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61.
A triple-resonance NMR technique suitable for the determination ofcarbonyl-related couplings in polypeptide systems is introduced. Theapplication of three novel pulse sequences to uniformly13C/15N-enriched proteins yields E.COSY-likemultiplet patterns exhibiting either one of the3J(Ci–1,Hi ), 3J(Ci–1,Ci ) and3J(Ci–1,Ci)coupling constants in the indirectly detected 13Cdimension, depending on the passive spin selected. The experiments aredemonstrated with oxidized flavodoxin from Desulfovibrio vulgaris. On thebasis of the J-values measured and the backbone -angles derived from ahigh-resolution X-ray structure of the protein, the three associated Karplusequations were reparametrized. The root-mean-square differences between theexperimental coupling constants and those predicted by the optimized Karpluscurves are 0.41, 0.33 and 0.32 Hz for3J(Ci–1,Hi ),3J(Ci–1,Ci ) and3J(Ci–1,Ci),respectively. The results are compared with the Karplus parameters previouslypublished for the same couplings.  相似文献   
62.
63.
Abstract: Given the extreme lability and the facile inactivation of the messenger nitric oxide (NO) by many reactive biochemical species, it has been suggested that some intermediate compounds, for example, S -nitrosothiols, may act to stabilize NO and at the same time to preserve its biological activity. To test this hypothesis, we investigated if the S -nitrosothiol of glutathione, which is the predominant low molecular weight thiol in CNS, is present in the rat brain. The HPLC analysis of cerebellar extract from [35S]cysteine-prelabeled slices suggested that S -nitrosoglutathione (GSNO) was indeed present in rat brain. To detect endogenous GSNO, a methodology based on liquid chromatography-mass spectrometry was developed. Besides an unequivocal identification of the endogenous GSNO, this method also permitted its precise quantification using 15N-labeled GSNO ([15N]-GSNO) as internal standard. GSNO level in adult cerebellum amounts to 15.4 ± 1.4 pmol/mg of protein. This is the first direct demonstration of the presence of endogenous GSNO in CNS. The packaging of NO in the form of GSNO might serve to facilitate its transport, prolong its life, and target its delivery to specific effectors.  相似文献   
64.
The prediction experiment reveals that fold recognition has become a powerful tool in structural biology. We applied our fold recognition technique to 13 target sequences. In two cases, replication terminating protein and prosequence of subtilisin, the predicted structures are very similar to the experimentally determined folds. For the first time, in a public blind test, the unknown structures of proteins have been predicted ahead of experiment to an accuracy approaching molecular detail. In two other cases the approximate folds have been predicted correctly. According to the assessors there were 12 recognizable folds among the target proteins. In our postprediction analysis we find that in 7 cases our fold recognition technique is successful. In several of the remaining cases the predicted folds have interesting features in common with the experimental results. We present our procedure, discuss the results, and comment on several fundamental and technical problems encountered in fold recognition. © 1995 Wiley-Liss, Inc.  相似文献   
65.
High performance liquid chromatography (HPLC), a valuable tool for characterization of peptides, is frequently used in combination with sensitive radioimmunoassays (RIA). The shadow phenomenon, representing carry-over of the peptide from previous application of the standard, can appear to result in the presence of endogenous peptide in the test sample when none is actually there. With delta sleep-inducing peptide (DSIP), we found the shadowing to be as high as 10%, although it was only 1% with 125I-Tyr-DSIP. Thus, when HPLC-RIA systems are used for identification of peptides, caution must be used to avoid false positive results.  相似文献   
66.
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   
67.
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   
68.
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   
69.
A variant of the carcinoembryonic antigen (CEA) with lower molecular weight than a CEA reference preparation has been separated from CEA. Using a polyclonal, spleen absorbed anti-CEA antiserum, the variant crossreacts with reference CEA in immunodiffusion. The CEA-activity of the variant has been demonstrated using an enzyme-immunoassay with monoclonal CEA specific antibodies. There is sufficient immunological evidence that this variant is a distinct antigen different from the crossreactive antigens described so far. The reactivity of the polyclonal anti-CEA antiserum with the CEA variant was abolished by absorption against the immobilized variant.  相似文献   
70.
In this study we examined the effect of the phorbol ester 12-O-tetradecanoyl-phorbol-13-acetate (TPA) on the bumetanide-sensitive Na+/K+/Cl- transporter in quiescent BALB/c 3T3 cells. We have shown that exposure of quiescent BALB/c 3T3 cultures to phorbol ester did not inhibit the basal bumetanide-sensitive Rb+ influx or efflux. In fact, at high concentration (100 ng/ml), TPA slightly stimulated the bumetanide-sensitive Rb+ influx and efflux. However, when the quiescent cultures were stimulated by serum or by defined growth factors, the stimulated fraction of the bumetanide-sensitive Rb+ influx was drastically inhibited by exposure of the cells to the phorbol ester TPA. Based on the above findings, we propose that activation of protein kinase C by the phorbol ester TPA does not inhibit the Na+/K+/Cl- cotransport activity; however it does suppress only the growth-factors-stimulated fraction of the cotransport in quiescent BALB/c 3T3 cells. These data propose that activation of kinase C has a regulatory feedback effect on the stimulation of the Na+/K+/Cl- cotransport activity by growth factors.  相似文献   
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