = 0.8, Km = 9.6 microM), AppA (V = 0.4), ApppppA (V = 0.6), and AppppppA (V = 0.34).     

Purification and demonstration of the enzymatic character of the nicking-closing protein from mouse L cells

A rapid procedure for the purification of the nicking-closing enzyme from mouse L cells is described. The procedure reproducibly provides high yields of enzyme. A purity of greater than 90% is obtained in five steps. The enzymatic character of the nicking-closing activity has been demonstrated. On the average 20 PM2 DNA I molecules are completely relaxed by one enzyme molecule. The enzyme releases superhelical turns from closed circular DNA by providing a swivel through a sequence of successive nicking and closing events. It is not known yet whether the release of superhelical turns proceeds via a one hit or a multiple hit mechanism.     

Purification and some properties of bovine liver cytosol thioltransferase

A cytosol thioltransferase was purified 37,000-fold from bovine liver by essentially the same procedure as reported for rat liver enzyme by Axelsson et al. [1978) Biochemistry 17, 2978-2984). The purified enzyme appears to be homogeneous on sodium dodecyl sulfate (SDS)-gel electrophoresis and has a molecular weight (Mr) of 11,000, an isoelectric point (pI) of 8.1, and an optimum pH with S-sulfocysteine and GSH as substrates of 8.5. It is specific for disulfides including L-cystine, S-sulfocysteine, ribonuclease A, trypsin, soybean kunitz trypsin inhibitor, soybean Bowman Birk trypsin inhibitor and insulin, and converts Bowman Birk trypsin inhibitor to an inactive form. The enzyme does not act as a protein : disulfide isomerase, as measured by reactivation of "scramble" ribonuclease and Kunitz soybean trypsin inhibitor. Thioltransferase activity was found in cytosol of various bovine tissues.     

Forms and intracellular distribution of alpha-D-mannosidases in murine liver and spleen

1. The intracellular distribution of alpha-D-mannosidase in homogenates of murine liver and spleen was investigated by differential and gradient density centrifugation. 2. In both tissues an enzyme with a neutral pH optimum was found in the cytosol together with an alpha-D-mannosidase with optimal activity between pH 5.5 and 6.0 which was also partially membrane-bound. 3. In liver the acidic alpha-D-mannosidase was obtained almost entirely in a particulate form distributed equally between a heterogeneous low density region and heavy density lysosomes. 4. The lysosomal form of the liver enzyme was purified to electrophoretic homogeneity and shown to be a glycoprotein composed of four identical subunits of molecular weight 65 kDa. 5. Antibody raised against the purified liver alpha-D-mannosidase immunoprecipitated a polypeptide from spleen which had the same molecular size. This acidic enzyme was the predominant type of alpha-D-mannosidase in spleen, but in contrast to liver, it was obtained mainly in a cytosoluble form, the remaining activity being present in the heterogeneous light density compartment. 6. Although both tissues contain the same molecular form of the acidic alpha-D-mannosidase, in murine spleen this enzyme does not appear to be associated with stable heavy density lysosomes.