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1.
Specific binding of the calcium antagonist [3H]verapamil to membrane fractions from plants 总被引:8,自引:0,他引:8
Specific binding of the Ca2+ channel blocker [3H] verapamil to a membrane fraction from plants has been characterized. Binding to zucchini membranes was saturable and reversible. The apparent equilibrium dissociation constant is KD = 102 nM and the maximum number of binding sites is Bmax = 60 pmol/mg of protein. The KD determined from the association and dissociation rate constants is 130 nM. [3H]Verapamil binding to zucchini membranes could not be inhibited by the Ca2+ antagonists nifedipine and diltiazem. However, [3H]verapamil could be displaced by diltiazem but not by nifedipine from corn membranes. Sucrose density fractionation of zucchini membrane preparations revealed that [3H]verapamil binding sites are located primarily at the plasma membrane. 相似文献
2.
1. The reversible proton dissociation equilibria of peroxidase, apoperoxidase and haem-recombined apoperoxidase have been explored in 150mm-potassium chloride at 20 degrees C at pH3-11.5. 2. Complementary heat measurements have been made of the classes of titratable groups to determine their intrinsic DeltaH dissociation. 3. These curves are interpreted as showing that there are two histidine residues capable of titration in peroxidase whereas there are three such in apoperoxidase. 4. Concomitant spectroscopic investigations indicate profound differences in the tyrosine ionizations in the two proteins. In peroxidase one group only of the five residues ionizes up to pH11.5. In apoperoxidase four residues are titratable. 5. Spectroscopic titration in 6m-guanidinium chloride and 150mm-potassium chloride reveal one tyrosine residue fewer in peroxidase than in apoperoxidase. 6. These findings are discussed in terms of the ;side chain' groups responsible for binding the haem group in peroxidase. A proximal imidazole group seems probable as is also the involvement of a distally placed tyrosine. 7. The differences between apo- and holo-peroxidase are stressed, particularly in respect of abnormal carboxyl group titration in the former. 相似文献
3.
The effects of calcium ions and pH on bovine prothrombin fragment 1. Intrinsic fluroescence studies.
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The effects of pH and Ca2+ on the intrinsic fluorescence of bovine prothrombin fragment 1 were investigated to deduce the nature of protein functional groups involved in Ca2+ binding to fragment 1. From pH values of 9 to 3, increasing the H3O+ concentration results in quenching of the fluorescence of fragment 1. Reversible pH-titration curves are obtained which appear to consist of two regions. From pH 4 to pH6.5 a broad titration curve is obtained, whereas from pH6.5 to 9 a more pronounced titration behaviour is evidenced by a group or groups on fragment 1 with an apparent pKa of approx. 7.5. In contrast, the apparent association constant for Ca2+ and fragment 1 shows a sharp pH-dependence in the region between pH7 and 8 with tighter Ca2+ binding at higher pH values. A PKa of approx. 7.5 can be estimated for the group or groups on fragment 1 linked to the tight binding of Ca2+. Both H3O+ and Ca2+ result in blue-shifts in the wave-lengths of fragment-1 emission. These results are interpreted in terms of H+ - and Ca2+ - induced changes in the conformation of fragment 1 as a result of surface-charge neutralization. 相似文献
4.
Endogenous divalent cations, such as Mg2+, Ca2+, and Zn2+, differentially affected the binding of (+)-[3H]5-methyl-10,11-dihydro-5H-dibenzo[a,d]cyclohepten-5,10-imi ne maleate ([3H]MK-801) to an ion channel associated with an N-methyl-D-aspartate-sensitive subclass of excitatory amino acid receptors in different preparations of brain synaptic membranes. Both Mg2+ and Ca2+ were weak inhibitors of the binding in membranes which had not been extensively washed (nonwashed membranes), over a concentration range effective in markedly potentiating the binding in the absence of any added stimulants in membranes which had been extensively washed, but not treated with a detergent (untreated membranes). In membranes extensively washed and treated with Triton X-100 (Triton-treated membranes), both cations significantly potentiated the binding in the presence of added glutamate alone. In contrast, Zn2+ was invariably active as a potent inhibitor of the binding irrespective of the membrane preparations used. In untreated membranes, Ca2+ markedly accelerated the initial association rate of [3H]MK-801 binding without affecting the binding at equilibrium in a manner similar to that found with glycine, as well as with glutamate; Mg2+, however, facilitated the initial association rate with a concomitant reduction of the binding at equilibrium. Zn2+ was effective in accelerating the initial rapid phase of association, with the initial slow phase being delayed, and in markedly reducing the binding at equilibrium. Both Mg2+ and Ca2+ also facilitated dissociation of the bound [3H]MK-801 and Zn2+ slowed the dissociation in untreated membranes.(ABSTRACT TRUNCATED AT 250 WORDS) 相似文献
5.
Reiner Dolle 《Physiologia plantarum》1988,73(1):7-14
Chlorophyll-free plasma membranes of the unicellular green alga Chlamydomonas reinhardtii Dangeard were purified from a microsomal fraction using an aqueous polymer two-phase system of 6.5% (w/w) dextran T500, 6·5% (w/w) polyethylene glycol 3350, 60 mM NaCI, 0 33 M sucrose and 5 mM potassium phosphate (pH 7·8). The plasma membrane fraction contained only 2·4% of the microsomal membrane protein. Specific activity of the plasma membrane marker enzyme, K*, Mg2+-ATPase (EC 3.6.1.3). was enriched 9-fold over the microsomal fraction, and 22% of total activity was recovered in the upper, polyethylene glycol-rich phase. Contamination from intracellular membranes was minimal. K*, Mg2+-ATPase showed a pH optimum at about 6·5, and addition of 0·05% (w/v) Triton X-100 stimulated the activity 3-fold. [3H]-Nimodipinc was employed to characterize 1,4-dihydropyridine-specific membrane receptors. Two apparent binding sites with different affinities to nimodipine were found in the crude microsomal fraction. The separation of plasma membranes from intracellular membranes revealed that one binding site with higher affinity (KD= 9 nM) was located on the plasma membrane and a second binding site with lower affinity (KD= 36 nM) on an intracellular membrane The apparent dissociation constants determined from the association and dissociation rate constants in kinetic experiments were comparable to those determined by equilibrium experiments. The maximum number of binding sites of the plasma membrane fraction and the intracellular membrane fraction was Bmax= 440 and 470 fmol (mg protein)-1, respectively. [3H]-Nimodipinc binding was inhibited by (±) verapamil and stimulated by D-cis-diltiazem in both fractions. Moreover, ethyle-neglycol-bis(2-aminoethylcther)-N, N'-tetraacctic acid (EGTA) inhibited [3H]-nimo-dipinc binding in the plasma membrane fraction but not in the intracellular membrane fraction This effect was cancelled by the addition of CaCl2. 相似文献
6.
Anomalous driving force for renal brush border H+/OH-transport characterized by using 6-carboxyfluorescein 总被引:2,自引:0,他引:2
The pH, delta pH, and membrane potential dependences of H+/OH-permeability in renal brush border membrane vesicles (BBMV) were studied by using the entrapped pH indicator 6-carboxyfluorescein (6CF). Quantitative H+/OH-fluxes (JH) were obtained from a calibration of the fluorescence response of 6CF to intravesicular pH using vesicles prepared with varying intravesicular and solution pHs. Intravesicular buffer capacity, determined by titration of lysed vesicles, increased monotonically from 140 to 260 mequiv/L in the pH range 5-8. JH was measured by subjecting voltage-clamped BBMV (K+/valinomycin) to preformed pH gradients over the pH range 5-8 and measuring the rate of change of intravesicular pH. For small preformed pH gradients (0.4 pH unit) JH [6 nequiv s-1 (mg of protein)-1] was nearly independent of pH (5-8), predicting a highly pH dependent H+ permeability coefficient. JH increased in a curvilinear manner from 6 to 104 nequiv s-1 (mg of protein)-1 as delta pH increased from 0.4 to 2.5. JH increased linearly [1.6-7.3 nequiv s-1 (mg of protein)-1] with induced K+ diffusion potentials (21-83 mV) in the absence of a pH gradient. These findings cannot be explained by simple diffusion of H+ or OH- or by mobile carrier models. Two mechanisms are proposed, including a lipid diffusion mechanism, facilitated by binding of H+/OH- to fixed sites in the membrane, and a linear H2O strand model, where dissociation of H2O in the membrane fixes H+ and OH- concentrations in strands, which can result in net H+/OH- transport. 相似文献
7.
Kinetic evidence for two interconvertible forms of the folate transport protein from Lactobacillus casei.
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Lactobacillus casei cells contain a folate transport protein which exhibits a high affinity for folate. The dissociation constant (KD) for folate derived from binding parameters at the steady state (at 0 degree C) is 0.4 nM at pH 7.5 and 0.1 nM at pH 6.0. In the present study, folate binding to this protein at pH 7.5 (and 0 degree C) was shown to follow second-order kinetics and to proceed with an association constant (k+1) of 4.9 X 10(7) liter/mol per min. K+1 was not affected by preincubation conditions which alter the energetic state of the cell. Measurements on the extent of binding showed further that (at 0 degree C) essentially all unoccupied folate-binding sites reside at or are readily accessible to the outer surface of the membrane. In contrast, after saturating the binding site with [3H]folate, the first-order rate constant (k-1) for dissociation of the bound substrate (at 0 degree C) was found to vary substantially with the conditions employed. k-1 was 0.028/min in freshly harvested cells, but it increased by 2.8-fold in cells preincubated at 23 degrees C for 60 min and by 5.4-fold in isolated membranes. In addition, the faster rate observed in preincubated cells (k-1 = 0.077/min) returned to a slower rate after brief exposure of the cells to pH 6.0 (k-1 = 0.041/min), glucose (k-1 = 0.050/min), or both (k-1 = 0.012/min). k-1 was twofold lower at pH 6.0 than at pH 7.5 and was less dependent on the preincubation conditions, although it also increased substantially (5.5-fold) when the cells were converted to plasma membranes. The proposed explanation for these results is that folate transport protein of L. casei exists in two forms which can be distinguished by the accessibility of the binding site to the external medium and whose amounts are dependent upon the presence of bound folate, the pH, and the energetic state of the cell. It is suggested that these forms are transport proteins with binding sites oriented towards the inner and outer surfaces of the membrane. 相似文献
8.
Common channels for water and protons at apical and basolateral cell membranes of frog skin and urinary bladder epithelia. Effects of oxytocin, heavy metals, and inhibitors of H(+)-adenosine triphosphatase
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We have compared the response of proton and water transport to oxytocin treatment in isolated frog skin and urinary bladder epithelia to provide further insights into the nature of water flow and H+ flux across individual apical and basolateral cell membranes. In isolated spontaneous sodium-transporting frog skin epithelia, lowering the pH of the apical solution from 7.4 to 6.4, 5.5, or 4.5 produced a fall in pHi in principal cells which was completely blocked by amiloride (50 microM), indicating that apical Na+ channels are permeable to protons. When sodium transport was blocked by amiloride, the H+ permeability of the apical membranes of principal cells was negligible but increased dramatically after treatment with antidiuretic hormone (ADH). In the latter condition, lowering the pH of the apical solution caused a voltage-dependent intracellular acidification, accompanied by membrane depolarization, and an increase in membrane conductance and transepithelial current. These effects were inhibited by adding Hg2+ (100 microM) or dicyclohexylcarbodiimide (DCCD, 10(-5) M) to the apical bath. Net titratable H+ flux across frog skin was increased from 30 +/- 8 to 115 +/- 18 neq.h-1.cm-2 (n = 8) after oxytocin treatment (at apical pH 5.5 and serosal pH 7.4) and was completely inhibited by DCCD (10(-5) M). The basolateral membranes of the principal cells in frog skin epithelium were found to be spontaneously permeable to H+ and passive electrogenic H+ transport across this membrane was not affected by oxytocin. Lowering the pH of the basolateral bathing solution (pHb) produced an intracellular acidification and membrane depolarization (and an increase in conductance when the normal dominant K+ conductance of this membrane was abolished by Ba2+ 1 mM). These effects of low pHb were blocked by micromolar concentrations of heavy metals (Zn2+, Ni2+, Co2+, Cd2+, and Hg2+). Lowering pHb in the presence of oxytocin (50 mU/ml) produced a transepithelial current (3 microA.cm-2 at pHb 5.5) which was blocked by 100 microM of Hg2+, Zn2+, or Ni2+ at the basolateral side, and by DCCD (10(-5) M) or Hg2+ (100 microM) from the apical side. The net hydroosmotic water flux (JH2O) induced by oxytocin in frog bladder sacs was blocked by inhibitors of H(+)-adenosine triphosphatase (ATPase). Diethylstilbestrol (DES 10(-5) M), oligomycin (10(-8) M), and DCCD (10(-5) M) prevented JH2O when present in the lumen. These effects cannot be attributed to inhibition of metabolism since cyanide (10(-4) M), or 2-deoxyglucose (10(-3) M) had no effect on JH2O.(ABSTRACT TRUNCATED AT 400 WORDS) 相似文献
9.
A pH titration study of cytochrome c peroxidase and apocytochrome c peroxidase was carried out at 25 degrees C and 0.1 M ionic strength. The net charge on cytochrome c peroxidase due to proton association and dissociation varies from +32 at pH 2 to --50.2 at pH 12, while that of apocytochrome c peroxidase varies between +24.5 at pH 3 to --48 at pH 12. The apoprotein tented to aggregate below pH 3. Between pH 4 and 8, the titration behavior of both the native enzyme and the apoenzyme are consistent with the semi-empirical Linderstr?m-Lang theory. Between pH 9 and 12, the titration behavior of both the holo- and apoproteins suggest they assume a more extended conformation which reduces the electrostatic interaction charged groups on the surface. In the acid region, between pH 4 and 3, a similar transition occurs in which the protein expands 40% based on the electrostatic factor of the Linderstr?m-Lang theory. 相似文献
10.
Sarcoplasmic reticulum (SR) and plasma membranes from canine left ventricle were used to evaluate the presence of the enzyme CDPdiglyceride-inositol transferase in these membranes. (K+,-Ca2+)-ATPase activity, a marker for SR, was 79.2 +/- 5.0 (SE) and 11.2 +/- 2.0 mumol.mg-1.h-1 in SR and plasma membrane preparations, respectively, and (Na+,K+)-ATPase activity, a marker for plasma membranes, was 5.6 +/- 1.2 and 99.2 +/- 8.0 mumol.mg-1.h-1, respectively. Contamination of SR and plasma membrane preparations by mitochondria was estimated to be 2% and 8%, respectively, and by Golgi membranes, 0.9% and 1.8%, respectively. Transferase activity, measured at pH 6.8, was 1.32 +/- 0.04 (SE) and 0.28 +/- 0.04 nmol of [3H]phosphatidylinositol ([3H]PtdIns).mg-1.min-1 in three SR and plasma membrane preparations, respectively. The transferase activity detected in the plasma membrane preparation could be accounted for largely, but not entirely, by contaminating SR membranes. The pH optimum for the SR transferase activity was between 8.0 and 9.0; little or no activity was detectable at pH 6.3 and 5.5, the lowest pH tested. Ca2+ inhibited the enzyme, half-maximal inhibition occurring at about 10 microM Ca2+; removal of the Ca2+ by addition of ethylene glycol bis(beta-aminoethyl ether)-N,N,N',N'-tetraacetic acid restored activity. No loss of [3H]PtdIns could be detected when membranes were incubated in the presence or absence of Ca2+. The Ca2+ inhibition of the transferase was noncompetitive with respect to CDP-dipalmitin while that with respect to myo-inositol was slightly noncompetitive at low [Ca2+] and became uncompetitive at higher [Ca2+].(ABSTRACT TRUNCATED AT 250 WORDS) 相似文献
11.
The cytosol fraction of human erythrocytes as well as 1 mM ATP decreased the rate constant (Kl) of association of [3H]cAMP with human erythrocyte membranes. However, the effect of 1 mM ATP on the association kinetics was much greater than that of the cytosol fraction. Accordingly, the cytosol fraction and 1 mM ATP increased the rate constant (k-l) of dissociation of bound [3H]cAMP from the human erythrocyte membranes. However, the cytosol fraction affected the dissociation kinetics to a far greater extent than 1 mM ATP. Thus, although both cytosol and 1 mM ATP decreased the affinity of [3H]cAMP for binding sites on human erythrocyte membranes at equilibrium, the detailed mode of their action on the membrane [3H]cAMP binding sites appears to differ. 相似文献
12.
The kinetics of binding and dissociation for the progesterone-binding globulin (PBG)-progesterone complex have been measured as a function of pH. The association rate constant appears to be independent of pH from pH to 10 with an average value of kon = 8.5 X 10(7)M-1 S-1. The dissociation rate constant is strongly pH dependent with the dependency defined by: koff = k0 (1 + [H+]/K1 + K2/[H+])(1 + K3*/[H+])/(1 + K3/[H+]). The best values for the various parameters were k0 = 0.0785 s-1, pK1 = 5.30, pK2 = 10.54, pK3* = 7.41, and pK3 = 7.21. Simpler expressions were inadequate to fit the data, and it was concluded that at least three ionizing residues are responsible for the stability of the PBG-progesterone complex. The affinity constant was determined by equilibrium dialysis over the range of pH 3 to 12. The ratio of the association and dissociation rate constants is in agreement with the affinity constant from pH 6.5 to 10.5. The influence of pH on the conformation and binding activity of PBG was also investigated. Denaturation by acid, base, or guanidine hydrochloride leads to a reversible loss of binding activity. Regain of binding activity in all cases is slow with half-times of 0.5 to 2.7 h, depending on conditions. The rate of acid denaturation was found to be incompletely protonated at pH 1.4, suggesting a buried carboxylic acid residue. The slow renaturation of PBG might be due to the difficulty of burying a charged residue in the protein's interior coupled with steric hindrance by the large carbohydrate moiety of PBG. 相似文献
13.
Both rat liver and Escherichia coli rRNA in 0.1m-sodium chloride were titrated with acid or alkali over the range pH3-7 at approx. 0 degrees C. rRNA did not bind acid reversibly and hysteresis was observed, i.e. the plot of acid bound to rRNA against pH had the form of a loop showing that the amount of acid bound at a particular pH depended on the direction of the titration. Although the boundary curves were reproducibly followed on titration from pH7 to 3 and from pH3 to 7, points within the loop were ;scanned', e.g. by titration from pH7 to a point in the range pH3-4 followed by titration with alkali to pH7. It is inferred that the ;lag' in the release of certain bound protons is at least 1 pH unit, that at least about 9-15% of the titratable groups (adenine and cytosine residues) that are involved in this process and that the free energy dissipated in completing a cycle is approx. 4.2kJ/mol (1kcal/mol) of nucleotide involved in hysteresis. The interpretation of the ;scanning' curves was illustrated by means of a cycle of possible changes in the conformation of a hypothetical nucleotide sequence that allows formation of poly(A).poly(AalphaH(+))-like regions in acidic solutions. It is also inferred that the extent of ;hysteresis' might depend on the primary nucleotide sequence of rRNA as well as on secondary structure. 相似文献
14.
Dissociation of the glycoprotein IIb-IIIa complex in isolated human platelet membranes. Dependence of pH divalent cations 总被引:4,自引:0,他引:4
The platelet membrane glycoproteins IIb and IIIa normally exist as a complex which forms a predominant immunoprecipitate after crossed immunoelectrophoresis of Triton-X-100-solubilized platelets. Dissociation of the complex occurs by solubilization in the presence of EDTA or EGTA at pH 8.7 and is readily verified by crossed immunoelectrophoresis. Incubations of isolated membranes with EDTA or EGTA at various pH levels were performed. Removal of the chelators and solubilization showed no dissociation of the glycoprotein IIb-IIIa complex in membranes incubated at pH below 8.0. At pH above 8.0 a dissociation which increased with increasing pH was seen. Under these conditions, dissociation appears to take place already in the intact membranes. The tendency of the glycoprotein IIb-IIIa complex to become dissociated with EDTA or EGTA at increasing pH seems to be due to increased chelating capacity of the chelators concomitant with a decreased chelating capacity of glycoprotein IIb and IIIa. The divalent cations Ca2+ and Mg2+, but not Cu2+, Zn2+, Mn2+ or Sr2+, in molar concentrations below that of EGTA were able to prevent the dissociation of the glycoprotein IIb-IIIa complex by the chelator at pH 9.0, indicating that Ca2+ as well as Mg2+ can be used to keep the complex together. In some experiments it was possible to reverse the dissociation in the membranes after removal of EDTA. At pH 7.5 reassociation occurred within 15 min whether divalent cations were added or not. At pH 9.0. reassociation occurred within 2 h provided Ca2+ was present. The tendency of glycoprotein IIb and IIIa to form a complex thus appeared to be most pronounced over the physiological pH range and to be a rapid process in platelet membranes under such conditions. 相似文献
15.
Electrically silent anion transport through lipid bilayer membranes containing a long-chain secondary amine 总被引:2,自引:2,他引:0
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The permeability properties of planar lipid bilayers made from egg lecithin, n-decane and a long-chain secondary amine (n-lauryl [trialkylmethyl]amine) are described. Membranes containing the secondary amine show halide selectivity and high conductance at pH less than 6, as estimated by measurements of zero-current potentials generated by NaBr activity gradients. In the absence of halide ions, the membranes show H+ selectivity, although the total membrane conductance is relatively low. In 0.1 M NaBr both the membrane conductance (Gm) and the Br- self-exchange flux (JBr) are proportional to H+ concentration over the pH range of 7 to 4, and both JBr and Gm saturate at pH less than 4. However, JBr is always more than 100 times the flux predicted from Gm and the transference number for Br-. Thus, greater than 99% of the observed (tracer) flux is electrically silent and is not a Br2 or HBrO flux because the reducing agent, S2O3=, has no effect on JBr. At pH 7, JBr is proportional to Br- concentration over the range of 1-340 mM, with no sign of saturation kinetics. Both urea and sulfate tracer permeabilities are low and are unaffected by pH. The results can be explained by a model in which the secondary amine behaves as a monovalent, titratable carrier which exists in three chemical forms (C, CH+, and CHBr). Br- crosses the membrane primarily as the neurtal complex (CHBr). The positively charged carrier (CH+) crosses the membrane slowly compared to CHBr, but CH+ is the principal charge carrier in the membrane. At neurtal pH greater than 99% of the amine is in the nonfunctional form (C), which can be converted to CH+ or CHBr by increasing the H+ or Br- concentrations. The permeability properties of these lipid bilayers resemble in many respects the permeability properties of red cell membranes. 相似文献
16.
The role of histidyls in lobster arginine kinase (EC 2.7.3.3) has been studied by 1H-NMR spectroscopy of the enzyme and its complexes with substrates or their analogues and 31P-NMR spectroscopy of complexes with ADP. Five histidyls were detected by 1H-NMR in native enzyme (His 1 to His 5). Three of them appeared possibly to be implicated in catalysis: His 3, whose pH/titration was affected by arginine binding, and His 1 and 4, shown from paramagnetic relaxation by Mn2+ to be close (less than or equal to 1.2 and less than or equal to 1.27 nm respectively) to the metal cofactor. His 4 was broadened beyond detection in the presence of any adenine nucleotide. In the enzyme reversibly inactivated by histidine ethoxyformylation, the modified histidyl was His 1. In the transition state analogue complex (in which NO3- mimics the transferred phosphoryl), Hill plots of histidyl pH/titration curves showed that His 1 and His 3 were both interacting with the same set of three titratable groups and hence spatially close. 31P-NMR demonstrated that ADP binding in this complex was unaffected by the chemical modification of His 1. It is concluded that His-ethoxyformyl-enzyme is inactive because ethoxyformyl-His 1 is unable to titrate. This is consistent with His 1 acting as the acid-base catalyst. However our results, which do not indicate any catalytic role of His 3, exclude any H-bonding of His 1 on either substrate. Involvement is needed of at least one other titratable residue for the proton evolved in the catalysis to exchange directly with the guanidino substrate. 相似文献
17.
1H nuclear magnetic resonance titration curves and microenvironments of aromatic residues in bovine pancreatic ribonuclease A 总被引:1,自引:0,他引:1
M Tanokura 《Journal of biochemistry》1983,94(1):51-62
The aromatic region of the NMR spectrum of bovine pancreatic ribonuclease A was analyzed in order to clarify the nature of the microenvironments surrounding the individual histidine, tyrosine, and phenylalanine residues and the interactions with inhibitors. The NMR titration curves of ring protons of six tyrosine and three phenylalanine residues as well as four histidine residues were determined at 37 degrees C between pH 1.5 and pH 11.5 under various conditions. The titration curves were analyzed on the basis of a scheme of a simple proton dissociation sequence and the most probable values were obtained for the macroscopic pK values and intrinsic chemical shifts. The microenvironments surrounding the residues and the effects of inhibitors are discussed on the basis of these results. Based on the titration curves of ring protons, the six tyrosine residues were classified into the following four groups: (1) titratable and different chemical shifts for C(delta) and C(epsilon) protons (two tyrosine residues), (2) titratable but similar chemical shifts for C(delta) and C(epsilon) protons (two tyrosine residues), (3) not titratable and different chemical shifts for C(delta) and C(epsilon) protons (one tyrosine residues), and (4) not titratable and similar chemical shifts for C(delta) and C(epsilon) protons (one tyrosine residue). The resonance signals of ring protons were tentatively assigned to tyrosine and phenylalanine residues. The NMR titration curves of His-48 ring protons were continuous in solution containing 0.2 M sodium acetate but were discontinuous in solution containing 0.3 M NaCl because the NMR signals disappeared at pH values between 5 and 6.5. The effects of addition of formate, acetate, propionate, and ethanol were investigated in order to elucidate the mechanism of the continuity of the titration curves of His-48 in the presence of acetate ion. The NMR signal of His-48 C(2) protons was observed at pH 6 in the presence of acetate and propionate ions but was not observed in the presence of formate ion or ethanol. This indicated that both the alkyl chain and the anionic carboxylate group are necessary for the continuity of the titration curves of His-48 ring protons. Based on the results, the mechanism of the effects of acetate ion is discussed. 相似文献
18.
The purification of axonal membranes of crustaceans was followed by measuring enrichment in [3H]tetrodotoxin binding capacity and in Na+, K+-ATPase activity. A characteristic of these membranes is their high content of lipids and their low content of protein as compared to other types of plasmatic membranes. The axonal membrane contains myosin-like, actin-like, tropomyosin-like, and tubulin-like proteins. It also contains Na+, K+-ATPase and acetylcholinesterase. The molecular weights of these two enzymes after solubilization are 280,000 and 270,000, respectively. The molecular weights of the catalytic subunits are 96,000 for ATPase and 71,000 for acetylcholinesterase. We confirmed the presence of a nicotine binding component in the axonal membrane of the lobster but we have been unable to find [3H]nicotine binding to crab axonal membranes. The binding to axonal membranes og of the sodium channel, has been studied in detail. The dissociation constant for the binding of [3H]tetrodotoxin to the axonal membrane receptor is 2.9 nM at pH 7.4. The concentration of the tetrodotoxin receptor in crustacean membranes is about 10 pmol/mg of membrane protein, 7 times less than the acetylcholinesterase, 30 times less than the Na+, K+-ATPase, and 30 times less than the nicotine binding component in the lobster membrane. A reasonable estimate indicates that approximately only one peptide chain in 1000 constitutes the tetrodotoxin binding part of the sodium channel in the axonal membrane. Veratridine, which acts selectively on the resting sodium permeability, binds to the phospholipid part of the axonal membrane. [3H]Veratridine binding to membranes parallels the electrophysiological effect. Veratridine and tetrodotoxin have different receptor sites. Although tetrodotoxin can repolarize the excitable membrane of a giant axon depolarized by veratridine, veratridine does not affect the binding of [3H]tetrodotoxin to purified axonal membranes. Similarly, tetrodotoxin does not affect the binding of [3H]veratridine to axonal membranes. Scorpion neurotoxin I, a presynaptic toxin which affects both the Na+ and the K+ channels, does not interfere with the binding of [3H]tetrodotoxin or [3H]veratridine to axonal membranes. Tetrodotoxin, veratridine, and scorpion neurotoxin I, which have in common the perturbation of the normal functioning of the sodium channel, act upon three different types of receptor sites. 相似文献
19.
Nigericin-mediated H+, K+ and Na+ transports across vesicular membrane: T-jump studies. 总被引:1,自引:0,他引:1
The decay of delta pH across vesicular membranes by nigericin-mediated H+ and metal ion (M+) transports has been studied at 25 degrees C after creating delta pH by temperature jump (T-jump). In these experiments K+ or Na+ were chosen as M+ for the compensating flux. Theoretical expressions derived to analyse these data suggest a method for estimating the intrinsic rate constants for the translocation of nig-H (k1) and for the translocation of nig-M (k2) across membrane, from the pH dependence of the delta pH decay. The following could be inferred from the analysis of data. (a) At pH approximately 7.5 and 250 mM ion concentrations, nigericin-mediated H+ and M+ transport rates are lower in a medium of K+ than in a medium of Na+, although ionophore selectivity of nigericin towards K+ is 25-45-times higher than that towards Na+. However, at lower [M+] (approximately 50 mM) the transport rates are higher in a medium of K+ than in a medium of Na+. Such behaviours can be understood with the help of parameters determined in this work. (b) The intrinsic rate constants k1 and k2 associated with the translocations of nig-H and nig-K or nig-Na across membrane are similar in magnitude. (c) At pH approximately 7.5 translocation of nig-H is the dominant rate-limiting step in a medium containing K+. In contrast with this, at this pH, translocation of nig-M is the dominant rate-limiting step when metal ion is Na+. (d)k1 approximately k2 approximately 6.10(3) s-1 could be estimated at 25 degrees C in vesicles prepared from soyabean phospholipid, and lipid mixtures of 80% phosphatidylcholine (PC) + 20% phosphatidylethanolamine and 92% PC + 8% phosphatidic acid. (e) The apparent dissociation constants of nig-M in vesicles were estimated to be approximately 1.5.10(-3) M for K+ and 6.4.10(-2) M for Na+ (at 50 mM ion concentrations) using approximately 10(-8.45) M for the apparent dissociation constant of nig-H. 相似文献
20.
Specific angiotensin binding to rat hepatocytes and purified liver plasma membranes was measured by using biologically active [(3)H]angiotensin (sp. radioactivity 14Ci/mmol). The kinetic parameters for angiotensin binding to hepatocytes are: K(+1) (association rate constant). 100mum(-1).min(-1); K(-1) (dissociation rate constant), 2min(-1); K(d) (dissociation constant). 30nm; maximal binding capacity, 0.42pmol/10(6) cells or 260000 sites/cell. Angiotensin binding to membranes is profoundly affected by GTP (0.1mm) and NaCl (100mm); these regulatory compounds greatly enhance both the rate of association and of dissociation and also the extent of dissociation. K(d) amounts to 10nm in the presence of GTP+NaCl and to 1.5nm in their absence; maximal binding capacity is 0.70pmol/mg of protein, both with or without GTP+NaCl. The relative affinities of 11 angiotensin structural analogues were deduced from competition experiments for [(3)H]angiotensin binding to hepatocytes and to membranes (in the latter case, GTP + NaCl were not included, in order to study the higher affinity state of the receptor). These are highly correlated with their biological activity (activation of glycogen phosphorylase in hepatocytes). Binding to membranes occurs in the same concentration range as the biological effect. On the other hand, the existence of numerous spare receptors is suggested by the observation that binding of the agonists to hepatocytes requires 25-fold higher concentrations than those needed for their biological activity. These data clearly suggest that the detected binding sites correspond to the physiological receptors involved in the glycogenolytic action of angiotensin on rat liver. 相似文献