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1.
Isocitrate lyase (threo-DS-isocitrate glyoxylate-lyase, EC 4.1.3.1) has been purified to homogeneity from castor endosperm. The enzyme is a tetrameric protein (molecular weight about 140,000; gel filtration) made up of apparently identical monomers (subunit molecular weight about 35,000; gel electrophoresis in the presence of sodium dodecyl sulfate). Thermal inactivation of purified enzyme at 40 and 45 °C shows a fast and a slow phase, each accounting for half of the intitial activity, consistent with the equation: , where A0 and At are activities at time zero at t, and k1 and k2 are first-order rate constants for the fast and slow phases, respectively. The enzyme shows optimum activity at pH 7.2–7.3. Effect of [S]on enzyme activity at different pH values (6.0–7.5) suggests that the proton behaves formally as an “uncompetitive inhibitor.” A basic group of the enzyme (site) is protonated in this pH range in the presence of substrate only, with a pKa equal to 6.9. Successive dialysis against EDTA and phosphate buffer, pH 7.0, at 0 °C gives an enzymatically inactive protein. This protein shows kinetics of thermal inactivation identical to the untreated (native) enzyme. Full activity is restored on adding Mg2+ (5.0 mm) to a solution of this protein. Addition of Ba2+ or Mn2+ brings about partial recovery. Other metal ions are not effective. 相似文献
2.
(1) The polymorphic phase behaviour of aqueous dispersions of various synthetic phosphatidylethanolamines, both singly and in mixtures, has been investigated by 31P-NMR. (2) PE remains in the lamellar phase up to 90°C. PE exhibits a lamellar to hexagonal (HII) transition between 60°C and 63°C. For PE, the lamellar to hexagonal (HII) transition occurs between 7 and 12°C, whereas for PE, the hexagonal (HII) phase is the preferred structure above ?15°C. (3) Mixtures of PE and PE exhibit near-ideal miscibility behaviour. For mixtures of PE and PE there is evidence of fluid-solid immiscibility at temperatures below the gel-liquid crystalline transition temperature of the PE component. Mixtures of PE and PE exhibit complex phase behaviour involving limited fluid-solid immiscibility at low temperatures and formation of a phase allowing isotropic motional averaging at higher temperatures. (4) 31P-NMR provides a graphic method for investigating the miscibility properties of mixed PE systems. 相似文献
3.
Binding of the chromogenic ligand p-nitrophenyl α-d-mannopyranoside to concanavalin A was studied in a stopped-flow spectrometer. Formation of the protein-ligand complex could be represented as a simple one-step process. No kinetic evidence could be obtained for a ligand-induced change in the conformation of concanavalin A, although the existence of such a conformational change was not excluded. The entire change in absorbance produced on ligand binding occurred in the monophasic process monitored in the stopped-flow spectrometer. The value of the apparent second-order rate constant (ka) for complex formation (ka = 54,000 s?1m? at 25 °C, pH 5.0, Γ/2 0.5) was independent of the protein concentration when the protein was in the range of 233–831 μm in combining sites and in excess of the ligand. The apparent first-order rate constant (k?a) for dissociation of the complex was obtained from the rate constant for the decomposition of the complex upon the addition of excess methyl α-d-mannopyranoside (k?a = 6.2 s?1 at 25 °C, pH 5.0, Γ/2 0.5). The ratio (0.9 × 104m?1) was in reasonable agreement with value of 1.1 ± 0.1 × 104m?1 determined for the equilibrium constant for complex formation by ultraviolet difference spectrometry. Plots of ln() and ln() vs were linear (T is temperature) and were used to evaluate activation parameters. The enthalpies of activation for formation and dissociation of the complex are 9.5 ± 0.3 and 16.8 ± 0.2 kcal/mol, respectively. The unitary entropies of activation for formation and dissociation of the complex are 2.8 ± 1.1 and 1.3 ± 0.7 entropy units, respectively. These entropy changes are much less than those usually associated with substantial changes in the conformation of proteins. 相似文献
4.
The enzymically active, semisynthetic complex formed by residues 1 through 118 and residues 111 through 124 of bovine pancreatic ribonuclease has been crystallized at pH 5.7 from solutions. The crystals belong to space group P3221, have unit cell dimensions γ = 120° and are isomorphous with form M of ribonuclease A as well as forms W and R of ribonuclease S. They diffract well and may be expected to yield a structure defined to at least 3.0 Å resolution. 相似文献
5.
G.D. Holman 《生物化学与生物物理学报:生物膜》1978,508(1):174-183
10?5 M cyclic AMP has high permeability in human erythrocyte ghosts (). Saturation of influx and efflux occurs. . . . at 30°C. Equilibrium exchange entry of cyclic AMP has similar kinetics to zero trans influx, though the system does show counterflow. Cythochalasin B is an apparent competitive inhibitor of cyclic AMP exit. ().Control experiments indicated that cyclic AMP remains intact during incubation with red blood cell ghosts and is contained within the intravesicular space during the transport experiments. 相似文献
6.
A complete titration of phosphatidic acid bilayer membranes was possible for the first time by the introduction of a new anaologue, acid, which has the advantage of a high chemical stability at extreme pH values. The synthesis of this phosphatidic acid is described and the phase transition behaviour in aqueous dispersions is compared with that of three ester phosphatidic acids; acid, 1,3-dimyristoylglycerol-2-phosphoric acid and acid.The phase transition temperatures () of aqueous phosphatidic acid dispersions at different degrees of dissociation were measured using fluorescence spectroscopy and 90° light scattering. The values are comparable to the melting points of the solid phosphatidic acids in the fully protonated states, but large differences exist for the charged states.The vs. pH diagrams of the four phosphatidic acids are quite similar and of a characteristic shape. Increasing ionisation results in a maximum value for the transition temperatures at pH 3.5 (). The regions between the first and the second of the phosphatidic acids are characterised by only small variations in the transition temperatures (extended plateau) in spite of the large changes occurring in the surface charge of the membranes. The slope of the plateau is very shallow with increasing ionisation. A further decrease in the H+ concentration results in an abrupt change of the transition temperature. The slope of the vs. pH diagram beyond becomes very steep. This is the 相似文献
7.
Determination of delta psi, delta pH and the proton electrochemical gradient in isolated cholinergic synaptic vesicles 总被引:1,自引:0,他引:1
The electrical potential (Δψ) of intact cholinergic synaptic vesicles was measured in the presence and absence of the proton translocator carbonyl cyanide p-trifluoromethoxy-phenylhydrazone (FCCP), and the results were utilized to calculate the vesicular proton chemical gradient (ΔpH) and proton electrochemical potential . At external pH = 7.4 the vesicles maintain a proton electrochemical gradient of (positive inside) which is composed of (negative inside) and (acidic inside). The proton chemical gradient (ΔpH) increases as a function of pHout whereas the vesicular electrical potential (Δψ) is only slightly affected by the external pH. Consequently, is larger at basic external pH values ( at pHout = 9.0) and smaller at acidic external pH values ( at (pHout = 5.6). The possible physiological role of the electrochemical potentials in maintaining high concentrations of acetylcholine within the cholinergic synaptic vesicle is discussed. 相似文献
8.
William C. Okulicz Robert A. Boomsma Richard G. MacDonald Wendell W. Leavitt 《Biochimica et Biophysica Acta (BBA)/General Subjects》1983,757(1):128-136
This study was undertaken to determine optium conditions for the extraction and measurement of uterine nuclear estrogen receptor at low temperature. We measured the influence of glycero, 0.5 M KCl, 10 mM pyridoxal 5′-phosphate, and 0.5 M NaSCN on the dissocation of estradiol from the receptor at 0°C. The half-time () of estradiol dissociation from the receptor in 0.5 M KCl nuclear extracts containing 30% glycerol was very slow (greater than 250 h). Exclusion of glycerol from the extract (Tris buffer) increased the dissociation rate (). The inhibitory effect of glycerol on estradiol dissociation kinetics predominated over the mild stimulatory effect of KCl; and both effects were independent of the electrical conductivity of the buffer. When pyridoxal phosphate was added to a nuclear KCl extract (barbital fubber) lacking glycerol, dissociation of the estrogen-receptor complex increased such that the ) decreased from 20 to 7.6 h; the receptor extracted from nuclei with 10 mM pyridoxal phosphate exhibited these same rapid dissociation kinetics. The of estradiol dissociation from the receptor at 0°C in the presence of 0.5 M NaSCN was 5.6 h. Following extraction of uterine receptro by KCl, pyridoxal phosphate, or NaSCN, we measured the number of estradiol binding sites at each of two incubation temperatures: 30°C for 1 hr and 0°C for 24 h. We verified that unoccupied receptors was measured reliability in KCl extract during incubation at 0°C in the presence of glycerol. Total receptor can be determined using either pyridoxal phosphate extract or NaSCN extract at low temperature. However, the number of sites recovered in either pyridoxal phosphate or NaSCN extract was twice the number obtained with the KCl procedure at elevated temperature. It is noteworthy that pyridoxal phosphate and NaSCN increased the number of sites when added directly to nuclear KCl extract, and the effect of pyridoxal phosphate and NaSCN was reversed by treatment with L-lysine and dialysis against KCl, respectively. Thus, the lower receptor recovery with the KCl procedure is not due to the inability of KCl to extract these sites from the nucleus but rather is ascribable to the assay procedure itself. Although total receptor can be measured at low temperature with either NaSCN or pyridoxal phosphate, the pyridoxal phosphate method can be used to assay nuclear progesterone receptor in tha same extract. 相似文献
9.
Robert P. le Borgne 《Journal of experimental marine biology and ecology》1979,37(2):127-137
Respiration (O), ammonium (NH4), phosphate (PO4), total nitrogen (NT) and phosphorus (PT) excretions were measured on mixed zooplankton during 3-, 6-, 9-, 12-, 21-, and 24-h incubation periods at 20–23 C. The excretion rates of PO4, NT. and PT decrease during a 21-h period, while rates of respiration and excretion of NH{IN4} are constant. The percentage of inorganic nitrogen excreted increases regularly from 3 h (30–40% of total nitrogen) to 21 h (70–80%) and it could be either due to a bacterial activity which was measured or to a decrease with time of organic nitrogen excreted because of starvation. , , , and ratios increase during the first 9 h of incubation; the percentage of inorganic phosphorus excreted is higher at the very beginning and then remains constant from 6 to 24 h. and ratios are constant during a 24-h term, which makes them useful metabolic indexes. 相似文献
10.
William L. Mock Jin-Tann Chen Joseph W. Tsang 《Biochemical and biophysical research communications》1981,102(1):389-396
Substitution of the active site zinc ion of carboxypeptidase A by cadmium yields an enzyme inactive towards ordinary peptide substrates. However, a substrate analog (BzGlyNHCH2CSPheOH) containing a thioamide linkage at the scissile position is cleaved to the thioacid. The kinetic parameters and their pH dependencies are , decreasing with either acid or base (PKE1 = 5.64, pKE2 = 9.55), and kcat = 1.02 × 102 min?1, decreasing with acid (pKES = 6.61). The thiopeptide is less efficiently cleaved by native (zinc) carboxypeptidase A. This cadmium-sulfur synergism supports a mechanism wherein the substrate amide is activated by metal ion coordination to its (thio) carbonyl. 相似文献
11.
Diethelm Kleiner 《生物化学与生物物理学报:生物膜》1982,688(3):702-708
Klebsiella pneumoniae can accumulate methylammonium up to 80-fold by means of a transport system as indicated by the energy requirement, saturation kinetics and a narrow pH profile around pH 6.8. Methylammonium transport (apparent , per g dry weight at 15°C) is competitively inhibited by ammonium (apparent ). The low value and the finding that methylammonium cannot serve as a nitrogen source indicate that ammonium rather than methylammonium is the natural substrate. Uphill transport is driven by a component of the protonmotive force, probably the membrane potential. The transport system is under genetic control; it is partially repressed by amino acids and completely by ammonium. Analysis of mutants suggest that the synthesis of the ammonium transport system is subject to the same ‘nitrogen control’ as nitrogenase and glutamine synthetase. 相似文献
12.
Ulrich Quast Alex M. Labhardt Valerie M. Doyle 《Biochemical and biophysical research communications》1984,123(2):604-611
The kinetics of the Quin 2-Ca2+ interaction have been studied using stopped-flow fluorimetry. Mixing the Quin 2-Ca2+ complex with a large excess of EGTA, EDTA or MgCl2 resulted in first order dissociation kinetics. The observed dissociation rate increased slightly with increasing EGTA concentration yielding a limiting value of 83±4 s?1 for the dissociation rate constant (k?) at pH 7.2, 37°C, ± 3mM Mg2+. The temperature dependence of the dissociation was weak (activation energy = 22±1 kJ/mol) and around neutral pH the pH dependence was negligible. The association reaction was too fast to be monitored directly. From this and the instrument dead-time, the second order rate constant k+ was estimated to be ≥109 M?1s?1, in agreement with the calculation from . These data should be useful in evaluating the potential of Quin 2 to measure fast intracellular Ca2+ transients. 相似文献
13.
J K Ladha P Rowell W D Stewart 《Biochemical and biophysical research communications》1978,83(2):688-696
5-hydroxylysine, an analogue of glutamate and lysine, causes production by N2-fixing A. cylindrica; it also reversibly inhibits GS activity in vitro but has no effect on alanine dehydrogenase or GOGAT. On adding 5-hydroxylysine intracellular pools of glutamine, glutamate and aspartate decrease; those of alanine and serine increase. 5-hydroxylysine alleviates the inhibitory effect of on heterocyst production and C2H2 reduction and in cultures results in heterocyst synthesis and in C2H2 reduction. The data suggest that the GS-GOGAT pathway is the sole route of importance in primary assimilation in A. cylindrica, that alone does not inhibit nitrogenase and heterocyst production, and that GS and/or a product is involved in regulating the production of both. 相似文献
14.
Hepatocytes prepared by collagenase perfusion from Antarctic nototheniid fish of genus Trematomus are active in uptake of [14C]leucine at 0, 5, and 10°C. The system is saturable with apparent about 1.0 mM. Isoleucine and phenylalanine were major competitors, valine was about one-half as effective, while alanine, glycine and histidine had no effect. Temperature dependency of rates in the 0–10°C range yielded (). The average first-order rate constant at 0°C was 0.1 min?1, one-third the value of 0.3 min?1 estimated for clearance of [14C]leucine by liver of these species in vivo. Affinity and specificity agreed well with in vivo data on liver clearance of leucine, both in Antarctic fish at 0°C and in temperate fish acclimated to 10°C and 20°C. The results indicate similar modifications of leucine transport associated with evolutionary cold adaptation and seasonal acclimation in fish. 相似文献
15.
Conditions for the production of a complementary DNA sequence for use in studies of ribosomal RNA are described. . DNA polymerase I is used to transcribe highly purified 28S ribosomal RNA from rat liver. The reaction is sensitive to the tertiary structure of the rRNA template-primer. The complementary DNA hybridizes to its rRNA template with a of 0.02. The hybrid formed between 28S ribosomal RNA and complementary DNA has a Tm of 73°C. The probe reacts with total rat nuclear RNA with a of 1.0. 相似文献
16.
Glucoamylase (EC 3.2.1.3) was coupled to controlled pore glass by using titanium(IV) chloride. The drying conditions used during the activation step were studied, and the highest activity (237 units/g of matrix) of immobilized enzyme was obtained when the support and the titanium(IV) chloride solution were dried at 45°C in vacuo for 16 h. After several washing cycles, the specific activity of the immobilized enzyme was ~13 units/mg of protein irrespective of the washing cycle used. However, this immobilized enzyme preparation was also the least stable (). Investigation of the possibility of the stabilization of the linkage of the enzyme to the support by crosslinking with bifunctional reagents showed that the stabilization of the enzyme () was achievable by treatment with a 5% glutaraldehyde solution at pH 7.0 for 2 h (product activity 67 units/g of matrix, specific activity 4 units/mg of protein); this product also showed no release of protein during use. A higher activity (296 units/g of matrix was achieved by stabilization by treatment with a 5% tannic acid solution at pH 7.0 for 2 h. The combined use of glutaraldehyde and tannic acid was effective in stabilizing the bound enzyme () with an initial activity of 116 units/g of matrix. When use was made of the same support in presilanized (3-aminopropyltriethoxy silane) form followed by glutaraldehyde coupling a similar initial activity (112 units/g of matrix) was obtained, but the operational stability was much better (. 相似文献
17.
The phase transition temperature () of dipalmitoyl phosphatidic acid multilamellar liposomes is depressed 10°C by the inhalation anesthetic methoxyflurane at a concentration of 100 mmol/mol lipid. Application of 100 atm of helium pressure to pure phosphatidic acid liposomes increased only 1.5°C. However, application of 100 atm helium pressure to dipalmitoyl phosphatidic acid lipsomes containing 100 mmol methoxyflurane/mol lipid almost completely antagonized the effect of the anesthetic. A nonlinear pressure effect is observed. In a previous study, a concentration of 60 mmol methoxyflurane/mol dipalmitoyl phosphatidylcholine depressed only 1.5°C, exhibiting a linear pressure effect. The completely different behavior in the charged membrane is best explained by extrusion of the anesthetic from the lipid phase. 相似文献
18.
W R Scowcroft A H Gibson J D Pagan 《Biochemical and biophysical research communications》1976,73(2):516-523
Nitrogenase activity in agar cultures of cowpea rhizobia, strain 32H1, was rapidly inhibited by but this was relieved by increased O2 tension. Inhibition was more rapid than that caused by inhibitors of protein synthesis and was not relieved by methionine sulfoximine or methionine sulfone. Under conditions were nitrogenase activity was inhibited by , glutamine synthetase and glutamate synthase were substantially unaffected. Glutamate dehydrogenase was undetected in either nitrogenase active or inhibited cultures. These results indicate that inhibition of nitrogenase activity in strain 32H1 is not effected through glutamine synthetase regulation of nitrogenase synthesis. 相似文献
19.
Ta-Min Chang Deborah W. Kullberg 《Biochimica et Biophysica Acta (BBA)/Molecular Cell Research》1984,805(3):268-276
Diacytosis of 125I-asialoorosomucoid by rat hepatocytes was studied by preincubating the cells with the labelled ligand at 37°C for 30 min or 18°C for 2 h, washing free of cell surface receptor-bound tracer at 4°C and then reincubating at 37°C. The cells preloaded at 37°C released a maximum of 18% of the total intracellular ligand as undegraded molecules after 1 h of incubation with an apparent first-order rate constant of 0.018 min?1 (). When the preloaded cells were incubated in the presence of 100 μg/ml unlabelled asialoorosomucoid or 5 mM ethylene glycol bis(β-aminoethyl ether)-N,N,N′,N′-tetraacetic acid, the amount of the released ligand increased to 32 and 37%, respectively, without apparent change in kinetics, indicating that these agents prevented rebinding of the released ligand. In the presence of 5 μM colchicine, 20 μM cytochalasin B, 20 μM chloroquine, 10 mM NH4Cl, 10 μM monensin or 20 μM leupeptin, degradation of the preloaded ligand was inhibited, whereas the release of the ligand was either slightly increased or unchanged. Similar effects of leupeptin, colchicine and asialoocrosomucoid were observed with cells preloaded at 18°C. These results indicate that diacytosis of 125I-asialoorosomucoid occurs from a prelysosomal compartment via a route insensitive to inhibition by the inhibitors of ligand degradation. 相似文献
20.
The effect of prostaglandins E1 and E2 on the human erythrocyte as monitored by spin labels 总被引:8,自引:0,他引:8
P G Kury P W Ramwell H M McConnell 《Biochemical and biophysical research communications》1974,56(2):478-483
The effects of the prostaglandins PGE1 and PGE2 on the deformability of the human erythrocyte were studied using spin-labeled erythrocytes. Two magnetic resonance parameters were measured: (1) The orientation relaxation time, , for the erythrocyte, and (2) the order parameter, S, for a fatty acid spin label bound to the membrane. Prostaglandins PGE1 and PGE2 exhibited opposite effects on both and S. PGE2 made the cell less deformable (increases of and S) and PGE1 made the erythrocyte more deformable (decrease of and S). 相似文献