Showing papers in "Acta Biochimica Polonica in 1979"

Oxidative and demethylating activity of multiple forms of laccase from Pholiota mutabilis.

Abstract: 1. The inducible and constitutive forms of laccase from the fungus Pholiota mutabilis show both the oxidative and demethylating activity, which proves the bifunctional character of the enzyme. 2. The oxidative/demethylating activity ratio of the forms induced either with ferulic or syringic acid is different from that shown by the constitutive form. 3. Splitting of one methoxyl group from the methoxyphenol substrate is associated with the release of one molecule of methanol.

Structure and tautomerism of the neutral and monoanionic forms of 2-thiouracil, 2,4-dithiouracil, their nucleosides, and some related derivatives.

Abstract: Ultraviolet and infrared spectrophotometric techniques have been utilized to demonstrate that the monoanionic form of 2-thiouracil in aqueous medium consists of an equilibrium mixture of two tautomeric monoanions, one due to dissociation of the N1 proton, the other to dissociation of the N3 proton, in the approximate ratio 1:1 In contrast to 2,4-diketopyrimidines, and 4-thiouracil, where monoanion formation involves charge delocalization, the two tautomeric monoanions of 2-thiouracil appear to have the charge localized on the O4 position The neutral forms of 2,4-dithiouracil and 2,4-dithiouridine are in the dithione form in both aqueous and non-aqueous media The monoanionic form of 2,4-dithiouracil consists of a mixture of two tautomeric monoanions, the predominant one of which is that with the proton on the ring N3, and with charge delocalization on both isomeric monoanions Such charge delocalization is also present in the monoanion of 2,4-dithiouridine For the reference compound 2-methylthiopyrimidone-4, the dominant, virtually exclusive, form in chloroform is that with the hydrogen localized on the ring N3, whereas in aqueous medium there is a 1:1 equilibrium mixture of two neutral tautomeric forms, one with the hydrogen on N3, the other with the hydrogen on N1

Methylation and tautomerism of 1-substituted 5-fluorocytosines.

Abstract: 1. Methylation and thiation of 5-fluorouracil led to 1,3-dimethyl-5-fluoro-4-thiouracil, the amination of which was examined under various conditions. At high dilution, and in the presence of a large excess of NH3, 1,3-dimethyl-5-fluoro-4-thiouracil was converted to 1,3-dimethyl-5-fluorocytosine in 60% yield. 2. Two procedures were employed for methylation of 5-fluoro-1-methylcytosine and 5-fluorocytidine. Treatment of each of these with diazomethane in alcohol-ether gave a complex mixture of products, the major one of which was the desired 3-methyl derivative. By contrast, treatment with methyl iodide in dimethylsulphoxide led exclusively to the 3-methyl derivatives in much better yeilds. 3. Ultraviolet absorption spectra and pKa values are presented for 1-substituted N-methyl-5-fluoro- and 5-bromo-cytosines. The basicity method was applied to determine the tautomeric equilibrium constants of the 1-substituted 5-halogenocytosines. The results show that the proportion of the imino forms of these compounds is of the same order of magnitude as for 1-substituted cytosines, and hence is unlikely to account for the observed mutagenic effects of 1-substituted 5-halogenocytosines.

Metal--glutathione interaction in aqueous solution. Nickel(II), cobalt(II) and copper(II) complexes with oxidized glutathione.

Abstract: The interaction of copper(II), nickel(II) and cobalt(II) ions with oxidized glutathione in aqueous solutions have been examined by spectroscopic methods. Cu(II) is the only ion which interacts with disulphide bridge and forms dimeric species containing the Cu(II)-S-S-Cu(II) unit. Ni(II) and Co(II) bind mainly with the terminal NH2 and COO- groups of glutamic acid, and the complexes formed are of nearly octahedral symmetry. At high pH, in the Co(II)-GSSG solution Co(II) is oxidized to Co(III) with the concomitant reduction of GSSG to GSH. Considerable differences were observed between the oxidized and reduced form of glutathione in the coordination ability towards metal ions.

Chemical modification of rat liver arginase.

Abstract: Chemical modifications were used to search for catalytically important residues of rat liver arginase. The results of carbamoylation, nitration and diazotization suggest that lysyl and tyrosyl residues are not involved in the catalytic function of arginase. The modification of 5--6 tryptophanyl residues by N-bromosuccinimide or 2-hydroxy-5-nitrobenzyl bromide led to about 90% inhibition of the enzyme activity. Photooxidation of 21 histydyl residues also led to considerable inactivation of arginase. The modification of tryptophanyl and histidyl residues did not cause dissociation of the enzyme into subunits.

Analyses of infrared amide bands of chitin.

Abstract: Infrared spectra of chitin isolated from various biological species were measured by Fourier transform technique. The recorded spectra were decomposed into component bands within 1500--1750 cm-1 and 3000--3500 cm-1 spectral regions; it allowed us to establish the precise position of amide I and amids II bands. It was shown that the positions of amide I and amide II bands are independent of the source from which chitin was isolated.

Properties of cysK mutants of Escherichia coli K12

Abstract: Triazole and azaserine resistant mutants of E. coli K12 affecting cysK gene coding for O-acetylserine sulphydrylase were isolated. The cysK gene in E. coli is located in the same region of chromosome as the cycK gene in Salmonella typhimurium. All azaserine and some triazole resistant mutants require cysteine for growth at a normal rate. The cysK mutants have reduced sulphate uptake. Stability and transfer by conjugation of triazole resistant phenotype were checked. Differences in sulphate metabolism between closely related organisms E. coli and S. typhimurium are discussed.

On the different response of Salmonella typhimurium hisG46 and TA1530 to mutagenic action of base analogues.

Abstract: Various mutagens are known to induce more his+ revertants in TA1530 than in hisG46 strain. To test whether the mutator effect shown by TA1530 is limited to the his mutation, the lysA8 marker was introduced into both the TA1530 and hisG46 strain, and its reversibility, after induction by N4-hydroxycytidine, was estimated. The ability to reverse the lys marker was tenfold higher in the TA1530/lysA8 transductants than in the hisG46/lysA8 transductants or in the donor for lys, the lysA8 strain.

Acyl esters of polyprenols: specificity of microsomal transacylase for polyprenols of different chain length and saturation.

Abstract: Transfer of fatty acids from phospholipids to polyprenols, catalysed by the transacylase from rat liver microsomes, was investigated. The specificity of the enzyme for polyprenols of different chain length and different degree of saturation was studied using individual isoprenologues, the preparation of which in highly tritiated form is described. It was found that short-chain polyprenols are better substrates for the enzyme than long-chain polyprenols, and alpha-saturated better than unsaturated or multiply saturated polyprenols. Short-chain, alpha-saturated single isoprenologues were several-fold more active as acyl acceptors than natural dolichol.

Conformation of the N(CH3)2 group in cytosine and in simple model pyrimidines and pyridines. Steric effects of ortho-methyl substitution on infrared spectra and molecular dipole moments

Abstract: Infrared spectra of amino and dimethylamino derivatives with and without an ortho-methyl group of 4- and 5-substituted pyrimidines, 4-substituted pyridine, benzene and of the respective cytosines were recorded in the region of skeletal ring vibrations. Integrated intensities of ring vibration(s) v8 at about 1600 cm-1 sensitive to the presence of electron-donating substituents were used for elucidation of the steric effects of ortho-methyl on the mesomeric interaction between the -N(CH3)2 group and the ring. Molecular dipole moments were also determined experimentally in benzene for simple pyrimidine and pyridine derivatives and analysed vectorially with the use of component group moments in terms of the N(CH3)2 group conformation. The data point to a progressive twist of the dimethylamino group in hindered derivatives in the order: pyrimidine-5 greater than pyridine-4 greater than pyrimidine-4. They are also in agreement with the essential planarity of sterically crowded m41,4,4,5cytosine.

Effect of temperature and 4,6'-diamidine-2-phenylindole on restriction of supercoiled Col E1 DNA by Eco RI endonuclease.

Abstract: Supercoiled Col E1 DNA is split by Eco RI endonuclease at 37 degrees C without intermediate formation of open circular DNA. Accumulation of this restriction product is observed at low temperature. The fluorescent dye, 4,6'-diamidine-2-phenylindole (DAPI) inhibits restriction by Eco RI endonuclease. This effect is due to the DAPI:DNA rather than to the DAPI:Eco RI interactions.

Induction by ferulic acid of multiple forms of peroxidase in the fungus Trametes versicolor (Basidiomycetes).

Abstract: Two forms of peroxidase (EC 1.11.1.7) were induced in mycelium of Trametes versicolor by ferulic acid, with an about 2,5-fold increase of their specific activity. Both inducible forms of peroxidase were isolated and partially purified by ion-exchange chromatography on CM-Sephadex C-50 and DEAE-Sepharose Cl 6B; some of their properties were also characterized.

Interaction of metal ions with nucleic acids. Interaction of copper(II) with pyrimidine nucleosides and their derivatives

Abstract: 1. In aqueous and non-aqueous solutions, copper(II) interacts with the N-3 of cytidine but not with the carbonyl group oxygens of pyrimidine nucleosides. 2. In aqueous solution, copper(II) interacts with the phosphate group and ribose of pyrimidine nucleotides, and additionally with N-3 of 5'-CMP. 3. Broadening of resonance signals of the H-5 proton of 5'-UMP and C-5 of 5'-UMP and 5'-TMP results probably from the interaction between metal ion and the phosphate group situated in direct vicinity of the above atoms. 4. In the copper(II)-pyrimidine nucleotide complexes in solid state, copper is coordinated with the phosphate group, and in 5'-CMP additionally with the pyrimidine moiety of the nucleotide.

Partial purification and some properties of a liver alkaline ribonuclease from the frog Rana esculenta.

Abstract: 1 RNAases varying in pH optimum, activation with pCMB, sensitivity towards temperature and acid treatment, as well as electrophoretic mobility were found in Rana esculenta liver extract 2 Of the three activity peaks of alkaline ribonuclease separated on CM-cellulose with 2000-fold purification, RNAase of peak C is thermo- and acid-stable and exhibits specificity for pyrimidine bases, preferring poly(U) over poly(C) 3 Differences in the specific "inhibitory effect" of frog liver supernatant on the frog liver alkaline RNAase were observed

Protein-cationic detergent interaction. Interaction of bovine serum albumin and other proteins with alkylpyridinium bromides studied by viscosity, gel filtration and spin-label methods.

Abstract: Viscosity, gel filtration and spin-labelling methods have been used to study the influence of alkylpyridinium bromides on the conformation of bovine serum albumin and other proteins. Cationic detergents cause partial unfolding of the native protein molecules. The magnitude of these changes increases with increasing length of the detergent hydrocarbon chain. When cationic detergents are added to reduced and carboxymethylated bovine serum albumin the observed changes are opposite to those found in native protein.

Adenine cycle in hepatopancreocytes of Helix pomatia (Gastropoda).

Abstract: Intact hepatopancreocytes were obtained from hibernating or active purinotelic snails, H. pomatia (Gastropoda). When incubated with [14C]glycine or [14C]formate, they synthesized de novo purine compounds, including also adenylates, adenosine and adenine. Hepatopancreocytes resynthesized also adenylates and other purine compounds from [3H]adenine or from [3H]adenosine split by the H. pomatia cell enzyme to adenine; the resynthesis of ADP+ATP was proportional to adenine concentration. Thus all reactions of the postulated adenine cycle: AMP leads to adenosine leads to adenine leads to AMP occur in the intact hepatopancreocytes; this cycle could probably be responsible for maintenance of the high level of adenylates during winter sleep.

The presence of deoxyribonucleolytic activity in cytoplasmic ribosomes of rye (Secale cereale L) germs.

Abstract: Deoxyribonucleolytic activity was found to be associated with cytoplasmic ribosomes and ribosomal subunits of rye germs. The activity has the pH optimum at 5.0. Treatment of ribosomes and 60S subunits with 0.5 M-ammonium chloride released a considerable part of deoxyribonucleolytic and ribonucleolytic activity; treatment of 40S subunits resulted in a complete release of deoxyribonucleolytic activity and partial release of ribonucleolytic activity. This suggests the presence in ribosomes of rye germs of two types of nucleolytic enzymes: an enzyme of the nuclease I type with deoxyribonuclease and ribonuclease activities, and typical ribonucleases hydrolysing RNA only.

Specificity of elastases: degradation of the oxidized beta-chain of insulin by porcine pancreatic elastase II and dog leucocyte elastase.

Abstract: Porcine elastase II (EC 3.4.21.-), a pancreatic proteinase with elastolytic activity, hydrolyses the oxidized beta-chain of insulin with major cleavages occurring at Leu17-Val18, Phe24-Phe25, Phe25-Tyr26 and Tyr26-Thr27. Canine leucocytic elastase splits the same substrate with major sites at Val12-Glu13 and Val18-Cys19 O3H. This indicates similarity of elastase II to chymotrypsins (EC 3.4.21.1 or 3.4.21.2) and of dog leucocyte enzyme to human granulocyte elastase and porcine pancreatic elastase I (EC 3.4.21.11).

Effect of protein deficiency on the metabolism of glycoproteins and glycosaminoglycans in albino rat skin.

Abstract: 1. The total content of neutral sugars in skin of the weanling albino rats kept on the protein-deficient diet was increased by about 40%; this was mainly due to the increased concentration of galactose. The content of sialic acid was increased by about 20%. The collagen nitrogen was decreased significantly, with a concomitant increase of non-collagen nitrogen. At the same time, the content of sulphated glycosaminoglycans in skin was significantly decreased and that of non-sulphated glycosaminoglycans was increased. 2. Protein-deficient diet enhanced the activities of the protein-bound carbohydrate-degrading lysosomal hydrolases, viz. cathepsin D (EC 3.4.4.23), N-acetyl-beta-D-glucosaminidase (EC 3.2.1.30) and beta-D-glucuronidase (EC 3.2.1.31) both in liver and skin. The activity of liver hyaluronidase (EC 3.2.1.35) was also increased upon limitation of protein supply. 3. The changes observed in skin were accompanied by increased concentration of the protein-bound hexoses, hexosamines and sialic acids in serum, and of hexosamine and uronic acid in urine. The serum fucose remained unchanged.

Solution conformations of the antimetabolite 9-beta-D-xylofuranosyladenine and its 8-bromo analogue.

Abstract: An analysis has been made, with the aid of 1H NMR spectroscopy, of the solution conformation of the known antimetabolite, 9-beta-D-xylofuranosyladenine (xyloA), and of its 8-bromo analogue. For xyloA, the results point to a strong preference for the sugar ring of the conformation type N (C(3') endo), a relatively low population of the gauche-gauche rotamer of the exocyclic 5'-CH2OH, and a preference for the conformation anti about the glycosidic bond. For 8-bromo-xyloA, the preference for the type N conformation of the sugar ring is less marked, and the preferred conformation about the glycosidic bond is syn. The conformation of the sugar ring in the foregoing xylonucleosides consequently differs appreciably from that for the corresponding ribonucleosides, which adopt preferentially the type S (C(2')endo) and gauche-gauche conformations. Comparison with previously reported results for O'-methyl derivatives of xyloA points to the similarity in conformational properties of all of these. In contrast to arabinonucleosides with free 2' and 5' hydroxyls, the conformation of xyloA is relatively unaffected in strongly alkaline medium where the sugar hydroxyl(s) dissociate. Under these conditions, there is no formation of an intramolecular hydrogen bond such as might have been anticipated from X-ray diffraction studies in the solid state.

Purification and properties of DNA polymerase gamma from rabbit intestinal epithelial cells.

Abstract: 1. DNA polymerase gamma from the cytoplasmic fraction of rabbit intestinal epithelial cells has been purified 120 000-fold and was free of phosphatase and nuclease activities towards deoxyribonucleoside-5'-triphosphates and polynucleotides. 2. The enzyme exhibited maximal activity for activated DNA and poly(A) . oligo(dT)12--18 at pH 8.5 IN 0.25 AND 0.15 M-KCl, respectively. Km values for dTTP with these two templates were 0.5 and 3.8 microM, respectively. 3. In contrast to DNA polymerases alpha and beta, the enzyme replicated poly(A) . oligo(dT)12--18 10 times faster and poly(dA) . oligo(dT)12--18 5 times slower than activated DNA. 4. DNA polymerase gamma did not replicate poly(C) . oligo(dG)12--18 or poly(Cm) . oligo(dT)12--18. The reaction with poly(I) and poly(U) did not exceed 1% of that observed with poly(A). 5. The enzyme was inhibited in 60% by antiserum against DNA polymerase gamma from human lymphoblasts. 6. The nuclear fraction of rabbit intestinal epithelial cells contained DNA polymerase gamma with the same characteristics.

Protein-cationic detergent interaction. Equilibrium dialysis study of the interaction of bovine serum albumin and other proteins with alkylpyridinium bromide.

Abstract: The binding isotherms of native bovine serum albumin with cationic detergents, such as octyl, decyl, dodecyl and tetradecylpyridinium bromides were determined at pH 6.8 and 3.4 at 25 degrees C. The isotherms for dodecyl and tetradecylpyridinium bromides were also determined at 3 degrees C. The average number of detergent cations bound increased with increasing hydrocarbon chain length. At low detergent concentration the binding of all alkylpyridinium bromides was smaller at pH 3.4 than at pH 6.8. Dodecylpyridinium bromide was bound to native beta-lactoglobulin, aldolase, ovalbumin, haemoglobin, myoglobin, lysozyme, trypsin and ribonuclease at pH 6.8. No binding occurred to alpha-chymotrypsin and chymotrypsinogen. The free enthalpy change, --delta G degrees, calculated from intrinsic association constants K was determined.

Effect of anaerobiosis and glucose on the content of haem and its precursors in intact yeast cells.

Abstract: The content of haem and its precursors was determined in yeast cells grown under various conditions. The cells grown aerobically on 2% galactose contain about three times more haem (about 300 nmoles/g dry wt.) than the cells grown on 10% glucose. A trace amount of haem was found in anoxia irrespective of the carbon source used. The "efficiency" of the first enzyme of the haem biosynthetic pathway--delta-aminolevulinic acid (ALA) synthetase--expressed as the sum of all intermediates of the pathway in the cells grown on galactose, is similar in anaerobic and aerobic cells. The "efficiency" of the second enzyme--ALA dehydratase--is lower about three times both in anoxia and under conditions of glucose repression. In anoxia, not haem but delta-aminolevulinic acid is the main biosynthetic product. The role of glucose repression and of the feedback mechanisms in regulation of haem synthesis in yeast is discussed. A method for haem determination in the intact yeast cells, based on the formation of pyridine haemochrome, is described.

Partial purification and characterization of the oxytocin-inactivating enzymes from chicken liver.

Abstract: 1. Two enzymes acting on the linear portion of oxytocin: carboxamidopeptidase (releasing Gly . NH2) and prolyl peptidase (releasing Leu-Gly . NH2) were identified in the cytoplasmic fraction of chicken liver. 2. Carboxamidopeptidase was purified 134-fold with a 23% yield, and prolyl peptiase 71-fold with a 20% yield. The specific activity of the final preparations was 181 and 96 microU/mg protein, respectively. 3. The optimum pH for carboxamidopeptidase was 6.0--6.5 and for prolyl peptidase, 7.5. Carboxamidopeptidase activity was inhibited by Mn2+, Zn2+, Ca2+, Co2+, and stimulated by EDTA; the activity of prolyl peptidase was inhibited by Zn2+ and Mn2+. The Km value of both enzymes for oxytocin was 1.5--2.4 microM.

Purification of cobalt-activated acylase by affinity chromatography.

Abstract: alpha-Hydroxyisocaproyltyrosine (HyIc-Tyr-OH), a potent competitive inhibitor of the cobalt-activated acylase form 2, was synthesized. Its derivative, alpha-aminopentyl-HyIc-Tyr-OEt was coupled to cyanogen bromide-activated Sepharose 4B and was used for about 100-fold purification of the acylase from human liver by affinity chromatography. The preparation obtained did not show aminoacylase, aspartyl acylase or alanylarylamidase activities. The same chromatographic method was also applied to isolate form 2 of the serum acylase from patients with viral hepatitis and guinea pig placenta.

Isolation of wheat ribosomes free of high molecular weight inhibitors of the natural messenger translation.

Abstract: The cell-free extract from wheat germ contains an inhibitor interfering with translation of a natural template (BMV RNA). The inhibitor affects neither the translation of poly(U) nor the aminoacylation of tRNA. It exhibits the activity of protein kinase (ATP:protein phosphotransferase, EC 2.7.1.37). The inhibitor is found in lipoprotein aggregates which can be separated from ribosomes on Sepharose 2B column. Ribosomes purified on the Sepharose are several times more active in translation of BMV RNA than those isolated by conventional methods.