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1.
O L Kon 《Biochimica et biophysica acta》1985,843(3):245-253
Isolated, intact rat liver nuclei have high-affinity (Kd = 10(-9) M) binding sites that are highly specific for nonsteroidal antiestrogens, especially for compounds of the triphenylethylene series. Nuclear [3H]tamoxifen binding capacity is thermolabile, being most stable at 4 degrees C and rapidly lost at 37 degrees C. More [3H]tamoxifen, however, is specifically bound at incubation temperatures of 25 degrees C and 37 degrees C than at 4 degrees C although prewarming nuclei has no effect, suggesting exchange of [3H]tamoxifen for an unidentified endogeneous ligand. Nuclear antiestrogen binding sites are destroyed by trypsin but not by deoxyribonuclease I or ribonuclease A. The nuclear antiestrogen binding protein is not solubilized by 0.6 M potassium chloride, 2 M sodium chloride, 0.6 M sodium thiocyanate, 3 M urea, 20 mM pyridoxal phosphate, 1% (w/v) digitonin or 2% (w/v) sodium cholate but is extractable by sonication, indicating that it is tightly bound within the nucleus. Rat liver nuclear matrix contains high-affinity (Kd = 10(-9) M) [3H]tamoxifen binding sites present in 5-fold higher concentrations (4.18 pmol/mg DNA) than in intact nuclei (0.78 +/- 0.10 (S.D.) pmol/mg DNA). Low-speed rat liver cytosol (20 000 X g, 30 min) contains high-capacity (955 +/- 405 (S.D.) fmol/mg protein), low-affinity (Kd = 10.9 +/- 4.5 (S.D.) nM) antiestrogen binding sites. In contrast, high-speed cytosol (100 000 X g, 60 min) contains low-capacity (46 +/- 15 (S.D.) fmol/mg protein), high-affinity (Kd = 0.61 +/- 0.20 (S.D.) nM) binding sites. Low-affinity cytosolic sites constitute more than 90% of total liver binding sites, high-affinity cytosolic sites 0.3%-3.2%, and nuclear sites less than 0.5% of total sites. 相似文献
2.
Evidence was obtained that four ionizable residues in the alpha and beta subunits of thermophilic ATP synthase (TF0F1), corresponding to Lys-21 and Asp-119 in the MgATP binding segments of adenylate kinase, are essential for the normal catalytic activity. TF0F1 was used because it is the only ATP synthase whose alpha-, beta- and gamma-subunits can be reassembled into an active complex in the absence of both ATP and Mg. Lys-164 and Asp-252 of its beta-subunit were modified to isoleucine and asparagine, respectively, by site-directed mutagenesis using a multifunctional plasmid, and these genes were over-expressed in Escherichia coli. The resulting beta I164 and beta N252 subunits were both noncatalytic after re-assembly into the alpha beta gamma-complex, even though both subunits bound significant amounts of ADP. When Lys-175 and Asp-261 of the alpha-subunit were similarly replaced by isoleucine and asparagine, respectively, the resulting alpha I175 subunit reassembled weakly into an oligomer, while the alpha N261 subunit showed an increased dissociation constant for ADP and was reconstituted into an alpha beta gamma-complex that showed no inter-subunit cooperativity. 相似文献
3.
4.
Two proteins serving as substrates for ADP-ribosylation catalyzed by islet-activating protein (IAP), pertussis toxin, and binding guanosine 5'-(3-O-thio)triphosphate (GTP gamma S) with high affinities were purified from the cholate extract of rat brain membranes. The purified proteins had the same heterotrimeric structure (alpha beta gamma) as the IAP substrates previously purified from rabbit liver and bovine brain and differed from each other in alpha only; the molecular weight of alpha was 41,000 (alpha 41 beta gamma) and 39,000 (alpha 39 beta gamma). Both were further resolved into alpha (alpha 41 or alpha 39) and beta gamma which were also purified to homogeneity to compare the activities of alpha-monomers with the original trimers. The maintenance of the rigid trimeric structure by combining alpha 41 or alpha 39 with beta gamma in the absence of Mg2+ was essential for the alpha-subunit to be ADP-ribosylated by IAP. The alpha-subunit was very stable but displayed the only partial GTP gamma S-binding activity under these conditions. Isolated alpha-monomers exhibited high GTPase activities when assayed in the presence of submicromolar Mg2+ but were very unstable at 30 degrees C and not ADP-ribosylated by IAP. The most favorable conditions for the GTP gamma S binding to alpha-subunits were achieved by combining alpha 41 or alpha 39 with beta gamma in the presence of millimolar Mg2+, probably due to the increase in stability and unmasking of the GTP-binding sites. There was no qualitative difference in these properties between alpha 41 beta gamma (alpha 41) and alpha 39 beta gamma (alpha 39). But alpha 39 beta gamma (or alpha 39) was usually more active than alpha 41 beta gamma (or alpha 41), at least partly due to its higher affinity for Mg2+ and lower affinity for beta gamma. Relation of these differences in activity between alpha 41 beta gamma and alpha 39 beta gamma to their physiological roles in signal transduction is discussed. 相似文献
5.
6.
S T Cooper P C Harkness E R Baker N S Millar 《The Journal of biological chemistry》1999,274(38):27145-27152
The predominant nicotinic acetylcholine receptor (nAChR) expressed in vertebrate brain is a pentamer containing alpha4 and beta2 subunits. In this study we have examined how temperature and the expression of subunit chimeras can influence the efficiency of cell-surface expression of the rat alpha4beta2 nAChR. Functional recombinant alpha4beta2 nAChRs, showing high affinity binding of nicotinic radioligands (K(d) = 41 +/- 22 pM for [(3)H]epibatidine), are expressed in both stably and transiently transfected mammalian cell lines. Despite this, only very low levels of alpha4beta2 nAChRs can be detected on the cell surface of transfected mammalian cells maintained at 37 degrees C. At 30 degrees C, however, cells expressing alpha4beta2 nAChRs show a 12-fold increase in radioligand binding (with no change in affinity), and a 5-fold up-regulation in cell-surface receptors with no increase in total subunit protein. In contrast to "wild-type" alpha4 and beta2 subunits, chimeric nicotinic/serotonergic subunits ("alpha4chi" and "beta2chi") are expressed very efficiently on the cell surface (at 30 degrees C or 37 degrees C), either as hetero-oligomeric complexes (e.g. alpha4chi+beta2 or alpha4chi+beta2chi) or when expressed alone. Compared with alpha4beta2 nAChRs, expression of complexes containing chimeric subunits typically results in up to 20-fold increase in nicotinic radioligand binding sites (with no change in affinity) and a similar increase in cell-surface receptor, despite a similar level of total chimeric and wild-type protein. 相似文献
7.
The hyperthermophilic bacterium, Thermotoga maritima, grows up to 90 degrees C by fermenting carbohydrates and it disposes of excess reductant by H(2) production. The H(2)-evolving cytoplasmic hydrogenase of this organism was shown to consist of three different subunits of masses 73 (alpha), 68 (beta) and 19 (gamma) kDa and to contain iron as the only metal. The genes encoding the subunits were clustered in a single operon in the order hydC (gamma), hydB (beta), and hydA (alpha). Sequence analyses indicated that: (a) the enzyme is an Fe-S-cluster-containing flavoprotein which uses NADH as an electron donor; and (b) the catalytic Fe-S cluster resides within the alpha-subunit, which is equivalent to the single subunit that constitutes most mesophilic Fe-hydrogenases. The alpha- and beta-subunits of the purified enzyme were separated by chromatography in the presence of 4 M urea. As predicted, the H(2)-dependent methyl viologen reduction activity of the holoenzyme (45-70 U mg(-1)) was retained in the alpha-subunit (130-160 U mg(-1)) after subunit separation. However, the holoenzyme did not contain flavin and neither it nor the alpha-subunit used NAD(P)(H) or T. maritima ferredoxin as an electron carrier. The holoenzyme, but not the alpha-subunit, reduced anthraquinone-2,6-disulfonate (apparent K(m), 690 microM) with H(2). The EPR properties of the reduced holoenzyme, when compared with those of the separated and reduced subunits, indicate the presence of a catalytic 'H-cluster' and three [4Fe-4S] and one [2Fe-2S] cluster in the alpha-subunit, together with one [4Fe-4S] and two [2Fe-2S] clusters in the beta-subunit. Sequence analyses predict that the alpha-subunit should contain an additional [2Fe-2S] cluster, while the beta-subunit should contain one [2Fe-2S] and three [4Fe-4S] clusters. The latter cluster contents are consistent with the measured Fe contents of about 32, 20 and 14 Fe mol(-1) for the holoenzyme and the alpha- and beta-subunits, respectively. The T. maritima enzyme is the first 'complex' Fe-hydrogenase to be purified and characterized, although the reason for its complexity remains unclear. 相似文献
8.
P Tuohimaa J Mester J M Renoir M G Catelli N Binart T Buchou E E Baulieu 《Journal of steroid biochemistry》1984,20(1):429-432
The chick oviduct cytosol progesterone receptor can be transformed to a small form (Rs = 21A, S20,w:2.9) denoted "mero-receptor" by incubation in the presence of Ca2+ [8]. In the molybdate-free cytosol all the progestin binding components could be completely transformed to mero-form by 1 h treatment with 100 mM Ca2+ at 0 degrees C. If EDTA was secondarily added, the ligand was rapidly released. If molybdate (20 mM) containing cytosol was incubated with Ca2+, no radioactivity was found in the meroposition on the Agarose A 0.5 m column, but the bound steroid sedimented at 2.9 S in sucrose gradients containing Ca2+ (and no molybdate). When 20 nM molybdate was added to cytosol containing receptor activated by 0.3 M KCl, complete mero-transformation by Ca2+ was obtained also by the gel filtration criterion, indicating that molybdate does not inhibit the mero-transforming factor. Ligand-free progesterone receptor could also be completely converted to mero-form by endogenous cytosolic transforming factor and calcium. The transforming factor was completely inactivated, when cytosol was run through Agarose A 0.5 m gel. Mero-transformation was found to be irreversible. The purified progesterone receptor subunit 110 K (B) was partially converted to smaller forms by calcium alone (100 mM, 0 degrees C, 1 h) whereas addition of a small amount of cytosol allowed complete conversion to mero-form. 相似文献
9.
Kinetics of activation of the respiratory burst oxidase in a fully soluble system from human neutrophils 总被引:12,自引:0,他引:12
In a fully soluble system from resting human neutrophils, activation of the respiratory burst oxidase under defined conditions was found to follow first-order kinetics. The manner in which this first-order activation process varied with the concentrations of the individual components in the activating system suggested the following. 1) The respiratory burst oxidase occurs in two forms that can be distinguished by their Km values for NADPH. The low-affinity form contains one component (M) from the membrane and two components (S and C alpha) from the cytosol, while the high-affinity form contains an extra cytosolic component (C beta). 2) The active forms of the oxidase are generated in the following reactions: (formula; see text) where S is a stabilizing component and where M.S is an activated form of M.S that is capable of binding C alpha and C beta to produce the active oxidase species M.S.C alpha (the low-affinity form) and M.S.C alpha C beta (the high-affinity form). 3) SDS activates the oxidase by mediating the conversion of M.S to M.S. 相似文献
10.
Effects of sodium tungstate on various properties of rat liver glucocorticoid receptor were examined at pH7 and pH 8. At pH 7, [3H]triamcinolone acetonide binding in rat liver cytosol preparations was completely blocked in the presence of 10--20 mM-sodium tungstate at 4 degrees C, whereas at 37 degrees C a 30 min incubation of cytosol receptor preparation with 1 mM-sodium tungstate reduced the loss of unoccupied receptor by 50%. At pH 8.0, tungstate presence during the 37 degrees C incubation maintained the steroid-binding capacity of unoccupied glucocorticoid receptor at control (4 degrees C) levels. In addition, heat-activation of cytosolic glucocorticoid-receptor complex was blocked by 1 mM- and 10 mM-sodium tungstate at pH 7 and pH 8 respectively. The DNA-cellulose binding by activated receptor was also inhibited completely and irreversibly by 5 mM-tungstate at pH 7, whereas at pH 8 no significant effect was observed with up to 20 mM-tungstate. The entire DNA-cellulose-bound glucocorticoid-receptor complex from control samples could be extracted by incubation with 1 mM- and 20 mM-tungstate at pH 7 and pH 8 respectively, and appeared to sediment as a 4.3--4.6 S molecule, both in 0.01 M- and 0.3 M-KCl-containing sucrose gradients. Tungstate effects are, therefore, pH-dependent and appear to involve an interaction with both the non-activated and the activated forms of the glucocorticoid receptor. 相似文献
11.
S Takahashi K Hashida K Yatsunami T Fukui M Negishi T Katada M Ui Y Kanaho T Asano A Ichikawa 《Biochimica et biophysica acta》1991,1093(2-3):207-215
We have characterized a soluble pertussis toxin (PT)-sensitive GTP-binding protein (G-protein) present in mouse mastocytoma P-815 cells. 65% of total ADP-ribosylation of PT substrate having a molecular mass of 40 kDa on SDS-polyacrylamide gel electrophoresis in cell homogenate was detected in the supernatant after centrifugation at 100,000 x g for 90 min. [32P]ADP-ribosylation of cytosolic PT substrate was significantly enhanced on the addition of exogenous beta gamma complex. The molecular mass of the cytosolic PT substrate was estimated to be about 80 kDa on an Ultrogel AcA 44 column, but the beta gamma complex was not detected in the cytosol by using the anti-beta gamma complex antibody. Furthermore, the cytosolic PT substrate was found to have some unique properties: [35S]GTP gamma S binding was not inhibited by GDP and [32P]ADP-ribosylation was not affected by GTP gamma S treatment. Only after the cytosolic PT substrate had been mixed with exogenous beta gamma complex, did it copurify with exogenous beta gamma complex by several column chromatographies including an Octyl-Sepharose CL-4B column. The PT substrate was identified as Gi2 alpha by Western blot analysis and peptide mapping with S. aureus V8 protease. These results suggest that Gi2 alpha without beta gamma complex exists with an apparent molecular mass of about 80 kDa in the cytosolic fraction of P-815 cells. 相似文献
12.
Reconstitution of galactosyl receptor inactivation in permeabilized rat hepatocytes is ATP-dependent. 总被引:1,自引:0,他引:1
A subpopulation of galactosyl receptors (GalRs) on isolated rat hepatocytes undergo a reversible inactivation and reactivation process during constitutive recycling (McAbee, D. D., and Weigel, P. H. (1988) Biochemistry 27, 2061-2069). Here, we report the reconstitution of this GalR inactivation in digitonin-permeabilized rat hepatocytes. Permeabilization of freshly isolated cells at 4 degrees C with 0.002% (w/v digitonin releases cytosol containing 35-40% of the total cellular protein, 10-15% of a lysosomal marker, and 5-10% of an early endosomal marker. Incubation of permeabilized cells with cytosol at 37 degrees C results in a time-dependent reduction of total 125I-asialoorosomucoid binding activity, which proceeds with first order kinetics (t 1/2 = 11.3 min). Only half of the total cellular GalRs are affected; maximal GalR activity loss, obtained by 30 min, is 50.5 +/- 9.5% (n = 21) of the control (4 degrees C) value. Increasing the digitonin concentration up to 0.055% does not increase the extent of inactivation. Permeabilized cells with reduced GalR activity were assessed for GalR protein content by Western blot analysis and by binding of anti-GalR antibody. The results show that the reduced 125I-asialoorosomucoid binding is due to GalR inactivation rather than receptor protein degradation. GalR inactivation does not occur in the absence of cytosol or in the presence of dialyzed cytosol. The cytosol also loses its GalR inactivating ability in the presence of an ATP-depleting system. GalR inactivation in the absence of cytosol is achieved by incubating permeabilized washed cells at 37 degrees C with ATP but not with ADP, AMP, or other NTPs. The rate and extent of inactivation are dependent on the ATP concentration. Half-maximal and maximal GalR inactivation are obtained at 0.3 and 3.0 mM ATP, respectively. In the presence of cytosol, permeabilized hepatocytes could replenish cytosolic ATP by oxidative phosphorylation. As a result, similar levels of GalR inactivation were obtained with 500-fold lower ATP concentrations. We conclude that ATP is the only cytosolic component necessary for GalR inactivation in permeabilized rat hepatocytes. 相似文献
13.
Transformation of calf uterine progesterone receptor: analysis of the process when receptor is bound to progesterone and RU38486 总被引:2,自引:0,他引:2
Effects of different transforming agents were examined on the sedimentation characteristics of calf uterine progesterone receptor (PR) bound to the synthetic progestin [3H]R5020 or the known progesterone antagonist [3H]RU38486 (RU486). [3H]R5020-receptor complexes [progesterone-receptor complexes (PRc)] sedimented as fast migrating 8S moieties in 8-30% linear glycerol gradients containing 0.15 M KCl and 20 mM Na2MoO4. Incubation of cytosol containing [3H]PRc at 23 degrees C for 10-60 min, or at 0 degrees C with 0.15-0.3 M KCl or 1-10 mM ATP, caused a gradual transformation of PRc to a slow sedimenting 4S form. This 8S to 4S transformation was molybdate sensitive. In contrast, the [3H]RU486-receptor complex exhibited only the 8S form. Treatment with all three activation agents caused a decrease in the 8S form but no concomitant transformation of the [3H]RU486-receptor complex into the 4S form. PR in the calf uterine cytosol incubated at 23 or at 0 degrees C with 0.3 M KCl or 10 mM ATP could be subsequently complexed with [3H]R5020 to yield the 4S form of PR. However, the cytosol PR transformed in the absence of any added ligand failed to bind [3H]RU486. Heat treatment of both [3H]R5020- and [3H]RU486-receptor complexes caused an increase in DNA-cellulose binding, although the extent of this binding was lower when RU486 was bound to receptors. An aqueous two-phase partitioning analysis revealed a significant change in the surface properties of PR following both binding to ligand and subsequent transformation. The partition coefficient (Kobsd) of the heat-transformed [3H]R5020-receptor complex increased about 5-fold over that observed with PR at 0 degrees C.(ABSTRACT TRUNCATED AT 250 WORDS) 相似文献
14.
Human C5 is composed of two nonidentical polypeptide chains, alpha and beta (m.w. 130,000 and 80,000, respectively) linked together by disulfide bonds and noncovalent forces. Cleavage of C5 by trypsin fragments with increased anodic mobilities. Limited digestion of C5 by trypsin (substrate to enzyme ratio 10:1 w/w at 37 degrees C for 1 min) resulted in the release of a small terminal alpha-chain peptide (alpha1, m.w. 15,000) probably analogous to C5a, from a large fragment, C5b (m.w. 195,000) composed of an intact beta-chain disulfide linked to an alpha-chain that has a lower m.w. (alpha' 115,000). Further digestion (37 degrees C, 5 min) resulted in cleavage of the alpha-chain at multiple sites with the production of three peptides from the alpha'-chain (alpha2I, 23,500; alpha2II 15,700 and alpha2III 10,200) and a residual fragment, C5c (m.w. 144,000). The alpha1 and alpha2 peptides are not covalently linked to the beta-chain nor to one another. The C5c fragment on the other hand is composed of small peptides of the alpha'c chain (alpha3 14,000; alpha4I 9,000; ALPHA 4II 11,000; alpha 5 23,000 to 30,000) which are linked to the beta-chain and also probably to one another by covalent bonds. Secondary cleavage occurred upon prolonged digestion with trypsin (37 degrees C, 20 min), and this resulted in the progressive erosion of the alpha'c peptides and the conversion of C5c to smaller C5c-like species. 相似文献
15.
B M Raaka M Finnerty E Sun H H Samuels 《The Journal of biological chemistry》1985,260(26):14009-14015
Treatment of intact GH1 cells with sodium molybdate inhibits the subsequent rate of nuclear accumulation of hormone-occupied glucocorticoid and estrogen receptors. Cells were incubated at 23 degrees C for 1 h with 30 mM molybdate and then for up to 30 min with [3H]triamcinolone acetonide or [3H]estradiol in the continued presence of molybdate. Although molybdate did not affect the rate of receptor occupancy with either steroid, cells treated with molybdate had more occupied cytosolic and fewer occupied nuclear receptors than control cells. For the glucocorticoid receptor, cells treated with molybdate had more 10 S and fewer 4 S cytosolic receptors than control cells. In low salt cytosol molybdate inhibits the temperature-mediated subunit dissociation of occupied 10 S glucocorticoid receptor. These results suggest that a hormone-mediated dissociation of an intracellular 10 S oligomeric glucocorticoid receptor form to its 4 S subunits is required prior to accumulation of occupied receptors in the nuclear fraction. In cells incubated at 37 degrees C for 1 h or longer with [3H]triamcinolone acetonide, molybdate shifts the steady state intracellular distribution of receptor toward the 10 S cytosolic receptor form, consistent with the interpretation that molybdate affects the rapidly exchanging subunit equilibrium between the 10 S and 4 S cytosolic forms by slowing the rate of 10 S receptor dissociation. Molybdate prevents loss of glucocorticoid-occupied 10 S but not 4 S receptors in heated cytosol by stabilizing the relatively protease-resistant 10 S receptor. Since molybdate stabilizes 10 S oligomeric steroid receptors in vitro, the effects of molybdate on nuclear accumulation of occupied receptors in intact cells support the intracellular existence and physiological relevance of 10 S glucocorticoid and estrogen receptors. These results support a general model for steroid receptor activation in which binding of hormone promotes dissociation of intracellular 8-10 S oligomeric receptors to their DNA-binding subunits. 相似文献
16.
Lifshitz Y Petrovich E Haviv H Goldshleger R Tal DM Garty H Karlish SJ 《Biochemistry》2007,46(51):14937-14950
Human alpha1 and alpha2 isoforms of Na,K-ATPase have been expressed with porcine 10*Histidine-tagged beta1 subunit in Pichia pastoris. Methanol-induced expression of alpha2 is optimal at 20 degrees C, whereas at 25 degrees C, which is optimal for expression of alpha1, alpha2 is not expressed. Detergent-soluble alpha2beta1 and alpha1beta1 complexes have been purified in a stable and functional state. alpha2beta1 shows a somewhat lower Na,K-ATPase activity and higher K0.5K compared to alpha1beta1, while values of K0.5Na and KmATP are similar. Ouabain inhibits both alpha1beta1 (K0.5 24.6 +/- 6 nM) and alpha2beta1 (K0.5 102 +/- 14 nM) with high affinity. A striking difference between the isoforms is that alpha2beta1 is unstable. Both alpha1beta1 and alpha2beta1 complexes, prepared in C12E8 with an added phosphatidyl serine, are active, but alpha2beta1 is rapidly inactivated at 0 degrees C. Addition of low concentrations of cholesterol with 1-stearoyl-2-oleoyl-sn-glycero-3-[phospho-l-serine] (SOPS) stabilizes strongly, maintaining alpha2beta1 active up to two weeks at 0 degrees C. By contrast, alpha1beta1 is stable at 0 degrees C without added cholesterol. Both alpha1beta1 and alpha2beta1 complexes are stabilized by cholesterol at 37 degrees C. Human FXYD1 spontaneously associates in vitro with either alpha1beta1 or alpha2beta1, to form alpha1beta1/FXYD1 and alpha2beta1/FXYD1 complexes. The reconstituted FXYD1 protects both alpha1beta1 and alpha2beta1 very strongly against thermal inactivation. Instability of alpha2 is attributable to suboptimal phophatidylserine-protein interactions. Residues within TM8, TM9 and TM10, near the alphabeta subunit interface, may play an important role in differential interactions of lipid with alpha1 and alpha2, and affect isoform stability. Possible physiological implications of isoform interactions with phospholipids and FXYD1 are discussed. 相似文献
17.
V Prakash 《Indian journal of biochemistry & biophysics》1991,28(5-6):374-380
The ribosomal proteins S4 and S9 were isolated from the 30S ribosomal subunit of Escherichia coli to greater than 95% purity and characterized in the reconstitution buffer. Neither of the proteins indicated any tendency to self associate at 3 degrees C in the concentration range studied. At higher temperatures (greater than 20 degrees C), protein S9 forms a significant amount of a soluble aggregate as seen from the sedimentation velocity and sedimentation equilibrium experiments. From an analysis of the solution mixture of S4 and S9 at 1:1.08 molar concentration ratio by sedimentation velocity experiment, an s20,w value of 1.77 +/- 0.02S was obtained. A fast moving component which accounts for approximately 20% of the mass was also observed. Increasing the concentration of S9 does not alter the observed s20w value significantly for that component which could be followed. A detailed analysis of the data obtained at 3 degrees C from sedimentation equilibrium experiments on mixtures of the proteins indicated that a species of molecular weight greater than either of the two proteins was present. The proteins were found to interact with a mean equilibrium constant of association of 3.66 +/- 2.39 x 10(4) M-1 and a Gibbs free energy of interaction, delta Go = -5.8 kcal/mole at 3 degrees C in TMKD buffer. This information helps in understanding the energetics of the 30S ribosomal subunits of E. coli. 相似文献
18.
M Kuo C R Jones T H Mahn P R Miller L J Nicholls W A Gibbons 《The Journal of biological chemistry》1979,254(20):10301-10306
Biosynthesis of specifically deuterated molecules and difference scalar decoupling permitted an analysis of all C alpha-C beta spin systems of gramicidin S. Proof is presented that proton magnetic resonance spectra obtained by difference scalar decoupling yield not only spectral assignments and simplification but also accurate chemicals shifts and scalar coupling constants. The variations in (3J alpha beta) and in proton chemical shifts at temperatures over the range of -54 degrees -+66 degrees C are consistent with the internal rotation around the C alpha-C beta bonds of Val1, Orn2, Leu3, and Phe4 residues discovered using carbon 13 spectroscopy. The value (3J alpha beta) = 1.5 Hz for the proline residue is consistent with there being only one C alpha-C beta conformer. This is supported by the small temperature dependence of (3J alpha beta). However, it cannot be rigorously excluded that oscillation between a major and a minor C alpha-C beta conformation occurs for proline. 相似文献
19.
del Campo R Criado JJ García E Hermosa MR Jiménez-Sánchez A Manzano JL Monte E Rodríguez-Fernández E Sanz F 《Journal of inorganic biochemistry》2002,89(1-2):74-82
We have synthesized two thiourea derivatives of methyl anthranilate (1, 2) and their complexes with nickel (3) and platinum(II) (4). We have also prepared the complexes of nickel(II) with two benzoylthiourea derivatives (5, 6). The obtained compounds were characterized by elemental analysis, spectroscopic methods (FT-IR, UV-vis, NMR), mass spectrometry and thermal analysis. Compound 1, C(20)H(23)N(3)O(2)S, crystallizes in monoclinic space group P21/n, with Z=4, and unit cell parameters, a=8.8042(4) A, b=7.6608(3) A, c=28.834(2) A, alpha=gamma=90 degrees, beta=90.94(1) degrees. Compound 2, C(20)H(21)N(3)O(3)S, crystallizes in monoclinic space group P21/c, with Z=4, and unit cell parameters, a=7.7345(4) A, b=8.6715(4) A, c=29.113(2) A, alpha=gamma=90 degrees, beta=90.67(1) degrees. Compound 5, C(24)H(30)N(4)NiO(2)S(2), crystallizes in monoclinic space group P21/n, with Z=4, and unit cell parameters, a=10.4317(8) A, b=18.517(2) A, c=13.299(1) A, alpha=gamma=90 degrees, beta=104.53(1) degrees. Compound 6, C(25)H(28)Cl(2)N(4)NiO(4)S(2), crystallizes with a molecule of CH(2)Cl(2) in triclinic space group P-1, with Z=2, and unit cell parameters, a=10.362(1) A, b=11.849(2) A, c=12.536(2) A, alpha=90.04(2) degrees, beta=84.73(1) degrees, gamma=113.43(2) degrees. Compounds 1 and 2 show antifungal activity against the major pathogens responsible for important plant diseases (Botrytis cinerea, Colletotrichum fragariae, Fusarium oxysporum and Phoma betae). The antifungal activity is practically the same for morpholine and ethyl derivatives. 相似文献
20.
Temperature-Dependent Kinetic Correlates of the Activation of the Glucocorticoid-Receptor Complex 总被引:1,自引:1,他引:0
The effects of temperature on the kinetics of activation were studied in [3H]triamcinolone acetonide[( 3H]TA)-labeled cytosol preparations from mouse whole brain. After removal of unbound [3H]TA and molybdate (which prevents activation) from the unactivated steroid-receptor complex by gel exclusion chromatography, activation was initiated by incubation at 6-30 degrees C for 0.75-24 min and then rapidly quenched at -5 degrees C with Na2MoO4 (20 mM final concentration). The loss of the 9.2S (unactivated) form of the [3H]TA-receptor complex and the concomitant formation of the 3.8S (activated) form increased dramatically with increases in the activation temperature. These hydrodynamic changes were correlated directly with rapid time- and temperature-dependent increases in the binding of [3H]TA-labeled cytosol to DNA-cellulose (DNA-C). Further analyses of these data revealed a greater than 50-fold increase in the apparent first-order rate constant for the increased binding to DNA-C as the activation temperature was increased from 6 degrees C to 30 degrees C. An Arrhenius plot of these temperature-dependent kinetic constants revealed an energy of activation of 116 kJ. These data support a proposed model for activation of the glucocorticoid-receptor complex that includes the splitting of a 297 kDa, unactivated species into a 92 kDa, activated species. 相似文献