Properties of 7-substituted deoxyguanosines formed by cis-diamminedichloroplatinum(II)

cis-Diamminedichloroplatinum(II) (cis-Pt) was reacted with deoxyguanosine and guanosine at pH 6 and the reaction products were purified by HPLC. The products were characterized by UV and 1H-NMR spectra and by incubating cis-Pt with 7-methylguanosine, N2-methylguanosine, [8-3H]guanosine and [U-14C]guanosine. The main product was shown to be a cross-link, in which cis-Pt was linked to the N-7 atoms of two guanines. In the other product cis-Pt was bound monofunctionally to the N-7 atom of guanine. The cis-Pt adducts had many unique properties compared to other N-7 alkylated guanosines. When cis-Pt was incubated with [8-3H]guanosine, the products still had their 8-3H-radioactivity. At pH 10 at room temperature the imidazole ring of the monoadduct was not opened in 20 days, while the half-life of imidazole ring-opening for 7-methyldeoxyguanosine was 21.5 min. The half-lives of acid-catalyzed depurination in 0.1 M HCl at 70 degrees C for deoxyguanosine, 7-methyldeoxyguanosine and the monoadduct were 30 s, 48 s and 35 min, respectively.     

Stabilities of 7-alkylguanosines and 7-deoxyguanosines formed by phosphoramide mustard and nitrogen mustard

7-Alkylguanosine and 7-alkyldeoxyguanosine were prepared from phosphoramide mustard and nitrogen mustard in nonaqueous conditions. The guanosine products were N-(2-chloroethyl)-N-[2-(7-guanosinyl)ethyl] phosphorodiamidic acid, and N-(2-chloroethyl)-N-[2-(7-guanosinyl)ethyl]methylamine, respectively. These were also formed in aqueous reactions but they rapidly underwent secondary reactions. The half-life of the phosphoramide mustard-guanosine adduct was 3.1 h (37 degrees C, pH 7.4) and that of the nitrogen mustard adduct 1 h (25 degrees C, pH 7.4), as determined by HPLC. The half-lives of the respective adducts for imidazole ring-opening were 9.5 h and 0.78 h (37 degrees C, pH 7.4). The respective deoxyguanosine derivatives depurinated with half-lives of 8.5 h and 1.6 h (25 degrees C, pH 4.2). The mustard adducts are notably more labile than simple alkyl substituted guanosines and deoxyguanosines.     

Depurination and imidazole ring-opening in nucleosides and DNA alkylated by styrene oxide

Styrene-7,8-oxide was reacted with guanosine and deoxyguanosine and four isomeric 7-alkylguanosines were isolated, two of each being substitutions through the alpha and beta carbon of styrene oxide. The diastereomeric adducts imidazole ring-opened at an identical rate but the alpha- and beta-adducts differed (half-lives 90 and 56 min, respectively, pH 10, 24 degrees C). The 7-beta alkyl-deoxyguanosine derivatives ring-opened at a six times slower rate, which was similar to 7-methyldeoxyguanosine. The diastereomeric guanosine products also depurinated at the same rate but the beta-derivatives depurinated faster than the alpha-derivatives (t1/2 35 vs. 79 min, respectively, pH 1, 70 degrees C). The differences in the ring-opening and depurination of the alpha- and beta-isomers corresponded to their respective pK alpha values (7.31-7.32 vs. 7.16-7.19). The 7-alkyldeoxyguanosine derivatives of styrene oxide depurinated equally fast as 7-methyldeoxyguanosine. By contrast, the depurination of 7-alkylguanine was 15 times slower in the single-stranded DNA and 55 times slower in the double-stranded DNA.     

Alkylation by propylene oxide of deoxyribonucleic acid, adenine, guanosine and deoxyguanylic acid

1. Propylene oxide reacts with DNA in aqueous buffer solution at about neutral pH to yield two principal products, identified as 7-(2-hydroxypropyl)guanine and 3-(2-hydroxypropyl)adenine, which hydrolyse out of the alkylated DNA at neutral pH values at 37 degrees C. 2. These products were obtained in quantity by reactions between propylene oxide and guanosine or adenine respectively. 3. The reactions between propylene oxide and adenine in acetic acid were parallel to those between dimethyl sulphate and adenine in neutral aqueous solution; the alkylated positions in adenine in order of decreasing reactivity were N-3, N-1 and N-9. A method for separating these alkyladenines is described. 4. Deoxyguanylic acid sodium salt was alkylated at N-7 by propylene oxide in neutral aqueous solution. 5. The nature of the side chain in the principal alkylation products was established by mass spectrometry, and the nature of the products is consistent with their formation by the bimolecular reaction mechanism.     

Purification and characterization of rat liver transglutaminase

1. Transglutaminase (EC 2.3.2.13) was purified from rat liver. 2. The enzyme was stable at 25 degrees C in the pH range of 6.0-9.0, with the optimum at pH 9.0. 3. The enzyme was inactivated after incubation for 20, 4 and 1 min at 44 degrees C, 52 degrees C, and 60 degrees C, respectively. 4. Activation energies were 30.4 kcal/mol for denaturation and 19.9 kcal/mol for substrate conversion to products. 5. The enzyme was inactivated by sulfhydryl modification with hydroxymercuribenzoate (99.1%) and N-ethylmalemide (78.5%). 6. Calcium, required for the activity, was replaced to a lesser extent, by Mg2+, Sr2+, Zn2+ and Mn2+ (31.8, 27.0, 24.6 and 3.5%). 7. Steady-state kinetics showed: Vmax = 10 microM-min-1, Km = 0.05 mM (N-dimethylated casein), kcat = 31.9 min-1 kcat/Km = 560 min-1 mM-1.     

The influence of N-7 platination and methylation on the stability of deoxyguanosine and deoxyguanylyl-(3'-5')-deoxyguanosine

The rates of degradation of N-7 platinated deoxyguanosine (dG) and deoxyguanylyl-(3'-5')-deoxyguanosine (dGpdG) were followed in 66 mM Tris-HCl (pH 7.4) at 100 degrees C. The half-life for the intramolecular cross-link of cis-diamminedichloroplatinum(II) (cis-Pt) between two neighboring guanines (Pt-dGpdG) was 86 min, and the half-life of the intermolecular cross-link of cis-Pt on two guanines (dG-Pt-dG) was 54 min. For comparison the half-lives of dGpdG and the N-7 methylated dGpdG (Me-dGpdG) were 636 min and 11 min, respectively. The end product of the degradation of dGpdG, Pt-dGpdG and dG-Pt-dG was guanine, while Me-dGpdG was degraded to 7-methylguanine. In the case of dG-Pt-dG the main reaction pathway was through the depurination of one of the deoxyguanosines; in the case of Pt-dGpdG the degradation occurred either through the cleavage of one of the N-glycosidic bonds or through the cleavage of one of the Pt-bonds.     

Purification and some properties of 1-aspartamido-beta-N-acetylglucosamine amidohydrolase from human liver.

Human liver 1-aspartamido-beta-N-acetylglucosamine amidohydrolase (aspartylglucosylaminase, EC 3.5.1.26) was purified 17 500-fold to apparent homogeneity as judged from polyacrylamide-gel disc electrophoresis. A pH optimum of 7.7-9.0 was found. The Km value was pH- and temperature-dependent. At 37 degrees C and pH 7.7, Km was 0.16 mM and it increased to 0.29 at pH 6.0 and 0.23 at pH 9.0. At 25 degrees C and pH 7.7, a Km value of 0.99 mM was obtained. When the substrate concentration was varied, apparent Michaelis-Menten kinetics were obtained. p-Hydroxymercuribenzoate, glutathione or cysteine had no effect on the enzyme activity; 5 mM-N-acetylcysteine inhibited about 47% of the total enzyme activity. Apart from Cu2+, other bivalent ions were virtually ineffective at 1 mM. The kinetic study differentiates this enzyme from aspartylglucosylaminase from other sources.     

Novel substrate specificity of a membrane-bound beta-glycosidase from the hyperthermophilic archaeon Pyrococcus horikoshii

A beta-glycosidase gene homolog of Pyrococcus horikoshii (BGPh) was successfully expressed in Escherichia coli. The enzyme was localized in a membrane fraction and solubilized with 2.5% Triton X-100 at 85 degrees C for 15 min. The optimum pH was 6.0 and the optimum temperature was over 100 degrees C, respectively. BGPh stability was dependent on the presence of Triton X-100, the enzyme's half-life at 90 degrees C (pH 6.0) was 15 h. BGPh has a novel substrate specificity with k(cat)/K(m) values high enough for hydrolysis of beta-D-Glcp derivatives with long alkyl chain at the reducing end and low enough for the hydrolysis of beta-linked glucose dimer more hydrophilic than aryl- or alkyl-beta-D-Glcp.     

Production and properties of alpha-amylase from Penicillium chrysogenum and its application in starch hydrolysis

Fungi were screened for their ability to produce alpha-amylase by a plate culture method. Penicillium chrysogenum showed high enzymatic activity. Alpha-amylase production by P. chrysogenum cultivated in liquid media containing maltose (2%) reached its maximum at 6-8 days, at 30 degrees C, with a level of 155 U ml(-1). Some general properties of the enzyme were investigated. The optimum reaction pH and temperature were 5.0 and 30-40 degrees C, respectively. The enzyme was stable at a pH range from 5.0-6.0 and at 30 degrees C for 20 min and the enzyme's 92.1% activity's was retained at 40 degrees C for 20 min without substrate. Hydrolysis products of the enzyme were maltose, unidefined oligosaccharides, and a trace amount of glucose. Alpha-amylase of P. chrysogenum hydrolysed starches from different sources. The best hydrolysis was determined (98.69%) in soluble starch for 15 minute at 30 degrees C.     

Expression of recombinant Bacillus licheniformis xylanase A in Pichia pastoris and xylooligosaccharides released from xylans by it

The mature peptide of Bacillus licheniformis xylanase A (BlxA) was successfully expressed in Pichia pastoris under the control of AOX1 promoter. After 96-h 0.25% methanol induction, the activity of recombinant B. licheniformis xylanase A (reBlxA) in culture supernatant was 122.9 U/mg. Enzymatic properties assays showed that the optimum temperature and pH for reBlxA were 60 degrees C and pH 6.0, respectively. When treated at 70 degrees C, pH 6.0 for 2 min, the residual activities of the reBlxA were 76%. Over 80% of reBlxA activity was retained after treatment of the enzyme by preincubation over a pH range of 5.0-9.0 for 1h at 25 degrees C. High performance liquid chromatography (HPLC) analysis revealed that xylotriose (X3) was the main hydrolysis product released from birchwood xylan and wheat bran insoluble xylan by reBlxA. The mode of action studies showed that reBlxA was an endo-acting xylanase and xylobiose (X2), xylotriose, xylotetraose (X4), xylopentaose (X5), and xylohexaose (X6) could be hydrolyzed by it. This is the first report on the expression of reBlxA in yeast and on determining and quantifying the hydrolysis products released from xylans by reBlxA.     

Alkylation by 1,2:5,6-dianhydrogalactitol of deoxyribonucleic acid and guanosine.

DNA was alkylated in neutral solution at 37 degrees C with 1,2:5,6-dianhydrogalactitol and hydrolysed to yield two principal products, identified as 7-galactitylguanine and 1,6-dideoxy-1,6-di(guanin-7-yl)galactitol. The reaction products were separated by chromatography on Sephadex G-10 and Dowex 50 (H+ form). The two compounds were also obtained by reaction between dianhydrogalactitol and guanosine in acetic acid. The products were characterized from their u.v.-spectral data by comparison with those of the 7-alkylguanines and were also identified by mass spectrometry.     

Various physico-chemical properties of the amylolytic complex from Bacillus mesentericus]

Some physico-chemical properties of the Bacillus mesentericus amylolytic complex were studied, and optimal conditions of starch hydrolysis (pH 7.5-8.0; 45 degrees C) were found. The half-life of amylases at 50 degrees was 75 min. The heat stability of the enzymes increased in the presence of Ca2+ ions. Amylase was stable at pH 7-9 and readily inactivated at pH below 6.0. By physical and chemical characteristics the complex was close to analogous preparations from Bacillus alkalophilic strains. Isoelectrofocusing revealed that the complex consisted at least of two amylolytic enzymes.     

Studies on a thermostable alpha-amylase from the thermophilic fungus Scytalidium thermophilum

An alpha-amylase produced by Scytalidium thermophilum was purified using DEAE-cellulose and CM-cellulose ion exchange chromatography and Sepharose 6B gel filtration. The purified protein migrated as a single band in 6% PAGE and 7% SDS-PAGE. The estimated molecular mass was 36 kDa (SDS-PAGE) and 49 kDa (Sepharose 6B). Optima of pH and temperature were 6.0 and 60 degrees C, respectively. In the absence of substrate the purified alpha-amylase was stable for 1 h at 50 degrees C and had a half-life of 12 min at 60 degrees C, but was fully stable in the presence of starch. The enzyme was not activated by several metal ions tested, including Ca(2+) (up to 10 mM), but HgCl(2 )and CuCl(2) inhibited its activity. The alpha-amylase produced by S. thermophilum preferentially hydrolyzed starch, and to a lesser extent amylopectin, maltose, amylose and glycogen in that order. The products of starch hydrolysis (up to 6 h of reaction) analyzed by thin layer chromatography, showed oligosaccharides such as maltotrioses, maltotetraoses and maltopentaoses. Maltose and traces of glucose were formed only after 3 h of reaction. These results confirm the character of the enzyme studied to be an alpha-amylase (1,4-alpha-glucan glucanohydrolase).     

Synthesis, characterization and properties of uridine 5′-(α-d-apio-d-furanosyl pyrophosphate)

1. A method was developed for synthesizing UDP-apiose [uridine 5'-(alpha-d-apio-d-furanosyl pyrophosphate)] from UDP-glucuronic acid [uridine 5'-(alpha-d-glucopyranosyluronic acid pyrophosphate)] in 62% yield with the enzyme UDP-glucuronic acid cyclase. 2. UDP-apiose had the same mobility as uridine 5'-(alpha-d-xylopyranosyl pyrophosphate) when chromatographed on paper and when subjected to paper electrophoresis at pH5.8. When [(3)H]UDP-[U-(14)C]glucuronic acid was used as the substrate for UDP-glucuronic acid cyclase, the (3)H/(14)C ratio in the reaction product was that expected if d-apiose remained attached to the uridine. In separate experiments doubly labelled reaction product was: (a) hydrolysed at pH2 and 100 degrees C for 15min; (b) degraded at pH8.0 and 100 degrees C for 3min; (c) used as a substrate in the enzymic synthesis of [(14)C]apiin. In each type of experiment the reaction products were isolated and identified and were found to be those expected if [(3)H]UDP-[U-(14)C]apiose was the starting compound. 3. Chemical characterization established that the product containing d-[U-(14)C]apiose and phosphate formed on alkaline degradation of UDP-[U-(14)C]apiose was alpha-d-[U-(14)C]apio-d-furanosyl 1:2-cyclic phosphate. 4. Chemical characterization also established that the product containing d-[U-(14)C]apiose and phosphate formed on acid hydrolysis of alpha-d-[U-(14)C]apio-d-furanosyl 1:2-cyclic phosphate was d-[U-(14)C]apiose 2-phosphate. 5. The half-life periods for the degradation of UDP-[U-(14)C]apiose to alpha-d-[U-(14)C]apio-d-furanosyl 1:2-cyclic phosphate and UMP at pH8.0 and 80 degrees C, at pH8.0 and 25 degrees C and at pH8.0 and 4 degrees C were 31.6s, 97.2min and 16.5h respectively. The half-life period for the hydrolysis of UDP-[U-(14)C]-apiose to d-[U-(14)C]apiose and UDP at pH3.0 and 40 degrees C was 4.67min. After 20 days at pH6.2-6.6 and 4 degrees C, 17% of the starting UDP-[U-(14)C]apiose was degraded to alpha-d-[U-(14)C]apio-d-furanosyl 1:2-cyclic phosphate and UMP and 23% was hydrolysed to d-[U-(14)C]apiose and UDP. After 120 days at pH6.4 and -20 degrees C 2% of the starting UDP-[U-(14)C]apiose was degraded and 4% was hydrolysed.     

A Study of the cellulases produced by three mesophilic actinomycetes grown on bagasse as substrate

The cellulases that strains of Streptomyces albogriseolus, S. nitrosporeus, and Micromonospora melanosporea produce when grown on untreated ballmilled bagasse were investigated. Optimum conditions for extracellular cellulase production and activity were determined to be growth at pH 6.7-7.4 and 25-35 degrees C for 4-5 days and assay at pH 5.0-6.0 and 45-55 degrees C, respectively. The endoglucanases were thermally stable at 50 degrees C, but the Avicelases had a half-life of approximately 24 h at this temperature. Nearly half of the endoglucanases and almost all of the Avicelases were absorbed on ballmilled bagasse after 15 min incubation at 50 degrees C. The beta-glucosidases were found to be mainly intracellular or cell wall bound. These mesophilic actinomycetes concomitantly produced xylanases and beta-xylosidases with cellulases that, apart from cellobiose and glucose, also release xylose from bagasse. This feature may be advantageous in the commerical application of the enzymes of mesophilic actinomycetes for the saccharification of natural cellulosic substrates.     

Effects of step changes in pH on isometric tetanic tension of toad sartorius muscle

The effect of a rapid change in pHe (pH of bathing solution) on the isometric tetanic tension developed by sartorius muscles of toads acclimated to 5 and 25 degrees C was measured at 5 and 25 degrees C. The pH was altered by changing the carbon dioxide concentration of a bicarbonate buffered physiological solution. Acclimation temperature did not modify the response to a rapid change in pH, but test temperature did. Following a pH decrease from 9.0 to 6.0, tetanic tension decreased at a faster rate at 5 degrees C than at 25 degrees C. A new steady state was reached in 15 min at 5 degrees C but in 40 min at 25 degrees C. Following a pH increase from 6.0 to 8.5, tetanic tension increased at a faster rate at 25 degrees C than at 5 degrees C. A new steady state was reached in 60 min at 5 degrees C but in 10 min at 25 degrees C. We conclude that the rate of carbon dioxide diffusion through the sartorius muscle is only one factor that determines how rapidly tetanic tension changes following the step change in pH, and that muscle resists pH change more effectively at higher temperatures.     

The role of epsilon-amino groups of lysine of human serum albumin in heat-induced protein denaturation. Increased stability of glycosylated and alkylated albumin in aggregation during heating

Human serum albumin was glycosidated by prolonged protein incubation in phosphate buffer, pH 6.8-7.0, with excess glucose at 37 degrees C. epsilon-amino groups of lysine residues of the albumin molecule were alkylated by pyridoxal-5-phosphate in the presence of NaBH4. The solutions of glycosidated and alkylated serum albumin were incubated at different temperature values in the range of 20 to 80 degrees C in phosphate buffer, pH 7.0, over 30 min. The nondenatured monomer and the resulting aggregated were isolated by TSK-HW-55-gel column chromatography and polyacrylamide gel electrophoresis. The stability of modified proteins elevated in parallel to the increase in the number of the ligand molecules covalently bound to albumin amino groups. The 1-3% aqueous solutions of glycosidated serum albumin containing 3-4 glucose residues and those of alkylated albumin containing 6-7 residues of pyridoxal-5-phosphate were stable on heating up to 80 degrees C and did not form aggregates. Under these conditions the initial serum albumin completely aggregated. Preincubation of the aggregated albumin with glucose at 37 degrees C resulted in protein "renaturation" to the monomeric form with a small number of dimers and trimers.     

The purine-2-deoxyribonucleosidase from Crithidia luciliae. Purification and trans-N-deoxyribosylase activity

Crude extracts of Crithidia luciliae catalysed a deoxyribosyl transfer from purine deoxynucleosides to free purine bases. Fractionation of a 0-80% (NH4)2SO4 fraction from C. luciliae on DEAE-cellulose resulted in the separation of three nucleosidase activities. Two of these were ribonucleosidases, one specific for inosine, uridine and xanthosine and the other for inosine and guanosine, whereas the third activity was specific for purine deoxyribonucleosides. This pattern is similar to that found in Leishmania donovani. Significant deoxyribosyltransferase activity was, however, associated with the purine-2'-deoxyribonucleosidase from C. luciliae. The purine-2'-deoxyribonucleosidase was purified to homogeneity by a six-step procedure involving (NH4)2SO4 fractionation and chromatography on DEAE-cellulose, hydroxyapatite, Sephadex G-75, and a chromatofocusing resin. The purified enzyme migrated as a single band of 17 kDa on SDS/polyacrylamide gel electrophoresis. The enzyme catalysed the hydrolysis of deoxyinosine, deoxyguanosine and deoxyadenosine with Km values of 80 +/- 10.5 microM, 20.7 +/- 3.2 microM and 17.3 +/- 5.3 microM, respectively, and V values for these substrates in the ratio 1:0.5:0.39. The pH optimum for deoxyribosyl transfer from deoxyinosine to guanine was at pH 7.7, while deoxyinosine hydrolysis in the presence of guanine was optimal in the range pH 6-7. During the synthesis of deoxyinosine from hypoxanthine and deoxyadenosine two products were formed. One of these coeluted with deoxyinosine on HPLC, while the second was tentatively identified as the positional isomer, 7-(beta-D-2'-deoxyribofuranosyl)hypoxanthine.     

Kinetic properties and storage stability of catalase immobilized on to florisil

The covalent immobilization of bovine liver catalase (CAT) on to florisil via glutaraldehyde was investigated. Optimum immobilization pH and temperature were determined as pH 6.0, 10 degrees C respectively, while the amount of initial CAT per g of carrier and immobilization time was determined as 5 mg g(-1) and 120 min, respectively. The Vmax values for free and immobilized CAT were found to be 1.7 x 10(5) and 2.0 x 10(4) micromol H2O2 min(-1) mg protein(-1), respectively, whereas KM values were 33.3 mM and 1722.0 mM respectively. Operational stability was determined by using a stirred batch-type column reactor. Immobilized CAT retained about 40% of its initial activity after 50 uses. It showed higher storage stability than free CAT at 4 degrees C and 25 degrees C. Its storage stability increased with increasing relative humidity (RH) from 0 to 20% of the medium. The highest storage stability was obtained in 20% RH, however, further increase in RH from 40 to 100% significantly decreased the storage stability.     

Production and properties of the linamarase and amygdalase activities of Penicillium aurantiogriseum P35.

The effects of medium composition on the production of beta-glucosidase (amygdalase and linamarase) by Penicillium aurantiogriseum P35 were studied and the medium optimized as follows (g/l of deionized water): pectin, 10.0; (NH4)2SO4, 8.0; KH2PO4, 8.0; Na2HPO4, 2.8; MgSO4.7H2O, 0.5; yeast extract, 4.0; initial pH 6.0. When grown in a bench fermenter on this medium, the fungus produced 50.5 mU of amygdalase and 9.4 mU of linamarase per ml of culture broth. Two beta-glucosidases (PGI and PGII), each having amygdalase and linamarase activities, were recovered from the culture broth and purified; their relative molecular weights, as native enzymes, were estimated to be about 247,000 and 147,000, respectively. Both enzymes showed the same optimum pH (6.0) but different optimum temperatures (55 and 60 degrees C for PGI and PGII, respectively). Thermostability (10 min at 60 degrees C) and half-life of enzyme activity (7 hours at 60 degrees C) of PGII were higher than those of PGI (10 min at 50 degrees C and 2 hours at 55 degrees C, respectively). A wide range of cyanogenic glycosides (such as tetraphyllin B, epivolkenin, gynocardin, passibiflorin, prunasin, taxiphyllin, amygdalin, lucumin, sambunigrin, dhurrin, linamarin and cardiospermin sulfate) were hydrolyzed by both enzymes.