The catalysis of nucleotide polymerization by compounds of divalent lead

Summary Soluble lead salts and a number of lead-containing minerals catalyze the formation of oligonucleotides from nucleoside 5-phosphorimidazolides. The effectiveness of lead compounds correlates strongly with their solubility. Under optimal conditions we were able to obtain 18% of pentamer and higher oligomers from ImpA. Reactions involving ImpU gave smaller yields.Abbreviations A adenosine - U uridine - Im imidazole - MeIm 1-methyl-imidazole - EDTA ethylenediaminetetraacetic acid - pA adenosine 5-phosphate - pU uridine 5-phosphate - Ap adenosine cyclic 2:3-phosphate - ATP adenosine 5-triphosphate - AppA P₁,P₂-diadenosine 5-diphosphate - pNp (N = A,U) nucleotide 2(3), 5-diphosphate - ImpA adenosine 5-phosphoreimidazolide - ImpU uridine 5-phosphorimidazolide - A ²pA adenylyl-[25]-adenosine - A ³pA adenylyl-[35]-adenosine - pA ²pA 5-phospho-adenylyl-[25]-adenosine - pA ³pA 5-phospho-adenylyl-[35]-adenosine - pUpU 5-phospho-uridylyl-uridine - pApU 5-phospho-adenylyl-uridine - pUpA 5-phospho-uridylyladenine - (pA)n (n, 2,3,4,) oligoadenylates with 5 terminal phosphate - ImpApA 5-phosphorimidazolide of adenylyl adenosine - (pA) ₅₊ pentamer and higher oligoadenylates with 5 terminal phosphate - (Ap)nA (n = 2,3,4) oligoadenylates without terminal phosphates In the following we do not specify the nature of the internucleotide linkageIn the following we do not specify the nature of the internucleotide linkage     

Preparations ofN,N′-ethylene-bridged dipeptides(eXX) constructed from (S)-methionine, -tryptophan, -tyrosine and-N(ɛ)-benzyloxycarbonyllysine through acid-catalyzed cyclization

Summary N, N-Ethylene-bridged bis-(S)-methionine[(2S, 7S)-2, 7-bis(2-methyl-thioethyl)-3,6-diazaoctanedioic acid] derived from (S)-methionine and 1,2-dibromoethane was cyclized and esterified simultaneously in boiling ethanol in the presence of an appropriate amount of strong acid such asp-toluenesulfonic acid, affording a cyclic compound,N, N-ethylene-bridged (S)-methionyl-(S)-methionine ethyl ester {ethyl(2S, 3S)-4-(methylthio)-2-[2-oxo-3-(2-methylthioethyl)-1-piperazinyl] butanoate}, exclusively in 80–90% yields. It was also found that, by applying this method, 70–80% yields of the otherN, N-ethylenebridged dipeptides containing (S)-tryptophan, -tyrosine and -N()-benzyloxycarbonyllysine were obtained.     

Enzymic synthesis,chemical characterisation and Sda activity of GalNAcß1-4[NeuAcα2-3]Galß1-4GlcNAc and GalNAcß1-4[NeuAcα2-3]Galß1-4Glc

The tetrasaccharides GalNAcß1-4[NeuAc2-3]Galß1-4Glc and GalNAcß1-4[NeuAc2-3]Galß1-4GlcNAc were synthesised by enzymic transfer of GalNAc from UDP-GalNAc to 3-sialyllactose (NeuAc2-3Galß1-4Glc) and 3-sialyl-N-acetyllactosamine (NeuAc2-3Galß1-4GlcNAc). The structures of the products were established by methylation and¹H-500 MHz NMR spectroscopy. In Sd^a serological tests the product formed with 3-sialyl-N-acetyllactosamine was highly active whereas that formed with 3-sialyllactose had only weak activity.     

Regioselective alkylation of benzylβ-d-lactoside and its derivatives by stannylation

The preparation of benzyl 2,3,6,2,6-penta-O-benzyl--d-lactoside, which is a key intermediate for chemical synthesis of oligosaccharide components of glycosphingolipids, was achieved by an improved method. The 3-O-p-methoxybenzyl and 3-O-methyl derivatives were prepared from benzyl 2,3,6,2,6-penta-O-benzyl--d-lactoside through stannylation. By using benzyl -d-lactoside as starting material, benzyl 3-O-methyl-, 3-O-benzyl- and 3-O-p-methoxybenzyl--d-lactoside were regioselectively synthesized using the same procedure.     

Localization of adenosine 5′-phosphosulfate sulfotransferase in spinach leaves

Roots of spinach (Spinacia oleracea L.) seedlings contained only a very low activity of adenosine 5-phosphosulfate sulfotransferase compared to the cotyledons. Adenosine 5-phosphosulfate sulfotransferase activity increased about tenfold in cotyledons during greening. Preparation of organelle fractions from spinach leaves by a combination of differential and isopycnic density gradient centrifugation showed that adenosine 5-phosphosulfate sulfotransferase banded with NADP-glyceraldehyde-3-phosphate dehydrogenase, a marker enzyme for intact chloroplasts. In the fractions of peroxisomes, mitochondria and broken chloroplasts virtually no adenosine 5-phosphosulfate sulfotransferase activity was measured. Comparison with the chloroplast enzyme NADP-glyceraldehyde-3-phosphate dehydrogenase indicates that in spinach, adenosine 5-phosphosulfate sulfotransferase is localized almost exclusively in the chloroplasts.Abbreviations APS Adenosine 5-phosphosulfate - APSSTase Adenosine 5-phosphosulfate sulfotransferase - BSA Bovine serum albumin - BRIJ58 Polyethylene glycolmonostearylether - DTE Dithioerythritol - DTT Dithiothreitol - EDTA Ethylenediaminetetraacetic acid - ME 2-Mercaptoethanol - NADP-GPD NADP-linked glyceraldehyde-3-phosphate dehydrogenase - PAPS Adenosine 3-phosphate 5-phosphate 5-phosphosulfate - POPOP 1,4 Di [2-(5-phenyloxazolyl)]-benzene - PPO 2,5-Diphenyloxazol The results presented in this paper are taken from the Ph. D. thesis of H.F.     

Genetic and biochemical studies on flavonoid 3′-hydroxylation in flowers of Petunia hybrida

Summary In flower extracts of defined genotypes of Petunia hybrida, an enzyme activity was demonstrated which catalyses the hydroxylation of naringenin and dihydrokaempferol in the 3-position. Similar to the flavonoid 3-hydroxylases of other plants, the enzyme activity was found to be localized in the microsomal fraction and the reaction required NADPH as cofactor. A strict correlation was found between 3-hydroxylase activity and the gene Ht1, which is known to be involved in the hydroxylation of flavonoids in the 3-position in Petunia. Thus, the introduction of the 3-hydroxyl group is clearly achieved by hydroxylation of C₁₅-intermediates, and the concomitant occurrence of the 3,4-hydroxylated flavonoids quercetin and cyanidin (paeonidin) in the presence of the functional allele Ht1 is due to the action of one specific hydroxylase catalysing the hydroxylation of common precursors for both flavonols and anthocyanins.     

Chemoenzymatic galactosialylation with integrated cofactor regeneration

As a precursor for the chemical synthesis of sialylated oligosaccharides, the trisaccharide glycoside Neu5Ac (2-8)Gal(1-4)GlcNAc(1-O)-pent-4-ene was synthesized starting from GlcNAc(1-O)-pent-4-ene, UDP-glucose andN-acetylneuraminic acid in a one pot reaction employing galactosyltransferase and (2-6)sialyl-transferase in a complete cofactor regeneration system.Abbreviations Neu5Ac N-acetylneuraminic acid - CMP-Neu5Ac cytidine 5-monophosphosialate - CMP cytidine 5-monophosphate - CDP cytidine 5-diphosphate - CTP cytidine 5-triphosphate - Gal galactose - GlcNAc N-acetylglucosamine - UDP uridine 5-diphosphate - UDP-Glc uridine-5-diphosphoglucose - UDP-Gal uridine-5-diphosphogalactose - PEP phosphoenolpyruvate     

Purification and properties of the ATP: adenylylsulphate 3′-phosphotransferase from Chlamydomonas reinhardii

APS-kinase (ATP: adenylylsulphate 3-phosphotransferase, EC 2.7.1.25) has been purified from the alga Chlamydomonas reinhardii, strain CW 15 by means of chromatofocussing and affinity chromatography. The isolated protein showed an apparent molecular mass of 44,000 upon sodium dodecylsulphate polyacrylamide gel electrophoresis. The transfer of phosphate groups from ATP onto APS required a pH of 6.8, the presence of Mg²⁺ ions and a reducing thiol. Its catalytical activity was destroyed by sulphhydryl group inhibitors (phenyl-mercuri compounds, dithiopyridine) and alkylating reagents.The purified enzyme attained a V _max of 360 pkat under optimal reaction conditions declining to v _limit of 260 pkat in the presence of excess substrate APS. This sensitivity towards changes in substrate concentrations was parallelled by a high affinity and specificity: apparent K _m APS: 2 · 10^-6 mol · l^-1, and K _m ATP: 7 · 10^-6 mol · l^-1. The enzyme was found specific for ATP, d-ATP and CTP, while UTP, ITP and GTP showed marginal activity. The Hill coefficients suggested 4 binding sites for APS and 1 for ATP. Excessive APS resulted in a negative slope indicating 3 inhibiting sites of the substrate.Abbreviations APS Adenosine 5-phosphosulphate - dATP 2-deoxyadenosine 5-triphosphate - p-CMB p-chloromercuribenzoate - DTE dithioerythritol - DTT dithiothreitol - -MSH -mercaptoethanol - PAPS 3-phosphoadenosine 5-phosphosulphate - PAP 3-phosphoadenosine 5-phosphate - SDS sodium dodecyl sulphate This work is part of a dissertation submitted by H. G. J., Bochum 1982     

cDNA cloning and characterization of a 3′/5′-O-methyltransferase for partially methylated flavonols from Chrysosplenium americanum

Enzymatic O-methylation of plant secondary metabolites is an important mechanism for the inactivation of reactive hydroxyl groups and for the modification of their solubility. A cDNA clone (pFOMT3) encoding the gene for the 3/5-O-methylation of partially methylated flavonols was isolated from Chrysosplenium americanum (Saxifragaceae). We used a PCR fragment obtained with degenerate oligonucleotides designed from conserved regions of various O-methyltransferases (OMTs). The pFOMT3 cDNA sequence shows about 67–85% similarity to other plant OMT sequences. The recombinant protein expresses strict specificity for positions 3/5 (meta) of partially methylated flavonols, but does not accept quercetin or caffeic acid for further methylation. Southern blot analysis of the genomic DNA probed with an OMT sequence suggests the presence of a number of related genes in this species, consistent with the multiple enzymatic methylations involved in the biosynthesis of polymethylated flavonols in this plant.     

Isolation of trace amounts of 3′,4′-dehydro-4′-deoxydothistromin from peanut plants naturally infected with Cercospora personata

Trace amounts of the anthraquinonoid toxic metabolite, 3,4-dehydro-4-deoxydothistromin have been identified for the first time, from peanut tissues naturally infected by Cercospora personata. Characteristic uv spectral and Chromatographic properties of the metabolite and its tetraacetate as well as the mass spectrum of the latter have established its identity.     

Purification and characterization of the 5′ → 3′ exonuclease domain-deleted Thermus filiformis DNA polymerase expressed in Escherichia coli

The gene encoding 5 3 exonuclease domain-deleted Tfi DNA polymerase, named 5 3 Exo^– Tfi fragment, from Thermus filiformis was expressed in Escherichia coli under the control of the tac promoter on a high-copy plasmid, pJR. The expressed enzyme was purified 27-fold with a 19% yield and a specific activity of 2621 U mg^–1 protein. The 5 3 exonuclease domain of Tfi DNA polymerase was removed without significant effect on enzyme activity and stability. PCR conditions for the 5 3 Exo^– Tfi fragment were more tolerant to the buffer composition as compared to the full-length enzyme.     

A cyclic adenosine 3′,5′-monophosphate-dependent protein kinase mutant of Neurospora crassa

A cpk mutant of Neurospora crassa with morphological alteration was obtained spontaneously during the cross between the wild-type and a glycerol utilizing cr-l strain. The growth rate of cpk was intermediate between the wild-type and cr-1 mutant strains. The cpk conidia contained a reduced level of carotenoid pigments as compared to the wild-type conidia. The cpk mutant had no detectable amount of cyclic adenosine 3,5-monophosphate (cAMP)-binding protein at all stages of growth tested. On a DEAE-Sephacel column chromatogram, protein kinase activity of the wild type was eluted at two peaks; the first peak was cAMP-dependent, and the second one was not. In contrast, the cpk strain had two peaks of cAMP-independent enzymes. It is suggested that cAMP-dependent protein kinase may be altered in the cpk mutant into a cAMP-independent type by an alteration of the regulatory subunit of this enzyme.Abbreviations cAMP Cyclic adenosine 3,5-monophosphate - 8-N₃-[³H] cAMP 8-azido-[³H]cyclic adenosine 3,5-monophosphate     

Facile synthesis of 2′-substituted lactoses

Isopropylidenation of lactose with 2,2-dimethoxypropane in the presence ofp-toluenesulfonic acid gave two products, which were identified by¹H- and¹³C-NMR as 2,35,63,4-tri-O-isopropylidenelactose dimethyl acetal (1) and its 6-O-(2-methoxy)-isopropyl derivative (2). These products were used for the synthesis of 2-O-methyllactose (7), 2,6-di-O-methyllactose (9) and 2-O-benzyllactose (13).     

Adenosine-5′-phosphosulfate (APS) as sulfate donor for assimilatory sulfate reduction in Rhodospirillum rubrum

Crude extracts of Rhodospirillum rubrum catalyzed the formation of acid-volatile radioactivity from (³⁵S) sulfate, (³⁵S) adenosine-5-phosphosulfate, and (³⁵S) 3-phosphoadenosine-5-phosphosulfate. An enzyme fraction similar to APS-sulfotransferases from plant sources was purified 228-fold from Rhodospirillum rubrum. It is suggested here that this enzyme is specific for adenosine-5-phosphosulfate, because the purified enzyme fraction metabolized adenosine-5-phosphosulfate, however, only at a rate of 1/10 of that with adenosine-5-phosphosulfate. Further, the reaction with 3-phosphoadenosine-5-phosphosulfate was inhibited with 3-phosphoadenosine-5-phosphate whereas this nucleotide had no effect on the reaction with adenosine-5-phosphosulfate. For this activity with adenosine-5-phosphosulfate the name APS-sulfotransferase is suggested. This APS-sulfotransferase needs thiols for activity; good rates were obtained with either dithioerythritol or reduced glutathione; other thiols like cysteine, 2-3-dimercaptopropanol or mercaptoethanol are less effective. The electron donor methylviologen did not catalyze this reaction. The pH-optimum was about 9.0; the apparent K _m for adenosine-5-phosphosulfate was determined to be 0.05 mM with this so far purified enzyme fraction. Enzyme activity was increased with K₂SO₄ and Na₂SO₄ and was inhibited by 5-AMP. These properties are similar to assimilatory APS-sulfotransferases from spinach and Chlorella.Abbreviations APS adenosine-5-phosphosulfate - PAPS 3-phosphoadenosine-5-phosphosulfate - 5-AMP adenosine-5-monophosphate - 3-AMP adenosine-3-monophosphate - 3-5-ADP 3-phosphoadenosine-5-phosphate (PAP) - DTE dithiorythritol - GSH reduced glutathione - BAL 2-3-dimercaptopropanol     

Pyridoxal 5′-phosphate binds to a lysine residue in the adenosine 3′-phosphate 5′-phosphosulfate recognition site of glycolipid sulfotransferase from human renal cancer cells

In the course of characterization of glycolipid sulfotransferase from human renal cancer cells, the manner of inhibition of sulfotransferase activity with pyridoxal 5-phosphate was investigated. Incubation of a partially purified sulfotransferase preparation with pyridoxal 5-phosphate followed by reduction with NaBH₄ resulted in an irreversible inactivation of the enzyme. When adenosine 3-phosphate 5-phosphosulfate was co-incubated with pyridoxal 5-phosphate, the enzyme was protected against this inactivation. Furthermore, pyridoxal 5-phosphate was found to behave as a competitive inhibitor with respect to adenosine 3-phosphate 5-phosphosulfate with aK _i value of 287 µm. These results suggest that pyridoxal 5-phosphate modified a lysine residue in the adenosine 3-phosphate 5-phosphosulfate-recognizing site of the sulfotransferase.     

Evidence of β-carotene 7,8(7′,8′) oxygenase (β-cyclocitral,crocetindial generating) in Microcystis

A -carotene oxygenase is described which occurs in the Cyanobacterium Microcystis. It cleaves -carotene and zeaxanthin specifically at the positions 7,8 and 7,8, while echinenone and myxoxanthophyll are not affected. The oxidative cleavage of -carotene leads to the formation of -cyclocitral and crocetindial and that of zeaxanthin to hydroxy--cyclocitral and crocetindial in nearly stoichiometric amounts. Oxidant is dioxygen as has been demonstrated by high incroporation (86%) of ¹⁸O₂ into -cyclocitral. -Carotene oxygenase is membrane bound, sensitive to sulfhydryl reagents, antioxidants and chelating agents. Iron seems to be an essential part of the enzyme activity. Cofactors necessary for the reaction could not be detected.Abbreviations TLC thin layer-chromatography - PIPES piperazine-N,N-bis-(2-ethanesulfonate) Na - TES 2{[tris-(hydroxymethyl)-methyl]-amino} ethanesulfonic acid Dedicated to Professor G. Drews on occasion of his 60th birthday     

Cyanamide mediated syntheses under plausible primitive earth conditions

Summary When an aqueous solution (pH 7.0) of deoxythymidine 5-phosphate, 4-amino-5-imidazolecarboxamide and cyanamide was dried and heated for 18 h at 60°C, P¹, P²-dideoxythymidine 5-pyrophosphate (I) was formed in a 58% yield. Oligonucleotides were not detected in the reaction product. Under conditions employed in the above reaction, (I) was shown to be stable. In prebiotic polymerization reactions employing deoxythymidine 5-triphosphate as the polymerizing species, (I) could therefore function as a primer and minimize the formation of cyclic nucleotides.Abbreviations dT deoxythymidine - dTMP deoxythymidine 5-phosphate - dTppT P¹, P²-dideoxythymidine 5-pyrophosphate - dTTP deoxythymidine 5-triphosphate - AICA 4-amino-5-imidazolecarboxamide     

Influence of thyroid hormones on the human ATP synthase β-subunit gene promoter

The action of thyroid hormones on the expression of the mitochondrial ATP synthase -subunit gene (ATPsyn) is controversial. We detected a binding site for the thyroid hormone receptor between-366 and-380 in the human ATPsyn gene by DNase I footprint analysis and band-shift assays. However, expression vectors in which the chloramphenicol acetyl transferase (CAT) reporter gene is driven by the 5 upstream region of ATPsyn gene were unresponsive to T₃ when transiently transfected to HepG2 or GH4C1 cells. CAT constructs driven by the rat phosphoenolpyruvate carboxykinase (PEPCK) or the growth hormone (GH) promoters were stimulated several fold by T₃ in parallel experiments. It is proposed that the biological effects of thyroid hormones on the ATPsyn expression occur through indirect mechanisms.     

Nucleotide sequence and expression of the two genes encoding D2 protein and the single gene encoding the CP43 protein of Photosystem II in the cyanobacterium synechococcus sp. PCC 7002

The unicellular photoheterotrophic cyanobacterium Synechococcus sp. PCC 7002 was shown to encode two genes for the Photosystem II reaction center core protein D2 and one gene for the reaction center chlorophyhll-binding protein CP43. These three genes were cloned and their DNA sequences determined along with their flanking DNA sequences. Northern hybridization experiments show that both genes which encode D2, psbD1 and psbD2, are expressed at roughly equivalent levels. For each of the two psbD genes, there are 18 nucleotide differences among the 1059 nucleotides which are translated. The DNA sequences surrounding the coding sequences are nearly 70% divergent. Despite the DNA sequence differences in the genes, the proteins encoded by the two genes are predicted to be identical. The proteins encoded by psbD1 and psbD2 are 92% homologous to other sequenced cyanobacterial psbD genes and 86% homologous to sequenced chloroplast-encoded psbD genes.The single gene for CP43, psbC, overlaps the 3 end of psbD1 and is co-transcribed with it. Results from previous sequencing of psbC genes encoded by chloroplasts suggest that the 5 end of the psbC gene overlaps the 3 end of the coding sequence of psbD by 50 nucleotides. In Synechococcus sp. PCC 7002, the methionine codon previously proposed to be the start codon for psbC is replaced by an ACG (threonine) codon. We propose an alternative start for the psbC gene at a GTG codon 36 nucleotides downstream from the threonine codon. This GTG codon is preceded by a consensus E. coli-like ribosome binding sequence. Both the GTG start codon and its preceding ribosome binding sequence are conserved in all psbC genes sequenced from cyanobacteria and chloroplasts. This suggests that all psbC genes start at this alternative GTG codon. Based on this alternative start codon, the gene product is 85% identical to other cyanobacterial psbC gene products and 77% identical to eucaryotic chloroplast-encoded psbC gene products.