Comparative study of purine nucleoside phosphorylase from rabbit kidney, spleen, liver and embryos

Homogeneous preparations of purine nucleoside phosphorylase (EC 2.4.2.1) from rabbit kidney, spleen, liver and embryos were studied. The enzyme preparations do not differ in electrophoretic mobility. The molecular weight of the enzyme obtained from various sources was determined by gel filtration on Sephadex G-150 superfine and is about 90-92 kD. The enzyme subunits are identical in terms of molecular weight, as can be evidenced from sodium dodecyl sulfate polyacrylamide gel electrophoresis (Mr approximately 31 kD). The pH optima of these enzyme preparations for guanosine and xanthosine phosphorolysis are 6.2 and 5.7, respectively. The isoelectric point of purine nucleoside phosphorylase from rabbit kidney was determined in the presence of 9 M urea and is equal to 5.55. The enzyme is the most stable at pH 7.7; it is specific towards hypoxanthine and guanine nucleosides as well as towards xanthosine, but does not cleave adenine nucleosides. The Km values for guanosine and inosine are 1.4.10(-4) M and 1.2.10(-4) M, respectively. The enzyme does not catalyze the ribosyl transfer reaction in the absence of Pi.     

Purification, crystallization, and properties of D-ribose isomerase from Mycobacterium smegmatis.

D-Ribose isomerase, which catalyzes the conversion of D-ribose to D-ribulose, was purified from extracts of Mycobacterium smegmatis grown on D-ribose. The purified enzyme crystalized as hexagonal plates from a 44% solution of ammonium sulfate. The enzyme was homogenous by disc gel electrophoresis and ultracentrifugal analysis. The molecular weight of the enzyme was between 145,000 and 174,000 by sedimentation equilibrium analysis. Its sedimentation constant of 8.7 S indicates it is globular. On the basis of sodium dodecyl sulfate gel electrophoresis in the presence of Mn2+, the enzyme is probably composed of 4 identical subunits of molecular weight about 42,000 to 44,000. The enzyme was specific for sugars having the same configuration as D-ribose at carbon atoms 1 to 3. Thus, the enzyme could also utilize L-lyxose, D-allose, and L-rhamnose as substrates. The Km for D-ribose was 4 mM and for L-lyxose it was 5.3 mM. The enzyme required a divalent cation for activity with optimum activity being shown with Mn2+. the Km for the various cations was as follows: Mn2+, 1 times 10(-7) M, Co2+, 4 times 10(-7) M, and Mg2+, 1.8 times 10(-5) M. The pH optimum for the enzyme was 7.5 to 8.5. Polyols did not inhibit the enzyme to any great extent. The product of the reaction was identified as D-ribulose by thin layer chromatography and by preparation of the O-nitrophenylhydrazone derivative.     

Protonation state of a single histidine residue contributes significantly to the kinetics of the reaction of plasminogen activator inhibitor-1 with tissue-type plasminogen activator

Stopped-flow fluorometry was used to study the kinetics of the reactive center loop insertion occurring during the reaction of N-((2-(iodoacetoxy)ethyl)-N-methyl)amino-7-nitrobenz-2-oxa-3-diazole (NBD) P9 plasminogen activator inhibitor-1 (PAI-1) with tissue-(tPA) and urokinase (uPA)-type plasminogen activators and human pancreatic elastase at pH 5.5-8.5. The limiting rate constants of reactive center loop insertion (k(lim)) and concentrations of proteinase at half-saturation (K(0.5)) for tPA and uPA and the specificity constants (k(lim)/K(0.5)) for elastase were determined. The pH dependences of k(lim)/K(0.5) reflected inactivation of each enzyme due to protonation of His57 of the catalytic triad. However, the specificity of the inhibitory reaction with tPA and uPA was notably higher than that for the substrate reaction catalyzed by elastase. pH dependences of k(lim) and K(0.5) obtained for tPA revealed an additional ionizable group (pKa, 6.0-6.2) affecting the reaction. Protonation of this group resulted in a significant increase in both k(lim) and K(0.5) and a 4.6-fold decrease in the specificity of the reaction of tPA with NBD P9 PAI-1. Binding of monoclonal antibody MA-55F4C12 to PAI-1 induced a decrease in k(lim) and K(0.5) at any pH but did not affect either the pKa of the group or an observed decrease in k(lim)/K(0.5) due to protonation of the group. In contrast to tPA, the k(lim) and K(0.5) for the reactions of uPA with NBD P9 PAI-1 or its complex with the monoclonal antibody were independent of pH in the 6.5-8.5 range. Since slightly acidic pH is a feature of a number of malignant tumors, alterations in PAI-1/tPA kinetics could play a role in the cancerogenesis. Changes in the protonation state of His(188), which is placed closely to the S1 site and is unique for tPA, has been proposed to contribute to the observed pH dependences of k(lim) and K(0.5).     

Regulation of aromatic amino Acid biosynthesis in higher plants: I. Evidence for a regulatory form of chorismate mutase in etiolated mung bean seedlings

Etiolated mung bean seedlings were examined for chorismate mutase activity. Evidence for the occurrence of two forms of the enzyme (designated CM-1 and CM-2) was obtained by ammonium sulfate fractionation, anion exchange cellulose chromatography, and isoelectric focusing. The two forms showed distinctly different properties, as CM-1 was inhibited by phenylalanine and tyrosine and activated by tryptophan, but inhibition by phenylalanine and tyrosine was reversed by tryptophan. The other form, CM-2, was unaffected by any of the three aromatic amino acids. Isoelectric points of the two forms were CM-1, pH 4.6, and CM-2, pH 5.6. The molecular weights estimated by molecular sieving on Sephadex G-200 were CM-1, 50,000, and CM-2, 36,000.     

Enzymatic properties of pierisin-1 and its N-terminal domain, a guanine-specific ADP-ribosyltransferase from the cabbage butterfly

The cabbage butterfly, Pieris rapae, produces an ADP-ribosylating cytotoxic protein, pierisin-1. Unlike other ADP-ribosylating toxins, the acceptor site for ADP-ribosylation by pierisin-1 is the N-2 position of guanine bases in DNA. The present study was designed to characterize this novel guanine-specific ADP-ribosyltransferase, pierisin-1. The N-terminal polypeptide from Met-1 to Arg-233, but not the C-terminal Ser-234-Met-850 polypeptide, was found to exhibit guanine ADP-ribosyltransferase activity. Trypsin-treated pierisin-1, which is considered to be a "nicked" full-length form composed of associated N- and C-terminal fragments, also demonstrated such activity. Optimum conditions for the N-terminal polypeptide of pierisin-1 were pH 8-10, 37-40 degrees C, in the presence of 100-200 mM NaCl or KCl. Other metal ions such as Ca(2+) or Mg(2+) were not required. Kinetic studies demonstrated potent ADP-ribosyltransferase activity with a K(M) value for NAD of 0.17 mM and k(cat) of 55 per second. Under these optimum conditions, the specific activity of trypsin-treated pierisin-1 was about half (k(cat) = 25 per second). When the conditions were changed to pH 5-7 or 10-20 degrees C, some activity (6-55% or 5-20%, respectively, of that under optimal conditions) of the N-terminal polypeptide was still evident; however, almost all of the trypsin-treated enzyme activity disappeared. This implies the inhibition of the N-terminal enzyme domain by the associated C-terminal fragment. Long-term reactions indicated that a single molecule of pierisin-1 has the capacity to generate more than 10(6) ADP-ribosylated DNA adducts, which could cause the death of a mammalian cell.     

Monomeric purine nucleoside phosphorylase from rabbit liver. Purification and characterization.

Rabbit liver purine nucleoside phosphorylase (purine nucleoside: orthophosphate ribosyltransferase EC 2.4.2.1.) was purified to homogeneity by column chromatography and ammonium sulfate fractionation. Homogeneity was established by disc gel electrophoresis in presence and absence of sodium dodecyl sulfate, and isoelectric focusing. Molecular weights of 46,000 and 39,000 were determined, respectively, by gel filtration and by sodium dodecyl sulfate-polyacrylamide disc gel electrophoresis. Product inhibition was observed with guanine and hypoxanthine as strong competitive inhibitors for the enzymatic phosphorolysis of guanosine. Respective Kis calculated were 1.25 x 10(-5) M for guanine and 2.5 x 10(-5) M for hypoxanthine. Ribose 1-phosphate, another product of the reaction, gave noncompetitive inhibition with guanosine as variable substrate, and an inhibition constant of 3.61 x 10(-4) M was calculated. The protection of essential --SH groups on the enzyme, by 2-mercaptoethanol or dithiothreitol, was necessary for the maintenance of enzyme activity. Noncompetitive inhibition was observed for p-chloromercuribenzoate with an inhibition constant of 5.68 x 10(-6)M. Complete reversal of this inhibition by an excess of 2-mercaptoethanol or dithiothreitol was demonstrated. In the presence of methylene blue, the enzyme showed a high sensitivity to photooxidation and a dependence of photoinactivation on pH, strongly implicating histidine as the susceptible group at the active site of the enzyme. The pKa values determined for ionizable groups of the active site of the enzyme were near pH 5.5 and pH 8.5 The chemical and kinetic evidences suggest that histidine and cysteine may be essential for catalysis. Inorganic orthophosphate (Km 1.54 x 10(-2) M) was an obligatory anion requirement, and arsenate substituted for phosphate with comparable results. Guanosine (Km 5.00 x 10(-5) M), deoxyguanosine (Km 1.00 x 10(-4)M) and inosine (Km 1.33 x 10(-4)M), were substrates for enzymatic phosphorolysis. Xanthosine was an extremely poor substrate, and adenosine was not phosphorylyzed at 20-fold excess of the homogeneous enzyme. Guanine (Km 1.82 x 10(-5)M),ribose 1-phosphate (Km 1.34 x 10(-4) M) and hypoxanthine were substrates for the reverse reaction, namely, the enzymatic synthesis of nucleosides. The initial velocity studies of the saturation of the enzyme with guanosine, at various fixed concentrations of inorganic orthophosphate, suggest a sequential bireactant catalytic mechanism for the enzyme.     

Phosphorylation and guanine nucleotide exchange on polypeptide chain initiation factor-2 from Artemia embryos

Eukaryotic initiation factor-2 (eIF-2) from Artemia embryos is able to exchange guanine nucleotides at the same rate in the presence or absence of Mg2+ when the reaction is carried out with either purified eIF-2 at 30 degrees C or less purified preparations at any temperature (10-30 degrees C). No exchange factor appears to catalyze this reaction. However, with purified eIF-2 at lower temperatures (10 degrees C) the exchange is clearly impaired by Mg2+ and this impairment is overcome by the guanine nucleotide exchange factor (GEF) of rabbit reticulocytes. Thus, Artemia eIF-2 is able to exchange guanine nucleotides by two alternative mechanisms that may reflect two states of the protein. Phosphorylation of the eIF-2 alpha subunit by the heme-controlled inhibitor (HCI) of rabbit reticulocytes abolishes the GEF-dependent reaction, but has no effect on the factor-independent one. The search for eIF-2 alpha kinases in Artemia embryo led to the detection of only one such enzyme, which was identified as a casein kinase type II. None of the exchange reactions is affected by the phosphorylation of the eIF-2 alpha subunit by this kinase, suggesting that, irrespective of the kind of mechanism for guanine nucleotide exchange that is actually operating in Artemia, it might not be a target for regulation by eIF-2 alpha phosphorylation.     

Purification and characterization of a GTP-pyrophosphate exchange activity from vaccinia virions. Association of the GTP-pyrophosphate exchange activity with vaccinia mRNA guanylyltransferase . RNA (guanine-7-)methyltransferase complex (capping enzyme)

A core-associated enzyme, which catalyzes a nucleotide-pyrophosphate exchange with GTP, has been purified from vaccinia virions. The enzyme requires MgCl2 for activity, has an alkaline pH optimum, and specifically utilizes GTP as the exchanging nucleotide. The enzyme does not catalyze exchange of GMP with GTP. The GTP-PPi exchange enzyme co-purifies with vaccinia capping enzyme (RNA guanylyltransferase and RNA (guanine-7-)methyltransferase) through successive chromatography steps on DEAE-cellulose, DNA-cellulose, and phosphocellulose. GTP-PPi exchange and capping activities remain physically associated during sedimentation in a glycerol gradient. Under high salt conditions (1 M NaCl), GTP-PPi exchange, capping, and methylating activities co-sediment with an RNA triphosphatase activity and a nucleoside triphosphate phosphohydrolase activity as a 6.5 S multifunctional enzyme complex which contains two major polypeptides of 96,000 and 26,000 molecular weight. The characteristics of the various enzymatic reactions catalyzed by this complex are described. The GTP-PPi exchange reaction of vaccinia guanylyltransferase affords a simple, sensitive assay for capping enzyme function. The relevance of the GTP-PPi exchange reaction to the mechanism of transguanylylation is considered.     

5'-Iodothyronine deiodinase of rat liver: activity in microsomes prepared by various methods, solubilization by detergents and partial purification

Iodothyronine deiodinase activities of rat liver microsomes prepared by (1) differential centrifugation, (2) column chromatography, (3) precipitation with Ca2+, (4) precipitation at low pH, or combinations of these were compared. Method 2 or 2 followed by 4 provided microsomes with specific activities 4.6- and 7.4-times higher than method 1, respectively. Both Triton X-100 at 0.1% (w/v) and 3-[(3-cholamidopropyl)dimethylammonio]-1-propane sulfonate (Chaps) at 4-6 mM efficiently solubilized deiodinase and were not inhibitory at low concentrations. The Chaps-soluble enzyme could be moderately purified by fractionation with ammonium sulfate but more effectively with poly(ethylene glycol).     

Calcium-stimulated guanosine--inosine nucleosidase from yellow lupin (Lupinus luteus)

Guanosine-inosine-preferring nucleoside N-ribohydrolase has been purified to homogeneity from yellow lupin (Lupinus luteus) seeds by ammonium sulfate fractionation, ion-exchange chromatography and gel filtration. The enzyme functions as a monomeric, 80kDa polypeptide, most effectively between pH 4.7 and 5.5. Of various mono- and divalent cations tested, Ca(2+) appeared to stimulate enzyme activity. The nucleosidase was activated 6-fold by 2mM exogenous CaCl(2) or Ca(NO(3))(2), with K(a)=0.5mM (estimated for CaCl(2)). The K(m) values estimated for guanosine and inosine were 2.7+/-0.3 microM. Guanosine was hydrolyzed 12% faster than inosine while adenosine and xanthosine were poor substrates. 2'-Deoxyguanosine, 2'-deoxyinosine, 2'-methylguanosine, pyrimidine nucleosides and 5'-GMP were not hydrolyzed. However, the enzyme efficiently liberated the corresponding bases from synthetic nucleosides, such as 1-methylguanosine, 7-methylguanosine, 1-N(2)-ethenoguanosine and 1-N(2)-isopropenoguanosine, but hydrolyzed poorly the ribosides of 6-methylaminopurine and 2,6-diaminopurine. MnCl(2) or ZnCl(2) inhibited the hydrolysis of guanosine with I(50) approximately 60 microM. Whereas 2'-deoxyguanosine, 2'-methylguanosine, adenosine, as well as guanine were competitive inhibitors of this reaction (K(i) values were 1.5, 3.6, 21 and 9.7 microM, respectively), hypoxanthine was a weaker inhibitor (K(i)=64 microM). Adenine, ribose, 2-deoxyribose, 5'-GMP and pyrimidine nucleosides did not inhibit the enzyme. The guanosine-inosine hydrolase activity occurred in all parts of lupin seedlings and in cotyledons it increased up to 5-fold during seed germination, reaching maximum in the third/fourth day. The lupin nucleosidase has been compared with other nucleosidases.     

Acetyl coenzyme A: alpha-glucosaminide N-acetyltransferase. Evidence for a transmembrane acetylation mechanism

The lysosomal membrane enzyme acetyl-CoA: alpha-glucosaminide N-acetyltransferase catalyzes the transfer of an acetyl group from acetyl-CoA to terminal alpha-linked glucosamine residues of heparan sulfate. The reaction mechanism was examined using highly purified lysosomal membranes from rat liver. The reaction was followed by measuring the acetylation of a monosaccharide acetyl acceptor, glucosamine. The enzyme reaction was optimal above pH 5.5, and a 2-3-fold stimulation of activity was observed when the membranes were assayed in the presence of 0.1% taurodeoxycholate. Double reciprocal analysis and product inhibition studies indicated that the enzyme works by a Di-Iso Ping Pong Bi Bi mechanism. Further evidence to support this mechanism was provided by characterization of the enzyme half-reactions. Membranes incubated with acetyl-CoA and [3H]CoA were found to produce acetyl-[3H]CoA. This exchange was optimal at pH values above 7.0. Treating membranes with [3H] acetyl-CoA resulted in the formation of an acetyl-enzyme intermediate. The acetyl group could then be transferred to glucosamine, forming [3H]N-acetylglucosamine. The transfer of the acetyl group from the enzyme to glucosamine was optimal between pH 4 and 5. The results suggest that acetyl-CoA does not cross the lysosomal membrane. Instead, the enzyme is acetylated on the cytoplasmic side of the lysosome and the acetyl group is then transferred to the inside where it is used to acetylate heparan sulfate.     

Natural killer cell cytolytic granule-associated enzymes. I. Purification, characterization, and analysis of function of an enzyme with sulfatase activity

An enzyme with sulfatase activity has been isolated from the granules of a rat NK leukemia cell line, CRNK-16. The enzyme has been purified from crude preparation, with a specific activity of 52 nmol/min/mg of protein, by DEAE ion exchange and Con A-Sepharose affinity chromatography, resulting in a specific activity of 230 nmol/min/mg of protein. The molecular mass of the purified enzyme was estimated to be 40 kDa by gel filtration chromatography at pH 7.4, but the enzyme had the ability to complex to molecular masses of greater than 300 kDa at low pH when crude granule extract was used as the starting sample, suggesting that it associates with other granule components. The enzyme was determined to be an arylsulfatase by its ability to (a) hydrolyze p-nitrophenyl sulfate (Km = 26.0 mM) and p-nitrocatechol sulfate (pNC sulfate) (Km = 1.1 mM) and (b) be inhibited by sulfite (Ki = 6.0 x 10(-7) M), sulfate (Ki = 1 x 10(-3) M), and phosphate (Ki = 4 x 10(-5) M) in a competitive manner. The pH optimum for enzymatic activity was determined to be 5.6. The role of this enzyme in cytolytic function was investigated by examining the effect of its substrates and inhibitors on granule- and cell-mediated lysis. pNC sulfate was shown to cause a dose-dependent inhibition of target cell lysis by isolated cytolytic granules (complete inhibition at 12.5 mM). Sulfite induced an incomplete inhibition (50% at 1 mM), whereas phosphate was essentially without inhibitory effect. Sulfate, on the other hand, altered lytic activity in a biphasic manner, inasmuch as it induced an inhibition of lysis at high concentrations and an increase of lysis at low concentrations. Cell-mediated lysis was inhibited by pNC sulfate in a dose-dependent fashion at concentrations greater than 2.5 mM, with nearly complete inhibition at 50 mM. Sulfate also altered the lytic activity by intact cells in a biphasic manner, although the effect was much less pronounced. Sulfite and phosphate caused only a 30% inhibition of lytic activity. These results suggest that the sulfatase enzyme is involved in NK cytolytic function, presumably at the lethal hit stage.     

Purification and characterization of a novel caffeine oxidase from Alcaligenes species

Alcaligenes species CF8 isolated from surface water of a lake produced a novel serine type metallo-caffeine oxidase. The optimal medium for caffeine oxidase production by this strain was (w/v) NaNO(3), 0.4%; KH(2)PO(4), 0.15%; Na(2)HPO(4), 0.05%; FeCl(3).6H(2)O, 0.0005%; CaCl(2).2H(2)O, 0.001%; MgSO(4).7H(2)O, 0.02%; glucose, 0.2%; caffeine, 0.05%, pH 7.5. The enzyme was purified to 63-fold by using ammonium sulfate precipitation, dialysis, ion exchange (diethylaminoethyl-cellulose) and gel filtration (Sephadex G-100) chromatographic techniques. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis revealed that the purified caffeine oxidase was monomeric with a molecular mass of 65 kDa. The purified caffeine oxidase with a half-life of 20 min at 50 degrees C had maximal activity at pH 7.5 and 35 degrees C. The purified caffeine oxidase had strict substrate specificity towards caffeine (K(m) 8.94 microM and V(max) 47.62 U mg protein(-1)) and was not able to oxidize xanthine and hypoxanthine. The enzyme activity was not inhibited by para-chloromercuribenzoic acid, iodoacetamide, n-methylmaleimide, salicylic acid and sodium arsenite indicating the enzyme did not belong to xanthine oxidase family. The enzyme was not affected by Ca(+2), Mg(+2) and Na(+), but was completely inhibited by Co(+2), Cu(+2) and Mn(+2) at 1mM level. The novel caffeine oxidase isolated here from Alcaligenes species CF8 may be useful in biotechnological processes including waste treatment and biosensor development.     

Isolation and characterization of human liver guanine deaminase

Guanine deaminase (EC 3.5.4.3, guanine aminohydrolase [GAH]) was purified 3248-fold from human liver to homogeneity with a specific activity of 21.5. A combination of ammonium sulfate fractionation, and DEAE-cellulose, hydroxylapatite, and affinity chromatography with guanine triphosphate ligand were used to purify the enzyme. The enzyme was a dimer protein of a molecular weight of 120,000 with each subunit of 59,000 as determined by gel filtration and sodium dodecyl sulfate-gel electrophoresis. Isoelectric focusing gave a pI of 4.76. It was found to be an acidic protein, as evidenced by the amino acid analysis, enriched with glutamate, aspartate, alanine and glycine. It showed a sharp pH optimum of 8.0. The apparent Km for guanine was determined to be 1.53 X 10(-5) M at pH 6.0 and 2 X 10(-4) M for 8-azaguanine as a substrate at pH 6.0. The enzyme was found to be sensitive to p-hydroxymercuribenzoate inhibition with a Ki of 1.53 X 10(-5) M and a Ki of 5 X 10(-5) M with 5-aminoimidazole-4-carboxamide as an inhibitor. The inhibition with iodoacetic acid showed only a 7% loss in the activity at 1 X 10(-4) M and a 24% loss at 1 X 10(-3) M after 30 min of incubation, whereas p-hydroxymercuribenzoate incubation for 30 min resulted in a 91% loss of activity at a concentration of 1 X 10(-4) M. Guanine was the substrate for all of the inhibition studies. The enzyme was observed to be stable up to 40 degrees C, with a loss of almost all activity at 65 degrees C with 30 min incubation. Two pKa values were obtained at 5.85 and 8.0. Analysis of the N-terminal amino acid proved to be valine while the C-terminal residue was identified as alanine.     

The reaction of trans-7,8-dihydroxy-anti-9,10-epoxy-7,8,9,10-tetrahydrobenzo(a)pyrene with DNA involves attack at the N7-position of guanine moieties

The reaction of trans-7,8-dihydroxy-anti-9,10-epoxy-7,8,9,10-tetrahydrobenzo(a)pyrene (anti-BPDE) with DNA prelabelled with [14C] and [3H]-purine precursors has indicated that in addition to the N2-position of guanine previously reported [10--12] reaction also involves the N7-position of guanine. The hydrocarbon-N7-guanine product was not detected earlier because it is lost from the DNA very readily at pH 7. The same N7-product was obtained by reaction of anti-BPDE with guanine in dimethylformamide.     

Purine nucleoside phosphorylase of the malarial parasite, Plasmodium lophurae

Purine nucleoside phosphorylase (EC 2.4.2.1, purine nucleoside:orthophosphate ribosyltransferase) was purified and characterized from the malarial parasite, Plasmodium lophurae, using a chromatofocusing (Pharmacia) column and a formycin B affinity column. The apparent isoelectric point of the native protein, as determined by chromatofocusing, was 6.80. By gel filtration and both native and sodium dodecyl sulfate-polyacrylamide gel electrophoresis, the native enzyme appeared to be a pentamer with a native molecular weight of 125,300 and a subunit molecular weight of 23,900. The enzyme had a broad pH optimum, pH 5.5-7.5, with maximum activity at pH 6.0-6.5. The enzyme reaction was readily reversible with a Km for inosine of 33 microM and a Km for hypoxanthine of 82 microM. Thioinosine, guanosine, and guanine were also substrates for the plasmodial enzyme, but allopurinol and adenine were not. The parasite enzyme was competitively inhibited by formycin B (Ki = 0.39 microM). Formycin A, azaguanine, and 8-aminoguanosine were not inhibitors of the enzyme.     

Hypermodification of tRNA in Thermophilic archaea. Cloning, overexpression, and characterization of tRNA-guanine transglycosylase from Methanococcus jannaschii

tRNA is structurally unique among nucleic acids in harboring an astonishing diversity of modified nucleosides. Two structural variants of the hypermodified nucleoside 7-deazaguanosine have been identified in tRNA: queuosine, which is found at the wobble position of the anticodon in bacterial and eukaryotic tRNA, and archaeosine, which is found at position 15 of the D-loop in archaeal tRNA. From homology searching of the Methanococcus jannaschii genome, a gene coding for an enzyme in the biosynthesis of archaeosine (tgt) was identified and cloned. The tgt gene was overexpressed in an Escherichia coli expression system, and the recombinant tRNA-guanine transglycosylase enzyme was purified and characterized. The enzyme catalyzes a transglycosylation reaction in which guanine is eliminated from position 15 of the tRNA and an archaeosine precursor (preQ(0)) is inserted. The enzyme is able to utilize both guanine and the 7-deazaguanine base preQ(0) as substrates, but not other 7-deazaguanine bases, and is able to modify tRNA from all three phylogenetic domains. The enzyme shows optimal activity at high temperature and acidic pH, consistent with the optimal growth conditions of M. jannaschii. The nature of the temperature dependence is consistent with a requirement for some degree of tRNA tertiary structure in order for recognition by the enzyme to occur.     

Thermostable chitosanase from Bacillus sp. strain CK4: its purification, characterization, and reaction patterns

A thermostable chitosanase, purified 156-fold to homogeneity in an overall yield of 12.4%, has a molecular weight of about 29,000 +/- 2,000, and is composed of monomer. The enzyme degraded soluble chitosan, colloidal chitosan, and glycol chitosan, but did not degrade chitin or other beta-linked polymers. The enzyme activity was increased about 2.5-fold by the addition of 10 mM Co2+ and 1.4-fold by Mn2+. However, Cu2+ ion strongly inhibited the enzyme. Optimum temperature and pH were 60 degrees C and 6.5, respectively. The enzyme was stable after heat treatment at 80 degrees C for 30 min or 70 degrees C for 60 min and fairly stable in protein denaturants as well. Chitosan was hydrolyzed to (GlcN)4 as a major product, by incubation with the purified enzyme. The effects of ammonium sulfate and organic solvents on the action pattern of the thermostable chitosanase were investigated. The amounts of (GlcN)3-(GlcN)6 were increased about 30% (w/w) in DAC 99 soluble chitosan containing 10% ammonium sulfate, and (GlcN)1 was not produced. The monophasic reaction system consisted of DAC 72 soluble chitosan in 10% EtOH also showed no formation of (GlcN)1, however, the yield of (GlcN)3 approximately (GlcN)6 was lower than DAC 99 soluble chitosan-10% ammonium sulfate. The optimal concentration of ammonium sulfate to be added was 20%. At this concentration, the amount of hexamer was increased by over 12% compared to the water-salt free system.     

Physical Properties and Kinetic Behavior of a Cephalosporin Acetylesterase Produced by Bacillus subtilis

An esterase that deacetylates cephalosporins was recovered from the supernatant of a Bacillus subtilis culture. It was partially purified by ammonium sulfate fractionation and ultrafiltration. The enzyme had a temperature optimum between 40 and 50 C and a pH optimum of 7.0. The molecular weight was estimated by gel filtration to be 190,000. The enzyme was very stable and retained greater than 80% of its activity after storage in solution at 25 C for 1 month. The esterase exhibited Michaelis-Menton kinetics with the substrates 7-aminocephalosporanic acid (7-ACA) and 7-(thiophene-2-acetamido)cephalosporanic acid (cephalothin); the K(m) values were 2.8 X 10(-3) and 8.3 X 10(-3) M, respectively. The products of 7-ACA deacetylation were weak competitive inhibitors, and a K(i) value of 5.0 X 10(-2) M was determined for acetate and of 3.6 X 10-2 M for deacetyl-7-ACA. Weak product inhibition did not prevent the deacetylation reaction from going to completion. A 5-mg/ml solution of partially purified esterase completely hydrolyzed (greater than 99.5%) a 24-mg/ml solution of 7-ACA in 3 h. Because of the kinetic properties and excellent stability, this enzyme may be useful in an immobilized form to prepare large quantities of deacetylated cephalosporin derivatives.