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1.
The immobilization of Rhodopseudomonas capsulata chromatophores by entrapment in an alginate gel is described. Alginate beads were prepared with Ba2+, Sr2+ and Ca2+ as gel-forming agents and compared for their mechanical strength, chemical resistance against disruption by phosphate-induced swelling, and yield of photophosphorylation activity. Barium alginate beads proved to have better physico-chemical properties than the more commonly used calcium alginate beads. After embedding in barium alginate gel, R. capsulata chromatophores retained a high yield (up to 70%) of their photophosphorylation capacity. Alginate entrapment did not cause a large increase in the Michaelis constant for ADP and phosphate, the substrates of adenosinetriphosphatase (ATPase). These constants were and for free chromatophores and and for chromatophores entrapped in barium alginate gel. However, embedding gave no additional protection against rapid inactivation of chromatophores upon storage at 3°C. Preliminary results with a batch reactor for continuous ATP regeneration are presented. The barium alginate method has two features which are not generally encountered at the same time, extremely mild conditions for entrapment and excellent physical properties of the gels beads, which make this method a suitable tool for the construction of bioreactors with immobilized cells or organelles. 相似文献
2.
G.E. Breitwieser 《生物化学与生物物理学报:生物膜》1982,689(3):457-463
(1) A membrane fraction enriched in (EC 3.6.1.3) was obtained from optic ganglia of the squid (Loligo pealei) by density gradient fractionation of membranes followed by treatment with either SDS or Brij-58. The resulting membrane had an specific activity of approx. 2 units/mg and was ouabain-sensitive. (2) The had a for ATP of and a pH optimum of 7.0. It was inhibited by ouabain with a of . (3) Optimum monovalent cation concentrations were: 240 mM NaCl, 60 mM KCl, tested with . (4) The Mg2+ dependence of hydrolysis varied with the absolute ATP concentration. At 3 mM ATP, the for Mg2+ was , and at 6 mM ATP, the was . High levels of Mg2+ caused inhibition of hydrolysis. (5) The interactions of Na+ and K+ were examined over a range of conditions. K+ levels caused modulations in the Na+ dependence in the range of 1–150 mM. (6) The prepared from squid optic ganglion displays properties similar to those of the sodium pump in injected nerves. 相似文献
3.
Robert I. Gelb Lowell M. Schwartz John J. Bradshaw Daniel A. Laufer 《Bioorganic chemistry》1980,9(3):299-304
Acid dissociation constants of aqueous cyclohexaamylose (6-Cy) and cycloheptaamylose (7-Cy) have been determined at 10–47 and 25–55°C, respectively, by pH potentiometry. Standard enthalpies and entropies of dissociation derived from the temperature dependences of these pKa's are ΔH0 = 8.4 ± 0.3 kcal mol?1, for 6-Cy and ΔH0 = 10.0 ± 0.1 kcal mol?1, for 7-Cy. Intrinsic 13C nmr resonance displacements of anionic 6- and 7-Cy were measured at 30°C in 5% D2O (). These results indicate that the dissociation of 6- and 7-Cy involves both C2 and C3 20-hydroxyl groups. The thermodynamic and nmr parameters are discussed in terms of interglucosyl hydrogen bonding. 相似文献
4.
F.M.A.H. Schuurmans Stekhoven H.G.P. Swarts Y.-F. Fu G.A.J. Kuijpers J.J.H.H.M. de Pont S.L. Bonting 《生物化学与生物物理学报:生物膜》1984,774(2):277-287
(1) Treatment of from rabbit kidney outer medulla with the labeled thio-analogue of ATP in the presence of and the absence of K+ leads to thiophosphorylation of the enzyme. The value for [γ-S]ATP is 2.2 μM and for Na+ 4.2 mM at 22°C. Thiophosphorylation is a sigmoidal function of the Na+ concentration, yielding a Hill coefficient . (2) The thio-analogue () can also support overall activity, but at 37°C is only h?1 or 0.09% of the specific activity for ATP (). (3) The thiophosphoenzyme intermediate, like the natural phosphoenzyme, is sensitive to hydroxylamine, indicating that it also is an acylphosphate. However, the thiophosphoenzyme, unlike the phosphoenzyme, is acid labile at temperatures as low as 0°C. The acid-denatured thiophosphoenzyme has optimal stability at pH 5–6. (4) The thiophosphorylation capacity of the enzyme is equal to its phosphorylation capacity, indicating the same number of sites. Phosphorylation by ATP excludes thiophosphorylation, suggesting that the two substrates compete for the same phosphorylation site. (5) The (apparent) rate constants of thiophosphorylation (0.4 s?1 vs. 180 s?1), spontaneous dethiophosphorylation (0.04 s?1 vs. 0.5 s?1) and K+-stimulated dethiophosphorylation (0.54 s?1 vs. 230 s?1) are much lower than those for the corresponding reactions based on ATP. (6) In contrast to the phosphoenzyme, the thiophosphoenzyme is ADP-sensitive (with an apparent rate constant in ADP-induced dethiophosphorylation of 0.35 s?1, ) and is relatively K+-insensitve. The for K+ in dethiophosphorylation is 0.9 mM and in dephosphorylation 0.09 mM. The thiophosphoenzyme appears to be for 75–90% in the ADP-sensitive E1-conformation. 相似文献
5.
(1) A quantitative study has been made of the binding of ouabain to the ( in homogenates prepared from brain tissue of the hawk moth, Manduca sexta. The results have been compared to those obtained in bovine brain microsomes. (2) The insect brain ( will bind ouabain either in the presence of Mg2+ and Pi, (‘Mg2+, Pi’ conditions) or in the presence of Na+, Mg2+, and an adenine nucleotide (‘nucleotide’ conditions) as is the case for the bovine brain (. The binding conditions did not alter the total number of receptor sites measured at high ouabain concentrations in either tissue. (3) Potassium ion decreases the affinity (increases the ) of ouabain to the M. sexta brain ( under both binding conditions. However, ouabain binding is more sensitive to K+ inhibition under the nucleotide conditions. In bovine brain ouabain binding is equally sensitive to K+ inhibition under the both conditions. (4) The enzyme-ouabain complex has a rate of dissociation that is 10-fold faster in the M. sexta preparation than in the bovine brain preparation. Because of this, the M. sexta ( has a higher for ouabain binding and is less sensitive to inhibition by ouabain than the bovine brain enzyme. (5) This data supports the hypothesis that two different conformational states of the M. sexta ( can bind ouabain. 相似文献
6.
The concept of high- and low-affinity reactions in bovine cytochrome c oxidase steady-state kinetics
(1) Analysis of the data from steady-state kinetic studies shows that two reactions between cytochrome c and cytochrome c oxidase sufficed to describe the concave Eadie-Hofstee plots ( and ). It is not necessary to postulate a third reaction of . (2) Change of temperature, type of detergent and type of cytochrome c affected both reactions to the same extent. The presence of only a single catalytic cytochrome c interaction site on the oxidase could explain the kinetic data. (3) Our experiments support the notion that, at least under our conditions (pH 7.8, low-ionic strength), the dissociation of ferricytochrome c from cytochrome c oxidase is the rate-limiting step in the steady-state kinetics. (4) A series of models, proposed to describe the observed steady-state kinetics, is discussed. 相似文献
7.
Mutations at the bithoraxoid (bxd) and postbithorax (pbx) loci cause a transformation of posterior haltere to posterior wing. It has previously been shown that pbx and cause this transformation by affecting the maintenance (or cell heredity function) of determination so that the transformed cells are indistinguishable from normal wing cells, and have no “memory” of having been part of a haltere disk (Adler, 1978a). I report here that Tp(3) and bxd1, pbx, ew both cause the transformation of posterior haltere to posterior wing in the same way as pbx. On the other hand, bxd1, , bxd51j, , and cause this same transformation of posterior haltere to posterior wing by interfering with the expression of the determined state so that the developmental information of posterior haltere is “misread” as posterior wing. The transformed cells in these disks retain the memory of having been part of a haltere disk; that is, these posterior cells that would secrete wing cuticle during metamorphosis regenerate anterior haltere structures. Thus it appears clear that it is possible to uncouple the expression and cell heredity functions of determination in the haltere disk of Drosophila. 相似文献
8.
Photoconversion and regeneration of active protochlorophyll(ide) in mutants defective in the regulation of chlorophyll synthesis 总被引:1,自引:0,他引:1
O F Nielsen 《Archives of biochemistry and biophysics》1974,160(2):430-439
Seedlings carrying mutations in regulatory genes for protochlorophyll(ide) synthesis accumulate protochlorophyll(ide) in darkness in amounts exceeding the wildtype level. Thus, +/tig-d12 and accumulate 2-fold, 5- to 6-fold, and 15-fold more protochlorophyll(ide) than the wild type.The amount of photoconvertible protochlorophyll(ide) accumulated in darkness is the same in all genotypes, despite the large differences in total protochlorophyll(ide) content, indicating a constant number of photoconversion sites.When briefly illuminated leaves are returned to darkness, regeneration of active protochlorophyll(ide) from the pool of inactive protochlorophyll(ide) takes place in wild-type and mutant leaves. Compared to the wild type, the rate of protochlorophyll(ide) activation during 4- and 10-min dark periods is higher in +/tig-d12, , and , but lower in .There was no indication that the accumulation of protochlorophyll(ide) influences the conversion sites of the protochlorophyll(ide) holochrome, as the kinetics of photoconversion of initially active protochlorophyll(ide) in leaves with the genotypes +/+, +/tig-o34, and are similar or identical. 相似文献
9.
Bovine spermatozoa, maturing in the caudal epididymis, apparently have a low content of mobilizable Ca2+ (6 ± 1 nmoles cells) in situ, but will accumulate added Ca2+ when simply diluted into isotonic media. It is suggested that the low concentrations of Ca2+ (0.5 ± 0.1 mM) and O2 present in epididymal fluids prevent the accumulatin of Ca2+ by sperm prior to ejaculation. Although the seminal plasma that surrounds ejaculated sperm contains 9 ± 1 mM Ca2+, washed ejaculated sperm also have a low Ca2+ content (7 ± 1 nmoles cells). Moreover, in contrast to epididymal sperm, washed ejaculated sperm are incapable of accumulating Ca2+ supplied exogenously. Evidence is presented that bovine seminal fluids contain a component that is added to the surface membranes of the sperm at ejaculation which prevents or delays the active uptake of Ca2+ by these cells. 相似文献
10.
The interactions between calmodulin, ATP and Ca2+ on the red cell Ca2+ pump have been studied in membranes stripped of native calmodulin or rebound with purified red cell calmodulin. Calmodulin stimulates the maximal rate of by 5–10-fold and the rate of Ca2+-dependent phosphorylation by at least 10-fold. In calmodulin-bound membranes ATP activates along a biphasic concentration curve (), but in stripped membranes the curve is essentially hyperbolic (). In calmodulin-bound membranes Ca2+ activates at low concentrations () in stripped membranes the apparent Ca2+ affinities are at least 10-fold lower.The results suggest that calmodulin (and perhaps ATP) affect a conformational equilibrium between E2 and E1 forms of the Ca2+ pump protein. 相似文献
11.
The binding of the crustacean selective protein neurotoxin, toxin B-IV, from the nemertine Cerebratulus lacteus to lobster axonal vesicles has been studied. A highly radioactive, pharmacologically active derivative of toxin B-IV has been prepared by reaction with Bolton-Hunter reagent. Saturation binding and competition of 125I-labeled toxin B-IV by native toxin B-IV have shown specific binding of 125I-labeled toxin B-IV to a single class of binding sites with a dissociation constant of 5–20 nM and a binding site capacity, corrected for vesicle sidedness, of 6–9 pmol per mg membrane protein. This compares to a value of 3.8 pmol [3H]saxitoxin bound per mg in the same tissue. Analysis of the kinetics of toxin B-IV association () and dissociation () shows a nearly identical of about 3 nM. There is no competition of toxin B-IV binding by purified toxin from Leiurus quinquestriatus venom while Centruroides sculpturatus Ewing toxin I appears to cause a small enhancement of toxin B-IV binding. 相似文献
12.
Dennis W. Jung Guey-Yueh Shi Gerald P. Brierley 《Archives of biochemistry and biophysics》1981,209(2):356-361
Depletion of mitochondrial divalent cations by addition of the ionophore A23187 results in a marked increase in passive exchange activity. The exchange is activated by increasing pH and temperature and inhibited by added divalent cations. The reaction is independent of the amount of A23187 present, but depends on the concentration of external K+ (Km = 25 mm). Intramitochondrial 42K+ in cation-depleted mitochondria exchanges passively with external Na+ and Li+, but not with choline+. The evidence suggests that removal of mitochondrial divalent cations by A23187 activates the endogenous exchange component of the mitochondrion and that the activated exchanger promotes cation/cation exchange in the absence of a metabolic pH gradient. 相似文献
13.
Robert F. Anderson 《BBA》1983,723(1):78-82
The bimolecular decay rates (2k) of the flavosemiquinones () of riboflavin, FMN and FAD have been determined using pulse radiolysis. The rates (defined as ) for the neutral flavosemiquinones at zero ionic strength and pH 5.9 are (in units of mol?1·dm3·s?1): (1.2 ± 0.1)·109, (5.0 ± 0.2)·108 and (1.4 ± 0.1)·108; and for the anionic flavosemiquinones at pH 11.2 (5.4 ± 0.9)·108, (4.5 ± 0.3)·107 and (8.5 ± 1.3)·106, respectively. The kinetic salt effect has been used to formulate rate equations for each flavin to adjust for ionic strength effects. 相似文献
14.
Single spherical bilayer membranes of the Pagano-Thompson type (Pagano, R. and Thompson, T.E. (1967) Biochim. Biophys. Acta 144, 666–669), formed from monooleyl phosphate and cholesterol dissolved in CHCl3/CH3OH/n-decane, were subjected to a fast impedance analysis of high precision. Dielectric behavior of the whole system, as monitored from outside the spherical membrane, was sensitive to changes in the membrane state from the thick colored to the thin black state. With a spherical membrane 2–3 mm in diameter formed in the sample cavity containing 0.12 ml 10 mM NaCl, the former state was characterized by a dielectric dispersion having dielectric increment (Δ?) of some 102 and characteristic frequency () around 106 Hz, while the latter had . Complex plane plots for both dispersions traced semicircles, indicating that the present system may be unequivocally analyzed to yield spherical radius and membrane capacity (Cm) on the basis of a well-established dielectric theory. Cm for the thin membranes has thus been determined to be 0.54 μF · cm?2, in excellent agreement with a separate determination on planar membranes. The applicability of the present type of spherical membranes under dielectric monitoring to the study of membrane fusion or of exocytosis is suggested. 相似文献
15.
J.J. Schrijen A. Omachi W.A.H.M. Van Groningen-Luyben J.J.H.H.M. De Pont S.L. Bonting 《生物化学与生物物理学报:生物膜》1981,649(1):1-12
(1) The total phospholipid content of a gradient purified ()-ATPase preparation from pig gastric mucosa is 105 μmol per 100 mg protein, and consists of 29% sphingomyelin, 29% phosphatidylcholine, 28% phosphatidylethanolamine, 10% phosphatidylserine and 4% phosphatidylinositol. The cholesterol content corresponds to 50 μmol per 100 mg protein. (2) Treatment with phospholipase C (from Clostridium welchii and Bacillus cereus) results in an immediate decrease of the phosphate content. Up to 50% of the phospholipids are hydrolyzed by each phospholipase C preparation alone, without further hydrolysis by increased phospholipase concentration or prolonged incubation time. Combined treatment with the two phospholipase C preparations, sequentially or simultaneously, hydrolyzes up to 65% of the phospholipids. (3) The ()-ATPase and K+ stimulated activities are decreased proportionally with the total phospholipid content, indicating that these enzyme activities are dependent on phospholipids. (4) Phospholipase C treatment does not change optimal pH, value for ATP and temperature dependence of the gastric ()-ATPase, but slightly decreases the value for K+. (5) Phospholipase C treatment lowers the binding and phosphorylation capacities, suggesting that inactivation occurs primarily on the substrate binding level. (6) Most of the results can be understood by assuming that hydrolysis of the phospholipids by phospholipase C leads to aggregation of the membrane protein molecules and complete inactivation of the aggregated ATPase molecules. 相似文献
16.
Diploid embryos which are homozygous for the t12 mutation die at the morula stage. In the current studies, ova from heterozygous () females were fertilized in vitro with spermatozoa from males. The fertilized ova were immediately placed into media containing cytochalasin B to prevent second polar body formation, producing +/+/+, +/+/t12, +/t12/t12, and t12/t12/t12 embryos. The subsequent development of these triploid embryos was compared with that of diploid +/+, , and embryos developing from ova which were also fertilized in vitro with spermatozoa from males but which were not treated with cytochalasin B immediately following gamete coincubation. The data show that those triploid embryos which possess a wild-type allele and two mutant alleles are phenotypically wild type while those possessing three mutant alleles are not phenotypically distinguishable from their diploid () counterparts. Like embryos, t12/t12/t12 embryos die at the morula stage, prior to blastocoelic cavity formation. 相似文献
17.
(1) +/electron acceptor ratios have been determined with the oxidant pulse method for cells of denitrifying Paracoccus denitrificans oxidizing endogenous substrates during reduction of O2, NO?2 or N2O. Under optimal H+-translocation conditions, the ratios , , for reduction to N2 and for reduction to N2O were 6.0–6.3, 4.02, 5.79 and 3.37, respectively. (2) With ascorbate/N,N,N′,N′-tetramethyl-p-phenylenediamine as exogenous substrate, addition of NO?2 or N2O to an anaerobic cell suspension resulted in rapid alkalinization of the outer bulk medium. , for reduction to N2 and for reduction to N2O were ?0.84, ?2.33 and ?1.90, respectively. (3) The ratios, mentioned in item 2, were not altered in the presence of and the triphenylmethylphosphonium cation. (4) A simplified scheme of electron transport to O2, NO?2 and N2O is presented which shows a periplasmic orientation of the nitrite reductase as well as the nitrous oxide reductase. Electrons destined for NO?2, N2O or O2 pass two H+-translocating sites. The acceptor ratios predicted by this scheme are in good agreement with the experimental values. 相似文献
18.
Chana Vinkler 《Biochemical and biophysical research communications》1981,99(4):1095-1100
The of ADP for photophosphorylation in lettuce chloroplasts was measured both at various light intensities and in the presence of various uncoupler (nigericin + K+) concentrations. Lowering the light intensity results in both, a decrease in the rate of phosphorylation and a several fold in the of ADP for the reaction. However, when increasing concentrations of the uncoupler nigericin + K+ are employed, the rate of photophosphorylation is decreased but a several-fold in the of ADP for the reaction is observed. The results are discussed in terms of the chemiosmotic hypothesis. It is suggested that these effects might indicate the existence of a mechanism controlling the rate of ATP formation which is different than the formation of the electrochemical gradient. 相似文献
19.
P. Månsson Ö. Jacobsson K.A. Granath 《International journal of biological macromolecules》1985,7(1):30-32
The molecular weight of Na- and K-hyaluronate has been determined by low angle laser light scattering (LALLS) technique. Two preparations of hyaluronate from rooster comb () were investigated. The LALLS was carried out both in a static mode and on the effluent from a column filled with porous gel. In contrast to Sheehan et al.1, no significant difference was found in the molecular weight of viscosity of Na- and K-hyaluronate in 2.0 M salt solutions 相似文献
20.
A simple procedure for resolution of Escherichia coli RNA polymerase holoenzyme from core polymerase. 总被引:32,自引:0,他引:32
The association constant, KA, for myosin subfragment-1 binding to actin was measured as a function of ionic strength [KCl, LiCl, and tetramethylammonium chloride (TMAC)]and temperature by the method of time-resolved fluorescence depolarization. The following thermodynamic values were obtained from solutions of 0.20 × 10?6m S-1, 1.00 × 10?6m actin in 0.15 m KCl, pH 7.0, at 25 °C: ΔG ° = ?39 ± 1 kJ M?1, ΔH0 = 44 ± 2 kJ M?1 and . For measurements in KCl (0.05 to 0.60 m), In . Thus, the binding is endothermic and strongly inhibited by high ionic strength. When KCl was replaced by LiCl or TMAC the ionic effects on the binding were cation specific. The nature of actin-(S-1) binding in the rigor state is discussed in terms of these results. 相似文献