Reaction of optically active S- and R-forms of dolichyl phosphates with activated sugars

Chemical synthesis was used to produce optically active isomers of dolichol (S- and R-forms) with 18 and 19 isoprene residues. The phosphorylated polyprene was studied in rat liver microsomal GDP-mannosyl and UDP-N-acetylglucosaminyl transferase systems. The two dolichol-P forms in both transferase systems gave Vmax values which for the S-form exceeded 4-6 times what was obtained with the R-form. The Km values were also higher for the S-form. The hepatocyte appears to contain a large excess of dolichyl-P, by 100 times exceeding that of the Km values. For this reason the S-form of dolichyl-P seems to be one of the requirements for the normal establishment of the N-glycosidically linked oligosaccharide chain.     

Purification and Characterization of Glutamine Synthetase from the Basidiomycete Pleurotus ostreatus

The purification and some properties of glutamine synthetase (GS) from the mycelium of the basidiomycete Pleurotus ostreatus are described. The enzyme was purified to apparent homogeneity with ion exchange chromatography and a Dyematrex Green A column as the major purification steps. The GS has a molecular weight of 470 kDa and is composed of eight subunits with a molecular weight of 58 kDa. A tetrameric form of the enzyme may also be active. The apparent K _m values for the biosynthetic reaction varied in different mycelial extracts from 2.5 to 3.5 mM and from 0.02 to 0.06 for glutamate and ammonium respectively. In the transferase reaction, K _m values of 48 mM and 6.2 mM were found for L-glutamine and hydroxylamine, respectively. From the divalent cations tested, Mn²⁺ showed the strongest stimulatory effect both on the transferase and the biosynthetic reaction. ADP was the only nucleotide having an activating effect on the transferase reaction. The biosynthetic reaction was strongly inhibited by AMP and the transferase reaction by carbamoylphosphate. L-Alanine and glycine inhibited both reactions. Received: 21 February 1996/Accepted: 12 March 1996     

Purification and properties of glutamine synthetase from Bacillus polymyxa

Glutamine synthetase (EC 6.3.1.2) was purified to homogeneity from a free-living nitrogen fixing bacteria, Bacillus polymyxa. The holoenzyme, relative molecular mass (M_r) of 600 000 is composed of monomeric sub-units of 60 000 (M_r). The isoelectric point of the sub-units was 5.2. The pH optimum for the biosynthetic and transferase enzyme activity was 8.2 and 7.8, respectively. The apparent K _m values (K _m ^app ) in the biosynthetic reaction for glutamate, NH₄Cl and ATP were 3.2, 0.22 and 1 mM, respectively. In the transferase reaction the K _m values for glutamine, hydroxylamine and ADP were 6.5, 3.5 and 8×10^-4 mM respectively. L-Methionine-D-L-sulfoximine was a very potent inhibitor in both biosynthetic and transferase reactions. Similar to most Gram positive bacteria there was no evidence of in vivo adenylylation and the enzyme seemed to be mainly regulated by feed-back mechanism.Abbreviations PMSF phenylmethylsulfonylfluoride - TCA trichloroacetic acid - GS glutamine synthetase - MSO L-Methionine-D-L-sulfoximine - SDS-PAGE sodium dodecyl sulfatepolyacrylamide gel electrophoresis - SVPDE snake venum phosphodiesterase     

The influence of prolonged di(2-ethylhexyl)phthalate treatment on the dolichol and dolichyl-P content of rat liver

Dolichols and glycosyl transferase activities were studied in rat liver fractions after treatment with the plasticizer di(2-ethylhexyl)phthalate, an inducer of peroxisomes and mitochondria. After a few weeks of treatment with 2% plasticizer in the diet, the amount of dolichol is more than doubled in the lysosomes but not in the microsomes while dolichyl-P decreased by 50% in the microsomes but not in the lysosomes. The isoprenoid pattern for dolichol and dolichyl-P, respectively, is modified to longer polyprenols in the two fractions as seen in the percent distribution of the individual isoprenes. Dolichyl-P and protein glycosylation by N-acetylglucosamine and mannose decreased considerably. Incubation with mixtures containing exogenous dolichyl-P did not increase protein glycosylation. Phthalate ester treatment for 2 years increased dolichol content above the control values even when the dose was decreased a hundred times, to 0.02%. The results demonstrate a compartmentalization of dolichol and dolichyl-P distribution, and the induction studies suggest that hepatocytes possess separate regulating mechanisms for these two compounds.     

Changes in hepatic dolichol and dolichyl monophosphate caused by treatment of rats with inducers of the endoplasmic reticulum and peroxisomes and during ontogeny

Rats were treated with various inducers of the endoplasmic reticulum and peroxisomes and the properties and distributions of dolichol and dolichyl phosphate analyzed. The treatment of rats with carcinogenic agents 2-acetylaminofluorene, N-nitrosodiethylamine and 3-methylcholanthrene and with the compounds such as phenobarbital, terpentine, cholestyramine and di(2-ethylhexyl)phthalate have all caused changes in the microsomal or lysosomal contents of dolichol to various extents, but only the latter group influenced dolichyl-P concentration. Shortly after birth, the hepatic content of dolichyl-P reaches the adult level, whereas the level of the free alcohol is low at birth but increases continuously thereafter. Incorporation of [3H]mevalonate into dolichol was also dependent on factors other than de novo synthesis, e.g., the pool size. Rates of glycosylation reactions dependent on dolichyl-P exhibit considerable changes but are independent of the existing levels of lipid intermediate. GDP-mannosyl transferase activity increases greatly with birth, but the enzyme activity returns to the adult level within a day after birth. These results demonstrate that structural and functional modifications induced with drugs can greatly influence the content and distribution of dolichol which are independent of the existing levels of dolichyl-P.     

The half-lives of dolichol and dolichyl phosphate in rat liver

Rat liver dolichol and dolichyl-P were labeled by injection of [³H]mevalonate into the portal vein and their rates of synthesis and breakdown determined. In the initial phase the radioactivity appeared in -unsaturated polyprenols. Subsequent saturation required 90 min. The half-lives of dolichols in microsomes were between 80 and 118 h, and shorter dolichols had shorter values of T_1/2. The half-lives of dolichols in lysosomes were between 115 and 137 h, while microsomal dolichyl-P exhibited a T_1/2 of 32 h. Injected dolichol was recovered in the lysomes of hepatocytes and exhibited a rate of breakdown which was slower than that of the endogenous compound. These results indicate differences in the catabolism of dolichol at different subcellular locations, as well as differences between the catabolism of dolichol and dolichyl-P.     

Purification and properties of UDP-GlcNAc:dolichyl-phosphate GlcNAc-1-phosphate transferase. Activation and inhibition of the enzyme

The GlcNAc-1-P transferase was solubilized from pig aorta microsomal fractions using 0.5% Nonidet P-40. The activity of the solubilized enzyme was stimulated by exogeneously added phospholipids in the order phosphatidylglycerol greater than phosphatidylinositol greater than phosphatidylserine. When the enzyme was stored in 20% glycerol containing 20 micrograms of phosphatidylglycerol/mg of protein, more than 80% of the activity remained after storage for 6 days at 0-4 degrees C. On the other hand, in the absence of the stabilizers, the enzyme lost most of its activity within 24 h. The transferase was purified about 68-fold using ammonium sulfate and DEAE-cellulose fractionation. The DEAE-cellulose chromatography separated a heat-stable factor from the enzyme, which when added back to the partially purified enzyme stimulated about 5-fold. With this partially purified enzyme, the Km for UDP-GlcNAc was found to be 1 X 10(-7) M, and that for dolichyl-P about 1 X 10(-6) M. The stimulatory factor increased the Vmax for both UDP-GlcNAc and dolichyl-P 5-10-fold, but the Km values remained the same. The pH optimum for the enzyme was between 7.4 and 7.6, and either Mn2+ (1 mM) or Mg2+ (10 mM) was required for optimum activity. The GlcNAc-1-P transferase was also stimulated by the addition of GDP-mannose (or other purine sugar nucleotides) or dolichyl-phosphoryl-mannose to the incubation mixtures. These two compounds acted in different ways on the enzyme since their stimulatory effects were additive. The effect of GDP-mannose was found to be due to protection of the substrate, UDP-GlcNAc, from degradation, but the effect of dolichyl-P-mannose remains to be established. In addition, the stimulations shown by phosphatidylglycerol, GDP-mannose, and factor, or phosphatidylglycerol, dolichyl-P-mannose, and factor, were all additive, indicating that they were acting at different sites on the enzyme. The transferase was quite sensitive to the action of sulfhydryl reagents such as N-ethylmaleimide or p-chloromercuribenzene sulfonate, and was rapidly inactivated in their presence. The enzyme could be protected to the extent of about 50% when all of the substrates (UDP-GlcNAc, dolichyl-P, Mn2+) were added before the addition of the sulfhydryl reagents.     

Phosphoethanolamine and phosphocholine transferases in gray matter and white matter from developing rat brain

We have studied the activities of 2′,3′-cyclic nucleotide 3′-phosphohydrolase, 1,2-diacylglycerol: CDPethanolamine phosphoethanolamine transferase (EC 2.7.8.1), and 1,2-diacylglycerol: CDPcholine phosphocholine transferase (EC 2.7.8.2) in developing rat brain gray matter and white matter. The specific activity of cyclic nucleotide phosphohydrolase was 5–8 fold higher in white matter than in gray matter at all ages. No significant changes were observed during development. The specific activity of phosphocholine transferase was 2 to 3 fold higher than phosphoethanolamine transferase at all ages both in gray and white matter. Both phosphocholine transferase and phosphoethanolamine transferase increased more than 2 fold in specific activity between 14 and 90 days of age. The total activity of phosphocholine transferase also showed an increase during development. The apparentK _m values for nucleotides and dicaprin were similar in gray matter and white matter. Except for lowK _m values for nucleotides at 14 days of age, no significant changes were observed during development. Changes in rates of glycerophospholipid synthesis may be partly due to the specific activities of these enzymes but are also determined by the quantities of substrates and inhibitors and by affinities for the substrates. Special Issue dedicated to Dr. Eugene Kreps.     

Characteristics of galactokinase and galactose-1-phosphate uridyltransferase in cultivated fibroblasts and amniotic fluid cells

Summary The kinetic characteristics of galactose-1-phosphate uridyltransferase and galactokinase in cultivated fibroblasts and amniotic fluid cells were investigated. The K _m values of galactokinase for galactose at 2.0 mM ATP are 0.34 mM in amniotic fluid cells and 0.48 mM in fibroblasts. The K _m values for ATP at 0.5 mM galactose are 1.25 mM and 2.10 mM.Transferase and galactokinase activities and protein content increase logarithmically during the growth of cultivated cells. The specific activity of both enzymes also increases and reaches a maximum level 10–15 days after subculture. The specific activity of transferase increases faster than that of galactokinase in the case of amniotic fluid cells. In the case of fibroblasts the specific activity of galactokinase increases faster than that of transferase.     

SYNTHESIS OF GLYCOPROTEINS IN BRAIN: IDENTIFICATION, PURIFICATION AND PROPERTIES OF GLYCOSYL TRANSFERASES FROM PURIFIED SYNAPTOSOMES OF GUINEA PIG CEREBRAL CORTEX

Abstract— Four glycoprotein:glycosyl transferases (a fetuin:N-acetylglucosaminyl transferase; a bovine submaxillary mucin: N-acetylgalactosaminyl transferase; a collagen: glucosyl transferase and an orosomucoid: galactosyl transferase) were purified 34-, 45-, 37- and 47-fold, respectively, from synaptosomes prepared from guinea pig cerebral cortex. Purifications were achieved by centrifugation and by column chromatography on Sephadex G-100 and G-150 of 0 , 1% (w/v) Triton X-100 extractsof the purified cerebral cortical synaptosomes. The enzymes were separated from endogenous acceptors and were highly specific for specific macromolecular acceptors; small molecules were ineffective as acceptors. The fetuin: N-acetylglucosaminyl transferase functioned only with fetuin minus N-acetylneuraminic acid, galactose and N-acetylglucosamine; the bovine submaxillary mucin: N- acetylgalactosaminyl transferase with bovine submaxillary much minus N-acetylneuraminic acid and N-acetylgalactosamine; the collagen: glucosyl transferase with collagen minus glucose; and the orosomucoid: galactosyl transferase with either orosomucoid minus N-acetylneuraminic acid and galactose or fetuin minus N-acetylneuraminic acid and galactose. Each transferase required a specific (XDP)-monosaccharide for transfer. The transferases were entirely dependent on either Mn²⁺ or Mg²⁺ for activation and Fe²⁺ and Hg²⁺ inhibited each of the four enzymes. The optimum pH's for the enzymes were: for fetuin: N-acetylglucosaminyl transferase, 7 , 4–8.0; for bovine submaxillary mucin: N-acetylgalactosaminyl transferase, 7 , 7; for collagen: glucosyl transferase, 7 , 7 and for orosomucoid: galactosyl transferase, 6 , 6. The enzymes were distributed subsynaptosomally primarily in the synaptosomal plasma membrane and in the mitochondria of the synaptosome. The respective values for K_m (μM) and V_mex (pmoles/h/mg of protein) for the transferases were: fetuin: N-acetylglucosaminyl transferase, 12 and 143; for bovine submaxillary mucin: N-acetylgalactosaminyl transferase, 25 and 166; for collagen: glucosyl transferase, 4 and 10 and for orosomucoid:galactosyl transferase, 8 and 111.     

Activites of Enzymes Synthesizing Diacyl, Alkylacyl, and Alkenylacyl Glycerophosphoethanolamine and Glycerophosphocholine During Development of Chicken Brain

Abstract: Ethanolamine and choline glycerophospholipids are the major phospholipids of brain membranes. During brain development, the accumulation of these phospholipids is most intense when myelination occurs. In order to gain knowledge about the regulatory mechanisms for synthesis of these lipids in relation to membrane synthesis, we investigated the activities of the 1,2-diradyl-sn-gIycerol: CDPethanolamine phosphoethanolarnine transferase and 1,2-diradyl-sri-glycerol:CDPcholine phosphocholine transferase during chicken brain development. Diacyl, alkenylacyl, and alkylacylglycerols are substrates for both enzymes. The specific activities of microsomal phospho-ethanolamine and phosphocholine transferases are constant between the 8th and 18th day of embryonic life. The specific activities of both enzymes double around hatching, which is the period of intense myelination and marked ac-cumulation of ethanolamine and choline glycerophospholipids in brain. At the same time, the amount of microsomes increases by 50%; thus the total activities increase threefold. Four days after hatching the specific activities of both enzymes are at adult values. Similar results were obtained in the presence of exogenous diacyl or alkylacylglycerols. During brain development the apparent K_m, value of rnicrosomal phosphoethanolamine transferase for CDP ethanolamine increases when assayed with diaclyglycerols or alkylacyl-glycerols a s lipid substrates. The apparent K_m, value of phosphocholine trans-ferase for CDP choline does not change during brain development in the presence of exogenous diacylglycerols, but increases in the presence of exogenous alkylacylglycerols. These changes in K_m, values may be due to the appearance of glial isoenzyme at the beginning of myelination. The apparent K_m, values of diacylglycerol phosphocholine, alklyacylglycerol phosphocholine, and diacyl-glycerol phosphoethanolamine transferases for their CDP bases are similar in adult brain microsomes and are threefold higher than the apparent K_m, value of alkylacylglycerolphosphoethanolamine transferase. The high affinity of alkylacylglycerolphosphoethanolamine transferase for CDPethanolamine may be responsible for the preferential synthesis of ethanolamine plasmalogens in brain.     

Partial purification and characterisation of an aromatic amino acid aminotransferase from mung bean (Vigna radiata L. Wilczek)

An aromatic amino acid aminotransferase (aromAT) was purified over 33 000-fold from the shoots and primary leaves of mung beans (Vigna radiata L. Wilczek). The enzyme was purified by ammonium sulfate precipitation, gel filtration and anion exchange followed by fast protein liquid chromatography using Mono Q and Phenylsuperose. The relative amino transferase activities using the most active amino acid substrates were: tryptophan 100, tyrosine 83 and phenylalanine 75, withK _m values of 0.095, 0.08 and 0.07 mM, respectively. The enzyme was able to use 2-oxoglutarate, oxaloacetate and pyruvate as oxo acid substrates at relative activities of 100, 128 and 116 andK _m values of 0.65, 0.25 and 0.24 mM, respectively. In addition to the aromatic amino acids the enzyme was able to transaminate alanine, arginine, aspartate, leucine and lysine to a lesser extent. The reverse reactions between glutamate and the oxo acids indolepyruvate and hydroxyphenylpyruvate occurred at 30 and 40% of the forward reactions of tryptophan and tyrosine, withK _m, values of 0.1 and 0.8 mM, respectively. The enzyme was not inhibited by indoleacetic acid, although -naphthaleneacetic acid did inhibit slightly. Addition of the cofactor pyridoxal phosphate only slightly increased the activity of the purified enzyme. The aromAT had a molecular weight of 55–59 kDa. The possible role of the aromAT in the biosynthesis of indoleacetic acid is discussed.Abbreviations AAT aspartate aminotransferase - aromAT aromatic amino acid aminotransferase - FPLC fast protein liquid chromatography - IPyA indolepyruvate - OHPhPy hydroxyphenylpyruvate - PLP pyridoxal phosphate - TAT tryptophan aminotransferase     

Glutamine synthetase of the nitrogen-fixing alga Anabaena cylindrica

Summary High levels of glutamine synthetase, detected using both a biosynthetic assay (P _i release from ATP) and a -glutamyl transferase assay, are present in aerobically grown N₂-fixing cultures of Anabaena cylindrica. The enzyme is soluble, has a pH optimum of 6.5–7.5, with a peak at 7.1–7.2 (biosynthetic activity) or 6.9 (transferase activity), and a temperature optimum at 30°C–40°C. Partially purified preparations are stable in air at 5°C for at least 3 days. Mg²⁺, Mn²⁺, Co²⁺ and Ca²⁺ support high rates of biosynthetic activity, Zn²⁺ is less effective and Cu²⁺ and Ba²⁺ are ineffective.Enzyme activity is regulated at several levels: possibly by repression and derepression of the enzyme in response to NH₄ ⁺ level; by variation in the Mn²⁺: ATP ratio with optimum activity at a 1:1 ratio; by feed-back inhibition which may be of a cumulative type. The consensus of the evidence suggests the absence of a covalent enzyme modification of the type found in E. coli. Glutamine synthetase levels are almost twice as high on a protein basis in the heterocysts as in the vegetative cells. Apparent K _m values for whole filaments for NH₄ ⁺ and glutamate in the biosynthetic reactions are 1 mM and 2 mM respectively.     

Application of luminescence for quality evaluation of in vitro regenerated strawberry plants

The parameters of chlorophyll a fluorescence induction were measured: F_v/F_m, S_c/F_m, Rf_d and coefficient of L_d delayed luminescence decay kinetics, related with a course of primary photosynthesis reactions on leaves of strawberry plants, cultured in vitro by means of the micropropagation methods. Strawberry plants cv. Ananasowa from in vitro cultures in optimal condition show significantly higher values of luminescence parameters indicating better condition of plants of this variety in comparison with the variety Senga Sengana. After temperature lowering, however, these values were more reduced than for plants of Senga Sengana, which can be interpreted as higher susceptibility of this variety to chill. Addition of BAP caused disturbance of primary photosynthesis reactions rate, particularly in lower temperature. Auxin 2,4-D had no effect on the luminescence parameters in comparison with control cultures. Dehydration stress strongly diminished the values of measured parameters for Ananasowa variety what indicates the inhibition of primary photosynthesis reaction in leaves. The old culture of Senga Sengana variety showed higher tolerance on linuron in comparison with the new one.     

Hepatic and extra-hepatic glutathione-S-transferase activity in wild pigeons (Columba livia)

Glutathione-S-transferase (EC 2.5.1.18) activity was assayed in hepatic and extra-hepatic tissues of pigeons using l-chloro-2,4-dinitrobenzene and 1,2-dichloro-4-nitrobenzene as substrates. Gluthathione-S-transferase activity towards 1-chloro-2,4-dinitrobenzene in pigeon was in the order: kidney > liver > testes > brain > lung> heart. The enzyme activity with 1-chloro-2,4-dinitrobenzene as substrate was 40–44 times higher in pigeon liver and kidney than that observed with 1,2-dichloro-4-dinitrobenzene as substrate.K _m values of hepatic and renal glutathione transferase with l-chloro-2,4-dinitrobenzene as substrate were 2.5 and 3 mM respectively. Double reciprocal plots with varying reduced gluthathione concentrations resulted in biphasic curves with twoK _m values (liver 0.31 mM and 4mM; kidney 0.36 mM and 1.3 mM). The enzyme activity was inhibited by oxidized gluthathione in a dose-dependent pattern. 3-Methylcholanthrene elicited about 50% induction of hepatic glutathione transferase activity whereas phénobarbital was ineffective.     

Purification and characterization of a cytoplasmic enzyme component of the Na+-activated malonate decarboxylase system of Malonomonas rubra: acetyl-S-acyl carrier protein: malonate acyl carrier protein-SH transferase

Malonate decarboxylation by crude extracts of Malonomonas rubra was specifically activated by Na⁺ and less efficiently by Li⁺ ions. The extracts contained an enzyme catalyzing CoA transfer from malonyl-CoA to acetate, yielding acetyl-CoA and malonate. After about a 26-fold purification of the malonyl-CoA:acetate CoA transferase, an almost pure enzyme was obtained, indicating that about 4% of the cellular protein consisted of the CoA transferase. This abundance of the transferase is in accord with its proposed role as an enzyme component of the malonate decarboxylase system, the key enzyme of energy metabolism in this organism. The apparent molecular weight of the polypeptide was 67,000 as revealed from SDS-polyacrylamide gel electrophoresis. A similar molecular weight was estimated for the native transferase by gel chromatography, indicating that the enzyme exists as a monomer. Kinetic analyses of the CoA transferase yielded the following: pH-optimum at pH 5.5, an apparent K_m for malonyl-CoA of 1.9mM, for acetate of 54mM, for acetyl-CoA of 6.9mM, and for malonate of 0.5mM. Malonate or citrate inhibited the enzyme with an apparent K_i of 0.4mM and 3.0mM, respectively. The isolated CoA transferase increased the activity of malonate decarboxylase of a crude enzyme system, in which part of the endogenous CoA transferase was inactivated by borohydride, about three-fold. These results indicate that the CoA transferase functions physiologically as a component of the malonate decarboxylase system, in which it catalyzes the transfer of acyl carrier protein from acetyl acyl carrier protein and malonate to yield malonyl acyl carrier protein and acetate. Malonate is thus activated on the enzyme by exchange for the catalytically important enzymebound acetyl thioester residues noted previously. This type of substrate activation resembles the catalytic mechanism of citrate lyase and citramalate lyase.Abbreviations DTNB 5,5 Dithiobis (2-nitrobenzoate) - MES 2-(N-Morpholino)ethanesulfonic acid - TAPS N-[Tris(hydroxymethyl)-methyl]-3-aminopropanesulfonic acid - SDS-PAGE sodium dodecyl sulfate-polyacrylamide gel electrophoresis     

Characterization of mannosyl transferases during the pupal instar of Stomoxys calcitrans (L)

Particulate fractions (10,000g) from pupae of Stomoxys calcitrans transfer [¹⁴C]-mannose from GDP-[¹⁴C]-mannose to dolichol monophosphate and proteins. Production of the mannosyl lipid was inhibited by Mn²⁺, UDP, GMP, GDP, and EDTA. The insect growth regulator diflubenzuron had no effect on mannosyl transferase activity. Dolichol monophosphate and Mg²⁺ stimulated mannosyl transferase activity. The mannosyl lipid product was identified as mannosyl-phosphoryl-dolichol (Man-P-Dol). The apparent K_m and V_max values for the formation of Man-P-Dol using GDP-[¹⁴C]-Man while holding dolichol phosphate constant were 2.4 ± 0.9 μM and 9.4 ± 2.3 pmol Man-P-Dol·min^?1·mg^?1 protein, respectively. The apparent K_m and V_max values using dólichol phosphate while holding GDP-Man constant were 2.2 ± 1.2 μM and 18.5 ± 1.7 pmol Man-P-Dol·min^?1·mg^?1 protein.     

Serology and gene sequence analysis of rare subgroup A3 blood group

To investigate the serological phenotypic characteristics and possible mechanism of subgroup A₃, a blood donor's ABO phenotypes were detected by the conventional microcolumn gel method and classic tube method. N-acetylgalactosaminyl transferase activity was detected by the non-radioactive phosphate coupling method. ABO subtype genotyping was determined by PCR-SSP and exons 1-7 of ABO gene were analyzed by Sanger sequencing. The donor's blood type was subgroup A₃ as evaluated by serological test. There was no N-acetylgalactosaminyl transferase activity in the red blood cells and weak N-acetylgalactosaminyl transferase activity in the plasma. The ABO blood group genotyping result was ABO*AO1, and the gene sequencing result was confirmed as A221/O01. Sequencing results showed two mutations, 467C>T and 607G>A in exon 7 in ABO*A allele. In conclusion, it is suggested that the ABO blood group of the donor be subgroup A₃, which may be induced by mutations 467C>T and 607G>A, and led to a decrease in N-acetylgalactosaminyl transferase activity and resulted in weakened A antigen.     

N-Methylation of quinolizidine alkaloids: an S-adenosyl-l-methionine: cytisine N-methyltransferase from Laburnum anagyroides plants and cell cultures of L. alpinum and Cytisus canariensis

An S-adenosyl-l-methionine (SAM): cytisine N-methyltransferase could be demonstrated in crude enzyme preparations from Laburnum anagyroides plants and cell cultures of L. alpinum and Cytisus canariensis. The transferase specifically catalyzes the transfer of a methyl group from SAM to cytisine. The apparent K_m values are 60 mol l^-1 for cytisine and 17 mol l^-1 for SAM. Other quinolizidine alkaloids, e.g. angustifoline and albine, are N-methylated by only 10–15%. The transferase shows a pH optimum at pH 8.5. It is activated by dithioerythritol and inhibited by thiol reagents and Fe²⁺ and Fe³⁺. The reaction product S-adenosylhomocysteine is a powerful inhibitor of the transferase reaction. Cell cultures of L. alpinum which have an active SAM: cytisine N-methyltransferase and which are able to N-methylate exogenous cytisine in vivo, do not accumulate cytisine or N-methylcytisine to a detectable degree.Abbreviations GLC gas-liquid chromatography - SAM S-adenosylmethionine - TLC thin-layer chromatography     

Glycogen in thermoacidophilic archaebacteria of the genera Sulfolobus,Thermoproteus, Desulfurococcus and Thermococcus

Glycogen has been found in thermoacidophilic archaebacteria of the genera Sulfolobus, Thermoproteus, Desulfurococcus and Thermococcus. Thermoplasma acidophilum yielded a related, though less defined compound.Glycogen was identified by elementary analysis, infrared spectroscopy, the nature of the hydrolysis products, the iodine reaction, and the nature of the products of periodate oxydation and reduction. The average chain length was 7.From crude extracts of Sulfolobus and Thermoproteus complexes of glycogen with 4 respectively 2 proteins have been isolated by CsCl density gradient centrifugation. In either case, one of the proteins was identified as glucosyl transferase.The glucosyl transferase of Sulfolobus acidocaldarius strain B 12 utilizes UDP-glucose as well as ADP-glucose as substrates, with K _m values of 0.42 and 0.2 mM respectively and turnover numbers of 4.6 and 5.2 per second respectively.In electron micrographs the isolated glycogen protein complex appears as scale like aggregates, whereas in cell sections amorphous bodies fill large portions of the cells.