Evidence that the heterodisulfide of coenzyme M and 7-mercaptoheptanoylthreonine phosphate is a product of the methylreductase reaction in Methanobacterium

When 7-mercaptoheptanoylthreonine phosphate (HS-HTP) was used as the sole source of electrons for reductive demethylation of 2-(methylthio)-ethanesulfonic acid (CH3-S-CoM) by cell extracts of Methanobacterium thermoautotrophicum strain delta H, the heterodisulfide of coenzyme M and HS-HTP (CoM-S-S-HTP) was quantitatively produced: HS-HTP + CH3-S-CoM----CH4 + CoM-S-S-HTP. CH4 and CoM-S-S-HTP were produced stoichiometrically in a ratio of 1:1. Coenzyme M (HS-CoM) inhibited HS-HTP driven methanogenesis indicating that CH3-S-CoM rather than HS-CoM was the substrate for CoM-S-S-HTP formation.     

A simplified methylcoenzyme M methylreductase assay with artificial electron donors and different preparations of component C from Methanobacterium thermoautotrophicum delta H.

Different preparations of the methylreductase were tested in a simplified methylcoenzyme M methylreductase assay with artificial electron donors under a nitrogen atmosphere. ATP and Mg2+ stimulated the reaction. Tris(2,2'-bipyridine)ruthenium (II), chromous chloride, chromous acetate, titanium III citrate, 2,8-diaminoacridine, formamidinesulfinic acid, cob(I)alamin (B12s), and dithiothreitol were tested as electron donors; the most effective donor was titanium III citrate. Methylreductase (component C) was prepared by 80% ammonium sulfate precipitation, 70% ammonium sulfate precipitation, phenyl-Sepharose chromatography, Mono Q column chromatography, DEAE-cellulose column chromatography, or tetrahydromethanopterin affinity column chromatography. Methylreductase preparations which were able to catalyze methanogenesis in the simplified reaction mixture contained contaminating proteins. Homogeneous component C obtained from a tetrahydromethanopterin affinity column was not active in the simplified assay but was active in a methylreductase assay that contained additional protein components.     

An unusual thiol-driven fumarate reductase in Methanobacterium with the production of the heterodisulfide of coenzyme M and N-(7-mercaptoheptanoyl)threonine-O3-phosphate

An unusual fumarate reductase was purified from cell extracts of Methanobacterium thermoautotrophicum and partially characterized. Two coenzymes previously isolated from cell extracts, 2-mercaptoethane-sulfonic acid (HS-CoM) and N-(7-mercaptoheptanoyl)threonine-O3-phosphate (HS-HTP), were established as direct electron donors for fumarate reductase. By measuring the consumption of free thiol, we determined that fumarate reductase catalyzed the oxidation of HS-CoM and HS-HTP; by the direct measurement of succinate and the heterodisulfide of HS-CoM and HS-HTP (CoM-S-S-HTP), we established that these compounds were products of the fumarate reductase reaction. A number of thiol-containing compounds did not function as substrates for fumarate reductase, but this enzyme had high specific activity when HS-CoM and HS-HTP were used as electron donors. HS-CoM and HS-HTP were quantitatively oxidized by the fumarate reductase reaction, and results indicated that this reaction was irreversible. Additionally, by measuring formylmethanofuran, we demonstrated that the addition of fumarate to cell extracts activated CO2 fixation for the formation of formylmethanofuran. Results indicated that this activation resulted from the production of CoM-S-S-HTP (a compound known to be involved in the activation of formylmethanofuran synthesis) by the fumarate reductase reaction.     

Endothelin-1 and endothelin-3 stimulate calcium mobilization by different mechanisms in vascular smooth muscle.

The mechanisms by which endothelin-1 (ET-1) and endothelin-3 (ET-3) stimulate Ca2+ mobilization were investigated in rat aortic smooth muscle cells. Both ET-1 and ET-3 potently stimulated mobilization of Ca2+ from intracellular stores, however only ET-1-stimulated Ca2+ mobilization appeared to occur as a consequence of an elevation in cellular inositol trisphosphate (IP3) concentration. Neomycin, an inhibitor of phospholipase C, inhibited both the increase in [3H]IP3 formation and the mobilization of Ca2+ induced by ET-1, however it did not affect Ca2+ mobilization induced by ET-3. Together these findings indicate that ET-1 stimulates Ca2+ mobilization via an increase in IP3, whereas the effect of ET-3 appears to be mediated by a separate, IP3-independent signalling pathway.     

Effects of noradrenergic neuronal activity on 3,4-dihydroxyphenylethylene glycol (DHPG) levels. Quantitation by high performance liquid chromatography

We describe a sensitive specific and simple high pressure liquid chromatographic procedure for determining 3,4-dihydroxyphenylethylene glycol (DHPG) in both plasma and tissue. DHPG is extracted from plasma or tissue extracts by adsorption onto alumina. DHPG in the alumina eluate is detected electrochemically following chromatography on a C₁₈ reverse phase column. The method is sensitive enough to detect approximately 20 pg/ml of plasma DHPG. Both clonidine (100 μg/kg) and desmethylimipramine (2.5 mg/kg) when administered to rabbits for 3 days induced significant falls in both cardiac and plasma DHPG concentrations. These experiments indicate that both tissue and plasma DHPG concentrations may be of value in assessing both the release and re-uptake of norepinephrine at peripheral sympathetic nerve endings in vivo.     

Growth of Rhodospirillum rubrum on synthesis gas: conversion of CO to H2 and poly-beta-hydroxyalkanoate

To examine the potential use of synthesis gas as a carbon and energy source in fermentation processes, Rhodospirillum rubrum was cultured on synthesis gas generated from discarded seed corn. The growth rates, growth and poly-beta-hydroxyalkanoates (PHA) yields, and CO oxidation/H(2) evolution rates were evaluated in comparison to the rates observed with an artificial synthesis gas mixture. Depending on the gas conditioning system used, synthesis gas either stimulated or inhibited CO-oxidation rates compared to the observations with the artificial synthesis gas mixture. Inhibitory and stimulatory compounds in synthesis gas could be removed by the addition of activated charcoal, char-tar, or char-ash filters (char, tar, and ash are gasification residues). In batch fermentations, approximately 1.4 mol CO was oxidized per day per g cell protein with the production of 0.75 mol H(2) and 340 mg PHA per day per g cell protein. The PHA produced from R. rubrum grown on synthesis gas was composed of 86% beta-hydroxybutyrate and 14% beta-hydroxyvalerate. Mass transfer of CO into the liquid phase was determined as the rate-limiting step in the fermentation.     

Functional genomic, biochemical, and genetic characterization of the Salmonella pduO gene, an ATP:cob(I)alamin adenosyltransferase gene

Salmonella enterica degrades 1,2-propanediol by a pathway dependent on coenzyme B12 (adenosylcobalamin [AdoCb1]). Previous studies showed that 1,2-propanediol utilization (pdu) genes include those for the conversion of inactive cobalamins, such as vitamin B12, to AdoCbl. However, the specific genes involved were not identified. Here we show that the pduO gene encodes a protein with ATP:cob(I)alamin adenosyltransferase activity. The main role of this protein is apparently the conversion of inactive cobalamins to AdoCbl for 1,2-propanediol degradation. Genetic tests showed that the function of the pduO gene was partially replaced by the cobA gene (a known ATP:corrinoid adenosyltransferase) but that optimal growth of S. enterica on 1,2-propanediol required a functional pduO gene. Growth studies showed that cobA pduO double mutants were unable to grow on 1,2-propanediol minimal medium supplemented with vitamin B(12) but were capable of growth on similar medium supplemented with AdoCbl. The pduO gene was cloned into a T7 expression vector. The PduO protein was overexpressed, partially purified, and, using an improved assay procedure, shown to have cob(I)alamin adenosyltransferase activity. Analysis of the genomic context of genes encoding PduO and related proteins indicated that particular adenosyltransferases tend to be specialized for particular AdoCbl-dependent enzymes or for the de novo synthesis of AdoCbl. Such analyses also indicated that PduO is a bifunctional enzyme. The possibility that genes of unknown function proximal to adenosyltransferase homologues represent previously unidentified AdoCbl-dependent enzymes is discussed.     

Regulation of inositol-1,4,5-triphosphate receptor by G(i)-protein

The pertussis toxin (PT) inhibits thrombin-induced mobilising of Ca2+ but does not affect formation of inositol-1, 4, 5-triphosphate (IPh3). The latter being related to Gi and G0, the possibility of a direct effect of these proteins on the IPh3-induced release of Ca2+ from sarcoplasmic reticulum, was studied. The data obtained suggest existence of an association of the Gi3 protein with the IPh3-P on the sarcoplasmic reticulum membrane, as well as a direct modulation by the Gi protein of the Ca2+ release from intracellular stores.