Three monofluorocinnamoylchymotrypsins have been examined at pH 4 by fluorine NMR spectroscopy. Protein-induced fluorine chemical shifts are quite large (~7 ppm) when fluorine is present at the para position but nearly zero for ortho fluorine. The shifts roughly parallel those observed in complexes formed between the enzyme and the analogous N-acetylfluorophenylalanines, suggesting a similarity in molecular environment for the aromatic ring in both systems. Little correlation is found, however, between the shifts for the acylenzymes and those of the corresponding enzyme-cinnamate complexes, indicating that the environment for the aromatic ring in the complexes is dissimilar from that experienced by the aromatic group in the acylated enzyme. Solvent isotope effects () on the fluorine chemical shifts for the fluorocinnamoylchymotrypsins are small and downfield. Fluorine NMR observations suggest that the presence of the fluorocinnamoyl group greatly stabilizes the enzyme toward denaturation in 8 m urea. 相似文献
Protein-induced distortion is a dramatic but not universally observed feature of sequence-specific DNA interactions. This is illustrated by the crystal structures of restriction enzyme–DNA complexes: While some of these structures exhibit DNA distortion, others do not. Among the latter is PvuII endonuclease, a small enzyme that is also amenable to NMR spectroscopic studies. Here 31P NMR spectroscopy is applied to demonstrate the unique spectral response of DNA to sequence-specific protein interactions. The 31P NMR spectrum of a noncognate DNA exhibits only spectral broadening upon the addition of enzyme. However, when enzyme is added to target DNA, a number of 31P resonances shift dramatically. The magnitudes of the chemical shifts (2–3 ppm) are among the largest observed. Site-specific substitution with phosphoramidates and phosphorothioates are used analyze these effects. While such spectral features have been interpreted as indicative of DNA backbone distortions, FRET analysis indicates that this does not occur in PvuII-cognate DNA complexes in solution. The distinct 31P spectral signature observed for cognate DNA mirrors that observed for the enzyme, underscoring the unique features of cognate complex formation. 相似文献
Actin polymerization in 2 mM MgCl2 is known to be inhibited by profilin. We found that small amounts of cytochalasin-binding complexes from human red cell membranes or actin nuclei cross-linked by -phenylenebismaleimide can reverse the inhibitory action of profilin, leading to the rapid polymerization of the actin. This type of polymerization is inhibited by low concentrations of cytochalasin B. These results indicate that (a) the complexes and nuclei promote actin polymerization in the presence of profilin by providing sites onto which actin monomers can be added, and (b) profilin and cytochalasin B affect two distinct steps (i.e. nucleus formation and filament elongation, respectively) in the polymerization reaction. 相似文献
Scalar couplings between 31P and isotopes of cadmium, mercury, lead, and tin are reported for the respective metal complexes with the chelating agent (ethylenedinitrilo) - tetramethylenephosphonic acid. 相似文献
The 31 P NMR chemical shift of β-P of adenosine triphosphate (ATP) undergoes a substantial change (2̃–3 ppm) upon chelation of divalent ions such as Mg2+ or Ca2+. In the presence of nonsaturating amounts of Mg2+ or Ca2+, the lineshape of this resonance depends on the characteristic association and dissociation rates of these metal-ATP complexes. A procedure for computer simulation of this lineshape is outlined. A comparison of computer-simulated lineshapes with the experimental lineshapes obtained at 121 MHz was used to determine the following dissociation rate of Mg2+ and Ca2+ from their ATP complexes at 20°C and pH 8.0: Ca2+, > 3 × 105 s?1 (Hepes buffer); Mg2+, 1200 s-1 (no buffer), 1000 s-1 (Tris buffer) and 2100 s?1 (Hepes buffer). The limits of error are ± 10% in these values. For the Mg2+ complexes, the rates were determined as a function of temperature to obtain activation energies (with a maximum deviation of 10% in the least-squares fit): 8.1 (no buffer and Hepes buffer) and 6.8 (Tris buffer). Lineshapes of the β-Presonance simulated as a function of Mg2+ concentration, using 2100 s?1 for the dissociation rate, are also presented. The computer simulation of lineshapes offers a reliable and straightforward method for the determination of exchange rates of diamagnetic cations from their ATP complexes, under a variety of sample conditions. 相似文献
Since coupling between phosphorylation and electron transport cannot be measured directly in intact chloroplasts capable of high rates of photosynthesis, attempts were made to determine ratios from the quantum requirements of glycerate and phosphoglycerate reduction and from the extent of oxidation of added NADH via the malate shuttle during reduction of phosphoglycerate in light. These different approaches gave similar results. The quantum requirement of glycerate reduction, which needs 2 molecules of ATP per molecule of NADPH oxidized was found to be pH-dependent. 9–11 quanta were required at pH 7.6, and only about 6 at pH 7.0. The quantum requirement of phosphoglycerate reduction, which consumes ATP and NADPH in a ratio, was about 4 both at pH 7.6 and at 7.0. ratios calculated from the quantum requirements and the extent of phosphoglycerate accumulation during glycerate reduction were usually between 1.2 and 1.4, occasionally higher, but they never approached 2.Although the chloroplast envelope is impermeable to pyridine nucleotides, illuminated chloroplasts reduced added NAD via the malate shuttle in the absence of electron acceptors and also during the reduction of glycerate or CO2. When phosphoglycerate was added as the substrate, reduction of pyridine-nucleotides was replaced by oxidation and hydrogen was shuttled into the chloroplasts to be used for phosphoglycerate reduction even under light which was rate-limiting for reduction. This indicated formation of more ATP than NADPH by the electron transport chain. From the rates of oxidation of external NADH and of phosphoglycerate reduction at very low light intensities ratios were calculated to be between 1.1 and 1.4.Fully coupled chloroplasts reduced oxaloacetate in the light at rates reaching 80 and in some instances 130 μmoles · mg?1 chlorophyll · h?1 even though ATP is not consumed in this reaction. The energy transfer inhibitor phlorizin did not significantly suppress this reduction at concentrations which completely inhibited photosynthesis. Uncouplers stimulated oxaloacetate reduction by factors ranging from 1.5 to more than 10. Chloroplasts showing little uncoupler-induced stimulation of oxaloacetate reduction were highly active in photoreducing CO2. Measurements of light intensity dependence of quantum requirements for oxaloacetate reduction gave no indication for the existence of uncoupled or basal electron flow in intact chloroplasts. Rather reduction is brought about by loosely coupled electron transport. It is concluded that coupling of phosphorylation to electron transport in intact chloroplasts is flexible, not tight. Calculated ratios were obtained under conditions, where coupling should be expected to be optimal, i.e. at low phosphorylation potentials . Flexible coupling implies, that ratios should decrease with increasing phosphorylation potentials inside the chloroplasts. 相似文献
When 10?6 M oubain is added to human red cells that have been incubated without glucose for two hours, there is a significant shift in the 31P nuclear magnetic resonances of both phosphate groups of cellular 2,3-diphosphoglycerate, which is not found in control cells incubated with glucose. This means that an effect induced by ouabain on the outside of the red cell membrane is transmitted through the membrane to alter the environment of an intracellular metabolite. Experiments with glycolytic cycle inhibitors have indicated that the intracellular ligand responsible for the resonance shifts is monophosphoglycerate mutase which requires 2,3-diphosphoglycerate as a cofactor for the reaction it catalyzes. To account for this finding a hypothesis is presented that the (Na+ + K+)-ATPase in human red cells is linked to monophosphoglycerate mutase through the agency of phosphoglycerate kinase. Evidence is presented for the existence of phosphoglycerate kinase/monophosphoglycerate mutase in solution. It is shown that this complex can interact with the cytoplasmic face of (Na+ + K+)-ATPase at the outside surface of inside out red cell vesicles, and that this interaction is inhibited when 10?6 M ouabain is contained within the vesicle. Neither monophosphoglycerate mutase nor phosphoglycerate kinase is significantly bound to the inside surface of the intact human red cell, but glyceraldehyde 3-phosphate dehydrogenase is; it is shown that this enzyme also interacts with the cytoplasmic face of the (Na+ + K+)-ATPase and that the interaction is inhibited by 10?6 M ouabain. 相似文献
When human red cells are treated with the mercurial sulfhydryl reagent, , osmotic water permeability is suppressed and only diffusional water permeability remains (Macey, R.I. and Farmer, R.E.L. (1970) Biochim. Biophys. Acta 211, 104–106). It has been suggested that the route for the remaining water permeation is by diffusion through the membrane lipids. However, after making allowance for the relative lipid area of the membrane, the water diffusion coefficient through lipid bilayers which contain cholesterol is too small by a factor of two or more. We have measured the permeability coefficient of normal human red cells by proton NMR and obtained a value of 4.0 · 10?3 cm · s?1, in good agreement with published values. In order to study permeation-through red cell lipids we have perturbed extracted red cell lipids with the lipophilic anesthetic, halothane, and found that halothane increases water permeability. The same concentration of halothane has no effect on the water permeability of human red cells, after maximal pCMBS inhibition. In order to compare halothane mobility in extracted red cell membrane lipids with that in red cell ghost membranes, we have studied halothane quenching of by equilibrium fluorescence and fluorescence lifetime methods. Since halothane mobility is similar in these two preparations, we have concluded that the primary route of water diffusion in pCMBS-treated red cells is not through membrane lipids, but rather through a membrane protein channel. 相似文献
The time at which dosage compensation of an X-linked gene in the mouse is established has been estimated by measuring the activity levels of two glycolytic enzymes, phosphoglycerate kinase (EC 2.7.2.3) and triosephosphate isomerase (EC 5.3.1.1), during early development of embryos from and mice. During preimplantation development the level of phosphoglycerate kinase in embryos from mice was constant for the first 48 hr of development (2.55–2.71 nmoles/hr/embryo) and then dropped to one-half the earlier level (1.44 nmoles/hr/embryo) by 72 hr of development. The general developmental profile of phosphoglycerate kinase was similar in embryos from mice; however, the absolute level of enzyme activity was reduced to approximately 1.4 nmoles/hr/embryo during the first 48 hr of development and to 0.9 nmoles/hr/embryo by 72 hr of development. These differences in phosphoglycerate kinase levels between embryos from and mice disappeared between the blastocyst and egg cylinder stages (150 hr postplug) during which time the activity levels had increased to 183 and 193 nmoles/hr/egg cylinder for embryos from and mice, respectively.The activity level for triosephosphate isomerase in embryos from and mothers did not differ at any stage of development; this suggests that the gene coding for triosephosphate isomerase is autosomal. In addition the level of activity remained constant during preimplantation development (approximately 3 nmoles/hr/embryo) and then, like phosphoglycerate kinase, increased 100-fold between the blastocyst and egg cylinder stages.The frequency distribution of the activity ratio (triosephosphate isomerase to phosphoglycerate kinase) in the egg cylinder of individual embryos from both and mothers was determined in order to detect differences among and embryos. These frequency distributions were unimodal, and hence these results coupled with the gene dosage differences observed during preimplantation development indicate that dosage compensation for an X-linked gene has been established in the 6-day mouse embryo. 相似文献
When young intact forespores of Bacillus megaterium were incubated with either Mn++ or the ionophore X-537A, the pool of 3-phosphoglyceric acid (3-PGA) was stable. However, incubation of forespores with Mn++ plus the ionophore X-537A resulted in rapid and complete utilization of the 3-PGA. This effect was not seen with Ca++ or Mg++, and was also not observed with older forespores or fresh dormant spores. Since the phosphoglycerate mutase of has an absolute and specific requirement for Mn++, it is possible that phosphoglycerate mutase in developing forespores may be inactive because of a low intrasporal level of free Mn++. 相似文献
α,ω-Bis(diphenylphosphino)alkane and α,ω-bis(diphenylphosphino)(poly)ether ligands can be prepared in very high yields via reaction of the appropriate dihalide with two equivalents of LiPPh2. For the [Rh(COD)()][ClO4] complexes of these ligands, the ligands with five or less atoms in the alkane or ether bridge form monomeric complexes via η2-coordination. In general the ligands with eight or more atoms in the bridge give di- or polynuclear species. In addition the long chain diphosphino-polyethers form – to a small extent – monomeric species by η2-coordination. 相似文献
The kinetics of the sodium binding to the ionophore monensin (Mon) in methanol has been studied by 23Na NMR spectroscopy. Fast quadrupole relaxation of the bound sodium affected the relaxation rate of the free sodium through an exchange process between these two species. The exchange was found to be dominated by the reaction: Na+ + Mon? ? MonNa. The dissociation rate constant at 25°C is 63 s?1, with an activation enthalpy of 10.3 and activation entropy of ?15.8 deg. These results indicate that the specificity of the binding of sodium ions to monensin is reflected in the relatively slow dissociation process. The entropy changes indicate that the activated monensin-sodium complex undergoes a conformational change, but the existence of a conformational change in monensin anion prior to complexation is excluded. 相似文献
31P-NMR is used to characterize the phase behavior of phosphonolipids in both model and biological membranes. ( gives rise to static chemical shift tensor elements (?87, 5 and 63 ppm) which differ considerably from those reported for the analogous phospholipid, (?81, ?20 and 105 ppm). Phosphonolipid, as well as a mixture of phosphonolipid and , in aqueous dispersion gives rise to 31P spectra which may be interpreted in terms of lamellar structures. A mixture of phosphonolipid and egg phosphatidylethanolamine exhibits a bilayer-to-hexagonal phase transition with a concomitant decrease by one-half in the value of the 31P chemical shift anisotropies of both the phosphonate and phosphate resonances. The chemical shift anisotropy associated with phosphonolipid has been found to be consistently smaller than that observed for the analogous phospholipid. 31P-NMR spectra of total lipid extracts of Tetrahymena sp. indicate that both phospho- and phosphonolipids have a bilayer organization between ?20 and 20°C. 相似文献
1H NMR spectra of ten (25)-steroidal sapogenins (I–X) and their acetates (Ia–Xa) in deuteriochloroform and pentadeuterio-pyridine were examined. The C-20 and C-25 methyl signal ass?ignments were revised. Some other features including benzene-induced shifts are described. 相似文献
Carbohydrate moieties derived from the G glycoprotein of Vesicular Stomatitis Virus (VSV) grown in parental Chinese hamster ovary (CHO) cells and the glycosylation mutant Lec4 have been analyzed by high-field 1H NMR spectroscopy. The major glycopeptides of and were purified by their ability to bind to concanavalin A-Sepharose. The carbohydrates in this fraction are of the biantennary, complex type with heterogeneity in the presence of α(2,3)-linked sialic acid and α(1,6)-linked fucose residues. A minor glycopeptide fraction, which does not bind to concanavalin A-Sepharose but which binds to pea lectin-agarose, was also investigated by 1H NMR spectroscopy. These carbohydrates are complex moieties which appear to contain N-acetylglucosamine in β(1,6) linkage. Their spectral properties are most similar to those of a triantennary complex oligosaccharide containing a 2,6-disubstituted mannose α(1,6) residue. Carbohydrates of this type are not found among the glycopeptides of VSV grown in the Lec4 CHO glycosylation mutant. 相似文献
ATPase preparations from rat brain, dog kidney, and human red blood cells also catalyze a K+-dependent phosphatase reaction. K+ activation and Na+ inhibition of this reaction are described quantitatively by a model featuring isomerization between E1 and E2 enzyme conformations with activity proportional to E2K concentration: Differences between the three preparations in for K+ activation can then be accounted for by differences in equilibria between E1K and E2K with dissociation constants identical. Similarly, reductions in produced by dimethyl sulfoxide are attributable to shifts in equilibria toward E2 conformations. Na+ stimulation of K+-dependent phosphatase activity of brain and red blood cell preparations, demonstrable with KCl under 1 mM, can be accounted for by including a supplementary pathway proportional to E1Na but dependent also on K+ activation through high-affinity sites. With inside-out red blood cell vesicles, K+ activation in the absence of Na+ is mediated through sites oriented toward the cytoplasm, while in the presence of Na+ high-affinity K+-sites are oriented extracellularly, as are those of the ATPase reaction. Dimethyl sulfoxide accentuated Na+-stimulated K+-dependent phosphatase activity in all three preparations, attributable to shifts from the E1P to E2P conformation, with the latter bearing the high-affinity, extracellularly oriented K+-sites of the Na+-stimulated pathway. 相似文献
Transport of Pr3+ across phosphatidylcholine vesicles, monitored through 31P nmr, is first-order in monensin (), second-order in etheromycin () or in lasalocid A (). When and (or and ) are incorporated in 1:1 ratio into the lipidic phase, transport is faster than with each ionophore alone. For instance, assuming that the complexes .Pr3+., .Pr3+., and .Pr3+ are equiprobable, they effect transport at intrinsic relative rates of 1, 2, and 13.5, a remarkable synergism is set up. 相似文献
The activity of the protein kinase that phosphorylates the light-harvesting chlorophyll-protein of Photosystem II (LHCP) has been investigated in intact chloroplasts isolated from maize mesophyll cells. Measurements of 32P incorporation into LHCP, ATP concentration, ratio, ΔpH, chlorophyll fluorescence and oxygen evolution were made in the presence of different metabolic substrates. Without added substrate a high level of LHCP phosphorylation was observed which was suppressed by addition of oxaloacetate or phosphoglycerate but stimulated by pyruvate. Whereas no correlation was observed between LHCP phosphorylation and adenylate status, a clear effect of redox state on protein kinase activity was observed. A correlation between a highly reduced electron-transfer chain (produced under conditions which favour cyclic electron flow) and the maximum level of protein phosphorylation was observed. The regulation of kinase activity and its dependence on electron transfer and carbon assimilation are discussed. 相似文献
Pulsed NMR spectroscopy has been used to study Na+ binding to several simple carbohydrates in aqueous solution. Changes in the 23Na spin-lattice relaxation time (T1) were monitored to indicate complex formation between sodium ions and a ligand. It was found that Na+ interacts with these hydroxy-compounds in a manner similar to other metal cations, but very weakly. Among the sugars investigated, -inositol forms the strongest complexes with the stability constant about 1.2 M?1 (if 1:1 complexes are assumed). A qualitative study of competition between Na+ and Ca2+ was done, indicating that both cations have the same binding sites. 相似文献
The steroidogenic activity of complexes of [biocytin25]-corticotropin1–25 amide (biotinylcorticotropin) with avidin (1:1), (4:1) and (1:10) was compared to that of biotinylcorticotropin using isolated rat adrenal cells. Parallel log-dose responses and maximal stimulation were observed with all these materials. The 1:1 complex is approximately 25% as active as biotinylcorticotropin (ED5022.5 and 5.6 respectively). The 4:1 complex is more active than the 1:1 complex (ED509.0 ). The presence of an excess of avidin (1:10 complex) does not interfere with the ability of biotinylcorticotropin to stimulate steroidogenesis (ED5018.0 ). It is concluded that biotinylcorticotropin attached to avidin binds specifically to receptors on the rat adrenal cell and elicits its biological response. These results indicate that biotinylcorticotropin can be noninvasively labeled with 125I-avidin. 相似文献