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1.
For the type I cGMP-dependent protein kinases (cGKIalpha and cGKIbeta), a high affinity interaction exists between the C2 amino group of cGMP and the hydroxyl side chain of a threonine conserved in most cGMP binding sites. To examine the effect of this interaction on ligand binding and kinase activation in the type II isozyme of cGMP-dependent protein kinase (cGKII), alanine was substituted for the conserved threonine or serine. cGKII was found to require the C2 amino group of cGMP and its cognate serine or threonine hydroxyl for efficient cGMP activation. Of the two binding sites, disruption of cGMP-specific binding in the NH(2)-terminal binding site had the greatest effect on cGMP-dependent kinase activation, like cGKI. However, ligand dissociation studies showed that the location of the rapid and slow dissociation sites of cGKII was reversed relative to cGKI. Another set of mutations that prevented cyclic nucleotide binding demonstrated the necessity of the NH(2)-terminal, rapid dissociation binding site for cyclic nucleotide-dependent activation of cGKII. These findings suggest distinct mechanisms of activation for cGKII and cGKI isoforms. Because cGKII mediates the effects of heat-stable enterotoxins via the cystic fibrosis transmembrane regulator Cl(-) channel, these findings define a structural target for drug design. 相似文献
2.
Smith JA Reed RB Francis SH Grimes K Corbin JD 《The Journal of biological chemistry》2000,275(1):154-158
The role of each of the two different cGMP-binding sites (referred to as slow and fast sites) of type I cGMP-dependent protein kinase (PKG) in altering the rate of catalysis of phosphorylation of exogenous substrates (heterophosphorylation) or the rate of autophosphorylation has not been resolved. In the present study, the cGMP concentration required for half-maximal activation (A(50)) of wild-type PKG type Ibeta (WT) was 5-fold higher for heterophosphorylation than for autophosphorylation. cGMP occupation of the slow site was associated with an increase in the autophosphorylation rate, whereas occupation of the fast and slow site together was associated with a decrease in the autophosphorylation rate compared with the rate observed with occupation of the slow site alone. The contributions of each cGMP-binding site were investigated using PKG mutants containing substitutions of an invariant threonine residue that is critical for high affinity cGMP-binding in each site. Site-directed mutagenesis of Thr-317 of the fast site (T317A) increased the cGMP A(50) for heterophosphorylation 4-fold at 30 degrees C, with nominal effect on cGMP A(50) for autophosphorylation compared with WT. The analogous slow site mutation (T193A) increased the cGMP A(50) for heterophosphorylation and autophosphorylation 32- and 64-fold, respectively. Compared with WT, the cGMP A(50) of the double mutant (T193A/T317A) for heterophosphorylation was increased 300-fold, whereas the cGMP A(50) for autophosphorylation was similar to that of T193A. Thus, occupation of both cGMP-binding sites of PKG is required for maximal stimulation of heterophosphorylation, whereas occupation of the slow site alone is sufficient for stimulation of the rate of autophosphorylation, and additional occupation of the fast site reduces this rate. 相似文献
3.
cGMP-dependent protein kinase. Autophosphorylation changes the characteristics of binding site 1 总被引:1,自引:0,他引:1
cGMP-dependent protein kinase binds 4 mol cGMP/mol enzyme to two different sites. Binding to site 1 (apparent Kd 17 nM) shows positive cooperativity and is inhibited by Mg . ATP, whereas binding to site 2 (apparent Kd 100-150 nM) is non-cooperative and not affected by Mg . ATP. Autophosphorylation of the enzyme abolishes the cooperative binding to site 1 and the inhibitory effect of Mg . ATP. The association (K1) and dissociation (K-1) rate constant for site 2 and K1 for site 1 are not affected significantly by Mg . ATP or autophosphorylation. The dissociation rate from site 1 measured in the presence of 1 mM unlabelled cGMP is decreased threefold and over tenfold by Mg . ATP and autophosphorylation, respectively. In contrast, the dissociation rate from site 1 measured after a 500-fold dilution of the enzyme-ligand complex is 100-fold faster than that determined in the presence of 1 mM cGMP and is only slightly influenced by Mg . ATP or autophosphorylation. Only Kd values calculated with the latter K-1 values are similar to the Kd values obtained by equilibrium binding. These results suggest that autophosphorylation of cGMP-dependent protein kinase affects mainly the binding characteristics of site 1. 相似文献
4.
Studies of two different intrachain cGMP-binding sites of cGMP-dependent protein kinase 总被引:3,自引:0,他引:3
The binding of [3H]cGMP to purified beef lung cGMP-dependent protein kinase (cG kinase) was examined using two methods of membrane filtration which avoided loss of bound [3H]cGMP. The enzyme bound 1.6-2.0 mol of [3H]cGMP/mol of monomer. If the kinase was saturated with [3H]cGMP and then excess unlabeled cGMP was added, [3H]cGMP dissociated from the enzyme as two approximately equal components (Sites 1 and 2). When 8-bromo-cGMP or cIMP was added to the [3H]cGMP-binding reaction at a concentration sufficient to competitively inhibit binding by greater than 50%, the relative amount of the slower or faster component, respectively, of [3H]cGMP dissociation decreased during the cGMP chase. The data indicated that the cG kinase, like its cAMP-dependent protein kinase homologue, possesses two highly conserved intrachain cyclic nucleotide-binding sites which have different dissociation rates and analog specificity. The Ka of the kinase for cGMP was about 20-fold lower using histone instead of heptapeptide as substrate. Aging of the enzyme caused conversion to a higher Ka form of the kinase and an apparent increase in the Site 1 cGMP dissociation rate. Using fresh enzyme and heptapeptide as substrate, Site 1 occupation occurred at lower concentrations of cGMP than did Site 2 occupation, and was associated with an increase in protein kinase activity. However, kinase activity appeared to correlate better with total cGMP binding than with binding to either of the two sites, and the activation by cGMP exhibited positive cooperativity (n = 1.57). It is suggested that both intrachain sites are involved in protein kinase activation. E2 + 4 cGMP in equilibrium E2 . cGMP4 The cG kinase could be photoaffinity-labeled using 8-azido-[32P]cAMP. When the labeled cG kinase was trypsin-treated followed by sodium dodecyl sulfate-slab gel electrophoresis, a single major peptide of approximate Mr = 12,000 was resolved. 相似文献
5.
We study the dissociation of preformed bivalent ligand-bivalent receptor aggregates in solution, where the ligand is a symmetric bivalent hapten with two identical 2,4-dinitrophenyl (DNP) groups and the receptor is a fluorescein-labeled monoclonal anti-DNP IgE. We promote dissociation in two ways: by the addition of high concentrations of a monovalent hapten that competes for IgE binding sites with the bivalent hapten and by the addition of high concentrations of unlabeled IgE that binds almost all ligand binding sites that dissociate from labeled IgE. We investigate both theoretically and experimentally the two types of dissociation and find them to be quite different. Theory predicts that their kinetics will depend differently on the fundamental rate constants that characterize binding and aggregation. Using monovalent ligand to promote dissociation, we find that the fraction of labeled IgE sites bound to bivalent ligand decays with a slow and fast component. The fast decay corresponds to the dissociation of a singly bound DNP hapten. The interpretation of the slow decay depends on the detailed way in which ligand-receptor aggregates break up. We show that one possible explanation of these data is that small stable rings form before the addition of monovalent ligand. Other possible explanations are also presented. 相似文献
6.
Studies of cGMP analog specificity and function of the two intrasubunit binding sites of cGMP-dependent protein kinase 总被引:4,自引:0,他引:4
J D Corbin D Ogreid J P Miller R H Suva B Jastorff S O D?skeland 《The Journal of biological chemistry》1986,261(3):1208-1214
The specificity of the two intrasubunit cGMP binding sites of cGMP-dependent protein kinase was determined by measuring the ability of 46 cGMP analogs to compete with [3H]cGMP. Both sites of the enzyme exhibited high specificity for the ribose cyclic phosphate moiety, and lower specificity for the guanine moiety. Effects of modifications in the ribose cyclic phosphate moiety suggested that cGMP is bound at both sites by three hydrogen bonds at 2'-OH, 3'-O, and 5'-O. A negative charge in the cyclic phosphate is apparently required. Modifications of the pyrimidine part of guanine, particularly at C-1, generally caused selectivity for the rapidly exchanging site while modifications of the imidazole part of guanine at C-7 and C-8 caused selectivity for the slowly exchanging site. These increases in selectivity for a site were mainly due to losses in affinity of the other site. There was an apparent requirement of the intact amino group at C-2, particularly for the slowly exchanging site. Comparison of the molecular interactions of cAMP and cGMP with their specific protein kinases showed that both nucleotides are bound by similar forces in the 2', 3' and 5' region, both bases may be bound in syn conformation, but that each base moiety is bound by different molecular interaction, thus leading to the selectivity of the two enzymes. cGMP analogs which possessed strong selectivity for the rapidly exchanging site, but not those selective for the slowly exchanging site, stimulated the binding of [3H]cGMP. Only a few cGMP analogs were more potent than cGMP in stimulating protein kinase activity. The potency of cGMP analogs as stimulators of kinase activity correlated better with the mean binding affinity for both binding sites than with the affinity for either site alone. Two analogs added in combination were synergistic in kinase activation, particularly if one analog was selective for the slowly exchanging site and the other for the rapidly exchanging site. These observations are suggestive that cGMP binding at the rapidly exchanging site stimulates cGMP binding at the slowly exchanging site and that both sites are involved in the activation process. 相似文献
7.
Low-temperature studies of the sarcoplasmic reticulum calcium pump. Mechanisms of calcium binding 总被引:4,自引:0,他引:4
Y Dupont 《Biochimica et biophysica acta》1982,688(1):75-87
The mechanism of the sarcoplasmic reticulum Ca2+-ATPase was investigated at low temperatures (0 to -12 degrees C). Transient states of the enzyme were studied by two complementary techniques: intrinsic protein fluorescence and rapid filtration on Millipore filters. Intrinsic fluorescence was used to distinguish conformational states of the protein and to evaluate the rate of conversion between these states. Filtrations were used to measure the evolution of the active sites during the transition; the time resolution was 2-5 s. At sub-zero temperatures this time is shorter than the lifetime of most of the enzymatic states which have been detected. In this paper the mechanism of Ca2+ binding to the protein is investigated in the absence of nucleotides. Two basic experiments are described; (1) Kinetics of calcium binding and dissociation over a wide range of calcium concentration. (2) Kinetics of calcium exchange (45Ca2+ in equilibrium 40Ca2+) at constant concentration. The results obtained in the first series of experiments are consistent with a sequential binding to two interacting Ca2+ binding sites. Calcium ions have very fast access to a site with low apparent affinity (Kd approximately 25 microM). Occupation of this site induces a slow conformational change which increased its apparent affinity and reveals a second site of high apparent affinity. At equilibrium the two sites are not equivalent in terms of rate of exchange. Two different rates were detected k fast greater than 0.2 s-1, k slow approximately 0.015 s-1 at -10 degrees C. Removal of Ca2+ from the fast exchanging site by addition of EGTA accelerates the rate of release of the slow exchanging one. A model is proposed with two interacting Ca2+-binding sites. A set of parameters has been obtained which produces correctly the Ca2+-binding curve and the fluorescence level at equilibrium as well as the rate constants of the calcium-induced fluorescence changes over a very wide range of Ca2+ concentrations (0.02 to 150 microM). The non-equivalence of the two classes of site and the meaning of the initial low-affinity binding are discussed. 相似文献
8.
A bovine lung cGMP-binding phosphodiesterase (cG-BPDE) was purified to homogeneity and exhibited specific cGMP hydrolytic (Km = 5.6 microM) and cGMP binding (half-maximum approximately 0.2 microM) activities which comigrated throughout the purification. A chimeric structure was suggested for cG-BPDE since DEAE chromatography of a partial alpha-chymotryptic digest of cG-BPDE separated cGMP-binding fragments from a cGMP hydrolytic fragment. Native cG-BPDE (178 kDa) appeared to be a homodimer comprised of two 93-kDa subunits. The order of potency of inhibitors of cG-BPDE hydrolysis of cGMP was as follows: zaprinast greater than dipyridamole greater than 3-isobutyl-1-methyl-8-methoxymethylxanthine greater than 3-isobutyl-1-methylxanthine greater than cilostamide greater than theophylline greater than rolipram. Minimum [3H]cGMP binding stoichiometry was 0.93 mol of cGMP bound/mol of monomer, but [3H]cGMP dissociation from cG-BPDE in the presence of excess unlabeled cGMP was curvilinear, suggesting multiple cGMP-binding sites. Two chymotryptic cGMP-binding fragments of 35 and 45 kDa were specifically photoaffinity labeled with [32P] cGMP, exhibited [3H]cGMP association and dissociation behavior indistinguishable from native cG-BPDE, and each had the amino-terminal sequence: Thr-Ser-Pro-Arg-Phe-Asp-Asn-Asp-Glu-Gly-. Cochromatography of the two cGMP-binding fragments suggested that both a dimerization domain and a cGMP-binding domain were located in a 35-kDa segment of cG-BPDE. Increased [3H]cGMP binding to or [32P]cGMP photoaffinity labeling of cG-BPDE binding sites in the presence of hydrolytic site-specific cyclic nucleotide analogs suggested communication between hydrolytic and binding sites. The principle of reciprocity thus predicts that cGMP binding to the binding sites may affect the hydrolytic site. In the presence of cGMP, the binding fragments or native cG-BPDE exhibited an electronegative shift on high performance liquid chromatography-DEAE, consistent with a cGMP-induced change in cG-BPDE conformation. 相似文献
9.
Calvert PD Govardovskii VI Arshavsky VY Makino CL 《The Journal of general physiology》2002,119(2):129-145
Vertebrate rod photoreceptors adjust their sensitivity as they adapt during exposure to steady light. Light adaptation prevents the rod from saturating and significantly extends its dynamic range. We examined the time course of the onset of light adaptation in bullfrog rods and compared it with the projected onset of feedback reactions thought to underlie light adaptation on the molecular level. We found that adaptation developed in two distinct temporal phases: (1) a fast phase that operated within seconds after the onset of illumination, which is consistent with most previous reports of a 1-2-s time constant for the onset of adaptation; and (2) a slow phase that engaged over tens of seconds of continuous illumination. The fast phase desensitized the rods as much as 80-fold, and was observed at every light intensity tested. The slow phase was observed only at light intensities that suppressed more than half of the dark current. It provided an additional sensitivity loss of up to 40-fold before the rod saturated. Thus, rods achieved a total degree of adaptation of approximately 3,000-fold. Although the fast adaptation is likely to originate from the well characterized Ca(2+)-dependent feedback mechanisms regulating the activities of several phototransduction cascade components, the molecular mechanism underlying slow adaptation is unclear. We tested the hypothesis that the slow adaptation phase is mediated by cGMP dissociation from noncatalytic binding sites on the cGMP phosphodiesterase, which has been shown to reduce the lifetime of activated phosphodiesterase in vitro. Although cGMP dissociated from the noncatalytic binding sites in intact rods with kinetics approximating that for the slow adaptation phase, this hypothesis was ruled out because the intensity of light required for cGMP dissociation far exceeded that required to evoke the slow phase. Other possible mechanisms are discussed. 相似文献
10.
Binding sites for [3H]cAMP on purified regulatory dimers of type II A-kinase (II-R2) are independent as assessed by equilibrium binding (KD = 6 +/- 1.3 nM at pH 7.2, 25 degrees; nH = 1.0) and by the lack of effect of unlabeled cAMP on dissociation rate (kd = 1.0 X 10(-3) sec -1 at pH 7.2, 25 degrees). In contrast, binding sites for [3H]cGMP on purified G-kinase displayed positively cooperative interactions in both equilibrium and dissociation assays with convex upward Scatchard plots, an nH of 1.6 and a dissociation rate (kd = 6.2 X 10(-3) sec-1 at pH 6.8, 0 degree) which was slowed by excess unlabeled cGMP (kd = 1.13 X 10(-3) sec-1 at pH 6.8, degree). Calculated transition state free energies of dissociation revealed that dissociation of nucleotide from G-kinase in the presence of cGMP was restrained by an energy barrier (20.8 kcal.mol-1) similar to that of II-R2 (20.9 kcal.mol-1), whereas dissociation from G-kinase without excess nucleotide occurred more easily (18.9 kcal.mol-1). 相似文献
11.
The cAMP-dependent protein kinase contains two different cAMP-binding sites referred to as the slow and fast sites. Mutation of Ala-334 to a threonine in the slow site of the bovine type I regulatory subunit created a site with marked increase in cGMP affinity without changing cAMP affinity (Shabb, J. B., Ng. L., Corbin, J. D. (1990) J. Biol. Chem. 265, 16031-16034). The corresponding fast site residue (Ala-210) was changed to a threonine by oligonucleotide-directed mutagenesis, and a double mutant containing a threonine in each site was also made. Holoenzymes were formed from native catalytic subunit and each recombinant regulatory subunit. The fast site mutant holoenzyme exhibited an improved cGMP activation constant and an impaired cAMP activation constant. The double mutant cGMP/cAMP selectivity was 200-fold greater than that of wild-type holoenzyme, making it as responsive to cGMP as native cGMP-dependent protein kinase. The increased intrinsic binding energies of mutated sites for cGMP were 2.7-3.0 kcal mol-1, consistent with the presence of an extra hydrogen bond. Cyclic nucleotide analog studies implied that this hydrogen bond was between the threonine hydroxyl and the 2-amino of cGMP. Comparisons of amino acid sequences and cyclic nucleotide specificities suggested that the Ala/Thr difference may also impart cAMP/cGMP binding selectivity to related proteins such as cyclic nucleotide-gated ion channels. 相似文献
12.
The binding of the bisubstrate ligand N-(phosphonacetyl)-L-aspartate (PALA) to the active sites of both the free catalytic subunit of aspartate transcarbamoylase and the intact holoenzyme causes conformational changes which have been studied extensively. However, no kinetic information has been available about the sequence of events occurring during the formation or dissociation of the complexes. Stopped flow kinetics, 31P saturation transfer NMR spectroscopy, and presteady-state kinetics were used to monitor the interaction of PALA with the catalytic subunit (or a derivative containing nitrotyrosyl chromophores which served as spectral probes). The various experimental approaches lead to a mechanism that includes a rapid binding of PALA with an "on" rate of about 10(8)M-1s-1 and an "off" rate of 28 s-1, followed by a much slower isomerization of the complex with a forward rate constant of 0.18 s-1. Analysis of the presteady-state bursts of enzyme activity when the protein is added to a mixture of substrates and PALA and of the lag in activity when the PALA complex with catalytic subunit is added to substrates yielded a rate constant for the reverse isomerization of 0.018s-1. Thus, the conformational change subsequent to PALA binding leads to a 10-fold increase in the equilibrium constant for complex formation. Stopped flow kinetic measurements of the spectral change resulting from mixing the complex of PALA and nitrated protein with native enzyme showed a slow process with a t1/2 of about 11 s, whereas 31P saturation transfer NMR experiments yielded at t1/2 of about 260 ms for the dissociation of PALA from the complex. This apparent disparity is understood in terms of the two-step binding scheme where rapid dissociation of the initial ligand X enzyme complex is measured by the NMR technique and the slow isomerization of the complex is responsible for the bulk of the stopped flow signal. 相似文献
13.
IL-12 receptor. I. Characterization of the receptor on phytohemagglutinin-activated human lymphoblasts. 总被引:7,自引:0,他引:7
R Chizzonite T Truitt B B Desai P Nunes F J Podlaski A S Stern M K Gately 《Journal of immunology (Baltimore, Md. : 1950)》1992,148(10):3117-3124
IL-12 is a 75-kDa heterodimeric cytokine composed of disulfide-bonded 35-kDa and 40-kDa subunits. Included among the biologic activities mediated by IL-12 is induction of proliferation of PHA-activated human PBL. The concentration of IL-12 required to stimulate maximum proliferation of PHA-activated lymphoblasts is 50 to 100 pM. In this study, highly purified 125I-labeled IL-12 (7 to 15 microCi/microgram; 50 to 100% bioactive) was used to characterize the receptor for IL-12 on 4-day PHA-activated lymphoblasts. The binding of 125I-labeled IL-12 to PHA-activated lymphoblasts was saturable and specific because the binding of radiolabeled ligand was only inhibited by IL-12 and not by other cytokines. The kinetics of [125I]IL-12 binding to PHA-activated lymphoblasts was rapid at both 4 degrees C and 22 degrees C; reaching equilibrium within 60 min. At 22 degrees C, the rate of dissociation of [125I]IL-12 was slow in the absence of competing IL-12 (t1/2 = 5.9 h) and more rapid in the presence of 25 nM competing IL-12 (t1/2 = 2.5 h). The kinetically derived equilibrium dissociation constant ranged from 10 to 83 pM. Analysis of steady state binding data by the method of Scatchard identified a single binding site with an apparent equilibrium dissociation constant of 100 to 600 pM and 1000 to 9000 sites/lymphoblast. The equilibrium dissociation constant for competing ligands and sites per cell calculated from unlabeled IL-12 competition experiments ranged from 164 to 315 pM and 1067 to 3336, respectively, which is in good agreement with the values determined from steady state binding. The variations in KD and sites per cell were dependent on the individual preparations of lymphoblasts. Although the steady state binding data were consistent with a single class of high affinity binding sites, the kinetic dissociation data indicates a cooperative interaction between receptors on PHA-activated lymphoblasts. Affinity cross-linking of surface bound [125I]IL-12 to PHA-activated lymphoblasts at 4 degrees C identified a major complex of approximately 210 to 280 kDa. Anti-IL-12 antibodies also immunoprecipitated a complex of approximately 210 to 280 kDa that was produced by cross-linking unlabeled IL-12 to 125I-labeled lymphoblast cell-surface proteins. Cleavage of this complex with reducing agent identified one radiolabeled protein of approximately 110 kDa. These data suggest that the IL-12 binding site on PHA-activated lymphoblasts may be composed of a single protein of approximately 110 kDa.(ABSTRACT TRUNCATED AT 400 WORDS) 相似文献
14.
Previous work with N-ethylmaleimide (NEM) has defined two sites on the Neurospora plasma membrane H+-ATPase. Modification of one (the "fast" site) by NEM is rapid but does not affect ATPase activity, while modification of the other (the "slow" site) inactivates the enzyme and is protectable by MgATP or MgADP. In the present study, a wider array of sulfhydryl reagents have been used to examine the properties of both sites. The results show the following. (a) Both fast and slow sites react preferentially with hydrophobic compounds (N-pyrenemaleimide, dithiobisnitropyridine greater than N-naphthylmaleimide, dithiobisnitrobenzoate greater than N-phenylmaleimide greater than N-ethylmaleimide) and are virtually insensitive to hydrophilic sulfhydryl reagents such as iodoacetamide and iodoacetic acid. (b) The reaction rate of the slow site with NEM is approximately 2000-fold less rapid than that of the fast site. The slow site also has an unusually high pKa (greater than 9.5). (c) Whether or not cysteine modification leads to inactivation of the ATPase depends upon the site and the reagent. For example, when the fast site reacts with NEM, enzymatic activity is retained; when it reacts with N-pyrenemaleimide, activity is lost. Likewise, when the slow site is modified by any of the maleimides or by dithiobisnitropyridine or dithiobisnitrobenzoate, the ATPase is inactivated; when it is modified by methylmethanethiosulfonate, activity remains intact. Thus, neither cysteine can be considered to play an essential role in the reaction cycle of the ATPase, but the introduction of a sufficiently bulky substituent at either site can disrupt activity. (d) Upon reaction of methylmethanethiosulfonate at the slow site, the K1/2 for MgATP hydrolysis is reduced from 0.65 to 0.25 mM. This result strengthens the evidence for a conformational relationship between the slow site cysteine and the nucleotide binding site of the ATPase. 相似文献
15.
The sarcoplasmic reticulum Ca2(+)-ATPase of skeletal muscle has two high affinity calcium sites, one of fast access ("f" site) and one of slow access ("s" site). In addition to Ca2+ these sites are able to interact with other cations like Mg2+ or K+. We have studied with a stopped-flow method the modifications produced by Mg2+ and K+ on the kinetics of the intrinsic fluorescence changes produced by Ca2+ binding to and dissociation from the Ca2(+)-ATPase of sarcoplasmic reticulum. The presence of Mg2+ ions (K1/2 = 0.5 mM at pH 7.2) leads to the appearance of a rapid phase in the Ca2+ binding, which represents half of the signal amplitude at optimal Mg2+. The presence of K+ greatly accelerates both the Ca2+ binding and the Ca2+ dissociation reactions, giving, respectively, a 4- and 8-fold increase of the rate constant of the induced fluorescence change. K+ ions also increase the rate of the 45Ca/40Ca exchange reaction at the s site measured by rapid filtration. These results lead us to build up a model for the Ca2(+)-binding mechanism of the sarcoplasmic reticulum Ca2(+)-ATPase in which Mg2+ and K+ participate at particular steps of the reaction. Moreover, we propose that, in the absence of Ca2+, this enzyme may be the pathway for monovalent ion fluxes across the sarcoplasmic reticulum membrane. 相似文献
16.
The effect of extracellular and intracellular Na(+) on the single-channel kinetics of Mg(2+) block was studied in recombinant NR1-NR2B NMDA receptor channels. Na(+) prevents Mg(2+) access to its blocking site by occupying two sites in the external portion of the permeation pathway. The occupancy of these sites by intracellular, but not extracellular, Na(+) is voltage-dependent. In the absence of competing ions, Mg(2+) binds rapidly (>10(8) M(-1)s(-1), with no membrane potential) to a site that is located 0.60 through the electric field from the extracellular surface. Occupancy of one of the external sites by Na(+) may be sufficient to prevent Mg(2+) dissociation from the channel back to the extracellular compartment. With no membrane potential; and in the absence of competing ions, the Mg(2+) dissociation rate constant is >10 times greater than the Mg(2+) permeation rate constant, and the Mg(2+) equilibrium dissociation constant is approximately 12 microM. Physiological concentrations of extracellular Na(+) reduce the Mg(2+) association rate constant approximately 40-fold but, because of the "lock-in" effect, reduce the Mg(2+) equilibrium dissociation constant only approximately 18-fold. 相似文献
17.
W A Hagopian H S Tager B Gysin D Trivedi V J Hruby 《The Journal of biological chemistry》1987,262(32):15506-15513
We have used glucagon and nine glucagon analogs to investigate the interactions of these ligands with glucagon-binding sites present on isolated canine hepatocytes. Curves reflecting the inhibition of 125I-labeled glucagon or 125I-labeled analog binding to cells by the 10 peptides spanned, overall, a 10(6)-fold range of hormone concentration, were consistent with hormone binding to two classes of binding sites in each case, and fell into two groups, one of which contained curves that were considerably more shallow than the other. Only conditions that emphasized prior binding to low affinity sites resulted in the rapid and extensive dissociation of receptor-bound ligand from isolated cells. Finally, all 10 peptides exhibited a concentration-dependent inhibition of the incorporation of [14C]fructose into hepatocyte glycogen that correlated best with dissociation constants for high affinity rather than for low affinity binding. We conclude that (a) the association of ligand with the high and low affinity glucagon-binding sites of isolated canine hepatocytes is a characteristic of analogs modified at diverse sites throughout the peptide hormone, (b) the different rates of dissociation of ligand from the two populations of binding sites most probably account for the biphasic dissociation of ligand from isolated cells and for the different affinities of the two receptor populations for ligand, and (c) the activity of glucagon and glucagon analogs to inhibit the incorporation of fructose into hepatocyte glycogen arises from the association of ligand with high affinity binding sites. 相似文献
18.
Nerve growth factor receptors. Characterization of two distinct classes of binding sites on chick embryo sensory ganglia cells 总被引:60,自引:0,他引:60
A Sutter R J Riopelle R M Harris-Warrick E M Shooter 《The Journal of biological chemistry》1979,254(13):5972-5982
Steady state and kinetic studies on the binding of 125I-beta nerve growth factor (NGF) to single cells from sensory ganglia of 8-day-old chick embryos show two distinct, saturable binding sites with dissociation constants of Kd(I) = 2.3 X 10(-11) M and Kd(II) = 1.7 X 10(-9) M. The difference in the affinities is due to different rate constants of dissociation (k-1(I) = 10(-3) s-1, k-1(II) = 2 X 10(-1 s-1). The association to both sites is apparently diffusion controlled (k+1(I) = 4.8 X 10(7) M-1s-1, k+2(II) = 10(7) to 10(8) M-1s-1). The binding of betaNGF to both sites is specific, since none of a number of hormones or proteins tested compete for the binding of 125I-betaNGF to either of those two sites. The heterogeneity of the binding of 125I-betaNGF is not due to heterogeneity of the 125I-betaNGF preparation nor to a negatively cooperative binding. In experiments where the dissociation of 125I-betaNGF is induced by the addition of saturating amounts of unlabeled betaNGF, the ratio of the 125I-betaNGF released with either of the two dissociation rate constants is solely dependent on the occupancy of the two sites before dissociation is started and is independent of the total occupancy of the sites during dissociation. The rate of dissociation of 125I-betaNGF from the higher affinity binding site I is accelerated by unlabeled betaNGF under conditions where the occupancy is both increased and decreased. Although the dissociation characteristics of 125I-beta NGF change with increasing times of exposure of the cells to the ligand, and 125I-beta NGF is degraded after it binds to the cells, these secondary processes do not interfere with the analysis of the binding data. At the lowest concentration of 125I-beta NGF used for the analysis less than 10% of the 125I-beta NGF is degraded. Both kinetic and steady state binding data reveal the two NGF binding sites at 2 degrees C as well as at 37 degrees C. 相似文献
19.
Analysis of glucagon-receptor interactions on isolated canine hepatocytes. Formation of reversibly and irreversibly cell-associated hormone 总被引:1,自引:0,他引:1
We have investigated the interactions of ligand with the canine hepatic glucagon receptor. Whereas time courses for radiolabeled glucagon binding to receptor and dissociation from receptor revealed fast and slow components at both 30 and 4 degrees C, time courses of ligand dissociation revealed a third component of irreversibly cell-associated (nondissociable) ligand only at the higher temperature. Related experiments identified that (a) the initial rate of formation of nondissociable ligand was slower than that of dissociably bound hormone; (b) the fraction of ligand bound to nondissociable sites achieved a plateau during extended incubations, whereas that bound to dissociable sites was seen to rise and then slowly to fall; (c) the kinetics of formation of a nondissociable ligand was consistent with linked, sequential reactions; (d) dissociable ligand-receptor complexes formed at 4 degrees C were converted to nondissociable complexes during subsequent incubation at 30 degrees C, and (e) nondissociable sites were filled by prior incubation of cells with unlabeled ligand. Analysis of receptor-bound hormone resulting from the incubation of cells with 125I-labeled glucagon and selected concentrations of either glucagon or [[127I]iodo-Tyr10]glucagon at steady state revealed in each case four components of receptor-bound ligand: those corresponding to high and low affinity components of dissociably bound ligand and to high and low affinity components of nondissociably bound ligand. Implications of these findings are considered in terms of mechanisms for the formation of irreversibly bound hormone and for the distribution of hormone among the various components of hepatic glucagon-binding sites. 相似文献
20.
A novel type of internal barium block of a maxi-K+ channel from human vas deferens epithelial cells.
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We have recently shown that a maxi-K+ channel from vas deferens epithelial cells contains two Ba2+-binding sites accessible from the external side: a "flickering" site located deep in the channel pore and a "slow" site located close to the extracellular mouth of the channel. Using the patch-clamp technique, we have now studied the effect of internal Ba2+ on this channel. Cytoplasmic Ba2+ produced a voltage- and concentration-dependent "slow" type of block with a dissociation constant of approximately 100 microM. However, based on its voltage dependence and sensitivity to K+ concentration, this block was clearly different from the external "slow" Ba2+ block previously described. Kinetic analysis also revealed a novel "fast flickering" block restricted to channel bursts, with an unblocking rate of approximately 310 s(-1), some 10-fold faster than the external "flickering" block. Taken together, these results show that this channel contains multiple Ba2+-binding sites within the conduction pore. We have incorporated this information into a new model of Ba2+ block, a novel feature of which is that internal "slow" block results from the binding of at least two Ba2+ ions. Our results suggest that current models for Ba2+ block of maxi-K+ channels need to be revised. 相似文献