共查询到20条相似文献,搜索用时 31 毫秒
1.
Franklin Fuchs 《BBA》1977,462(2):314-322
A double isotope technique and EGTA buffers were used to measure the binding of Ca2+ to rabbit psoas muscle fibers extracted with detergent and glycerol. These experiments were designed to test the effect of rigor complex formation, determined by the degree of filament overlap, on the properties of the Ca2+-binding sites in the intact filament lattice. In the presence of 5 mM MgCl2 (no ATP), reduction of filament overlap was associated with a reduced binding of Ca2+ over the entire range of free Ca2+ concentrations (5 · 10?8 – 2 · 10?5 M). With maximum filament overlap (sarcomere length 2.1–2.2 μm) the maximum bound Ca2+ was equivalent to 4 mol Ca2+/mol troponin and there was significant positive interaction between binding sites, as shown by Scatchard and Hill plots. With no filament overlap (sarcomere length 3.8–4.4 μm) the maximum bound Ca2+ was equivalent to 3 μmol Ca2+/mol troponin and graphical analysis indicated a single class of non-interacting sites. The data provide evidence that when cross-bridge attachments between actin and myosin filaments are formed not only does an additional Ca2+ binding site appear, but cooperative properties are imposed upon the binding sites. 相似文献
2.
Armand Malnoë Jos A. Cox Eric A. Stein 《Biochimica et Biophysica Acta (BBA)/General Subjects》1982,714(1):84-92
The binding parameters of 125I-labeled calmodulin to bovine cerebellar membranes have been determined and correlted with the activation of adenylate cyclase by calmodulin. In the presence of saturating levels of free Ca2+, calmodulin binds to a finite number of specific membrane sites with a dissociation constant (Kd) of 1.2 nM. Furthermore, Scatchard analysis reveals a second population of binding sites with a 100-fold lower affinity for calmodulin. The Ca2+-dependence of calmodulin binding and of adenylate cyclase activation varies with the amount of calmodulin present, as can be infered from the model of sequential equilibrium reactions which describes the activation of calmodulin-dependent enzymes. On the basis of this model, a quantitative analysis of the effect of free Ca2+ and of free calmodulin concentration on both binding and activation of adenylate cyclase was carried out. This analysis shows that both processes take place only when calmodulin is complexed with at least three Ca2+ atoms. The concentration of the active calmodulin ·Ca2+ species required for half-maximal activation of adenylate cyclase is very similar to the Kd of the high affinity binding sites on brain membranes. A Hill coefficient of approx. 1 was found for both processes indicating an absence of cooperativity. Phenothiazines and thioxanthenes antipsychotic agents inhibit calmodulin binding to membranes and calmodulin-dependent activation of adenylate cyclase with a similar order of potency. These results suggest that the Ca2+-dependent binding of calmodulin to specific high affinity sites on brain membranes regulates the activation of adenylate cyclase by calmodulin. 相似文献
3.
A simple double-isotope procedure has been developed for making simultaneous measurements of bound Ca2+ and relative force in glycerinated rabbit psoas bundles containing two fibers. With this preparation it is possible to study Ca2+-troponin interactions coincident with MgATP-induced force development. Over the free [Ca2+] range 6 · 10?8–1.2 · 10?5 M the bound Ca2+ varied from 0.25 to 1.65 μmol/g protein. The free [Ca2+] at half-maximal Ca2+ saturation was 2 · 10?7 M while that a half-maximal force was 5 · 10?7 M. Half-maximal Ca2+ saturation was associated with 20% maximal force. The force-[Ca2+] saturation curve showed a steep rise in slope at greater than half saturation. The observed relationship was consistent with a model in which multiple occupancy of troponin Ca2+-binding sites is essential for initiation of cross-bridge cycling. 相似文献
4.
Godfrey W. Amphlett Walter Kisiel Francis J. Castellino 《Archives of biochemistry and biophysics》1981,208(2):576-585
The binding isotherms of Ca2+ and Sr2+ to human blood coagulation Factor IX have been obtained at 25 °C and pH 7.4. In the case of both cations, a Scatchard plot of the data reveals that a single class of binding sites exist. For Ca2+, a total of 16.0 ± 1.0 sites, of KD 7.3 ± 0.2 × 10?4m, are present on human Factor IX. Similar analysis of the Sr2+ data indicates that Factor IX contains 11.0 ± 1.0 binding sites, with a KD of 1.9 ± 0.1 × 10?3m. Both Sr2+ and Mn2+ effectively displace Ca2+ from human Factor IX; whereas Mg2+ is considerably less potent in this regard. Conversely, Ca2+ is capable of nearly complete displacement of Sr2+ from its binding sites on human Factor IX. The activation of human Factor IX, by human Factor XIa, shows a complex dependence on the Ca2+ concentration. Sr2+ can substitute for Ca2+ in this activation process. Mn2+ cannot, in itself, substitute for Ca2+ in activation of Factor IX, but does significantly enhance the activation of Factor IX by Factor XIa at suboptimal levels of Ca2+. The rate of activation of human Factor IX by the coagulant protein of Russell's viper venom also shows a dependence on the presence of divalent cations. Here, however, a rigid specificity is not noted, since Ca2+, Sr2+, and Mn2+ all allow activation to proceed equally well. 相似文献
5.
David C. Pang 《生物化学与生物物理学报:生物膜》1980,598(3):528-542
Ca2+ binding to fragmented sarcolemma isolated from canine heart was measured by an ultracentrifugation technique. Two classes of binding site with dissociation constants of 2.0 · 10?5 and 1.2 · 10?3 M were identified. The capacities of the high- and low-affinity sites were 15 and 452 nmol/mg, respectively. These sites were not affected by treatment with neuraminidase. The effects of various cations and drugs on Ca2+ binding were studied. All cations tested inhibited Ca2+ binding with the following order of potency: trivalent > divalent > monovalent cations. The order of potency for the monovalent ions was: Na+ > K+ > Li+ ? Cs+ and for the divalent and trivalent ions: La3+ ? Mn2+ > Sr2+ ? Ba2+ > Mg2+. 1 · 10?3 M caffeine and 1 · 10?8 M ouabain increased the capacity of the low-affinity sites to 1531 and 837 nmol/mg, respectively. 1 · 10?7 M verapamil, acidosis (pH 6.4), 1?10?5 M Mn2+ and 1 · 10?4 M ouabain depressed the capacity of the low-affinity sites to a range of 154–291 nmol/mg. The dissociation constants of the high- and low-affinity sites and the capacity of the high-affinity sites were not affected by these agents. 相似文献
6.
7.
The combination of ultrafiltration and difference spectroscopy allows the quantitative determination of spectra of thionine bound to heparin. The spectra of the bound dye do not depend on the degree of coverage; this and the shape of the Scatchard plot show that “all-or-none” binding is valid. A calculus of variations based on a modification of the Hill plot shows that aggregates of seven thionine cations are bound. Tetrasaccharides with an average charge of two carboxylate and five sulfate groups are suggested to be the binding sites. The binding constant given for one mole thionine is 4.4 · 105 M?1. The Gibbs enthalpy for binding of one mole of thionine is ?31.7 kJ·M?1 at 20°C. 相似文献
8.
Olin D. Liang Marco Maccarana Jan-Ingmar Flock Marianne Paulsson Klaus T. Preissner Torker Wadström 《生物化学与生物物理学报:疾病的分子基础》1993,1225(1):57-63
Multiple interactions between human vitronectin and Staphylococcus aureus strain V8 were observed. An upward-curved Scatchard plot indicated both high-affinity binding (Kd1 = 7.4 · 10?10 M) with 260 binding sites per bacterial cell and moderate-affinity binding (Kd2 = 7.4 · 10?8 M) with 5240 copies per cell. Negative cooperativity of this binding was characterized by its Hill coeffiocient of less than unity (0.70 ± 0.08). Up to 60% of the vitronectin-bacteria interaction was unaffected by high ionic strength (i.e., 2.4 M NaCl), and was not inhibited by highly-charged heparin oligosaccharides. Various oligosaccharides (4–20 monosaccharide units) generated by partial deaminative cleavage of heparin were found to affect vitronectin binding to S. aureus. Short-chain-length oligosaccharides increase and long oligosaccharides inhibit vitronectin binding, in accordance with direct association of these saccharides with multimeric vitroectin. A protein having a molecular mass of 60 kDa was identified as a putative high-affinity staphylococcal vitronectic-binding protein. These results indicate that interaction of multimeric vitronectin, mostly present at extracellular matrix sites with multiple recognition sites on the S. aureus surface, may contribute to bacterial colonisation. 相似文献
9.
Jun Nakamura 《BBA》1983,723(2):182-190
The effects of ATP on Ca2+ binding in the absence of added Mg2+ to the purified sarcoplasmic reticulum Ca2+-ATPase were studied at pH 7.0 and 0°C. ATP increased the number of Ca2+-binding sites of the enzyme from 2 to 3 mol per mol of phosphorylatable enzyme. The association constant for the ATP-induced Ca2+ binding was 4·105 M?1, which was not significantly different from that obtained in the absence of ATP. AdoP[CH2]PP has little effect on the Ca2+-binding process. The amount of phosphoenzyme formed was equivalent to the level of ATP-induced Ca2+ binding. ADP decreased the level of ATP-induced Ca2+ binding and phosphoenzyme by the same amount. These results suggest that ATP-induced Ca2+ binding exists in the form of an ADP-reactive phosphoenzyme·Ca complex. In addition, the Ca2+ bound to the enzyme in the presence of ATP was released on the addition of 1 mM MgCl2; after the release of Ca2+, the phosphoenzyme decayed. These observations suggest that Mg2+, added after the ATP-induced Ca2+-binding process, may replace the Ca2+ on the phosphoenzyme and initiate phosphoenzyme decomposition. 相似文献
10.
Ca2+ binding by skeletal muscle microsomes in 5 mM ATP exhibited saturation kinetics in the range of Ca2+ concentrations between 3 · 10?8 and 10?5 M. Approximately 140 nmoles binding sites per mg protein were found. These had a Ca2+ binding constant of approximately 4.5 · 106 M?1 with half saturation at 2.2 · 10?7 M Ca2+. In the presence of oxalate, much larger amounts of Ca2+ (approx. 6 μmoles/mg protein) were taken up by the microsomes (Ca2+ uptake), but the rate of Ca2+ uptake increased linearly with [Ca2+] when ionized Ca2+ concentrations were below 3 · 10?6 M. At Ca2+ concentrations above 3 · 10?6, Ca2+ uptake was inhibited. Double reciprocal plots of the Ca2+ dependence of the initial rates of Ca2+ uptake in the concentration range between 3 · 10?7 M and 10?5 M, unlike those of Ca2+ binding, did not demonstrate saturation kinetics, but could be interpreted as representing a non-saturable system with inhibition at higher Ca2+ concentrations. In view of these differences, and because Ca2+-binding sites were almost fully saturated at 10?6 M Ca2+, whereas Ca2+ uptake rate increased linearly with increasing [Ca2+] to approximately 3 · 10?6 M, the Ca2+-binding sites are not shown kinetically to participate in oxalate-dependent Ca2+ uptake. 相似文献
11.
Ouabain binding was studied in isolated adult dog heart myocytes. The binding was correlated with the inhibition of K+-activated para-nitrophenylphosphatase (K+-PNPPase) activity and the beating response. It was shown that: (i) the specific binding was dependent upon Mg2+ and was inhibited by K+; (ii) the maximal binding capacity (Bmax) was 7.4 × 105 ouabain molecules per cell, or 410 pmol ouabain/K+-PNPPase unit (μmol/min); (iii) in the presence of Mg2+ (5 mm), there were two components in the Scatchard plot, i.e., a high-affinity component with a Kd value of 5.6 × 10?8m and a low-affinity component with a Kd value of 6.7 × 10?7m; (iv) the Hill coefficient (n′) for ouabain binding was 0.72 with a S0.5 value of 7.1 × 10?7m; these values were compatible with the values obtained from studies of K+-PNPPase inhibition by ouabain (n′ = 0.55, S0.5 = 3.6 × 10?7 m) and remained unchanged in the presence of physiological concentrations of Na+ plus K+; (v) in the presence of Mg2+ and K+, the high-affinity component tended to conform to the low-affinity component with an apparent decrease in Bmax; (vi) in the presence of Mg2+ and para-nitrophenylphosphate, the low-affinity component was changed to the high-affinity component with no change in Bmax; (vii) the dissociation rate of the labeled ouabain in the highly dilute medium was not altered in the presence of excess amounts of unlabeled ligand; this eliminated the possibility that the apparent negative cooperativity was due to a site-to-site interaction between receptors; (viii) ouabain increased the number of beating cells and the frequency of beating. Based on these findings, it is concluded that: (i) isolated myocytes possess functional receptors for ouabain; (ii) the binding of ouabain is associated with its inhibition of K+-PNPPase activity; (iii) ouabain receptors in isolated myocytes are of one class with at least two interconvertible conformational states. 相似文献
12.
Marie-José Prigent Patrice Boquet 《Biochimica et Biophysica Acta (BBA)/General Subjects》1982,717(3):445-452
The mode of binding of Vicia graminea125I-labelled lectin to human M and N erythrocytes at 4°C has been investigated. The labelled lectin retained the full activity of native lectin. Lectin association at 4°C was characterized by a of 3 to 5 min, reaching steady-state within 15 min. Incubation of cells for 15 min at 4°C with increasing concentrations of Vicia graminea125I-labelled lectin showed that saturation binding occurred. Scatchard analysis of equilibrium data determined over a wide range of lectin concentrations yielded a curvilinear plot with an upward concave slope; this representation indicated that there was not a single homogeneous class of noninteracting binding sites. This result could indicate two or more independent classes of binding sites or one class of interacting sites exhibiting negative cooperativity. Since unlabelled lectin, which at the concentration used, rapidly binds to available receptors, did not affect the dissociation rate of the labelled lectin and since identical Scatchard plots were found using native and formaldehyde-fixed erythrocytes we conclude that there are two classes of independent Vicia graminea binding sites on human erythrocytes. Computer analysis of the Scatchard plots gave high- and low-affinity constant (7.07±1.1) · 107 M?1 and (0.2±0.01) · 107 M?1, respectively, for N erythrocytes and (1.13±0.18) · 107 M?1 and (0.24±0.01) · 107 M?1, respectively for the M cells. N erythrocytes were estimated to have 0.085 · 105 high-affinity and 2.1 · 105 low-affinity sites and M erythrocytes, 0.011 · 105 high affinity and 0.13 · 105 low-affinity sites. N cells therefore have 10-times as many sites as M cells. Studies of the dissociation of 125I-labelled lectin from N and M cells in the presence of unlabelled lectin gave dissociation rate constants of 51 · 10?4 s?1 and 1.97 · 10?4 s?1 for the high- and low-affinity sites of N cells and 13 · 10?4 s?1 and 1.6 · 10?4 s?1 for the high- and low-affinitym sites of M cells, indicating that the binding of Vicia graminea lectin to human erythrocytes is reversible. 相似文献
13.
Lipoxygenbase-derived products of arachidonic acid mediate stimulation of hexose uptake in human polymorphonuclear leukocytes 总被引:3,自引:0,他引:3
D A Bass M J Thomas E J Goetzl L R DeChatelet C E McCall 《Biochemical and biophysical research communications》1981,100(1):1-7
The titration of metal-freed bovine α-lactalbumin with Mg2+ ions causes a two-stepped decrease in the tryptophan fluorescence quantum yield and a pronounced spectral shift towards shorter wavelengths, which seems to be a result of the binding of two magnesium ions to the protein molecule. The magnesium binding constants evaluated from the fluorimetric Mg2+-titration are 2·103 and 2·102 M?1. Mg2+ ions in millimolar concentrations almost do not influence the binding of Ca2+ ions to the protein. 相似文献
14.
Kikuo Kasai James B. Field 《Biochimica et Biophysica Acta (BBA)/General Subjects》1982,718(2):125-134
Bovine thyroid tissue exhibited cAMP-dependent and Ca2+-dependent protien kinase activities as well as a basal (cAMP- and Ca2+-independent) one, and phosphoprotein phosphatase activity. Although the former two protein kiniase activities were not clearly demonstrated using endogenous protein as substrate, they were clearly shown in soluble, particulate and plasma membrane fractions using exogenous histones as substrate. The highest specific activities were in the plasma membrane. The apparent Km values of cAMP and Ca2+ for the membrane-bound protein kinase were 5·10?8 M and 8.3·10?4M (in the presence of 1 mM EGTA), respectively. The apparent Km values of Mg2+ were 7·10?4 M (without cAMP and Ca2+, 5·10?4 M (with cAMP) and 1.3·10?3 M (with Ca2+), and those ATP were 3.5·10?5 M (with or without cAMP) and 8.5·10?5 M (with Ca2+). The Ca2+-dependent protein kinase could be dissociated from the membrane by EGTA-washing. The enzyme activity so released was further activated by added phospholipid (phosphatidylserine/1,3-diolein), but not by calmodulin. Phosphoprotein phosphatase activity was also clearly demonstrated in all of the fractions using 32P-labeled mixed histones as substrate. The activity was not modified by either cAMP or Ca2+, but was sitmulated by a rather broad range (5–25 mM) of Mg2+ and Mn2+. NaCl and substrate concentrations also influenced the activity. Pyrophosphate, ATP, inorganic phosphate and NaF inhibited the activity in a dose-dependent manner. Trifluoperazine, chlorpromazine, dibucaine and Triton X-100 (above 0.05%, w/v) specifically inhibited the Ca2+-dependent protein kinase in plasma membranes. Repetitive phosphorylation of intrinsic and extrinsic proteins by the membrane-bound enzyme activities clearly showed an important co-ordination of them at the step of protein phosphorylation. These findings suggest that these enzyme activities in plasma membranes may contribute to regulation of thyroid function in response to external stimuli. 相似文献
15.
ATPase was found in plasma membrane of cultured endothelial cells from bovine carotid artery. The activity of the enzyme solubilized by octaethyleneglycol mono-n-dodecyl ether was enhanced by the addition of Ca2+ or Mg2+ and was not affected by F-actin and ouabain. Vmax was 2.8 and 10.0 μmol Pi/mg protein per h for Ca2+- and Mg2+-dependent activity, respectively, and the corresponding Km was 4.8·10?4 M and 3.2·10?4 M. Molecular weight of the protein was estimated to be approx. 250 000, as determined by activity-staining electrophoresis with polyacrylamide gels. 相似文献
16.
Ernst Graf Adelaida G. Filoteo John T. Penniston 《Archives of biochemistry and biophysics》1980,203(2):719-726
Of several methods employed for preparing 125I-calmodulin, only the glucose oxidase-lactoperoxidase system under controlled conditions produced an iodinated derivative which retained complete biological activity. Unlabeled calmodulin and 125I-calmodulin stimulated cyclic nucleotide phosphodiesterase from bovine brain interchangeably and both proteins displaced 125I-calmodulin from high-affinity binding sites on human erythrocyte ghosts with equal effectiveness. This procedure yielded a labeling stoichiometry of 1.34. Scatchard plots of binding of 125I-calmodulin to ghosts were consistent with the presence of a single class of high-affinity binding sites with the properties expected of (Ca2+ + Mg2+)-ATPase molecules. The binding showed positive cooperativity and occurred only in the presence of Ca2+. The maximum amount of binding seen in Scatchard plots corresponded to 4.1 × 103 sites per ghost. 相似文献
17.
The kinetic plot (initial rate of Ca2+ transport versus concentration) of mitochondrial Ca2+ transport is hyperbolic in a sucrose medium. The plot becomes sigmoidal in the presence of competitive inhibitors of Ca2+ binding to low affinity sites of the membrane surface such as Mg2+ and K+. The plot also becomes sigmoidal in the presence of Ba2+. Ba2+ is a competitive inhibitor of both Ca2+ transport and Ca2+ binding to the low affinity sites. The Ki for the inhibition of Ca2+ transport by Ba2+ increases in the presence of K+ and Mg2+, which suggests a competition for the low affinity sites between the cations. The plot is still hyperbolic in the presence of La3+, which inhibits Ca2+ transport competitively. Ruthenium red which is a pure non-competitive inhibitor of mitochondrial Ca2+ transport, does not affect the shape of the kinetic plot. These results indicate that the surface potential, which depends on the ions bound to the low affinity sites, determines whether the kinetics of Ca2+ uptake in mitochondria is sigmoidal or hyperbolic. 相似文献
18.
Barbara E. Ehrlich Jared M. Diamond 《Biochimica et Biophysica Acta (BBA)/General Subjects》1978,543(2):264-268
A new method for Li+ analysis by flameless atomic absorption spectroscopy is several orders of magnitude more sensitive than previous methods. Li+ quantities as small as 1·10?12 mol, or Li+ concentrations as low as 1·10?8 M, can be determined with a coefficient of variation of 2–4%. The same technique can determine approx. 1·10?14 mol of Ca2+ and Mg2+, and approx. 1·10?13 mol of Na+ and K+. 相似文献
19.
Mg2+ and ATP effects on K+ activation of the Ca2+-transport ATpase of cardiac sarcoplasmic reticulum
Larry R. Jones 《生物化学与生物物理学报:生物膜》1979,557(1):230-242
ATP and the divalent cations Mg2+ and Ca2+ regulated K+ stimulation of the Ca2+-transport ATPase of cardiac sarcoplasmic reticulum vesicles. Millimolar concentrations of total ATP increased the K+-stimulated ATPase activity of the Ca2+ pump by two mechanisms. First, ATP chelated free Mg2+ and, at low ionized Mg2+ concentrations, K+ was shown to be a potent activator of ATP hydrolysis. In the absence of K+ ionized Mg2+ activated the enzyme half-maximally at approximately 1 mM, whereas in the presence of K+ the concentration of ionized Mg2+ required for half-maximal activation was reduced at least 20-fold. Second MgATP apparently interacted directly with the enzyme at a low affinity nucleotide site to facilitate K+-stimulation. With a saturating concentration of ionized Mg2+, stimulation by K+ was 2-fold, but only when the MgATP concentration was greater than 2 mM. Hill plots showed that K+ increased the concentration of MgATP required for half-maximal enzymic activation approx. 3-fold.Activation of K+-stimulated ATPase activity by Ca2+ was maximal at anionized Ca2+ concentration of approx. 1 μM. At very high concentrations of either Ca2+ or Mg2+, basal Ca2+-dependent ATPase activity persisted, but the enzymic response to K+ was completely inhibited. The results provide further evidence that the Ca2+-transport ATPase of cardiac sarcoplasmic reticulum has distinct sites for monovalent cations, which in turn interact allosterically with other regulatory sites on the enzyme. 相似文献
20.
Levorphanol inhibits Ca++ binding to synaptic membranes in a dose dependent fashion (0.5–10nM) and induces release of calcium previously bound to membranes. The inhibition is noncompetitive and exhibits a Ki of 9 × 10?9M. Naloxone (10 nM) blocks this inhibition while dextrophan (100nM) is without effects. The Ca++ binding exhibits a high affinity (KD=0.84μM) and a saturable capacity of 350 picomles/mg protein. The binding curve is sigmoid with a Hill coefficient of 2.2, while in the presence of levorphanol (0.5 to 50nM), the curve becomes hyperbolic with a Hill coefficient of 1.1. The data suggest the calcium and opiate receptor binding sites are in close proximity and may be functionally linked through sub-unit interactions. 相似文献