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101.
Methanogenesis from acetate by a rod-shaped enrichment culture grown at 60° C was found to require the presence of two organisms rather than a single aceticlastic methanogen. A thermophilic Methanobacterium which grew on H2/CO2 or formate was isolated from the enrichment. Lawns of this methanogen were used to co-isolate an acetate oxidizer in roll tubes containing acetate agar. The rod-shaped acetate oxidizer was morphologically distinct from the methanogen and did not show F420 autofluorescence. The coculture completely degraded 40 mol/ml acetate, and produced nearly equal quantities of methane, and methanogenesis was coupled with growth. The doubling time for the coculture at 60°C was 30–40 h and the yield was 2.7±0.3 g dry wt/mol CH4. Studies with 14C-labelled substrates showed that the methyl group and the carboxyl group of acetate were both converted primarily to CO2 by the coculture and that CO2 was concurrently reduced to CH4. During growth, there was significant isotopic exchange between CO2 and acetate, especially with thecarboxyl position of acetate. These results support a mechanism for methanogenesis from acetate by the coculture in which acetate was oxidized to CO2 and H2 by one organism, while H2 was subsequently used by a second organism to reduce CO2 to CH4. Since the H2 partial pressure must be maintained below 10-4 atm by the methanogen for acetate oxidation to be thermodynamically feasible, this is an example of obligate interspecies hydrogen transfer. This mechanism was originally proposed for a single organism by Barker in 1936. 相似文献
102.
Edward Bańkowski Wiktor Rzeczycki Henry K. F. Nowak Kazimierz J. Jodczyk 《Molecular and cellular biochemistry》1978,20(2):77-83
Summary Rat kidney fibroblasts transformed with SV-40 produce in vitro a significantly lower amount of hydroxyproline-containing material which is collagenase sensitive as compared to normal cells. In contrast to normal fibroblast cultures, no collagenous material was found by histochemical methods in intercellular spaces of transformed cultures. 相似文献
103.
M. A. Moskowitz Deborah Rubin J. Liebschutz H. N. Munro T. S. Nowak R. J. Wurtman 《Journal of neurochemistry》1977,28(4):779-782
Abstract— l -DOPA or d -amphetamine administration disaggregates brain polyribosomes in animals maintained in an environment warm enough (26°C) so that the drugs concurrently elevate their body temperatures to above 39°C. The production of equivalent hyperthermia (by keeping control rats at ambient temperatures of 40–44° C) does not cause similar disaggregation of brain polysomes. Hence, the role of hyperthermia in the drug-induced disaggregation is permissive. 相似文献
104.
The formation of multiple ligand complexes with muscle pyruvate kinase was measured in terms of dissociation constants and the standard free energies of formation were calculated. The binding of Mn2+ to the enzyme (KA = 55 +/- 5 X 10(-6) M; deltaF degrees = -5.75 +/- 0.05 kcal/mol) and to the enzyme saturated with phosphoenolpyruvate (conditional free energy) KA' = 0.8 +/- 0.4 X 10(-6) M; deltaF degrees = -8.22 +/- 0.34 kcal/mol) has been measured under identical conditions giving a free energy of coupling, delta(deltaF degrees) = -2.47 +/- 0.34 kcal/mol. Such a large negative free energy of coupling is diagnostic of a strong positively cooperative effect in ligand binding. The binding of the substrate phosphoenolpyruvate to free enzyme and the enzyme-Mn2+ complex was, by necessity, measured by different methods. The free energy of phosphoenolpyruvate binding to free enzyme (KS = 1.58 +/- 0.10 X 10(-4)M; deltaF degrees = -5.13 +/- 0.04 kcal/mol) and to the enzyme-Mn2+ complex (K3 = 0.75 +/- 0.10 X 10(-6)M; deltaF degrees = -8.26 +/- 0.07 kcal/mol) also gives a large negative free energy of coupling, delta(deltaF degrees) = -3.16 +/- 0.08 kcal/mol. Such a large negative value confirms reciprocal binding effects between the divalent cation and the substrate phosphoenolpyruvate. The binding of Mn2+ to the enzyme-ADP complex was also investigated and a free energy of coupling, delta(deltaF degrees) = -0.08 +/- 0.08 kcal/mol, was measured, indicative of little or no cooperativity in binding. The free energy of coupling with Mn2+ and pyruvate was measured as -1.52 +/- 0.14 kcal/mol, showing a significant amount of cooperativity in ligand binding but a substantially smaller effect than that observed for phosphoenolpyruvate binding. The magnitude of the coupling free energy may be related to the role of the divalent cation in the formation of the enzyme-substrate complexes. In the absence of the activating monovalent cation, the coupling free energies for phosphoenolpyruvate and pyruvate binding decrease by 40-60% and 25%, respectively, substantiating a role for the monovalent cation in the formation of enzyme-substrate complexes with phosphoenolpyruvate and with pyruvate. 相似文献
105.
Affinity chromatography purification of Pseudomonas aeruginosa exotoxin A on specifically linked NAD agarose.
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The specific binding of P. aeruginosa exotoxin A to NAD was exploited for the rapid purification of the toxin. Affinity chromatography on a column of agarose-N6-(aminohexyl)carbamoylmethyl-NAD resulted in an enzymatically, biologically, and immunologically active purified toxin preparation. Other NAD-agarose resins were not efficient substrates for toxin purification. 相似文献
106.
Duck and goose fibrinogen were isolated from fresh pooled plasma by three different methods. To minimize proteolytic activity, epsilon-aminocaproic acid and trasylol were used throughout the preparation procedures. Amino acid composition of fibrinogens and carbohydrate content (hexose, hexosamine, sialic acid) as well as phosphorus were analysed. Intact preparations showed single band on SDS-polyacrylamide gel electrophoresis. After reduction and modification of the thiol groups, the material could be separated by SDS-polyacrylamide gel electrophoresis into four bands corresponding to the gamma, partially degraded A alpha, B beta and intact A alpha chain. Intact polypeptide subunits were separated by ion-exchange chromatography or preparative SDS-polyacrylamide gel electrophoresis and their amino acid compositions were determined. Evidences supporting the view that bird fibrinogen is very sensitive to proteolytic degradation and that a partial degradation of the A alpha chain takes place even when inhibitors are used in all steps of the purification procedures are presented. 相似文献
107.
Analysis of progress curves. Rate law of pyruvate kinase type I from Escherichia coli. 总被引:3,自引:2,他引:1
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Progress curves of the reaction catalysed by pyruvate kinase from Escherichia coli K12, designed to cover the four-dimensional concentration space of phosphoenolpyruvate, ADP, Mg2+ and ATP in the regulatory region, were recorded with the pH-stat method (pH 7.0 and 25 degrees C). Additional initial-rate measurement were performed to assess specific points. Two methods for the evaluation of progress curves were used: fitting the rate law to the rates obtained from the tangents of the progress curves and fitting the integrated rate law directly to the curves. Two models, both extensions of the concerted model given by Monod, Wyman & Changeux [(1965) J. Mol. Biol. 12, 88--118] with four protomers, could be fitted to the data within the experimental error. Model discrimination in favour of one of these models was possible by proper experimental design. In the selected model one conformational state of the enzyme forms the active complex. The active site of a second conformational state forms abortive complexes with Mg2+, causing strong inhibition at high Mg2+ concentrations. In the absence of ligands, most of the enzyme is in a third state that binds ATP at an allosteric site. 相似文献
108.
James P. Kutney John Balsevich Robert Carruthers Arie Markus Michael J. McGrath Robert N. Young Brian R. Worth 《Bioorganic chemistry》1978,7(3):289-302
Syntheses of the C8 and C10 olefinic units cis- and trans-5-ethyl-1-iodo-hex-4-enes and cis- and trans-7-ethyl-3-iodo-oct-6-enes are described. The Wittig coupling of such units with derivatives of α- and β-thujaketonic acids to give analogs of insect juvenile hormones is discussed. 相似文献
109.
Systematic thermodynamic studies have been conducted for flavin (FMN, FAD) binding to purified riboflavin-binding proteins from hen egg white and egg yolk. These studies were conducted under a variety of temperature (14, 26, and 38 °C), pH (4.5, 5.5, 6.5, 7.4, and 9.0), and buffer conditions, and an extensive thermodynamic profile was constructed. Enthalpies of binding FMN to white riboflavin-binding protein and yolk riboflavin-binding protein were ?19.3 and ?14.4 kcal/mol, respectively, at pH 7.4 and 38 °C. FAD bound to white and yolk riboflavin-binding proteins under the same conditions with ΔH values of ?11.7 and ?6.0, respectively. Binding constants of about 105 and 104 were obtained for FMN and FAD, respectively, and were the same for both proteins under all conditions studied. Using established thermodynamic relationships, we were able to calculate entropy and free energy changes. Entropies indicated a large degree of ordering in the system upon flavin binding with FMN (about ?40 cal/mol/ °C) twice as large as FAD (about ?15 to ?25 cal/mol/ °C), which may indicate a structured solvent interaction with the charged phosphate group, or steric limitations placed on the ribityl side chain in the bound state. Our thermodynamic data support the idea that flavin binding is a mixture of forces, with no one predominant. Analysis of the data suggests that the nucleotide may bind both as the mono- or dianion, that flavin binding occurs with no significant change in the pK of any functional group in the system, except at low pH for FAD binding, and that the temperature variation of the enthalpy change is quite small. These findings are combined with other published data to outline a general scheme of flavin binding with a histidine residue implicated in hydrogen bonding to the adenine portion of FAD, which may be in the unstacked form. 相似文献
110.
S Gutowski A Smrcka L Nowak D G Wu M Simon P C Sternweis 《The Journal of biological chemistry》1991,266(30):20519-20524
The subfamily of guanine nucleotide-binding regulatory (G proteins) designated Gq has been shown to regulate the activity of phospholipase C by reconstitution. However, the role of these proteins in hormonal regulation of this activity has not been demonstrated. Two antisera were used in attempts to interrupt this pathway. Antiserum W082, developed against a peptide representing an internal sequence in alpha q, was specific for alpha q by immunoblots but did not recognize the native protein. Antiserum X384 was developed against a peptide representing the 12 amino acids of the common carboxyl termini of alpha q and alpha 11. It had a broader specificity for this subfamily of G protein alpha subunits and recognized the native proteins. Antiserum X384 specifically immunoprecipitated alpha q and its homologs from purified preparations and detergent extracts of membranes. Affinity-purified antibodies attenuated stimulation of phosphatidylinositide 4,5-bisphosphate hydrolysis by bradykinin, angiotensin, and histamine in membranes derived from NG108-15 cells, rat liver, and 1321N1 cells, respectively. Activation of the phospholipase C activity by guanosine 5'-3-O-(thio)triphosphate alone was also inhibited. Inclusion of the peptide to which the antisera were raised blocked the effect of the antibody. In contrast, affinity-purified W082, which did not recognize native proteins, did not alter regulation of phospholipase C. This indicates that the Gq family of signaling proteins can couple to several receptors and is responsible for the hormonal regulation of phospholipase C in these diverse systems. The further generality of this regulatory pathway remains to be established. 相似文献