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An acid urease from Providencia rettgeri JN-B815 was purified via ultrasonication, ethanol precipitation, and DEAE ion-exchange column chromatography. It was found that the enzyme exhibits not only urease activity, but also urethanase activity, which made it possible to reduce EC already existed or would produce and its precursor urea at the same time. Then, crosslinked enzyme aggregates of P. rettgeri urease (PRU-CLEAs) were prepared using genipin as crosslinking agent. The purification process of acid urease, the effects of genipin concentration, and crosslinking time on PRU-CLEAs activity were investigated. The crosslinking was performed at pH 4.5 for 2.5 h, using 0.3% genipin as crosslinking agent, and 0.3 g · L−1 bovine serum albumin as protein feeder. Using the obtained PRU-CLEAs, the removal rate of urea was up to 9.31 mg · L−1 · h−1. The removal rate of urea was still up to 7.56 mg · L−1 · h−1 after PRU-CLEAs was re-used for 6 times. When PRU-CLEAs were applied in a batch stirred and membrane reactor, the removal rate of urea in rice wine reached 5.16 mg · L−1 · h−1 and the removal rate of EC was 9.21 μg · L−1 · h−1. Furthermore, the treatment with PRU-CLEAs revealed no significant change of volatile flavor substances in Chinese rice wine. Thus PRU-CLEAs have great potential in the elimination of EC in Chinese rice wine. [ABSTRACT FROM AUTHOR]

Discusses research on the structure of macrophomate synthase (MPS) enzyme compared with structure of 2-dehydro-3-deoxygalactarate (DDG) aldolase. Ability of MPS crystal structure to catalyze chemical transformations oxalacetate and 2-pyrone; Purification and crystallization of MPS; Instruments used in the study; Analysis of MPS crystals.

Membrane-bound L-(+)-pantoyl lactone dehydrogenase, an enzyme that catalyzes the formation of ketopantoyl lactone from L-(+)-pantoyl lactone, was solubilized with Brij 35 and purified 78-fold to apparent homogeneity, with a 3.7% overall recovery, from Nocardia asteroides through purification procedures including successive ammonium sulfate fractionation, and DEAE-Sephacel, Sepharose CL-6B and Cellulofine GC-700-m column chromatography in the presence of Brij 35. The relative molecular mass of the native enzyme, as estimated on high-performance gel-permeation chromatography, is at least more than 600 kDa and its subunit molecular mass is 42 kDa. The enzyme shows high specificity for L-(+)-pantoyl lactone as a substrate (Km = 26.8 mM; Vmax = 4.22 µmol · min-1 · mg protein-1). Brij 35 acts as a stabilizer and also as an efficient activator of the enzyme. The prosthetic group of L-(+)-pantoyl lactone dehydrogenase was identified as noncovalently bound FMN. [ABSTRACT FROM AUTHOR]

A new cobalt-containing nitrile hydratase was purified from extracts of urea-induced cells from Rhodococcus rhodochrous J1 in seven steps. At the last step, the enzyme was crystallized by adding ammonium sulfate. Nitrile hydratase was a 500-530-kDa protein composed of two different subunits (α subunit 26 kDa, β subunit 29 kDa). The enzyme contained approximately 11-12 mol cobalt/mol enzyme. A concentrated solution of highly purified nitrile hydratase exhibited a broad absorption spectrum in the visible range, with an absorption maxima at 410 nm. The enzyme had a wide substrate specificity. Aliphatic saturated or unsaturated nitriles as well as aromatic nitriles, were substrates for the enzyme. The optimum pH of the hydratase was pH 6.5-6.8. The enzyme was more stable than ferric nitrile hydratases. The amino-terminal sequence of each subunit of R. rhodochrous J1 enzyme was determined and compared with that of ferric nitrile hydratases. Prominent similarities were observed with the β subunit. However, the amino acid sequence of the α subunit from R. rhodochrous J1 was quite different from that of the ferric enzymes. [ABSTRACT FROM AUTHOR]

The NADPH-linked acetoacetyl-CoA reductase, (R)-3-hydroxyacyl-CoA dehydrogenase (EC 1.1.1.36), from the bacterium Zoogloea ramigera, involved in the formation of D-3-hydroxybutyryl-CoA for poly(D-3hydroxybutyrate) biosynthesis, has been purified from an over-producing Escherichia coli strain. The purification was achieved in two steps, yielding an electrophoretically homogeneous enzyme of high specific activity (608 U/ mg). The enzyme is an α4 homotetramer of four 25-kDa subunits. It has a Km of 2 µM and a kcat/Km of 1.8 × 108 M-1 s-1 for acetoacetyl-CoA; it is inhibited by acetoacetyI-CoA above 10 µM. K is 10-10 M for the dehydrogenation. Kinetic studies of the back reaction revealed a sequential mechanism involving a ternary complex. The stereospecificity of the hydride-equivalent transfer was demonstrated using NMR techniques to be 4S (B side). Using the fingerprint method proposed by Wierenga et al. [(1986) J. Mol. Biol. 187, 101-107], we identified a 28-residue stretch (residues 3-31) as a possible NADPH fold. Finally the specificity of the reductase was examined using 3-oxo-acyl-CoA analogs and analogs lacking the adenosine 3',5'-bisphosphate moiety of CoA. Only the straight-chain C5 analog (3-oxo-propionyl-CoA) was found to be an alternative substrate (40%) for the reductase. [ABSTRACT FROM AUTHOR]

An improved procedure was developed for the isolation of pyruvate decarboxylase from wheat germ. Its final step, an electrophoresis of the native apoenzyme in concave pore gradient polyacrylamide gels, followed by superficial activity-staining, produced two bands of different molecular masses and chain compositions. The highmolecular-mass band occurred in Iow quantity and consisted of, probably eight, apparently identical chains of Mr = 33 000, as judged from sodium dodecyl sulfate electrophoreses. The low-molecular-mass band contained two types of chains with Mrα = 63 000-65 000 and Mrβ = 61 000-62 000. The N termini of both chains were threonine, whereas their C-terminal sequences were different: α, -(Val)-(Ser)-(Ala)-Leu; β, -(His)-(Asp)-(Ala)-Ser. Their amino acid composition was too different to be compatible with our original concept of one chain being produced from the other by proteolytic shortening. Limited proteolysis by Staphylococcus aureus V8 proteinase yielded peptides partly identical size and partly quite different. In all properties investigated, the low-molecular-mass enzyme largely resembled yeast pyruvate decarboxylase; the holoenzyme appeared to possess (αβ)2 structure, the apoenzyme αβ. SH reagents inactivated the enzyme. Binding and fluorescence of 2-p-toluidinonaphthalene-6-sulfonate indicated a similar lipophilicity of the active site as found earlier for the yeast enzyme. 2-Hydroxy-5-nitrobenzyl modification of exposed tryptophan residues left the holoenzyme intact, but in the apoenzyme it destroyed most of the cofactor-binding ability and hence the activity. The strength of cofactor binding and the maximal specific activity were found somewhat lower than in yeast pyruvate decarboxylase. [ABSTRACT FROM AUTHOR]

Reports on the purification and characterization of brain pyridoxal kinase from sheep brain. Use of ammonium sulphate fractionation, diethylaminoethanol-cellulose chromatography, affinity chromatography and Sephadex G-100 gel filtration; Molecular mass of the native enzyme.

A processing mannosidase acting on (Man)9(GlcNAc)2 oligosaccharides, Man9 mannosidase, has been purified 2190-fold from calf liver crude microsomes by a four-step procedure involving (a) differential salt/detergent extraction, (b) affinity chromatography on AH-Sepharose 4B with N-5-carboxypentyl-l-deoxymannojirimycin as ligand, (c) ConA-Sepharose and (d) DEAE-Sephacel chromatography. (Man)9 mannosidase has a subunit molecular mass of 56 kDa and does not bind to ConA-Sepharose, indicating the absence of high-mannose oligosaccharides. The enzyme has a pH optimum close to pH 6.0 and requires divalent cations for activity, Ca2+ being most effective. It is inhibited by 1-deoxymannojirimycin (dMM), N-methyl-dMM and N-5-carboxypentyl-dMM with Ki = 7 μM, 75 μM, and 140 μM, respectively. Man9 mannosidase cleaves three of the four αl,2-1inked mannose residues from the (Man)9(GlcNAc)2 oligosaccharide, does not hydrotyse the remaining (Man)6(GlcNAc)2 structure and is not active against aryl α-mannosides. This pronounced substrate specificity points to the participation of Man9 mannosidase in the N-linked processing pathway and, in addition, clearly distinguishes this enzyme from the mannosidases reported previously. As Man9 mannosidase appears to act in the processing sequence immediately after the three glucose residues have been removed from the (Glc)3(Man)9(GlcNAc)2 intermediate, we assume that the enzyme is located in the endoplasmic reticulum. [ABSTRACT FROM AUTHOR]

A membrane-bound α-L-fucosyltransferase, which is involved in the synthesis of a developmentally regulated carbohydrate antigen, SSEA-1, was purified about 2000-fold from F9 embryonal carcinoma cells. The procedures used were solubilization with Triton X-100, column chromatography on SP-Sephadex, DEAE-Sephadex, RCA-agarose and on G DP-agarose. Upon sodium dodecyl sulfate gel electrophoresis, the purified preparation gave a protein band with a relative molecular mass of 65000. The optimum pH of the enzyme was between 6.0 and 7.0 and the Km toward N-acetyllactosamine was 0.55 mM. The enzyme was active with asialofetuin, but not with intact fetuin. Susceptibility of the product to α-L-fucosidase I from almond emulsin verified that the enzyme transferred fucose to C-3 hydroxyl of N-acetylglucosamine in the N-acetyllactosamine structure. Activities of βgalactoside α1 → 2-fucosyltransferase and N-acetylglucosaminide α1 → 4-fucosyltransferase acting on synthetic substrates were not detected in the purified enzyme nor in the crude extract of F9 cells. PYS-2 parietal endoderm cells lacked all the fucosyltransferases mentioned above. [ABSTRACT FROM AUTHOR]

The gene that encodes for triosephosphate isomerase from Trypanosoma cruzi was cloned and sequenced. In T. cruzi, there is only one gene for triosephosphate isomerase. The enzyme has an identity of 72% and 68% with triosephosphate isomerase from Trypanosoma brucei and Leishmania mexicana, respectively. The active site residues are conserved: out of the 32 residues that conform the interface of dimeric triosephosphate isomerase from T. brucei, 29 are conserved in the T. cruzi enzyme. The enzyme was expressed in Escherichia coli and purified to homogeneity. Data from electrophoretic analysis under denaturing techniques and filtration techniques showed that triosephosphate isomerase from T. cruzi is a homodimer. Some of its structural and kinetic features were determined and compared to those of the purified enzymes from T. brucei and L. mexicana. Its circular dichroism spectrum was almost identical to that of triosephosphate isomerase from T. brucei. Its kinetic properties and pH optima were similar to those of T. brucei and L. mexicana, although the latter exhibited a higher Vmax with glyceraldehyde 3-phosphate as substrate. The sensitivity of the three enzymes to the sulfhydryl reagent methylmethane thiosulfonate (MeSO2-SMe) was determined; the sensitivity of the T. cruzi enzyme was about 40 times and 200 times higher than that of the enzymes from T. brucei and L. mexicana, respectively. Triosephosphate isomerase from T. cruzi and L. mexicana have the three cysteine residues that exist in the T. brucei enzyme (positions 14, 39, 126, using the numbering of the T. brucei enzyme); however, they also have an additional residue (position 117). These data suggest that regardless of the high identity of the three trypanosomatid enzymes, there are structural differences in the disposition of their cysteine residues that account for their different sensitivity to the sulthydryl reagent. The disposition of the cysteine in triosephosphate isomerase from T. cruzi appears to make it unique for inhibition by modification of its cysteine. [ABSTRACT FROM AUTHOR]

Acetoacetyl-CoA synthetase was purified to electrophoretic homogeneity from Zoogloea ramigera 1-16-M, a poly(3-hydroxybutyrate)-accumulating bacterium, which lacks 3-ketoacid CoA-transferase. The purified enzyme had a specific activity of 52.2 μmol acetoacetyl-CoA formed min-1 mg protein-1, which constituted a 680-fold purification compared to the crude extract, with a 5.1% yield. The enzyme absolutely required ATP, CoA, a monovalent cation (K+, Rb+, Cs+ or NH4+) and a divalent cation (Mg2+, Mn2+, Ca2+ or Ni2+) for the activation of acetoacetate, yielding acetoacetyl-CoA, AMP and pyrophosphate in equimolar amounts. The pH optimum of the enzyme reaction was 8.4. The molecular weight of the enzyme was approximately 70 000 as estimated by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate, and 72 000 by Sephadex G-200 gel filtration. The enzyme was active only on acetoacetate and to a lesser extent on L(+)-3-hydroxybutyrate, and the Km values for acetoacetate, L(-+-)-3-hydroxybutyrate, ATP and CoA were 7.6 x 10-5 M, 1.4 x 10-3 M, 3.3 x 10-5 M and 9.1 x 10-5 M respectively. [ABSTRACT FROM AUTHOR]

A diamine oxidase has been purified lo homogeneity from the latex of an herbaceous shrub, Euphorbia characias. This enzyme has a relative molecular mass of 144000 and is composed of two identical subunits. It contain two Cu(II) and two carbonyl-like groups per dinner. The purified enzyme is pink and shows a broad absorption in the visible region centered at 480 nm, which is modified by the addition of phenylhydrazine or semicarbazide. The electron paramagnetic resonance spectrum is typical of copper(II) in a tetragonal symmetry. This enzyme oxidizes putrescine and cadaverine at fairly high rate and, less efficiently a few related compounds, but not histamine, spermine or spermidine. [ABSTRACT FROM AUTHOR]

A chymotrypsin-like enzyme has been purified from sperm of the sea urchin, Hemicentrotus pulcherrimus, using tryptophan methyl ester (TrpOMe) linked to Sepharose 4B as an affinity column for chromatography and gel filtration. The isolated enzyme preparation is homogenous in sodium dodecylsulfate/polyacrylamide gel electrophoresis, the estimated molecular weight being 18500-19000. This enzyme hydrolyses N-acetyl-L-tyrosine ethyl ester (AcTyrOEt) and N-benzoyl-L-tyrosine ethyl ester (BzTyrOEt); the optimal pH is 8.0. It does not hydrolyse N-benzoyl-L-arginine ethyl ester, N-α-toluenesulfonyl-L-arginine methyl ester, N-α-benzoyl-DL-arginine-p-nitroanilide, hippuryl-L-arginine or hippuryl-L-phenylalanine. The Michaelis constants for AcTyrOEt and BzTyrOEt are 0.05 mM and 0.0106 mM, respectively. The enzyme activity is inhibited completely by phenylmethylsulfonyl fluoride (PhMeSO2F), chymostatin and L-1-tosylamido-2-phenylethyl chloromethyl ketone (TosPheCH2Cl), and partially by soybean trypsin inhibitor and N-α-p-tosyl-L-lysine chloromethyl ketone (TosLysCH2Cl). The enzyme is activated by CaCl2, MgCl2, NaCl and KCl, and loses its activity in 5 min at 67 °C. It digests the jelly coat and vitelline layer, not the fertilization membrane. The microvilli of unfertilized eggs elongate and decrease in number as the vitelline layer lyses. The vitelline layer lytic activity is inhibited completely by PhMeSO2F, TosPheCH2Cl, and chymostatin, and partially by soybean trypsin inhibitor, TosLysCH2Cl, and α1-antitrypsin. We have confirmed by transmission electron microscopy that our chymotrypsin-like enzyme completely digests the vitelline layer. A result implying release of this enzyme from the acrosome vesicle is also reported. [ABSTRACT FROM AUTHOR]

Reports on the purification of an alpha-amanitin-resistant DNA-dependent RNA polymerase II from the lower eukaryote Aspergillus nidulans to apparent homogeneity. Use of extraction of the enzyme at low salt concentration, polymin P fractionation, binding to ion-exchangers and density gradient centrifugation; Resolution of the subunit composition by polyacrylamide gel electrophoresis in the presence of sodium dodecylsulphate.

Glucose-6-phosphate dehydrogenase has been purified 192-fold from Methylomonas M15, grown on methanol as sole source for carbon and energy. The purification procedure involved N-cetyl- N,N,N,-trimethylammonium bromide precipitation, ion-exchange chromatography and affinity chromatography on blue-dextran-Sepharose. The final enzyme preparations were homogeneous as judged by acrylamide gel electrophoresis and by sedimentation in an ultracentrifuge. The molecular weight of the enzyme was calculated to be 108000 ± 5000 from sedimentation equilibrium experiments. Electrophoresis in sodium dodecylsulfate gels indicates that the enzyme is a dimer composed of two probably identical subunits each having a molecular weight of 55000. The enzyme exhibits activity with either NAD or NADP as electron acceptor. Mg2+ or an other bivalent cation is not essential for enzyme activity. The Km values were found to be 0.2 mM for NAD, 0.014 mM for NADP, 0.29 mM for glucose 6-phosphate with NAD and 0.11 mM for glucose 6-phosphate with NADP as electron acceptor. The reduced coenzymes NADPH and NADH as well as ATP are powerful inhibitors of the enzyme. The intracellular levels of glucose 6-phosphate, ATP, NAD and NADP were measured and found to be approximately 2.5 mM, 1.5 mM, 1 mM and 2 mM respectively, during exponential growth. From 3 consideration of the substrate pool sizes and types of inhibitors, it is concluded th3.t this enzyme may function in two roles m the cell: NADH production for energetics and NADPH production for reductive biosynthesis. [ABSTRACT FROM AUTHOR]

Bacillus subtilis delta protein is a 21 500-Mr polypeptide that can be isolated in association with RNA polymerase holoenzyme from uninfected bacteria and with modified forms of RNA polymerase from cells infected with phage SP01 [Pero, J., Nelson, J. and Fox, T. (1975) Proc. Natl Acad. Sci. U.S.A. 72, 1589]. Although no function has been assigned to δ protein in uninfected cells, this host polypeptide enhances the specificity of transcription by phage-modified forms of RNA polymerase that contain SP01-coded regulatory subunits. This report describes the purification of δ and σ proteins from uninfected B. subtilis and examines the comparative effects of these polypeptides on transcription by core RNA polymerase. Purified σ polypeptide was found to stimulate the transcription of phage DNA while having little effect on RNA synthesis with the synthetic DNA poly(dA-dT) as template. In contrast, purified δ protein markedly depressed the transcription of poly(dA-dT) while having little effect on enzyme activity with phage DNA as template. The inhibitory effect of δ protein on poly(dA-dT) transcription was strongly dependent on the presence of KCI in the RNA synthesis reaction mixture. [ABSTRACT FROM AUTHOR]

A new method for discovering the minimum degree of rate equations using only the experimental graphs and a straight-edge (transparent ruler) is presented. This method is then illustrated by an analysis of the wavy υ vs [S] curves and non-linear double-reciprocal plots reproducibly given by NAD-dependent 15-hydroxyprostaglandin dehydrogenase for which a new improved purification is described. It is shown by this analysis that the enzyme has a complex mechanism involving cooperatively linked dependent sites and requiring a rate equation of at least fourth degree in prostaglandin and NAD+ with no kinetically significant dead-end complexes. [ABSTRACT FROM AUTHOR]

An inducible membrane-bound L-4-hydroxymandelate oxidase (decarboxylating) from Pseudomonas convexa has been solubilized and partially purified. It catalyzes the conversion of L-4-hydroxymandelic acid to 4-hydroxybenzaldehyde in a single step with the stoichiometric consumption of 02 and liberation of CO2. The enzyme is optimally active at pH 6.6 and at 55 °C. It requires FAD and Mn2+ for its activity. The membrane-bound enzyme is more stable than the solubilized and purified enzyme. After solubilization it gradually loses its activity when kept at 5 °C which can be fully reactivated by freezing and thawing. The Km values for DL-4-hydroxymandelate and FAD are 0.44 mM and 0.038 mM respectively. The enzyme is highly specific for DL-4-hydroxymandelic acid. DL-3,4-Dihydroxymandelic acid competitively inhibited the enzyme reaction. From the Dixon plot the Ki for DL-3,4-dihydroxymandetic acid was calculated to be 1.8 × 10-4 M. The enzyme is completely inactivated by thiol compounds and not affected by thiol inhibitors. The enzyme is also inhibited by denaturing agents, heavy metal ions and by chelating agents. [ABSTRACT FROM AUTHOR]

Prolyl hydroxylase was purified from human foetal skin and from a mixture of human foetal tissues by the affinity chromatography procedure using poly(L-proline). The enzyme from both sources was pure, when examined by polyacrylamide gel electrophoresis, as a native protein or in the presence of sodium dodecylsulphate, and enzyme activity recovery varied from 38% to 70% with seven enzyme preparations. The enzyme synthesized from 61.0 µmol to 82.7 µmol hydroxyproline mg protein-1 h-1 at 37 °C with a saturating concentration of (Pro-Pro-Gly)5 as substrate. The molecular weight of the enzyme was identical with that of the chick prolyl hydroxylase when studied by gel filtration, and the molecular weights of the subunits of the enzyme were about 61 000 and 64 000 as determined by sodium dodecylsulphate—polyacrylamide gel electrophoresis. The amino acid composition of the human enzyme was very similar to that of the chick prolyl hydroxylase. Antisera tohuman and chicl prolyl hydroxylases were prepared in rabbits. A single precipitin line was seen between the antiserum to human prolyl hydroxylase and the human enzyme in double immunodiffusion, and no cross-reactivity was detected between the human and chick enzymes by this technique. However, a distinct cross-reactivity was observed between the human and chick enzymes in inhibition experiments. [ABSTRACT FROM AUTHOR]

1. A procedure has been described for the purification of the major isozyme of yeast phosphoglucomutase of highest known specific activity. 2. The native enzyme has a molecular weight of about 65 400 and was found to be homogeneous as judged by sucrose density gradient centrifugation, gel filtration, electrophoresis on acrylamide gel and ultracentrifugal analysis. In the presence of denaturing agents such as guanidine hydrochloride or sodium dodecyl sulfate, the enzyme dissociated into 32 000-molecular-weight subunits. 3. As isolated, the enzyme has one mole of phosphate bound per mole of enzyme. Preparations incubated with 1.0 mM EDTA in 10 mM citrate buffer, pH 5.5 and dialysed against 10 mM metal-free citrate buffer, pH 5.5, contain no intrinsically bound Zn2+ and were enzymatically inactive but fully active in the presence of 5 mM M2+ and 84% as active with 0.5 mM Zn2+. Simultaneous presence of both ions at these concentrations did not enhance activity. Enzyme was completely and irreversibly inactivated by preincubation with Be2+. Inactive enzyme had one mole of Be2+ bound per mole of enzyme. 4. Enzyme exhibited "ping-pong" kinetics rather than "random sequential". Km values for glucose 1-phosphate and for glucose 1,6-bisphosphate were calculated to be 2.34 × 10-5 M and 2.24 × 10-6 M, respectively. Rate of enzyme phosphate turnover was studied with rapid-mixing technique. The rates of 32P release from 32P-labeled enzyme and its appearance as glucose 6-[32P]phosphate were comparable and remained unaffected by addition of glucose 1,6-bisphosphate. [ABSTRACT FROM AUTHOR]

N-Acetylrnannosamine kinase (ATP: N-acetylrnannosamine 6-phosphotransferase) of Salmonella typhimurium has been purified about 260-fold. The purified enzyme is highly specific for N-acetylmannosamine and for the phosphoryl donor. ATP. The products of the enzyme catalyzed phosphorylation have been identified as N-acetylmannosamine-6-phosphate and ADP. The enzyme also requires a divalent cation such as Mg++ for its activity. The Km for N-acetyl- mannosamine is 0.75 mM. and for ATP is 1.15 mM. The purified enzyme is fairly stable and shows a broad pH optima between 6.5 and 8.0. The formation of this enzyme in bacteria is highly susceptible to "glucose effect". In the absence of glucose, N-acetyImannosamine kinase is synthesized constitutively in a large number of bacteria. [ABSTRACT FROM AUTHOR]

400 fold purification of GDP glucose pyrophosphorylase from pea seedlings concomitant with stabilization of enzyme activity has been accomplished by (NH4)2SO4 fractionation, followed by chromatography on Sephadex G200 and DEAE-cellulose columns. In this last step the enzyme is eluted with 0.20 M NaCl. The enzyme is specifically activated by Mn2+. The apparent Km value for glucose-1-phosphate (37°, pH 7.5) is about 3 × 10-4 M. The GDP glucose biosynthesis is found to be reversible, with the equilibrium in favour of pyrophosphorolysis. The purified enzyme is not active with GTP plus mannose-1-phosphate or galactose-1-phosphate. No formation of nucleotide sugar is observed in the presence of glucose-1-phosphate plus ATP, ITP, dTTP, or CTP. A small residual UDPG pyrophosphorylase activity is activated by Mg2+. [ABSTRACT FROM AUTHOR]

Crude extracts of Candida utilis contain two types of 6-phosphogluconate dehydrogenase. One can be obtained in the crystalline form and has already been described and studied. In the present paper the purification of the second one, together with the differences in the properties between the two purified proteins, is reported. The two types of enzyme have identical pH optimum, same Km for the substrates and same specificity. They differ in amino acid composition, molecular weight, electrophoretic mobility, stability to pH and heat treatment, sensitivity to chlorodinitrobenzene and proteolytic treatment. All evidence indicates a greater instability of the crystalline enzyme in respect to the non-crystalline one and seems to exclude that the two forms of enzyme are an artefact due to the extraction or the purification procedures. [ABSTRACT FROM AUTHOR]

The purification of ribose-5-phosphate isomerase from Candida utilis is described. The procedure used extends over six steps to a 27% yield of a homogeneous protein. The following properties have been elucidated: the enzyme has a molecular weight of 105000 and will dissociate into four subunits upon the addition of sodium dodecylsulfate. The specific activity is about 350 international units per mg protein. The pH optimum, Km for ribose-5-phosphate, amino acid composition and isoelectric point have been determined. The isomerase is extremely sensitive to organic mercurials. [ABSTRACT FROM AUTHOR]

Potato Q-enzyme (α-l,4-glucan: α-1,4-glucan 6-glycosyltransferase) has been purified at least 430-fold. Its instability in the purified state was reversed by Cleland's reagent (dithiothreitol) and the preparation was essentially free of other starch-metabolising enzymes (α-amylase, α-glucosidase, D-enzyme and R-enzyme). Studies involving successive and simultaneous actions of Q-enzyme and pullulanase on amylose showed that the final average unit-chain length and iodine stain of the products were almost identical, but gel filtration revealed that their chain-length distributions were different. Similarly, two amylopectin-like polysaccharides formed by the direct action of Q-enzyme on amylose and by the combined action of Q-enzyme and potato phosphorylase on glucose 1-phosphate were debranched with pullulanase and their unit-chain length distributions compared by gel filtration with that of a debranched native amylopectin. In neither case was the unit-chain profile of native amylopectin reproduced. The Q-enzyme preparation has been shown to introduce additional branch points into amylopectin with the formation of a significant proportion of maltohexaosyl side chains. Evidence is presented that Q-enzyme and not a second branching enzyme is responsible for this action and a mechanism for the formation of the maltohexaosyl chains is proposed. [ABSTRACT FROM AUTHOR]

The putative metal-dependent hydrolase gene TTE1006 from Thermoanaero- bacter tengcongensis strain MB4T (T = type strain; Genbank accession No. AE008691) was heterologously expressed in Escherichia coli. The 205-amino-acid gene product was purified and crystallized. The crystal used for data collection belongs to space group P21, with unit-cell parameters a = 85.2, b = 62.1, c = 172.4 Å, β = 104.2°. Using a synchrotron-radiation source, the resolution limit of the data reached 1.87 Å. Eight molecules were estimated to be present in the asymmetric unit, with a solvent content of 48%. Structure determination is ongoing using the multiple-wavelength anomalous diffraction (MAD) method and also the molecular-replacement (MR) method. [ABSTRACT FROM AUTHOR]

Pantothenate kinase is an essential enzyme in the bacterial life cycle. It catalyzes the phosphorylation of pantothenate (vitamin B5) to 4'-phosphopantothenate, the first step in the coenzyme A biosynthetic pathway. The enzyme from Mycobacterium tuberculosis, MW 35.7 kDa, has been cloned, expressed, purified and crystallized in two different trigonal crystal forms, both belonging to space group P3121. Two complete data sets of resolution 2.5 Å (form I) and 2.9 A (form II) from crystals with unit-cell parameters a = b = 78.3, c = 115.45 Å and a = b = 107.63, c = 89.85 Å, respectively, were collected at room temperature on a home X-ray source. Structures of both crystal forms were solved for one subunit in the asymmetric unit by molecular replacement. [ABSTRACT FROM AUTHOR]

The glycolytic enzyme phosphoglucose isomerase catalyses the reversible isomerization of glucose 6-phosphate to fructose 6-phosphate. The phosphoglucose isomerase from the hyperthemophilic archaeon Pyrococcus furiosus, which shows no sequence similarity to any known bacterial or eukaryotic phosphoglucose isomerase, has been cloned and overexpressed in Escherichia coli, purified and subsequently crystallized by the hanging-drop method of vapour diffusion using 1.6 M sodium citrate as the precipitant at pH 6.5. Multiple-wavelength anomalous dispersive X-ray data have been collected to a maximum resolution of 1.92 Å on a single seleno-methionine-incorporated crystal. This crystal belongs to space group C2, with approximate unit-cell parameters a=84.7, b=42.4,c=57.3 Å, β=120.6° and a monomer in the asymmetric unit. [ABSTRACT FROM AUTHOR]