共查询到20条相似文献,搜索用时 62 毫秒
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TE Roche SL Powers-Greenwood WF Shi WB Zhang SZ Ren ED Roche DJ Cox CM Sorensen 《Canadian Metallurgical Quarterly》1993,32(21):5629-5637
Quasielastic light scattering (QELS) measurements on several preparations of bovine heart and kidney pyruvate dehydrogenase complex yielded hydrodynamic radii (rH values) ranging from 25.7 to 30 nm. Gel filtration chromatography removed stable aggregates and generated preparations that gave essentially the same rH values of 24.3 +/- 0.6 nm for both complexes. The data were characteristic of a monodisperse system and agree with estimates using cryoelectron microscopy [Wagenknecht et al. (1991) J. Biol. Chem. 266, 24650-24656]. The equivalent hydrodynamic sizes for the heart and kidney complex indicate that the larger number of pyruvate dehydrogenase components in the heart complex (M(r) congruent to 9 x 10(6)) than the kidney complex (M(r) congruent to 7.5 x 10(6)) associate without radial expansion of the heart complex. That accommodation of additional mass is consistent with the space available since even in the more massive complex greater than 80% of the volume within the dimensions of the complex must be occupied by solvent. Preparations of the core of the complex are primarily composed of 60 dihydrolipoyl acetyltransferase (E2) subunits whose inner domains associate to form a pentagonal dodecahedron that is readily observed by electron microscopy (particle radius 10.7-11.3 nm). However, the bulk of E2's mass is present in an exterior multidomain structure. These mobile outer structures are very difficult to observe by standard electron microscopy techniques. Preparations of the core formed stable aggregates that were removed by gel filtration chromatography. QELS measurements gave an rH of 20.1 +/- 0.8 nm.(ABSTRACT TRUNCATED AT 250 WORDS) 相似文献
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The histidine triad (HIT) protein family is among the most ubiquitous and highly conserved in nature, but a biological activity has not yet been identified for any member of the HIT family. Fragile histidine triad protein (FHIT) and protein kinase C interacting protein (PKCI) were used in a structure-based approach to elucidate characteristics of in vivo ligands and reactions. Crystallographic structures of apo, substrate analog, pentacovalent transition-state analog, and product states of both enzymes reveal a catalytic mechanism and define substrate characteristics required for catalysis, thus unifying the HIT family as nucleotidyl hydrolases, transferases, or both. The approach described here may be useful in identifying structure-function relations between protein families identified through genomics. 相似文献
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Changes in the kinetic properties of homoserine dehydrogenase-I (HD-I) from Escherichia coli, caused by substitution of Na+ for the normal activating monovalent ion, K+, has been investigated by equilibrium isotope exchange kinetics (EIEK). HD-I, part of the aspartokinase/homoserine dehydrogenase-I complex, is one of the few dehydrogenases to exhibit allosteric feedback regulation and cation activation. EIEK methods are especially useful for definitively identifying which rate constants are altered by bound modifiers. Saturation curves for the [14C]Hse<-->ASA and [3H]NADP+<-->NADPH exchanges were compared in the presence of K+ vs Na+, varying different combinations of substrate pairs in constant ratio at equilibrium. Kinetic differences between the K+ and Na+ enzymes were analyzed systematically by simulations with the ISOBI program. This analysis clearly demonstrates that substituting Na+ for K+ shifts the kinetic mechanism from preferred order random to a nearly random order scheme, along with causing significant rate limitation at catalysis between the central complexes. Initial velocity kinetics demonstrate that HD-I has a 10-fold higher affinity for Na+ than K+, but that the Na(+)-enzyme is 10-fold less active and exhibits higher substrate Km values, especially for L-Hse. 相似文献
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Gln34, Gln224, Leu228, and Ser240 are conserved residues in the vicinity of bound IMP in the crystal structure of Escherichia coli adenylosuccinate synthetase. Directed mutations were carried out, and wild-type and mutant enzymes were purified to homogeneity. Circular dichroism spectroscopy indicated no difference in secondary structure between the mutants and the wild-type enzyme in the absence of substrates. Mutants L228A and S240A exhibited modest changes in their initial rate kinetics relative to the wild-type enzyme, suggesting that neither Leu228 nor Ser240 play essential roles in substrate binding or catalysis. The mutants Q224M and Q224E exhibited no significant change in KmGTP and KmASP and modest changes in KmIMP relative to the wild-type enzyme. However, kcat decreased 13-fold for the Q224M mutant and 10(4)-fold for the Q224E mutant relative to the wild-type enzyme. Furthermore, the Q224E mutant showed an optimum pH at 6.2, which is 1.5 pH units lower than that of the wild-type enzyme. Tryptophan emission fluorescence spectra of Q224M, Q224E, and wild-type proteins under denaturing conditions indicate comparable stabilities. Mutant Q34E exhibits a 60-fold decrease in kcat compared with that of the wild-type enzyme, which is attributed to the disruption of the Gln34 to Gln224 hydrogen bond observed in crystal structures. Presented here is a mechanism for the synthetase, whereby Gln224 works in concert with Asp13 to stabilize the 6-oxyanion of IMP. 相似文献
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A Kittaka T Kuze T Asakura K Ito T Miyasaka J Inoue 《Canadian Metallurgical Quarterly》1998,8(22):3207-3210
5-Formyl-2'-O-methyluridine was incorporated into the various positions of oligonucleotide 26-mers containing the NF-kappa B binding sequence. Some of them showed binding selectivity toward the homo- and heterodimers of subunits of NF-kappa B. 相似文献
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The fluorescent characteristic and emission spectra of Eu~(2+) in the cubic structureCa_8Zn(SiO_4)_4Cl_2 with three kinds of cation sites is reported.The influence of temperature,Eu~(2+) concentra-tion and excitation conditions on fluorescent properties of Eu~(2+) are studied at 77 and 298 K.Thecoordination number of Eu~(2+) at different sites is obtained.The green and red emission bands arise fromEu_(2+) ions locating on eight- and six- coordinated inequivalent sites respectively. 相似文献
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X Zhu X Zhao WF Burkholder A Gragerov CM Ogata ME Gottesman WA Hendrickson 《Canadian Metallurgical Quarterly》1996,272(5268):1606-1614
DnaK and other members of the 70-kilodalton heat-shock protein (hsp70) family promote protein folding, interaction, and translocation, both constitutively and in response to stress, by binding to unfolded polypeptide segments. These proteins have two functional units: a substrate-binding portion binds the polypeptide, and an adenosine triphosphatase portion facilitates substrate exchange. The crystal structure of a peptide complex with the substrate-binding unit of DnaK has now been determined at 2.0 angstroms resolution. The structure consists of a beta-sandwich subdomain followed by alpha-helical segments. The peptide is bound to DnaK in an extended conformation through a channel defined by loops from the beta sandwich. An alpha-helical domain stabilizes the complex, but does not contact the peptide directly. This domain is rotated in the molecules of a second crystal lattice, which suggests a model of conformation-dependent substrate binding that features a latch mechanism for maintaining long lifetime complexes. 相似文献
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Cytosolic group IV phospholipase A2 (cPLA2) plays a role in liberating arachidonic acid from the sn-2 position of mammalian cellular phospholipids. The enzyme consists of a catalytic domain joined to an N-terminal calcium-dependent, membrane binding domain (C2 domain). The interfacial binding properties of the full-length, nonphosphorylated enzyme and its C2 domain to phospholipid vesicles were studied as a function of vesicle phospholipid composition and calcium concentration. The binding of cPLA2 to phosphatidylcholine vesicles is mostly governed by its C2 domain; binding is relatively weak, and calcium enhances binding and interfacial catalysis by about 10-fold. Catalytically productive interfacial binding was measured by monitoring the increase in the rate of cPLA2-catalyzed hydrolysis of a fluorimetric substrate present in vesicles as a function of bulk vesicle concentration. Enzyme-vesicle binding was also measured by fluorescence as was enzyme-calcium binding. Compared to zwitterionic vesicles, cPLA2 binding to anionic phosphatidylmethanol vesicles is of higher affinity and calcium-independent, although calcium is required for the binding of the C2 domain to these anionic vesicles. cPLA2 is fully catalytically active on phosphatidylmethanol vesicles in the absence of calcium. Phosphatidylserine is not a good replacement for phosphatidylmethanol for inducing high-affinity, calcium-independent binding of cPLA2. These results reveal two modes of catalytically productive interfacial binding of cPLA2: calcium-dependent anchoring via the C2 domain and a calcium-independent component involving a phosphatidylmethanol recognition element in the catalytic domain. They also show that membrane binding of cPLA2 is not, in general, predicted by the interfacial binding properties of its C2 domain. 相似文献
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The aim of the work was to show the usefulness of neurological and radiological signs in the patients with L4 and L5 discopathy. The axial symptoms with the highest occurrence frequency and the differential symptoms closely connected with definite disc pathology: the type and/or the level of discopathy were defined. The problem of importance of the above-mentioned signs in the diagnostic management was discussed on the basis of literature. The significance of bilateral and polyradicular symptoms in the diagnosis of central lumbar disc and unilateral symptoms in the diagnosis of lateral lumbar disc were emphasized. 相似文献
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L Sonderbye S Meehan R Palsson N Ahsan J Ladefoged E Langhoff 《Canadian Metallurgical Quarterly》1998,65(7):1004-1008
The distribution of FST(i) estimates were studied in representative samples of ith genes for the gene pools of the major human populations, including the populations of Europe, Asia, Africa, Australia, America, North-eastern Eurasia, and five subregions of the latter. An average of 80 FST(i) estimates were analyzed for each sample of marker genes with a level of polymorphism (q) from 0.05 to 0.95. For each gene pool, the empirical distributions of FST(i) estimates were approximated by the main types of theoretical distributions--the normal, chi 2, Weibull, gamma, and beta distributions. In all gene pools, only beta distributions were good approximations of the empirical FST(i) distributions. The parameters of the beta distributions are reported in this study. It was demonstrated that the characteristics of beta distributions for all major gene pools studied could be interpolated to the smaller constituent gene pools. since the use of the traditional parametric tests starts from an assumption of normal distribution, they are inapplicable to analysis of FST statistics. Therefore, the obtained parameters of beta distributions should be used. These parameters allow the confidence intervals of the average FST values to be determined and permit correct comparison between the characteristics of both individual genes and gene pools to be performed. 相似文献
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