Haloanaerobacter salinarius sp. nov., a novel halophilic fermentative bacterium that reduces glycine-betaine to trimethylamine with hydrogen or serine as electron donors; emendation of the genus Haloanaerobacter.
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A novel halophilic fermentative bacterium has been isolated from the black sediment below a gypsum crust and a microbial mat in hypersaline ponds of Mediterranean salterns. Morphologically, physiologically and genetically this organism belongs to the genus Haloanaerobacter. Haloanaerobacter strain SG 3903T (T = type strain) is composed of non-sporulating long flexible rods with peritrichous flagella, able to grow in the salinity range of 5-30% NaCl, with an optimum at 14-15%. The strain grows by fermenting carbohydrates or by using the Stickland reaction with either serine or H2 as electron donors and glycine-betaine as acceptor, which is reduced to trimethylamine. The two species described so far in the genus Haloanaerobacter are not capable of Stickland reaction with glycine-betaine + serine; however, Haloanaerobacter chitinovorans can use glycine-betaine with H2 as electron donor. Strain SG 3903T thus represents the first described strain in the genus Haloanaerobacter capable of the Stickland reaction with two amino acids. Although strain SG 3903T showed 67% DNA-DNA relatedness to H. chitinovorans, it is physiologically sufficiently different from the two described species to be considered as a new species which has been named Haloanaerobacter salinarius sp. nov. (+info)
Mechanisms of hypoxic vasodilatation of isolated rat mesenteric arteries: a comparison with metabolic inhibition.
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1. Hypoxia (PO2 < 5 mmHg) decreased vessel tone in isolated rat mesenteric arteries precontracted with either high [K+] or the thromboxane analogue U46619. This response was not altered by N-nitro-L-arginine (L-NA) and indomethacin. 2. Simultaneous measurement of pHi and tension showed that the decrease in vessel tone was accompanied by an intracellular acidification. Similar reductions in tone and pHi were observed with the metabolic inhibitors 2,4-dinitrophenol (DNP) and sodium azide. 3. The presence of the lactate transport inhibitor alpha-cyano-4-hydroxy-cinnamic acid (CHC) increased the magnitude of the acidification and resulted in a significantly faster reduction in tone in response to hypoxia. Addition of CHC to normoxic tissues caused both a vasodilatation and a reduction of pHi. 4. A decrease in pHi induced on washout of ammonium chloride (NH4Cl) resulted in an increase in tone. 5. Relaxation to hypoxia or metabolic inhibition was unaffected when the change in pHi was neutralized by addition of the weak base trimethylamine (TMA). 6. It is concluded that severe hypoxia decreases tone in isolated rat mesenteric arteries by a mechanism which is independent of nitric oxide and prostaglandins. Both severe hypoxia and metabolic inhibition reduced pHi, although this does not appear to be contributing to the changes in tone observed. (+info)
Trimethylamine N-oxide-induced cooperative folding of an intrinsically unfolded transcription-activating fragment of human glucocorticoid receptor.
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A number of biologically important proteins or protein domains identified recently are fully or partially unstructured (unfolded). Methods that allow studies of the propensity of such proteins to fold naturally are valuable. The traditional biophysical approaches using alcohols to drive alpha-helix formation raise serious questions of the relevance of alcohol-induced structure to the biologically important conformations. Recently we illustrated the extraordinary capability of the naturally occurring solute, trimethylamine N-oxide (TMAO), to force two unfolded proteins to fold to native-like species with significant functional activity. In the present work we apply this technique to recombinant human glucocorticoid receptor fragments consisting of residues 1-500 and residues 77-262. CD and fluorescence spectroscopy showed that both were largely disordered in aqueous solution. TMAO induced a condensed structure in the large fragment, indicated by the substantial enhancement in intrinsic fluorescence and blue shift of fluorescent maxima. CD spectroscopy demonstrated that the TMAO-induced structure is different from the alpha-helix-rich conformation driven by trifluoroethanol (TFE). In contrast to TFE, the conformational transition of the 1-500 fragment induced by TMAO is cooperative, a condition characteristic of proteins with unique structures. (+info)
Inhibition of secretion by 1,3-Cyclohexanebis(methylamine), a dibasic compound that interferes with coatomer function.
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We noted previously that certain aminoglycoside antibiotics inhibit the binding of coatomer to Golgi membranes in vitro. The inhibition is mediated in part by two primary amino groups present at the 1 and 3 positions of the 2-deoxystreptamine moiety of the antibiotics. These two amines appear to mimic the epsilon-amino groups present in the two lysine residues of the KKXX motif that is known to bind coatomer. Here we report the effects of 1, 3-cyclohexanebis(methylamine) (CBM) on secretion in vivo, a compound chosen for study because it contains primary amino groups that resemble those in 2-deoxystreptamine and it should penetrate lipid bilayers more readily than antibiotics. CBM inhibited coatomer binding to Golgi membranes in vitro and in vivo and inhibited secretion by intact cells. Despite depressed binding of coatomer in vivo, the Golgi complex retained its characteristic perinuclear location in the presence of CBM and did not fuse with the endoplasmic reticulum (ER). Transport from the ER to the Golgi was also not blocked by CBM. These data suggest that a full complement of coat protein I (COPI) on membranes is not critical for maintenance of Golgi integrity or for traffic from the ER to the Golgi but is necessary for transport through the Golgi to the plasma membrane. (+info)
A novel mechanism of ion homeostasis and salt tolerance in yeast: the Hal4 and Hal5 protein kinases modulate the Trk1-Trk2 potassium transporter.
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The regulation of intracellular ion concentrations is a fundamental property of living cells. Although many ion transporters have been identified, the systems that modulate their activity remain largely unknown. We have characterized two partially redundant genes from Saccharomyces cerevisiae, HAL4/SAT4 and HAL5, that encode homologous protein kinases implicated in the regulation of cation uptake. Overexpression of these genes increases the tolerance of yeast cells to sodium and lithium, whereas gene disruptions result in greater cation sensitivity. These phenotypic effects of the mutations correlate with changes in cation uptake and are dependent on a functional Trk1-Trk2 potassium transport system. In addition, hal4 hal5 and trk1 trk2 mutants exhibit similar phenotypes: (i) they are deficient in potassium uptake; (ii) their growth is sensitive to a variety of toxic cations, including lithium, sodium, calcium, tetramethylammonium, hygromycin B, and low pH; and (iii) they exhibit increased uptake of methylammonium, an indicator of membrane potential. These results suggest that the Hal4 and Hal5 protein kinases activate the Trk1-Trk2 potassium transporter, increasing the influx of potassium and decreasing the membrane potential. The resulting loss in electrical driving force reduces the uptake of toxic cations and improves salt tolerance. Our data support a role for regulation of membrane potential in adaptation to salt stress that is mediated by the Hal4 and Hal5 kinases. (+info)
The reaction of trimethylamine dehydrogenase with trimethylamine.
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The reductive half-reaction of trimethylamine dehydrogenase with its physiological substrate trimethylamine has been examined by stopped-flow spectroscopy over the pH range 6.0-11.0, with attention focusing on the fastest of the three kinetic phases of the reaction, the flavin reduction/substrate oxidation process. As in previous work with the slow substrate diethylmethylamine, the reaction is found to consist of three well resolved kinetic phases. The observed rate constant for the fast phase exhibits hyperbolic dependence on the substrate concentration with an extrapolated limiting rate constant (klim) greater than 1000 s-1 at pH above 8.5, 10 degrees C. The kinetic parameter klim/Kd for the fast phase exhibits a bell-shaped pH dependence, with two pKa values of 9.3 +/- 0.1 and 10. 0 +/- 0.1 attributed to a basic residue in the enzyme active site and the ionization of the free substrate, respectively. The sigmoidal pH profile for klim gives a single pKa value of 7.1 +/- 0. 2. The observed rate constants for both the intermediate and slow phases are found to decrease as the substrate concentration is increased. The steady-state kinetic behavior of trimethylamine dehydrogenase with trimethylamine has also been examined, and is found to be adequately described without invoking a second, inhibitory substrate-binding site. The present results demonstrate that: (a) substrate must be protonated in order to bind to the enzyme; (b) an ionization group on the enzyme is involved in substrate binding; (c) an active site general base is involved, but not strictly required, in the oxidation of substrate; (d) the fast phase of the reaction with native enzyme is considerably faster than observed with enzyme isolated from Methylophilus methylotrophus that has been grown up on dimethylamine; and (e) a discrete inhibitory substrate-binding site is not required to account for excess substrate inhibition, the kinetic behavior of trimethylamine dehydrogenase can be readily explained in the context of the known properties of the enzyme. (+info)
The role of Tyr-169 of trimethylamine dehydrogenase in substrate oxidation and magnetic interaction between FMN cofactor and the 4Fe/4S center.
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Tyr-169 in trimethylamine dehydrogenase is one component of a triad also comprising residues His-172 and Asp-267. Its role in catalysis and in mediating the magnetic interaction between FMN cofactor and the 4Fe/4S center have been investigated by stopped-flow and EPR spectroscopy of a Tyr-169 to Phe (Y169F) mutant of the enzyme. Tyr-169 is shown to play an important role in catalysis (mutation to phenylalanine reduces the limiting rate constant for bleaching of the active site flavin by about 100-fold) but does not serve as a general base in the course of catalysis. In addition, we are able to resolve two kinetically influential ionizations involved in both the reaction of free enzyme with free substrate (as reflected in klim/Kd), and in the breakdown of the Eox.S complex (as reflected in klim). In EPR studies of the Y169F mutant, it is found that the ability of the Y169F enzyme to form the spin-interacting state between flavin semiquinone and reduced 4Fe/4S center characteristic of wild-type enzyme is significantly compromised. The present results are consistent with Tyr-169 representing the ionizable group of pKa approximately 9.5, previously identified in pH-jump studies of electron transfer, whose deprotonation must occur for the spin-interacting state to be established. (+info)
Common components of patch-clamp internal recording solutions can significantly affect protein kinase A activity.
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Common components of whole-cell internal recording solutions were tested both in vitro and in patch-clamp experiments for their effects on the activity of cAMP-dependent protein kinase. Potassium fluoride (KF), 440 mM trimethylamine chloride and exclusion of bovine serum albumin (BSA) decreased the activity of the enzyme, while ethylene glycol-bis (beta-aminoethyl ether) N,N,N',N'-tetraacetic acid (EGTA) and the potassium salts of aspartate, gluconate, methylsulfate and monobasic phosphate increased its activity. Addition of KF to the internal solution produced a hyperpolarizing shift in the V1/2 of Ih channel activation, consistent with the KF-induced reduction of protein kinase A activity. Therefore, consideration of the composition of internal solutions is warranted when studying channel physiology by patch-clamp techniques. (+info)