共查询到20条相似文献,搜索用时 31 毫秒
1.
Investigation of the forms of pig duodenal Ca2+-binding protein produced by limited tryptic hydrolysis.
下载免费PDF全文
![点击此处可从《The Biochemical journal》网站下载免费的PDF全文](/ch/ext_images/free.gif)
Vitamin D-dependent Ca2+-binding protein from pig duodenum was hydrolysed with trypsin in the presence of Ca2+ and two products were obtained: T1, which differed from the native protein by loss of Ac-Ser-Ala-Gln-Lys from the N-terminus and Ile-Ser-Gln-OH from the C-terminus, and T2, which differed from T1 by loss of a C-terminal lysine. The hydrolysis inactivated one of the two high-affinity Ca2+-binding sites on the native protein, and the remaining site was stable in T1 but labile in T2 when the proteins were Ca2+-free. Binding studies showed that T1 had Kd values of 2.8 +/- 0.1 nM, 57 +/- 13 microM and 0.8 +/- 0.3 microM for Ca2+, Mg2+ and Mn2+ respectively, and T2 had Kd 2.2 +/- 0.3 nM for Ca2+. The affinity for Mn2+, together with the other Kd values, identified the site on T1 as the site on the native protein previously found to have Kd 0.6 microM for Mn2+, rather than one with Kd 50 microM for Mn2+. In contrast with both the native protein and another form of the protein with a single Ca2+-binding site, the intrinsic fluorescence of T1 and T2 was little affected by the addition of Ca2+. It was concluded that the active binding site in T1 and T2, and also the site in the native protein with the higher affinity for Mn2+, was probably in the C-terminal half of the molecule. 相似文献
2.
Fluorimetric or spectrophotometric titrations with the appropriate cations gave Kd values of 2.9 +/- 0.2 nM and 89 +/- 5 microM respectively for the Ca2+ and Mg2+ complexes of quin 2 at pH 7.5. Mixtures of quin 2 and vitamin D-dependent Ca2+-binding protein from pig duodenum were titrated fluorimetrically with Ca2+ in the absence or presence of Mg2+. These measurements were used with the Kd values of the Ca2+ and Mg2+ complexes of quin 2 to obtain Kd or apparent Kd values for Ca2+-protein complexes ranging from 5 nM to 5 microM with good accuracy. 相似文献
3.
Rat intestinal Golgi-enriched membrane fractions take up Ca2+ by a vitamin D-dependent process that has been shown to recover within 15 min of repletion of vitamin D-deficient animals with intravenous 1,25-dihydroxycholecalciferol. The present paper reports studies characterizing the Ca2+-binding sites of these membrane fractions. Equilibrium binding of Ca2+ at concentrations between 5 and 400 microM showed significant decreases at all concentrations in membranes derived from vitamin D-deficient animals when compared with normal control-diet-fed animals. The predominant class of binding sites had a relatively high affinity for Ca2+ (KD approx. 3 microM). Vitamin D-deficiency did not change the affinity of this class of site, but decreased the number from 347 +/- 26 to 168 +/- 50 nmol of Ca2+ bound/mg of protein (means +/- S.D.). Mg2+ inhibited binding only at low Ca2+ concentrations, and the characteristics of this binding suggested positive co-operativity between two binding sites. Equimolar concentrations of Zn2+, La3+, Pb2+ and Mn2+ inhibited Ca2+ binding by over 50%. Increased ionic strength decreased Ca2+ binding by no more than half. Binding was maximal at pH 7.5 and half-maximal at pH 6.3. The large number of binding sites with relatively high affinity for Ca2+ suggests that it is unlikely that this binding is to any specific protein or to non-specific sites present on many proteins, and that the most likely sites are lipid molecules. 相似文献
4.
E M Wilson 《The Journal of biological chemistry》1985,260(15):8683-8689
The effects of divalent cations (Zn2+, Cd2+, Ca2+, Mg2+) on the cytosol androgen receptor were determined by sedimentation into sucrose gradients. At low ionic strength (25 mM KCl, 50 mM Tris, pH 7.4), Zn2+ (200 microM total, which calculates to 130 nM free Zn2+ in 10 mM mercaptoethanol) causes a shift in the sedimentation coefficient of the rat Dunning prostate tumor (R3327H) cytosol receptor and rat ventral prostate cytosol receptor from 7.5 +/- 0.3 S to 8.6 +/- 0.3 S. Zn2+ stabilizes the 8.6 S receptor form in salt concentrations up to 0.15 M KCl in 50 mM Tris, pH 7.2. In low ionic strength gradients containing Ca2+ (greater than or equal to 200 microM) or Mg2+ (greater than or equal to 1 mM), the receptor sediments as 4.7 +/- 0.3 S. The dissociating effects of Ca2+ and Mg2+ can be fully reversed by sedimentation into gradients containing Zn2+ (200 microM total) or Cd2+ (10 microM total). In the presence of Zn2+ (200 microM total), Ca2+ (10 microM to 3 mM) converts the receptor to an intermediate form with sedimentation coefficient 6.2 +/- 0.2 S, Stokes radius 73 A, and apparent Mr approximately 203,000. The potentiating effect of Zn2+ on formation of the 8.6 S receptor (in the absence of Ca2+) and the 6.2 S receptor (in the presence of Ca2+) requires both the 4.5 S receptor and the 8 S androgen receptor-promoting factor. Sodium molybdate stabilizes the untransformed cytosol receptor but, unlike Zn2+, does not promote reconstitution of the 8.6 S receptor from its partially purified components. These results indicate that divalent cations alter the molecular size of the androgen receptor in vitro and thus may have a role in altering the state of transformation of the receptor. 相似文献
5.
Recombinant IalA protein from Bartonella bacilliformis is a monomeric adenosine 5'-tetraphospho-5'-adenosine (Ap4A) pyrophosphatase of 170 amino acids that catalyzes the hydrolysis of Ap4A, Ap5A, and Ap6A by attack at the delta-phosphorus, with the departure of ATP as the leaving group [Cartwright et al. (1999) Biochem. Biophys. Res. Commun. 256, 474-479]. When various divalent cations were tested over a 300-fold concentration range, Mg2+, Mn2+, and Zn2+ ions were found to activate the enzyme, while Ca2+ did not. Sigmoidal activation curves were observed with Mn2+ and Mg2+ with Hill coefficients of 3.0 and 1.6 and K0.5 values of 0.9 and 5.3 mM, respectively. The substrate M2+ x Ap4A showed hyperbolic kinetics with Km values of 0.34 mM for both Mn2+ x Ap4A and Mg2+ x Ap4A. Direct Mn2+ binding studies by electron paramagnetic resonance (EPR) and by the enhancement of the longitudinal relaxation rate of water protons revealed two Mn2+ binding sites per molecule of Ap4A pyrophosphatase with dissociation constants of 1.1 mM, comparable to the kinetically determined K0.5 value of Mn2+. The enhancement factor of the longitudinal relaxation rate of water protons due to bound Mn2+ (epsilon b) decreased with increasing site occupancy from a value of 12.9 with one site occupied to 3.3 when both are occupied, indicating site-site interaction between the two enzyme-bound Mn2+ ions. Assuming the decrease in epsilon(b) to result from cross-relaxation between the two bound Mn2+ ions yields an estimated distance of 5.9 +/- 0.4 A between them. The substrate Ap4A binds one Mn2+ (Kd = 0.43 mM) with an epsilon b value of 2.6, consistent with the molecular weight of the Mn2+ x Ap4A complex. Mg2+ binding studies, in competition with Mn2+, reveal two Mg2+ binding sites on the enzyme with Kd values of 8.6 mM and one Mg2+ binding site on Ap4A with a Kd of 3.9 mM, values that are comparable to the K0.5 for Mg2+. Hence, with both Mn2+ and Mg2+, a total of three metal binding sites were found-two on the enzyme and one on the substrate-with dissociation constants comparable to the kinetically determined K0.5 values, suggesting a role in catalysis for three bound divalent cations. Ca2+ does not activate Ap4A pyrophosphatase but inhibits the Mn2+-activated enzyme competitively with a Ki = 1.9 +/- 1.3 mM. Ca2+ binding studies, in competition with Mn2+, revealed two sites on the enzyme with dissociation constants (4.3 +/- 1.3 mM) and one on Ap4A with a dissociation constant of 2.1 mM. These values are similar to its Ki suggesting that inhibition by Ca2+ results from the complete displacement of Mn2+ from the active site. Unlike the homologous MutT pyrophosphohydrolase, which requires only one enzyme-bound divalent cation in an E x M2+ x NTP x M2+ complex for catalytic activity, Ap4A pyrophosphatase requires two enzyme-bound divalent cations that function in an active E x (M2+)2 x Ap4A x M2+ complex. 相似文献
6.
To examine the role of divalent cations in the generation of superoxide anion (O2-) by the NADPH oxidase system of phagocytic cells, membrane-rich fractions were prepared from human neutrophils and monocytes. O2- generation by the fractions in sucrose was enhanced by addition of Ca2+ or Mg2+. EDTA inhibited most of the O2- generation; Ca2+ or Mg2+ reversed the inhibition. Zn2+, Mn2+, or Cu2+ completely inhibited O2- production. Neutrophil membrane fraction solubilized with Triton X-100, then passed through a chelating column, lost 80% of its oxidase activity; the loss could be reversed by addition of Ca2+ or Mg2+. Addition of 0.3 mM Ca2+ or Mg2+ protected against thermal instability of the enzyme. Kinetic analysis of the neutrophil oxidase activity as a function of NADPH and Ca2+ or Mg2+ concentrations showed that cation did not interact with NADPH in solution or affect the binding of NADPH to the oxidase; rather, cation bound directly to the oxidase, or to some associated regulatory component, to activate the enzyme. For the neutrophil oxidase, the Km for NADPH was 51 +/- 6 (S.D.) microM. Hyperbolic saturation was observed with Ca2+ and Mg2+, and the Kd values were 1.9 +/- 0.3 and 2.9 +/- 0.3 microM, respectively, suggesting that the oxidase, or some associated component, has a relatively high-affinity binding site for Ca2+ and Mg2+. 相似文献
7.
Ca2+ binding to chromaffin vesicle matrix proteins: effect of pH, Mg2+, and ionic strength 总被引:5,自引:0,他引:5
Recently we found that Ca2+ within chromaffin vesicles is largely bound [Bulenda, D., & Gratzl, M. (1985) Biochemistry 24, 7760-7765]. In order to explore the nature of these bonds, we analyzed the binding of Ca2+ to the vesicle matrix proteins as well as to ATP, the main nucleotide present in these vesicles. The dissociation constant at pH 7 is 50 microM (number of binding sites, n = 180 nmol/mg of protein) for Ca2+-protein bonds and 15 microM (n = 0.8 mumol/mumol) for Ca2+-ATP bonds. When the pH is decreased to more physiological values (pH 6), the number of binding sites remains the same. However, the affinity of Ca2+ for the proteins decreases much less than its affinity for ATP (dissociation constant of 90 vs. 70 microM). At pH 6 monovalent cations (30-50 mM) as well as Mg2+ (0.1-0.5 mM), which are also present within chromaffin vesicles, do not affect the number of binding sites for Ca2+ but cause a decrease in the affinity of Ca2+ for both proteins and ATP. For Ca2+ binding to ATP in the presence of 0.5 mM Mg2+ we found a dissociation constant of 340 microM and after addition of 35 mM K+ a dissociation constant of 170 microM. Ca2+ binding to the chromaffin vesicle matrix proteins in the presence of 0.5 mM Mg2+ is characterized by a Kd of 240 microM and after addition of 15 mM Na+ by a Kd of 340 microM.(ABSTRACT TRUNCATED AT 250 WORDS) 相似文献
8.
M Sumida M Hamada H Takenaka Y Hirata K Nishigauchi H Okuda 《Journal of biochemistry》1986,100(3):765-772
The effects of various divalent cations on the Ca2+ uptake by microsomes from bovine aortic smooth muscle were studied. High concentrations (1 mM) of Co2+, Zn2+, Mn2+, Fe2+, and Ni2+ inhibited neither the Ca2+ uptake by the microsomes nor the formation of the phosphorylated intermediate (E approximately P) of the Ca2+,Mg2+-ATPase of the microsomes. The cadmium ion, however, inhibited both the Ca2+ uptake and the E approximately P formation by the microsomes. Dixon plot analysis indicated Cd2+ inhibited (Ki = 135 microM) the Ca2+ dependent E approximately P formation in a non-competitive manner. The inhibitory effect of Cd2+ was lessened by cysteine or dithiothreitol. The strontium ion inhibited the Ca2+ uptake competitively, while the E approximately P formation increased on the addition of Sr2+ at low Ca2+ concentrations. At a low Ca2+ concentration (1 microM), Sr2+ was taken up by the aortic microsomes in the presence of 1 mM ATP. It is thus suggested that Sr2+ replaces Ca2+ at the Ca2+ binding site on the ATPase. 相似文献
9.
The binding of Ca2+ ions to pig heart NAD+-isocitrate dehydrogenase and the 2-oxoglutarate dehydrogenase complex. 总被引:2,自引:1,他引:1
下载免费PDF全文
![点击此处可从《The Biochemical journal》网站下载免费的PDF全文](/ch/ext_images/free.gif)
1. The binding of Ca2+ ions to purified pig heart NAD+-isocitrate dehydrogenase and 2-oxoglutarate dehydrogenase, freed of contaminating Ca2+ by parvalbumin/polyacrylamide chromatography, has been studied by flow dialysis and by the use of fura-2. 2. For the 2-oxoglutarate dehydrogenase complex, 3.5 mol of Ca2+-binding sites/mol of complex were apparent, with an apparent dissociation constant (Kd value) for Ca2+ of 2.0 microM. These values were little affected by Mg2+ ions, ADP or 2-oxoglutarate. 3. By contrast, binding of Ca2+ to NAD+-isocitrate dehydrogenase (Kd = 14 microM) required ADP, isocitrate and Mg2+ ions. The number of Ca2+-binding sites associated with NAD+-isocitrate dehydrogenase was then 0.9 mol/mol of tetrameric enzyme. 4. The 2-oxoglutarate dehydrogenase complex bound ADP (as ADP3-) to a group of tight-binding sites (Kd = 3.1 microM) with a stoichiometry, 3.3 mol/mol of complex, similar to that for the binding of Ca2+; a variable number of much weaker sites (Kd = 100 microM) for ADP3- was also apparent. 相似文献
10.
Ca2+ release from sarcoplasmic reticulum membranes, activated by alkaline pH occurs only when EGTA is present in the release medium. Addition of very low concentrations of Ca2+ to the medium inhibits Ca2+ release. The concentration of free Ca2+ required for 50% inhibition ranges from between 5 and 20 nM in different experiments and/or membrane preparations, irrespective of whether the free Ca2+ concentration is controlled by EGTA or CDTA. Other divalent cations such as Mn2+, Ba2+, Cu2+, Cd2+ and Mg2+ also exert an inhibitory effect on Ca2+ release, with higher or lower potency than that of Ca2+. The inactivation of Ca2+ release by Ca2+ is reversible. We suggest the involvement of high-affinity Ca2+-binding sites in the control of Ca2+ release. 相似文献
11.
Calmodulin from phosphorylase kinase (the delta subunit) was obtained as a homogeneous protein in a spectroscopically pure form, and its interaction with Ca2+ and Mg2+ was studied. 1. Determination of the binding of Ca2+ to calmodulin in a buffer of low ionic strength (0.001 M) show that it contained six binding sites for this divalent cation. 2. Employment of a buffer of high ionic strength (0.18 M) allowed two Ca2+/Mg2+-binding sites (KdCa2+ = 4.0 microM), which showed Ca2+ - Mg2+ competition (KdMg2+ = 0.75 mM), to be distinguished from two Ca2+-specific binding sites (KdCa2+ = 40 microM). The remaining two Ca2+-binding sites are not observed under these conditions and are probably Mg2+-specific binding sites. Thus, the binding sites on calmodulin are remarkably similar to those of the homologous Ca2+-binding protein, troponin C [Potter and Gergely (1975) J. Biol. Chem. 250, 4628, 4633]. 3. The conformational states of calmodulin are defined by Ca2+, Mg2+ and salt concentrations, which can be differentiated by their Ca2+ affinity and their relative tyrosine fluorescence intensity. In a buffer of high ionic strength, Mg2+ induces a conformation which enhances the apparent affinity for Ca2+. Addition of Ca2+ leads to an enhancement of the tyrosine fluorescence intensity, which remains enhanced even upon removal of Ca2+ by chelation with EGTA. Only additional chelation of Mg2+ with EDTA reduces the tyrosine fluorescence intensity. 4. Comparison of the Ca2+-binding parameters of phosphorylase kinase, which were previously determined under identical experimental conditions [Kilimann and Heilmeyer (1977) Eur. J. Biochem. 73, 191-197], with those reported here on calmodulin isolated from this enzyme, allows the conclusion that Ca2+ binding to the holoenzyme occurs by binding to the delta subunit exclusively. 5. Ca2+ binding and Ca2+ activation of phosphorylase kinase are compared and discussed in relation to the Ca2+ and Mg2+-induced conformation changes of calmodulin. 相似文献
12.
P J van Haastert 《Biochimica et biophysica acta》1985,846(3):324-333
cAMP induces a transient increase of cAMP and cGMP levels in Dictyostelium discoideum cells. Fast binding experiments reveal three types of cAMP-binding site (S, H and L), which have different off-rates (t0.5, 0.7-15 s) and different affinities (Kd, 15-450 nM). A time- and cAMP-concentration-dependent transition of H- to L-sites occurs during the binding reaction (Van Haastert, P.J.M. and De Wit, R.J.W. (1984) J. Biol. Chem. 13321-13328). Extracellular Ca2+ had multiple effects on cAMP-binding sites. (i) The number of H + L-sites increased 2.5-fold, while the number of S-sites was not strongly affected. (ii) The Kd of the S-sites was reduced from 16 nM to 5 nM (iii) The conversion of H-sites to L-sites was inhibited up to 80%. The kinetics of the cAMP-induced cAMP accumulation was not strongly altered by Ca2+, but the amount of cAMP produced was inhibited up to 80%. The kinetics of the cAMP-induced cGMP accumulation was strongly altered; maximal levels were obtained sooner, and the Ka was reduced from 15 to 3.5 nM cAMP. Ca2+, Mg2+ and Mn2+ increased the number of binding sites, all with EC50 = 0.5 mM. The S-sites and the cGMP response were modified by equal Ca2+ concentrations and by higher concentrations of Mg2+ and Mn2+ (EC50 are respectively 0.4 mM, 2.5 mM and about 25 mM). The conversion of H- to L-sites and the cAMP response were specifically inhibited by Ca2+ with EC50 = 20 microM. It is concluded that cAMP activates guanylate cyclase through the S-sites; adenylate cyclase is activated by the H + L-sites, in which the appearance of the L-sites during the binding reaction represents the coupling of occupied surface cAMP receptors to adenylate cyclase. 相似文献
13.
J J Robinson 《Biochimie et biologie cellulaire》1989,67(11-12):808-812
The interaction of metal ions with the sea urchin extraembryonic coat protein hyalin was investigated. Hyalin, immobilized on nitrocellulose membrane, bound Ca2+ and this interaction was disrupted by ruthenium red and selective metal ions. The divalent cations Cd2+ and Mn2+, when present at a concentration of 30 microM, displaced hyalin-bound Ca2+. In competition assays, 1 mM Cd2+ or 3 mM Mn2+ were effective competitors with Ca2+ for binding to hyalin. Cobalt, at a concentration of 30 microM, was unable to displace protein-bound Ca2+, but was effective in competition assays at a concentration of at least 10 mM. Magnesium and the monovalent cation Cs+ were unable to disrupt Ca2(+)-hyalin interaction. Interestingly, Cd2+, Mn2+, and Co2+ mimicked the biological effects of Ca2+ on the hyalin self-association reaction. These results clearly demonstrate that the Ca2(+)-binding sites on hyalin can selectively accommodate other divalent cations in a biologically active configuration. 相似文献
14.
Kinetics of rapid Ca2+ release by sarcoplasmic reticulum. Effects of Ca2+, Mg2+, and adenine nucleotides 总被引:24,自引:0,他引:24
A radioisotope flux-rapid-quench-Millipore filtration method is described for determining the effects of Ca2+, adenine nucleotides, and Mg2+ on the Ca2+ release behaviour of "heavy" sarcoplasmic reticulum (SR) vesicles. Rapid 45Ca2+ efflux from passively loaded vesicles was blocked by the addition of Mg2+ and ruthenium red. At pH 7 and 10(-9) M Ca2+, vesicles released 45Ca2+ with a low rate (k = 0.1 s-1). An increase in external Ca2+ concentration to 4 microM or the addition of 5 mM ATP or the ATP analogue adenosine 5'-(beta,gamma-methylenetriphosphate) (AMP-PCP) resulted in intermediate 45Ca2+ release rates. The maximal release rate was observed in media containing 4 microM Ca2+ and 5 mM AMP-PCP and had a first-order rate constant of 30-100 s-1. Mg2+ partially inhibited Ca2+- and nucleotide-induced 45Ca2+ efflux. In the absence of AMP-PCP, 45Ca2+ release was fully inhibited at 5 mM Mg2+ or 5 mM Ca2+. The composition of the release media was systematically varied, and the flux data were expressed in the form of Hill equations. The apparent n values of activation of Ca2+ release by ATP and AMP-PCP were 1.6-1.9. The Hill coefficient of Ca2+ activation (n = 0.8-2.1) was dependent on nucleotide and Mg2+ concentrations, whereas the one of Mg2+ inhibition (n = 1.1-1.6) varied with external Ca2+ concentration. These results suggest that heavy SR vesicles contain a "Ca2+ release channel" which is capable of conducting Ca2+ at rates comparable with those found in intact muscle. Ca2+, AMP-PCP (ATP), and Mg2+ appear to act at noninteracting or interacting sites of the channel. 相似文献
15.
Metal ions, such as Ca2+ and Mn2+, are necessary for the generation of cofactor activity following reconstitution of factor VIII from its isolated light chain (LC) and heavy chain (HC). Titration of EDTA-treated factor VIII with Mn2+ showed saturable binding with high affinity (K(d) = 5.7 +/- 2.1 microM) as detected using a factor Xa generation assay. No significant competition between Ca2+ and Mn2+ for factor VIII binding (K(i) = 4.6 mM) was observed as measured by equilibrium dialysis using 20 microM Ca2+ and 8 microM factor VIII in the presence of 0-1 mM Mn2+. The intersubunit affinity measured by fluorescence energy transfer of an acrylodan-labeled LC (fluorescence donor) and fluorescein-labeled HC (fluorescence acceptor) in the presence of 20 mM Mn2+ (K(d) = 53.0 +/- 17.1 nM) was not significantly different from the affinity value previously obtained in the absence of metal ion (K(d) = 53.8 +/- 14.2 nM). The sensitization of phosphorescence of Tb3+ bound to factor VIII subunits was utilized to detect Mn2+ binding to the subunits. Mn2+ inhibited the phosphorescence of Tb3+ bound to HC and LC, as well as the HC-derived A1 and A2 subunits with a relatively wide range of estimated inhibition constant values (K(i) values = 169-1147 microM), whereas Ca2+ showed no effect on Tb3+ phosphorescence. These results suggest that factor VIII cofactor activity can be generated by Mn2+ binding to site(s) on factor VIII that are different from the high-affinity Ca2+ binding site. However, like Ca2+, Mn2+ did not alter the affinity for HC and LC association. Thus, Mn2+appears to generate factor VIII cofactor activity by a similar mechanism as observed for Ca2+following its association at nonidentical sites on the protein. 相似文献
16.
Vacuoles of radish (Raphanus sativus) contained a Ca2+-binding protein (RVCaB) of 43 kDa. We investigated the Ca2+-binding properties of the protein. RVCaB was expressed in Escherichia coli and was purified from an extract by ion-exchange chromatography, nitrocellulose membrane filtration, and gel-filtration column chromatography. Ca2+-binding properties of the recombinant protein were examined by equilibrium dialysis with 45Ca2+ and small dialysis buttons. The protein was estimated to bind 19Ca2+ ions per molecule with a Kd for Ca2+ of 3.4 mM. Ca2+ was bound to the protein even in the presence of high concentrations of Mg2+ or K+. The results suggested that the protein bound Ca2+ with high ion selectivity, high capacity, and low affinity. 相似文献
17.
Effects of intracellular and extracellular concentrations of Ca2+, K+, and Cl- on the Na+-dependent Mg2+ efflux in rat ventricular myocytes
下载免费PDF全文
![点击此处可从《Biophysical journal》网站下载免费的PDF全文](/ch/ext_images/free.gif)
Intracellular Mg2+ concentration ([Mg2+]i) was measured in rat ventricular myocytes with the fluorescent indicator furaptra (25 degrees C). After the myocytes were loaded with Mg2+, the initial rate of decrease in [Mg2+]i (initial Delta[Mg2+]i/Deltat) was estimated upon introduction of extracellular Na+, as an index of the rate of Na+-dependent Mg2+ efflux. The initial Delta[Mg2+]i/Deltat values with 140 mM [Na+]o were essentially unchanged by the addition of extracellular Ca2+ up to 1 mM (107.3+/-8.7% of the control value measured at 0 mM [Ca2+]o in the presence of 0.1 mM EGTA, n=5). Intracellular loading of a Ca2+ chelator, either BAPTA or dimethyl BAPTA, by incubation with its acetoxymethyl ester form (5 microM for 3.5 h) did not significantly change the initial Delta[Mg2+]i/Deltat: 115.2+/-7.5% (seven BAPTA-loaded cells) and 109.5+/-10.9% (four dimethyl BAPTA loaded cells) of the control values measured in the absence of an intracellular chelator. Extracellular and/or intracellular concentrations of K+ and Cl- were modified under constant [Na+]o (70 mM), [Ca2+]o (0 mM with 0.1 mM EGTA), and membrane potential (-13 mV with the amphotericin-B-perforated patch-clamp technique). None of the following conditions significantly changed the initial Delta[Mg2+]i/Deltat: 1), changes in [K+]o between 0 mM and 75 mM (65.6+/-5.0% (n=11) and 79.0+/-6.0% (n=8), respectively, of the control values measured at 140 mM [Na+]o without any modification of extracellular and intracellular K+ and Cl-); 2), intracellular perfusion with K+-free (Cs+-substituted) solution from the patch pipette in combination with removal of extracellular K+ (77.7+/-8.2%, n=8); and 3), extracellular and intracellular perfusion with K+-free and Cl--free solutions (71.6+/-5.1%, n=5). These results suggest that Mg2+ is transported in exchange with Na+, but not with Ca2+, K+, or Cl-, in cardiac myocytes. 相似文献
18.
The Ca2+-binding properties of isolated brush-border membranes at physiological ionic strength and pH were examined by rapid Millipore filtration. A comprehensive analysis of the binding data suggested the presence of two types of Ca2+-binding sites. The high-affinity sites, Ka = (6.3 +/- 3.3) X 10(5) M-1 (mean +/- S.E.M.), bound 0.8 +/- 0.1 nmol of Ca2+/mg of protein and the low-affinity sites, Ka = (2.8 +/- 0.3) X 10(2) M-1, bound 33 +/- 3.5 nmol of Ca2+/mg of protein. The high-affinity site exhibited a selectivity for Ca2+, since high concentrations of competing bivalent cations were required to inhibit Ca2+ binding. The relative effectiveness of the competing cations (1 and 10 mM) for the high-affinity site was Mn2+ approximately equal to Sr2+ greater than Ba2+ greater than Mg2+. Data from the pH studies, treatment of the membranes with carbodi-imide and extraction of phospholipids with aqueous acetone and NH3 provided evidence that the low-affinity sites were primarily phospholipids and the high-affinity sites were either phosphoprotein or protein with associated phospholipid. Two possible roles for the high-affinity binding sites are suggested. Either high-affinity Ca2+ binding is involved with specific enzyme activities or Ca2+ transport across the luminal membrane occurs via a Ca2+ channel which contains a high-affinity Ca2+-specific binding site that may regulate the intracellular Ca2+ concentration and gating of the channel. 相似文献
19.
High-affinity binding of Ca2+ to bovine alpha-lactalbumin in the absence and presence of EGTA.
下载免费PDF全文
![点击此处可从《The Biochemical journal》网站下载免费的PDF全文](/ch/ext_images/free.gif)
Literature values for the Kd for Ca2+ in bovine alpha-lactalbumin range over 3 orders of magnitude. There is a difference between two results obtained with EGTA as a metal-ion buffer, partly because different values for the Kd of Ca2+-EGTA were used in the calculations, and a much wider difference between results obtained in the presence and absence of EGTA, which has been attributed to an interaction between EGTA and the protein. Titrations in a flow-dialysis cell showed that Mn2+ competed with Ca2+ for the high-affinity site on the protein, and the results, combined with a Kd for Mn2+ of 2.1 +/- 0.1 microM, which was determined fluorimetrically, gave a Kd for Ca2+ of 1.3 +/- 0.1 nM. When alpha-lactalbumin containing 45Ca2+ was titrated with EGTA in a flow-dialysis cell, and widely accepted metal-chelation data for EGTA were used in the calculations, a Kd for Ca2+ of 1.10 +/- 0.03 nM was obtained. The results from the two methods are so similar as to indicate that the affinity for Ca2+ was unaffected by the presence of EGTA. 相似文献
20.
An acid-stable phosphoprotein was formed in a microsomal membrane fraction isolated from bovine aortic smooth muscle in the presence of Mg2+ + ATP and Ca2+. The microsomes also showed Ca2+ uptake activity. The Ca2+ dependence of phosphoprotein formation and of Ca2+ uptake occurred over the same range of Ca2+ concentration (1-10 microM), and resembled similar findings from rabbit skeletal microsomes. The molecular weight of the phosphorylated protein, estimated by SDS-gel electrophoresis, was approximately 105,000. The phosphoprotein was labile at alkaline pH, and its decomposition was accelerated by hydroxylamine. Half-maximum incorporation of 32P in the presence of 10 microM Ca2+ occurred at 60 nM ATP. The calcium-dependent phosphoprotein formation was not affected by 5 mM NaN3, but was inhibited in a dose-dependent fashion by ADP with a 50% inhibition occurring at 180 microM. Fifty mM MgCl2 was required for the maximal phosphorylation. The rate of phosphoprotein decomposition after adding 2 mM EGTA was accelerated by varying the Mg2+ concentration from 10 microM to 3 mM. Alkaline pH (9.0) slowed the rate of phosphoprotein decay. Optimal Ca2+-dependent phosphoprotein occurred at 15 degrees C over a broad pH range (6.4 to 9.0). The activation energy of EGTA-induced phosphoprotein decomposition was 25.6 kcal/mol between 0 and 16 degrees C and 14.6 kcal/mol between 16 and 30 degrees C. The phosphoprotein formed by aortic microsomes was thus quite similar to the acid-stable phosphorylated intermediate of the Ca2+-transport ATPase of sarcoplasmic reticulum from skeletal and cardiac muscle. These data suggest that the Ca2+-dependent phosphoprotein is a reaction intermediate of the Ca2+,Mg2+-ATPase of the aortic microsomes. 相似文献