Inhibition of adenylate cyclase activity in the goldfish melanophore is mediated by α 2-adrenoceptors and a pertussis toxin-sensitive GTP-binding protein

The signal-transduction system that mediates the melanosome-aggregating response in melanophores of the black-moor goldfish, Carassius auratus, was investigated by examining the inhibition of adenylate cyclase activity mediated by -adrenoceptors in cultured cells. When the melanophores were incubated with 1 mmol·l^-1 3-isobutyl-1-methylxanthine for 5 min, the intracellular level of cyclic adenosine-3,5-monophosphate increased two- to three-fold. Norepinephrine at 100 nmol·l^-1 and naphazoline at 1 mol·l^-1 inhibited the 3-isobutyl-1-methylxanthine-induced accumulation of cyclic adenosine-3,5-monophosphate in the cells in both the presence and the absence of isoproterenol, a -adrenergic agonist. Methoxamine and phenylephrine also reduced the extent of accumulation of cyclic adenosine-3,5-monophosphate, but only when they were present at relatively high concentrations (above 100 mol·l^-1). The range of concentrations at which norepinephrine inhibited the accumulation of cyclic adenosine-3,5-monophosphate was consistent with the range at which it induced the aggregation of melanosomes. Pretreatment of the cells with pertussis toxin (1 g·ml^-1) for 15 h or treatment with 100 nmol·l^-1 yohimbine (an ₂-adrenergic antagonist) inhibited the effects of the -adrenergic agonists on both the aggregation of melanosomes and the 3-isobutyl-1-methylxanthine-induced accumulation of cyclic adenosine-3,5-monophosphate, but prazosin (an ₁adrenergic antagonist) at 100 nmol·l^-1 was not inhibitory. These results indicate that the melanosome-aggregating response of the goldfish melanophore is induced mainly via inhibition of the activity of adenylate cyclase, which occurs as result of stimulation of a pathway that involves ₁adrenergic and a inhibitory GTP-binding protein.Abbreviations A-kinase cAMP-dependent protein kinase - BSS balanced salts solution - CaM calmodulin - cAMP cyclic adenosine-3,5-monophosphate - Clo clonidine - EDTA ethylenediaminetetra-acetic acid - G-protein GTP-binding protein - HEPES N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid - IBMX 3-isobutyl-1-methylxanthine - IP₃ inositol 1,4,5-trisphosphate Mex, methoxamine - MSH melanocyte-stimulating hormone - Nap naphazoline - NE norepinephrine - Oxy oxymetazoline - Phe phenylephrine - PTX pertussis toxin     

IP3-and cAMP-induced responses in isolated olfactory receptor neurons from the channel catfish

Summary Olfactory receptor neurons enzymatically dissociated from channel catfish olfactory epithelium were depolarized transiently following dialysis of IP₃ or cAMP (added to the patch pipette) into the cytoplasm. Voltage and current responses to IP₃ were blocked by ruthenium red, a blocker of an IP₃-gated Ca²⁺-release channel in sarcoplasmic reticulum. In contrast, the responses to cAMP were not blocked by extracellularly applied ruthenium red, nor by l-cis-diltiazem or amiloride and two of its derivatives. The current elicited by cytoplasmic IP₃ in neurons under voltage clamp displayed a voltage dependence different from that of the cAMP response which showed marked outward rectification. A sustained depolarization was caused by increased cytoplasmic IP₃ or cAMP when the buffering capacity for Ca²⁺ of the pipette solution was increased, when extracellular Ca²⁺ was removed or after addition of 20–200 nm charibdotoxin to the bathing solution, indicating that the repolarization was caused by an increase in [Ca _i ] that opened Ca²⁺-activated K⁺ channels. The results suggest that different conductances modulated by either IP₃ or cAMP are involved in mediating olfactory transduction in catfish olfactory receptor neurons and that Ca²⁺-activated K⁺ channels contribute to the termination of the IP₃ and cAMP responses.Abbreviations ATP adenosine 5-triphosphate - BAPTA (bis-(o-aminophenoxy)-ethane-N-N-N-N)-tetraacetic acid - cAMP adenosine cyclic 3,5-monophosphate - cGMP guanosine cyclic 3,5-monophosphate - CTX charybdotoxin - DCB 3,4-dichlorobenzamil - EDTA ethylenediaminetetraacetic acid - EGTA ethylenglycol-bis-(b-aminoethyl)-N-N-N-N-tetraacetic acid - HEPES N-2-hydroxyethylpiperazine-N-2-ethanesulfonic acid - IP₃ inositol-1,4,5-triphosphate - NMDG N-methyl-d-glucamine We would like to thank the Tanabe Seiyaku Co., Ltd., for their gift of l-cis-diltiazem. This work was supported by National Institutes of Health grants DC00566 and BRSG S07RR05825.     

Excision and transposition of two Ds transposons from the bronze mutable 4 derivative 6856 allele of Zea mays L

Summary The regulatory mutation bronze mutable 4 Derivative 6856 (bz-m4 D6856) contains a complex 6.7 kb Dissociation (Ds) element tagged with a duplication of low copy bz 3 flanking sequences (Klein et al. 1988). This creates a unique opportunity to study the transposition of a single member of the repetitive family of Ds elements. Eighteen full purple revertants (Bz alleles) of bz-m4 were characterized enzymatically and by genomic mapping. For 17 of the Bz alleles, reversion to a wild-type phenotype was caused by excision of the 6.7 kb Ds transposon. Nine of these Bz alleles retained the transposon somewhere in their genome. In this study we show that like Ac (Schwartz 1989; Dooner and Belachew 1989), the 6.7 kb Ds element can transpose within a short physical distance, both proximal and distal to its original position. Additional bz sequences have been mapped immediately distal to the mutant locus in bz-m4 D6856; genetic evidence suggests these are flanked by two additional Ds elements. The remaining Bz revertant, Bz :107, arose from excision of a more complex 13 kb Ds element.     

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.     

Isolation,structure determination and biological activity of A-16686 factors A′ 1, A′ 2 and A′ 3 glycolipodepsipeptide antibiotics

Summary WhenActinoplanes strain ATCC 33076, the producer of A-16686 A1, A2 and A3 complex, is fermented in a suitable medium three additional factors, designated A1, A2 and A3 are produced. These were isolated and characterized, and were shown to differ from the parent components of the original complex by lacking one mannose unit. Bioconversion of A factors into A factors was achieved by incubation with the mycelium ofActinoplanes ATCC 33076. Factor A2 has better antibacterial activity than A2 against some bacteria.     

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     

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.     

A New Aerobic Gram-Positive Bacterium with a Unique Ability to Degrade ortho- and para-chlorinated Biphenyls

Strain B51 capable of degrading polychlorinated biphenyls (PCB) was isolated from soil contaminated with wastes from the chemical industry. Based on its morphological and chemotaxonomic characteristics, the strain was identified as a Microbacterium sp. Experiments with washed cells showed that strain B51 is able to degrade ortho- and para-substituted mono-, di-, and trichlorinated biphenyls (MCB, DCB, and TCB, respectively). Unlike the known PCB degraders, Microbacterium sp. B51 is able to oxidize the ortho-chlorinated ring of 2,2-DCB and 2,4-DCB and the para-chlorinated ring of 4.4-DCB. The degradation of 2,4-DCB and 4,4-DCB was associated with the accumulation of 4-chlorobenzoic acid (4-CBA) in the medium in amounts comprising 80–90% of the theoretical yield. The strain was able to utilize 2-MCB, 2,2-DCB, and their intermediate 2-CBA and to oxidize the mono(ortho)-chlorinated ring of 2,4,2-TCB and the di(ortho-para)-chlorinated ring of 2,4,4-TCB. A mixed culture of Microbacterium sp. B51 and the 4-CBA-degrading bacterium Arthrobacter sp. H5 was found to grow well on 1 g/l 2,4-DCB as the sole source of carbon and energy.     

Mutation spectra of N-ethyl-N''-nitro-N-nitrosoguanidine and 1-(2-hydroxyethyl)-1-nitrosourea in Escherichia coli

Summary DNA sequencing was used to determine the specific types of DNA base changes induced following in vivo exposure of Escherichia coli to the ethylating agent N-ethyl-N-nitro-N-nitrosoguanidine (ENNG) and the hydroxyethylating agent 1-(2-hydroxyethyl)-1-nitrosourea (HENU) using the xanthine guanine phosphoribosyltransferase (gpt) gene as the genetic target. We observed that 22/30 of the ENNG-induced mutations were GCAT transitions, 4/30 were ATGC transitions, 3/30 were ATTA transversions, and 1/30 was an ATCG transversion. We observed that 37/40 HENU-induced mutations were GCAT transitions and that the remaining 3/40 were ATGC transitions. A majority of the GCAT transitions induced by ENNG and HENU (68% and 73%, respectively) occurred at the second guanine of the sequence 5-GG(A or T)-3; this sequence specificity was similar to that previously seen with the alkylating agents N-methyl- and N-ethyl-N-nitrosourea (MNU and ENU) and N-methyl-N-nitro-N-nitrosoguanidine (MNNG). A DNA strand preference for the GA changes (antisense strand), previously noted for MNU, ENU, and MNNG, was observed following exposure to HENU and ENNG. The ATGC transitions induced by ENNG, HENU, and ENU also exhibit a sequence specificity with 13/13 mutations occurring at the T of the sequence 5-NTC-3. A strand preference was not apparent for these mutations.     

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.     

Properties of a high-affinity cytokinin-binding protein from wheat germ

A soluble protein that interacts with a range of cytokinins was extensively purified from wheat (Triticum aestivum L.) germ. This protein has a K _d for kinetin of 2×10^-7 M. The binding of kinetin to the protein is inhibited by low concentrations of synthetic and naturally-occurring cytokinins including N⁶-benzyladenine, N⁶-benzyladenosine, kinetin riboside, N⁶-dimethylallyladenine, N⁶-dimethylallyladenosine, zeatin, zeatin riboside, N⁶-dimethyladenine and N⁶-dimethyladenosine. Adenine, adenosine and several non-N⁶-substituted adenine derivatives were ineffective as inhibitors of kinetin binding. While N⁶-butyryl-3,5-cyclic AMP, N⁶,2-O-dibutyryl-3,5-cyclic AMP and 2,3-cyclic AMP inhibited binding of kinetin to the protein, 3,5-cyclic AMP was ineffective. The kinetin-binding protein is heat-labile and pronase-sensitive. Kinetin-binding activity exactly co-chromatographs with a single peak of carbohydrate and protein on gel-filtration and is displaced from concanavalin A-Sepharose 4B by -methylglucoside. On gel filtration, the kinetin-binding protein behaves as a soluble protein with an apparent molecular weight of 180,000 daltons.     

Evidence for a uridine-5′-diphosphate-glucose-protectedp-chloromercuribenzene sulfonic acid-binding site in sugarcane vacuoles

The uptake of uridine-5-diphosphate (UDP) glucose into vacuoles isolated fromSaccharum sp. cells was fully inhibited by pretreatment with 50 Mp-chloromercuribenzenesulfonic acid (PCMBS) and was not affected by N-ethylmaleimide up to a concentration of 5 mM. The addition of 10 mM UDP-glucose during the pretreatment partially protected the uptake mechanism from PCMBS inhibition, while the presence of adenosine-5-diphosphate (ADP) glucose or of various hexose-phosphates had no protective effect. Parallel experiments on the binding of [²⁰³Hg]PCMBS to the vacuoles showed that UDP-glucose and UDP added at 10 mM concentrations caused a 40% decrease in the binding of PCMBS while ADP-glucose did not inhibit the binding. The results indicate the presence in a previously proposed group translocator of at least one site that can bind UDP-glucose. This site, which is blocked by PCMBS, interacts with the nucleotide moiety of UDP-glucose.Abbreviations ADP-glucose adenosine-5-diphosphate glucose - PCMB p-chloromercuribenzoic acid - PCMBS p-chloromercuribenzenesulfonic acid - UDP uridine-5-diphosphate - UDP-glucose uridine-5-diphosphate glucose     

Export and activity of hybrid FhuA''-''Iut receptor proteins and of truncated FhuA'' proteins of the outer membrane of Escherichia coli

Summary The FhuA protein in the outer membrane of Escherichia coli serves as a multifunctional receptor for the phages T5, T1, 80, for colicin M, for ferrichrome (Fe³⁺-siderophore) and for the structurally related antibiotic, albomycin. To determine structural domains required for these receptor functions and for export, a fusion protein between FhuA and Iut (receptor for Fe³⁺-aerobactin and cloacin DF13) was constructed. In the FhuA-Iut hybrid protein, 24 amino acids of FhuA were replaced by 19 amino acids, 18 of which were from Iut. The number of plaque forming units of phage T5 and T1 on cells expressing FhuA-Iut was nearly as high as on cells expressing plasmid-encoded wild-type FhuA. However, 10⁷-fold higher concentrations of phage 80 and 10³ times more colicin M were required to obtain a zone of growth inhibition. Truncated FhuA proteins in which the last 24 amino acids at the carboxy-terminus were replaced by 16 (FhuA2) or 3 (FhuAT) amino acids could hardly be detected on polyacrylamide electrophoretograms of outer membrane proteins, due to proteolytic degradation. Sensitivity of cells expressing FhuA2 to phage T5 and T1 was reduced by several orders of magnitude and sensitivity to phage 80 and colicin M was totally abolished. In contrast, cells expressing FhuAT were nearly as sensitive to phage T5, T1, and 80 and to colicin M as cells containing FhuA-Iut. None of the constructs could grow on ferrichrome as sole iron source and none was sensitive to albomycin. Ferrichrome did not inhibit binding of T5 to TonB^– cells expressing FhuA-Iut (as it did in FhuA⁺ cells) due to the lack of ferrichrome binding. It is concluded that very small deletions (relative to the size of FhuA with 714 amino acids) at the C-terminal end render FhuA susceptible to proteolytic cleavage. The C-terminal alterations affect sensitivity to FhuA-specific agents very differently. Apparently, the C-terminus is a very important part of FhuA regarding stability and activity. Expression of active FhuA and partially inactive FhuA derivatives in the same cell revealed no negative complementation, suggesting a FhuA monomer as functional unit.     

Interaction between photosynthetic and respiratory electron-transfer chains in the membranes of Anabaena variabilis

The rate of CO₂- and p-benzoquione-dependent photosynthetic O₂ evolution by Anabaena variabilis cells remained unaltered and the rate of O₂ uptake observed after switching off the light (endogenous respiration) was enhanced by a factor of 6–8 when the O₂ concentration was increased from 200 to 400 M. Photosystem-I-linked O₂ uptake and respiration of the cells incubated with ascorbate and N,N,NN-tetramethyl-p-phenylenediamine was not appreciable influenced by the O₂ concentration. 2-Iodo-6-isopropyl-3-methyl-2,4,4-trinitrodiphenyl ether, blocking electron transfer at the plastoquinone level, suppressed O₂ evolution and had no influence on endogenous respiration. 2-n-Heptyl-4-hydroxyquinoline-N-oxide, an inhibitor of electron transfer between photosystems II and I, as well as the cytochrome-oxidase inhibitors N ₃ ^- , CN^- and NH₂OH, caused a 35–50% retardation of endogenous respiration and blocked photosynthetic O₂ evolution. The molar ratio of cytochromes b₆, f, c-553, aa₃ and photosystem-I reaction centers in the isolated membranes equalled approx. 2:1:2:0.7:2. It is inferred that endogenous respiration of A. variabilis cells is inhibited by the light-induced electron flow through both photosystems at the level of the plastoquinone-plastocyanin-oxidoreductase complex.Abbreviations DCMU 3-(3,4-dichlorophenyl)-1,1-dimethylurea - DNP-INT 2-iodo-6-isopropyl-3-methyl-2,4,4-trinitrodiphenyl ether - Hepes 4-(2-hydroxyethyl)-1-piperazine ethansulfonic acid - TMPD N,N,NN-tetramethyl-p-phenylenediamine     

Abscisic acid metabolism —vacuolar/extravacuolar distribution of metabolites

The biotransformation of abscisic acid (ABA) was studied in cell suspension cultures of Lycopersicon esculentum. The ABA was converted by the cells to phaseic acid, nigellic acid, dihydrophaseic acid, abscisic acid--D-glucopyranosyl ester (ABA-Glc) and other ABA and phaseic acid conjugates. Investigation of their cellular distribution showed that the conjugated forms were located only in the vacuoles whereas ABA and its acidic metabolites were found mainly in the extravacuolar fractions. Our results, together with a number of studies on the increase of ABA-Glc as a response to stress, allow us to propose that ABA-Glc is irreversibly compartmented in the vacuoles of plant cells.Abbreviations ABA abscisic acid - ABA-Glc -D-glucopyranosyl ester of ABA - DPA 4-dihydrophaseic acid; nigellic acid=3-methyl-5-(1-hydroxy-2-hydroxymethyl-6-dimethyl-4-oxo-cyclohex-2-enyl)-penta-2Z, 4E-dienoic acid - PA phaseic acid     

Carotenoid pigments in a red strain of Rhizobium from Lotononis bainesii Baker

The carotenoid pigments of a Rhizobium strain isolated from Lotononis bainesii were found to be diglucosyl-4,4-diapocarotene-4,4-dioate and glucosyl-4,4-diapocarotene-4-oate-4-oic acid.5th publication in the series Carotenoids of Rhizobia [4th publication: Helv. chim. Acta 62: 2551–2557 (1979)]     

Polynucleotides and Their Components in the Processes of Aromatic Nucleophilic Substitution: II.1 Nucleophilic Modification of 3′,5′-Bis-O- (α,β,α′,β′-tetrafluoropyrid-γ-yl)thymidine

The interaction of 3,5-bis-O-(,,,-tetrafluoropyrid--yl)thymidine with various nucleophilic reagents was studied to evaluate the possibility of molecular design of new types of nucleic acid analogues using S _NAr reactions. The reactions with morpholine and sodium azide led to the introduction of one and two nucleophilic residues into each of the polyfluorinated pyridine rings. The nucleophilic polycondensation with bifunctional reagents ethylenediamine and hexamethylenediamine depended on the nature of nucleophile and reaction conditions and resulted in the formation of supramolecules containing about five or more than 20 pyrimidine bases.     

Two-dimensional 1H NMR study of two cyclobutane type photodimers of thymidylyl-(3′→5′)-thymidine

2D NMR spectroscopy and J coupling constant analysis are applied to resolve the structure of two photoproducts of thymidylyl-(35)-thymidine. These products are cyclobutane type thymine dimers possessing the cis-syn (the predominant one) and trans-syn geometry. The cis-syn is formed in an ANTI-ANTI conformation about the N-glycosyl linkages and resembles the normal base-stacked configuration. The glycosidic conformation in solution of the 5 terminal fragment differs from the crystal in which the less common SYN conformation is observed. In this isomer only the sugar pucker of the 3 terminal fragment is changed substantially with respect to the dinucleotide. The trans-syn isomer is formed in a SYN-ANTI glycosidic conformation. In this isomer the sugar puckers of both deoxyribose rings are affected and a preference for a pure 2-endo conformation is observed.Abbreviations dTpdT 2-deoxythymidylyl-(35)-2-deoxythymidine - dTp[]dT cyclobutane type photodimers of dTpdT - dTp- and dTp[]- their 5' terminal fragments (fragment A) - -pdT and-[]pdT their 3 terminal fragments (fragment B) - RP-HPLC reversed-phase high-performance liquid chromatography - COSY two-dimensional correlated spectroscopy - 2D NOE two-dimensional nuclear Overhauser spectroscopy     

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.     

Reductive activation of the methyl-tetrahydromethanopterin: coenzyme M methyltransferase from Methanobacterium thermoautotrophicum strain ΔH

The enzymatic conversion of formaldehyde to CH₃S-CoM in crude extracts of Methanobacterium thermoautotrophicum was used as a means to investigate the methyl-tetrahydromethanopterin: HS-CoM methyltransferase reaction. All components necessary for formaldehyde conversion were shown to be present in a soluble protein fraction. This soluble cell fraction still contained a major amount of corrinoids. Apart from tetrahydromethanopterin no other soluble cofactors were required for formaldehyde conversion. The dependence of the system on catalytic amounts of ATP was shown to be specific. Several nucleoside triphosphates or ADP were unable to substitute for ATP. Remarkably, various strong reducing systems, especially titanium(III)citrate could replace ATP to a large extent. The ATP-dependent formaldehyde conversion to CH₃S-CoM was inhibited in the presence of nitrous oxide, detergents or 2,3-dialdehyde-ATP. The results support a role for a corrinoid protein in the methyl-tetrahydromethanopterin: HS-CoM methyltransferase reaction at which ATP is involved in the activation of this protein, probably in the conversion of inactive B_12a or B_12r to active B_12s.Abbreviations HS-CoM Coenzyme M, 2-mercaptoethanesulfonate - CH₃S-CoM methylcoenzyme M, 2-(methylthio)ethanesulfonate - H₄MPT 5,6,7,8-tetrahydromethanopterin - BES 2-bromoethanesulfonate - BCE boiled cell-free extract - DTT dithiothreitol - TCS 3,3,4,5-tetrachlorosalicylanilide - DNTB 2,2-dinitro-5,5-dithiobenzoic acid - TES N-tris(hydroxymethyl)methyl-2-aminoethanesulfonate - HEPES N-2-hydroxyethylpiperazine-N-2-ethanesulfonic acid - PIPES piperazine-N,N-bis[2-ethanesulfonic acid] - AMP-PNP 5-adenylyl imidophosphate