Demonstration of two different reactive sulfhydryl groups in the ATP-binding sites of Ca2+-ATPase of sarcoplasmic reticulum by disulfides of thioinosine triphosphates

1. The disulfide of thioinosine triphosphate, (SnoPPP)2, is a substrate of the Ca2+-pump and the Ca2+-ATPase of sarcoplasmic reticulum (Km = 400 microM). 2. Inactivation of Ca2+-ATPase by the beta,gamma-methylene diphosphonate analogue of the disulfide of thioinosine triphosphate, (SnoPP[CH2]P)2, in the presence of (Ca2+ + Mg2+ + K+) is preceeded by a dissociable enzyme inhibitor complex with a dissociation constant of 130 microM for a low-affinity binding site. ATP protected Ca2+-ATPase against the inactivation under these conditions with a dissociation constant of 140 microM. 3. Kinetic analysis of the inactivations of Ca2+-ATPase by (SnoPP[CH2]P)2 in the absence of Ca2+ and Mg2+ but the presence of K+ and EGTA led to the appearance of two nucleotide binding sites with two different inactivation velocities. Inactivation rate constants k2 were found for the rapid inactivating part (k2' = 1.44 X 10(-2) s-1) and the slow inactivating part (k2" = 1.15 X 10(-3) s-1). From the protective effect of ATP under these conditions a high-affinity (Kd = 48.78 microM) and a low-affinity ATP binding site (Kd = 114 microM) were apparent. 4. The affinity of the analogues to the enzyme is decreased in the sequence: (SnoPPP)2 > (SnoPP[NH]P)2 > (SnoPP[CH2]P)2 > (SnoP)2. 5. (SnoPPP)2-inactivated Ca2+-ATPase was reactivated by incubation with dithiothreitol. 6. Inactivation of Ca2+-ATPase by [gamma-32P](SnoPPP)2 in the presence of (Mg2+ + K+ + Ca2+) or (EGTA + K+) was accompanied by the incorporation of hydroxylamine-insensitive radioactivity into the acid-precipitable protein. The enzyme-bound [gamma-32P]SnoPPP was cleaved by dithiothreitol. 7. It is concluded that (SnoPPP)2 and its non-hydrolyzable analogues (SnoPP[NH]P)2 and (SnoPP[CH2]P)2 act as ATP affinity labels and form mixed disulfides with a sulfhydryl group within the active site.     

Interaction of guanosine cyclic 3',5'-phosphate dependent protein kinase with lin-benzoadenine nucleotides

Using the activated cGMP-dependent protein kinase in the presence of the phosphorylatable peptide [[Ala34]histone H2B-(29-35)], we found that lin-benzoadenosine 5'-diphosphate (lin-benzo-ADP) was a competitive inhibitor of the enzyme with respect to ATP with a Ki (22 microM) similar to the Kd (20 microM) determined by fluorescence polarization titrations. The Kd for lin-benzo-ADP determined in the absence of the phosphorylatable peptide, however, was only 12 microM. ADP bound with lower affinity (Ki = 169 microM; Kd = 114 microM). With [Ala34]histone H2B-(29-35) as phosphoryl acceptor, the Km for lin-benzo-ATP was 29 microM, and that for ATP was 32 microM. The Vmax with lin-benzo-ATP, however, was only 0.06% of that with ATP as substrate [0.00623 +/- 0.00035 vs. 11.1 +/- 0.17 mumol (min.mg)-1]. Binding of lin-benzo-ADP to the kinase was dependent upon a divalent cation. Fluorescence polarization revealed that Mg2+, Mn2+, Co2+, Ni2+, Ca2+, Sr2+, and Ba2+ supported nucleotide binding to the enzyme; Ca2+, Sr2+, and Ba2+, however, did not support any measurable phosphotransferase activity. The rank order of metal ion effectiveness in mediating phosphotransferase activity was Mg2+ greater than Ni2+ greater than Co2+ greater than Mn2+. Although these results were similar to those observed with the cAMP-dependent protein kinase [Hartl, F. T., Roskoski, R., Jr., Rosendahl, M. S., & Leonard, N. J. (1983) Biochemistry 22, 2347], major differences in the Vmax with lin-benzo-ATP as substrate and the effect of peptide substrates on nucleotide (both lin-benzo-ADP and ADP) binding were observed.     

The binding of the fluorescent ATP analogue 2'(3')-trinitrophenyladenosine-5'-triphosphate to rat liver fatty acid-binding protein.

The less polar fluorescent analogue of ATP, 2'(3')-trinitrophenyl-5'-triphosphate bound to rat liver fatty acid-binding protein with high affinity (Kd 6.3 x 10(-6) M) and 1:1 molar stoichiometry. This probe bound to the fatty acid binding site of the protein and was displaced by oleic acid and oleoyl CoA. High concentrations of ATP did not cause significant displacement of the fluorescent ATP analogue. Since the anionic part of this molecule is the triphosphate group it is difficult to envisage this group being accommodated at an anion binding site within the non-polar core of this protein as is the case with other fatty acid binding proteins. Therefore it is anticipated that the ligand must bind to liver fatty acid-binding protein with this triphosphate group surface exposed. Caution must be exercised when using the more hydrophobic fluorescent analogue of ATP to investigate the ATP binding properties of proteins.     

Two distinct classes of nucleotide binding sites in sarcoplasmic reticulum Ca-ATPase revealed by 2',3'-O-(2,4,6-trinitrocyclohexadienylidene)-ATP

It was previously reported that 2',3'-O-(2,4,6-trinitrocyclohexadienylidene) (TNP)-nucleotides bind with high affinity to the sarcoplasmic reticulum Ca-ATPase (Dupont, Y., Chapron, Y., and Pougeois, R. (1982) Biochem. Biophys. Res. Commun. 106, 1272-1279 and Watanabe, T., and Inesi, G. (1982) J. Biol. Chem. 257, 11510-11516). Here we report a study of the Ca-ATPase nucleotide binding sites using TNP-nucleotides. Competition at equilibrium between TNP-nucleotides and ATP was measured in the absence of calcium; it was found that TNP-nucleotides and ATP competitively bind to two classes of sites of equal concentration (3.5 nmol/mg). The ATP dissociation constants for the two classes of sites were found to be sensitive to H+ and Mg2+ concentrations. In the absence of Mg2+ (independently of pH) or at acid pH (independently of Mg2+ concentration), the nucleotide sites behave like one single family of sites of intermediate affinity (Kd = 20 microM). They split into two classes of sites of high (Kd = 2-4 microM) and low (Kd greater than 1 mM) affinity at pH values higher than neutral and in the presence of Mg2+. The calcium-activated ATP hydrolysis is accelerated by TNP-ATP (or TNP-AMP-PNP) binding on the phosphorylated enzyme. It is concluded 1) that the Ca-ATPase enzyme possesses two classes of ATP binding sites, 2) that the affinity of these two sites and the nature of their interaction is modulated by the H+ and Mg2+ concentrations, and 3) that the hydrolytic activity of the high affinity ATP binding site is activated by ATP or TNP-AMP-PNP (or TNP-ATP) binding in a low affinity ATP binding site.     

Ca2+ binding to chromaffin vesicle matrix proteins: effect of pH, Mg2+, and ionic strength

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)     

2',3'-O-(2,4,6-trinitrophenyl)-8-azido-adenosine mono-, di-, and triphosphates as photoaffinity probes of the Ca2+-ATPase of sarcoplasmic reticulum. Regulatory/superfluorescent nucleotides label the catalytic site with high efficiency

We have synthesized a new class of ATP photo-affinity analogs, 2',3'-O-(2,4,6-trinitrophenyl)-8-azido (TNP-8N3)-ATP, -ADP, and -AMP, and their radiolabeled derivatives, and characterized their interaction with sarcoplasmic reticulum vesicles. The nucleotides bind with high affinity (Kd = 0.04-0.4 microM) to the catalytic site of the Ca2+-ATPase. TNP-8N3-ATP and TNP-8N3-ADP, at low concentrations (less than 10 microM), accelerate ATPase activity 1.5- and 1.4-fold, respectively, indicating that they bind to a regulatory site. In the same concentration range, they all undergo a large increase in fluorescence ("superfluorescence") during enzyme turnover in the presence of ATP and Ca2+, or on phosphorylation from Pi in a Ca2+-depleted medium. Irradiation at alkaline pH results in specific covalent incorporation of the nucleotide at the catalytic site on the A1 tryptic subfragment. The efficiency of catalytic site labeling is greatest (up to 80% of available sites/irradiation period) in the presence of ATP, Ca2+, and Mg2+, conditions in which the probe binds only to the regulatory and superfluorescent sites. The covalently attached nucleotide exhibits fluorescence enhancement on enzyme turnover in the presence of acetyl phosphate plus Ca2+ or on phosphorylation from Pi in a Ca2+-depleted medium, but not in the presence of ATP plus Ca2+. The results suggest that the catalytic, regulatory, and superfluorescent nucleotide sites are at the same locus and that the binding domain includes portions of the A1 subfragment. The high efficiency with which the site is photolabeled during turnover is ascribed to water exclusion and possibly cleft closure in E2-P.     

Calcium binding to the H+,K(+)-ATPase. Evidence for a divalent cation site that is occupied during the catalytic cycle

The binding and conformational properties of the divalent cation site required for H+,K(+)-ATPase catalysis have been explored by using Ca2+ as a substitute for Mg2+. 45Ca2+ binding was measured with either a filtration assay or by passage over Dowex cation exchange columns on ice. In the absence of ATP, Ca2+ was bound in a saturating fashion with a stoichiometry of 0.9 mol of Ca2+ per active site and an apparent Kd for free Ca2+ of 332 +/- 39 microM. At ATP concentrations sufficient for maximal phosphorylation (10 microM), 1.2 mol of Ca2+ was bound per active site with an apparent Kd for free Ca2+ of 110 +/- 22 microM. At ATP concentrations greater than or equal to 100 microM, 2.2 mol of Ca2+ were bound per active site, suggesting that an additional mole of Ca2+ bound in association with low affinity nucleotide binding. At concentrations sufficient for maximal phosphorylation by ATP (less than or equal to 10 microM), APD, ADP + Pi, beta,gamma-methylene-ATP, CTP, and GTP were unable to substitute for ATP. Active site ligands such as acetyl phosphate, phosphate, and p-nitrophenyl phosphate were also ineffective at increasing the Ca2+ affinity. However, vanadate, a transition state analog of the phosphoenzyme, gave a binding capacity of 1.0 mol/active site and the apparent Kd for free Ca2+ was less than or equal to 18 microM. Mg2+ displaced bound Ca2+ in the absence and presence of ATP but Ca2+ was bound about 10-20 times more tightly than Mg2+. The free Mg2+ affinity, like Ca2+, increased in the presence of ATP. Monovalent cations had no effect on Ca2+ binding in the absence of ATP but dit reduce Ca2+ binding in the presence of ATP (K+ = Rb+ = NH4 + greater than Na+ greater than Li+ greater than Cs+ greater than TMA+, where TMA is tetramethylammonium chloride) by reducing phosphorylation. These results indicate that the Ca2+ and Mg2+ bound more tightly to the phosphoenzyme conformation. Eosin fluorescence changes showed that both Ca2+ and Mg2+ stabilized E1 conformations (i.e. cytosolic conformations of the monovalent cation site(s)) (Ca.E1 and Mg.E1). Addition of the substrate acetyl phosphate to either Ca.E1 or Mg.E1 produced identical eosin fluorescence showing that Ca2+ and Mg2+ gave similar E2 (extracytosolic) conformations at the eosin (nucleotide) site. In the presence of acetyl phosphate and K+, the conformations with Ca2+ or Mg2+ were also similar. Comparison of the kinetics of the phosphoenzyme and Ca2+ binding showed that Ca2+ bound prior to phosphorylation and dissociated after dephosphorylation.(ABSTRACT TRUNCATED AT 400 WORDS)     

Characterization of 2',3'-O-(2,4,6-trinitrocyclohexadienylidine)adenosine 5'-triphosphate as a fluorescent probe of the ATP site of sodium and potassium transport adenosine triphosphatase. Determination of nucleotide binding stoichiometry and ion-induced changes in affinity for ATP

The fluorescent ATP derivative 2',3'-O-(2,4,6-trinitrocyclohexadienylidine) adenosine 5'-triphosphate (TNP-ATP) binds specifically with enhanced fluorescence to the ATP site of purified eel electroplax sodium-potassium adenosine triphosphatase, (Na,K)-ATPase. A single homogeneous high affinity TNP-ATP binding site with a KD of 0.04 to 0.09 microM at 3 degrees C and 0.2 to 0.7 microM at 21 degrees-25 degrees C was observed in the absence of ligands when binding was measured by fluorescence titration or with [3H]TNP-ATP. ATP and other nucleotides competed with TNP-ATP for binding with KD values similar to those previously determined for binding to the ATP site. Binding stoichiometries determined from Scatchard plot intercepts gave one TNP-ATP site/175,000 g of protein (range: 1.64 X 10(5) to 1.92 X 10(5) when (Na,K)-ATPase protein was determined by quantitative amino acid analysis. The ratio of [3H]ouabain sites to TNP-ATP sites was 0.91. These results are inconsistent with "half-of-sites" binding and suggest that there is one ATP and one ouabain site/alpha beta protomer. (Na,K)-ATPase maintained a high affinity for TNP-ATP regardless of the ligands present. K+ increased the KD for TNP-ATP about 5-fold and Na+ reversed the effect of K+. The effects of Na+, K+, and mg2+ on ATP binding at 3 degrees C were studied fluorimetrically by displacement of TNP-ATP by ATP. The results are consistent with competition between ATP and TNP-ATP for binding at a single site regardless of the metallic ions present. The derived KD values for ATP were : no ligands, 1 microM; 20 mM NaCl, 3-4 microM; 20 mM KCl, 15-19 microM; 20 mM Kcl + 4 mM MgCl2, 70-120 microM. These results suggests that a single ATP site exhibits a high or low affinity for ATP depending on the ligands present, so that high and low affinity ATP sites observed kinetically are interconvertible and do not co-exist independently. We propose that during turnover the affinity for ATP changes more than 100-fold owing to the conformational changes associated with ion binding, translocation, and release.     

Evidence of a calcium-induced structural change in the ATP-binding site of the sarcoplasmic-reticulum Ca2+-ATPase using terbium formycin triphosphate as an analogue of Mg-ATP

Terbium ions and terbium formycin triphosphate have been used to investigate the interactions between the cation and nucleotide binding sites of the sarcoplasmic reticulum Ca2+-ATPase. Three classes of Tb3+-binding sites have been found: a first class of low-affinity (Kd = 10 microM) corresponds to magnesium binding sites, located near a tryptophan residue of the protein; a second class of much higher affinity (less than 0.1 microM) corresponds to the calcium transport sites, their occupancy by terbium induces the E1 to E2 conformational change of the Ca2+-ATPase; a third class of sites is revealed by following the fluorescence transfer from formycin triphosphate (FTP) to terbium, evidencing that terbium ions can also bind into the nucleotide binding site at the same time as FTP. Substitution of H2O by D2O shows that Tb-FTP binding to the enzyme nucleotide site is associated with an important dehydration of the terbium ions associated with FTP. Two terbium ions, at least, bind to the Ca2+-ATPase in the close vicinity of FTP when this nucleotide is bound to the ATPase nucleotide site. Addition of calcium quenches the fluorescence signal of the terbium-FTP complex bound to the enzyme. Calcium concentration dependence shows that this effect is associated with the replacement of terbium by calcium in the transport sites, inducing the E2----E1 transconformation when calcium is bound. One interpretation of this fluorescence quenching is that the E1----E2 transition induces an important structural change in the nucleotide site. Another interpretation is that the high-affinity calcium sites are located very close to the Tb-FTP complex bound to the nucleotide site.     

Mutations of either or both Cys876 and Cys888 residues of sarcoplasmic reticulum Ca2+-ATPase result in a complete loss of Ca2+ transport activity without a loss of Ca2+-dependent ATPase activity. Role of the CYS876-CYS888 disulfide bond.

Disulfide-containing peptides in pepsin digest of sarcoplasmic reticulum vesicles were identified by using a fluorogenic thiol-specific reagent 4-fluoro-7-sulfamoylbenzofurazan and a reductant tributylphosphine. Sequencing of the purified peptides revealed the presence of a Cys(876)-Cys(888) disulfide bond on the luminal loop connecting the 7th and 8th transmembrane helices (loop 7-8) of the Ca(2+)-ATPase (SERCA1a). We substituted either or both of these cysteine residues with alanine and made three mutants (C876A, C888A, C876A/C888A), in which the disulfide bond is disrupted. The mutants and the wild type were expressed in COS-1 cells, and functional analysis was performed with the microsomes isolated from the cells. Electrophoresis performed under reducing and non-reducing conditions confirmed the presence of Cys(876)-Cys(888) disulfide bond in the expressed wild type. All the three mutants possessed high Ca(2+)-ATPase activity. In contrast, no Ca(2+) transport activity was detected with these mutants. These mutants formed almost the same amount of phosphoenzyme intermediate as the wild type from ATP and from P(i). Detailed kinetic analysis showed that the three mutants hydrolyze ATP in the mechanism well accepted for the Ca(2+)-ATPase; activation of the catalytic site upon high affinity Ca(2+) binding, formation of ADP-sensitive phosphoenzyme, subsequent rate-limiting transition to ADP-insensitive phosphoenzyme, and hydrolysis of the latter phosphoenzyme. It is likely that the pathway for delivery of Ca(2+) from the binding sites into the lumen of vesicles is disrupted by disruption of the Cys(876)-Cys(888) disulfide bond, and therefore that the loop 7-8 having the disulfide bond is important for formation of the proper structure of the Ca(2+) pathway.     

Detection of conformational changes in chloroplast coupling factor 1 by 8-anilino-1-naphthalene-sulphonate fluorescence changes

Chloroplast coupling factor 1 (CF1) contains a high-affinity binding site for 8-anilino-1-napthalene sulphonate (ANS,Kd = 5-6 microM). The binding of ANS to the enzyme is associated with a fluorescence enhancement and a blue-shift in the emission spectrum. ANS only slightly inhibits ATP hydrolysis by CF1. Adenine nucleotides and inorganic phosphate induce a fast ANS fluorescence quenching of about 50% which is due to a decrease in the affinity of the enzyme for ANS (Kd increases from 6 microM to 22 microM) and in the fluorescence quantum yield of the bound probe (by 33%) but not in the number of ANS sites (n = 1). Conversely, Mg and Ca ions induce a fluorescence enhancement of bound ANS. Inactivation of the enzyme enhances ANS fluorescence, eliminates the response to adenine nucleotides and inorganic phosphate but increases the response to divalent metals. The affinity of latent CF1 for ADP (Kd = 12 microM) is considerably higher than for ATP (Kd = 95 microM) in buffer containing EDTA. The Kd for inorganic phosphate is 140 microM. Mg increases the apparent affinity for ATP (Kd = 28 microM) but not for ADP or Pi. Binding of ATP to the tight-sites does not inhibit the ADP or Pi-induced fluorescence quenching but decreases the affinity for ADP (Kd = 34 microM) and for inorganic phosphate (Kd = 320 microM). These results suggest that the ADP and phosphate binding sites are different but not independent from the tight sites. Activation of a Mg-specific ATPase in CF1 by octyl glucoside decreases the affinity for ADP and inorganic phosphate by about threefold but increases the affinity for ATP. ATPase activation of CF1 also increases the Ki for ADP inhibition of ATP hydrolysis. ATPase activation also influences the ANS responses to Ca and Mg. Ca-ATPase activation increases the fluorescence enhancement and the apparent affinity for Ca whereas Mg-ATPase activation specifically increases the Mg-induced fluorescence enhancement. The fluorescence of CF1-bound ANS is enhanced by Dio-9 and quenched by phloridzin, quercetin, Nbf-Cl and FITC. Nbf-Cl and FITC completely inhibit the ADP-induced fluorescence quenching whereas Dio-9 inhibits the Mg-induced fluorescence enhancement. ANS does not relieve the quercetin or phloridzin inhibition of ATP hydrolysis indicating that these inhibitors do not compete with ANS for a common binding site. ANS may be used, therefore, as a sensitive probe to detect conformational changes in CF1 in response to activation or inactivation and to binding of substrates and of inhibitors.     

Mechanism of eosin Y action of activity of solubilized Ca2+, Mg2+- ATPase from smooth muscle sarcolemma

The eosin Y inhibitory effect on the activity of smooth muscle plasma membrane Ca(2+)-transporting ATPase was studied: effect of this inhibitor on the maximal initial rate of ATP-hydrolase reaction, catalyzed by Ca2+, Mg(2+)-ATPase, on the affinity of enzyme for the reaction reagents (Ca2+, Mg2+, ATP). Dependence of eosin Y inhibitory effect on some physicochemical factors of incubation medium was studied too. It was determined that eosin Y inhibited reversibly and with high specificity purified Ca2+, Mg(2+)-ATPase solubilized from myometrial cell plasma membrane (Ki--0.8 microM), decreased the turnover rate of this enzyme determined both by Mg2+, ATP and Ca2+. This inhibitor had no effect on the enzyme affinity for Ca2+, increased affinity for Mg2+ and decreased affinity for ATP. It was determined that inhibition of Ca2+, Mg(2+)-ATPase by eosin Y depended on pH and dielectric permeability of the incubation medium: increasing of pH from 6.5 to 8.0 reduced the apparent Ki, decreasing of dielectric permeability from 74.07 to 71.19 increased the apparent Ki.     

Interaction of nucleotides with Asp(351) and the conserved phosphorylation loop of sarcoplasmic reticulum Ca(2+)-ATPase.

The nucleotide binding properties of mutants with alterations to Asp(351) and four of the other residues in the conserved phosphorylation loop, (351)DKTGTLT(357), of sarcoplasmic reticulum Ca(2+)-ATPase were investigated using an assay based on the 2', 3'-O-(2,4,6-trinitrophenyl)-8-azidoadenosine triphosphate (TNP-8N(3)-ATP) photolabeling of Lys(492) and competition with ATP. In selected cases where the competition assay showed extremely high affinity, ATP binding was also measured by a direct filtration assay. At pH 8.5 in the absence of Ca(2+), mutations removing the negative charge of Asp(351) (D351N, D351A, and D351T) produced pumps that bound MgTNP-8N(3)-ATP and MgATP with affinities 20-156-fold higher than wild type (K(D) as low as 0.006 microM), whereas the affinity of mutant D351E was comparable with wild type. Mutations K352R, K352Q, T355A, and T357A lowered the affinity for MgATP and MgTNP-8N(3)-ATP 2-1000- and 1-6-fold, respectively, and mutation L356T completely prevented photolabeling of Lys(492). In the absence of Ca(2+), mutants D351N and D351A exhibited the highest nucleotide affinities in the presence of Mg(2+) and at alkaline pH (E1 state). The affinity of mutant D351A for MgATP was extraordinarily high in the presence of Ca(2+) (K(D) = 0.001 microM), suggesting a transition state like configuration at the active site under these conditions. The mutants with reduced ATP affinity, as well as mutants D351N and D351A, exhibited reduced or zero CrATP-induced Ca(2+) occlusion due to defective CrATP binding.     

Investigation of the binding of Ca2+, Mg2+, Mn2+ and K+ to the vitamin D-dependent Ca2+-binding protein from pig duodenum.

The cation-binding properties of the vitamin D-dependent Ca2+-binding protein from pig duodenum were investigated, mainly by flow dialysis. The protein bound two Ca2+ ions with high affinity, and Mg2+, Mn2+ and K+ were all bound competitively with Ca2+ at both sites. The sites were distinguished by their different affinities for Mn2+, the one with the higher affinity being designated A (Kd 0.61 +/- 0.02 microM) and the other B (Kd 50 +/- 6 microM). Competitive binding studies allied to fluorimetric titration with Mg2+ showed that site A bound Ca2+, Mg2+ and K+ with Kd values of 4.7 +/- 0.8 nM, 94 +/- 18 microM and 1.6 +/- 0.3 mM respectively, and site B bound the same three cations with Kd values of 6.3 +/- 1.8 nM, 127 +/- 38 microM and 2.1 +/- 0.6 mM. For the binding of these cations, therefore, there was no significant difference between the two sites. In the presence of 1 mM-Mg2+ and 150 mM-K+, both sites bound Ca2+ with an apparent Kd of 0.5 microM. The cation-binding properties were discussed relative to those of parvalbumin, troponin C and the vitamin D-dependent Ca2+-binding protein from chick duodenum.     

Ca2(+)-induced conformational changes and location of Ca2+ transport sites in sarcoplasmic reticulum Ca2(+)-ATPase as detected by the use of proteolytic enzyme (V8)

Treatment of Ca2(+)-ATPase from sarcoplasmic reticulum with V8 protease from Staphylococcus aureus produced appreciable amounts of a Ca2(+)-ATPase fragment (p85) in the presence of Ca2+ (E1 conformation of the enzyme), along with many other peptide fragments that were also formed in the presence of [ethylenebis(oxyethylenenitrilo)]tetraacetic acid (E2 conformation). p85 was formed as a carboxyl-terminal cleavage product of Ca2(+)-ATPase by a split of the peptide bond between Glu-231 and Ile-232. Other conformation-dependent V8 splits were localized to the "hinge" region, involved in ATP binding, between the middle and COOH-terminal one-third of the Ca2(+)-ATPase polypeptide chain. Representative split products in this region (p48,p31) were identified as NH2-terminal and COOH-terminal cleavage products of p85. In the membrane p85 probably remains associated with its complementary NH2-terminal fragment(s) and retains the capacity to bind Ca2+ as evidenced by resistance to V8 degradation in Ca2+ and ability to become phosphorylated by ATP. However, the hydrolysis rate of the phosphorylated enzyme is reduced, indicating that peptide cleavage at Glu-231 interferes with Ca2+ transport steps after phosphorylation. Binding of Ca2+ to V8 and tryptic fragments of Ca2(+)-ATPase was studied on the basis of Ca2(+)-induced changes in electrophoretic mobility and 45Ca2+ autoradiography after transfer of peptides to Immobilon membranes. These data indicate binding by the NH2-terminal 1-198 amino acid residues (corresponding to the tryptic A2 fragment) and the COOH-terminal 715-1001 amino acid residues (corresponding to p31). By contrast the central portion of Ca2(+)-ATPase, including the NH2-terminal portion of p85, is devoid of Ca2+ binding. These results question an earlier proposition that Ca2(+)-binding is located to the "stalk" region of Ca2(+)-ATPase (Brandl, C. J., Green, N. M., Korczak, B., and MacLennan, D. H.) (1986) Cell 44, 597-607) but are in agreement with recent data obtained by oligonucleotide-directed mutagenesis of Ca2(+)-ATPase (Clarke, D. M., Loo, T. W., Inesi, G., and MacLennan, D. H. (1989) Nature 339, 476-478). These different studies suggest that Ca2+ translocation sites may have an intramembranous location and are formed predominantly by the carboxyl-terminal part of the Ca2(+)-ATPase polypeptide chain.     

Ca2+-ATPase activity in pancreatic acinar plasma membranes. Regulation by calmodulin and acidic phospholipids

A high degree of ATP hydrolytic activity present in purified rat pancreatic acinar cells was localized to plasma membranes. This activity was stimulated almost equally by Mg2+ or Ca2+. Kinetic analysis revealed that the enzyme had a higher affinity for Ca2+ (Kd = 1.73 microM) than Mg2+ (Kd = 2.98 microM) but a similar maximal rate of activity. A comparison of substrate requirements revealed very similar profiles for the Mg2+- and Ca2+-stimulated activities. Combinations of saturating concentrations of Mg2+ or Ca2+ produced the same degree of maximal activity. Investigation of the partial reactions of the ATPase activity revealed two phosphoprotein intermediates (Mr = 115,000 and 130,000) in the presence of Ca2+ and Mg2+. A significant stimulation of the Ca2+-ATPase activity by calmodulin was observed (Kd = 0.7 microM). Calmodulin increased the Ca2+-sensitivity of this enzyme system; Mg2+ appeared to be required for this effect. The Ca2+-ATPase activity was also stimulated by acidic phospholipids. Using an 125I-labeled calmodulin gel overlay technique, calmodulin was shown to bind in a Ca2+-dependent fashion to 133,000- and 230,000-dalton proteins present in the plasma membrane-enriched fraction. Under conditions that favor Ca2+-dependent kinase activity, calmodulin enhanced the phosphorylation of a 30,000- and 19,000-dalton protein. The major ATP hydrolytic activity in pancreatic acinar plasma membranes was present as an ectoenzyme.     

Proximate sulfhydryl groups in the acetylglutamate complex of rat carbamylphosphate synthetase I: their reaction with the affinity reagent 5'-p-fluorosulfonylbenzoyladenosine

A preparation of rat carbamylphosphate synthetase I, isolated in the presence of antipain and stable without glycerol, has been used to investigate the effect of the allosteric activator, N-acetyl-L-glutamate (AcGlu), on the sulfhydryl chemistry of the enzyme. The enzyme X AcGlu complex was rapidly inactivated by several sulfhydryl group reagents and the ATP analog, 5'-p-fluorosulfonylbenzoyladenosine (FSO2BzAdo), with the loss of two sulfhydryl groups per monomer. Inactivation was much slower without AcGlu, and ATP/Mg2+/K+ provided complete protection. Reaction with a 1.1 molar excess of 4,4'-dipyridyldisulfide resulted in an intramonomer disulfide bond between groups that are probably juxtaposed in the activated enzyme, because 1.1 equivalents of the vicinal dithiol reagent, phenylarsine oxide, eliminated the rapid reaction with the disulfide. Evidence is presented that the same disulfide bond was formed in the reactions with 5-thiocyano-2-nitrobenzoic acid and FSO2BzAdo. Inactivation by FSO2BzAdo was a pseudo-first-order reaction. The concentration dependence of the rate is consistent with the reaction proceeding through a noncovalent complex (KI = 67 microM and k2 = 0.23 min-1 at pH 7.0, 30 degrees C). Protection from FSO2BzAdo by ATP required Mg2+ in excess of ATP with KMgATP = 4.5 microM at saturating free Mg2+ (0.1 M K+) and KMg2+ = 6.5 mM. KMgATP is close to Kd for the molecule of ATP that contributes the phosphoryl group of carbamylphosphate (H.B. Britton, V. Rubio, and S. Grisolia, (1979) Eur. J. Biochem. 102, 521-530]; KMg2+ agrees with the minimum value for the steady-state kinetic parameter, Ki,Mg2+, obtained under the same conditions. Dissociation constants for adenosine (320 microM), MgADP (110 microM) at 10 mM Mg2+, and AcGlu (100 microM) were also estimated.     

Interaction of ATP with sarcoplasmic reticulum Ca2+-ATPase; effect on the conformational state of the enzyme

It was studied how temperature influences the NBD-Cl inactivation of sarcoplasmic reticulum Ca2+-ATPase and the protective effect of ATP under conditions preventing ATP hydrolysis. Two types of ATP-binding sites with Kd equal to 30 and 220 microM at 37 degrees C were found. ADP interacts with these sites with the (K'd = 20 and 200 microM). The temperature decrease from 25 degrees to 5 degrees C induces the abrupt increase in the Kd for the low affinity site. The possible reasons for heterogeneity of ATP-binding sites are discussed. The conclusion is made that interaction of monomers in oligomeric complex of Ca2+-ATPase induces heterogeneity of ATP-binding sites.     

The interaction of vanadate ions with the Ca-ATPase from sarcoplasmic reticulum

The interaction of vanadate ions with the Ca-ATPase from sarcoplasmic reticulum vesicles was studied in a native and a fluorescein-labeled ATPase preparation (Pick, U., and Karlish, S. J. D. (1980) Biochim. Biophys. Acta 626, 255-261). Vanadate induced a fluorescence enhancement in a fluorescein-labeled enzyme, indicating that it shifts the equilibrium between the two conformational states of the enzyme by forming a stable E2-Mg-vanadate complex (E2 is the low affinity Ca2+ binding conformational state of the sarcoplasmic reticulum Ca-ATPase). Indications for tight binding of vanadate to the enzyme (K1/2 = 10 microM) in the absence of Ca2+ and for a slow dissociation of vanadate from the enzyme in the presence of Ca2+ are presented. The enzyme-vanadate complex was identified by the appearance of a time lag in the onset of Ca2+ uptake and by a slowing of the fluorescence quenching response to Ca2+. Ca2+ prevented the binding of vanadate to the enzyme. Pyrophosphate (Kd = 2 mM) and ATP (Kd = 25 microM) competitively inhibited the binding of vanadate, indicating that vanadate binds to the low affinity ATP binding site. Binding of vanadate inhibited the high affinity Ca2+ binding to the enzyme at 4 degrees C. Vanadate also inhibited the phosphorylation reaction by inorganic phosphate (Ki = 10 microM) but had no effect on the phosphorylation by ATP. It is suggested that vanadate binds to a special region in the low affinity ATP binding site which is exposed only in the E2 conformation of the enzyme in the absence of Ca2+ and which controls the rate of the conformation transition in the dephosphorylated enzyme. The implications of these results to the role of the low affinity ATP binding sites are discussed.     

Adenosine inhibits the renal plasma-membrane (Ca2+ + Mg2+)-ATPase through a pathway sensitive to cholera toxin and sphingosine.

Adenosine, a potent autacoid produced and released in kidneys, affects nearly all aspects of renal function, and an increase in cytosolic calcium has been implicated in adenosine effects. The aim of this work was to investigate whether adenosine modifies the calcium pump present in basolateral membranes of kidney proximal tubule cells. Adenosine exerts a biphasic influence on (Ca2+ + Mg2+)-ATPase activity. Inhibition occurs up to 0.1 microM and then gradually disappears as the adenosine concentration increases to 100 microM, an effect mimicked by the adenosine analog N6-cyclohexyladenosine, which preferentially binds to A1-type receptors. In contrast, the A2 receptor agonist 5', N-ethylcarboxamideadenosine is ineffective. The A1 receptor antagonist 8-cyclopentyl-1,3-dimethylxanthine blocks the inhibitory effect of 0.1 microM adenosine and stimulates (Ca2+ + Mg2+)-ATPase activity in the presence of 1 mM adenosine, a concentration high enough to occupy the low-affinity A2 receptors. Inhibition by adenosine increases as medium ATP is lowered to micromolar concentrations, is maintained in the presence of pertussis toxin, and is completely abolished with 0.1 microM cholera toxin or 1 microM sphingosine. The inhibitory effect of adenosine can be reproduced by guanosine 5'-[gamma-thio]triphosphate, inositol 1,4, 5-trisphosphate or the diacylglycerol analog 12-O-tetradecanoylphorbol 13-acetate. In conjunction with the selectivity for its analogs and for its receptor agonist, the concentration profile of adenosine effects indicates that both inhibitory (A1) and stimulatory (A2) receptors are involved. The results obtained with the toxins indicate that a pathway that is modulated by G-proteins, involves a phospholipase C and a protein kinase C, and is affected by local variations in adenosine concentrations participates in the regulation of the (Ca2+ + Mg2+)-ATPase resident in basolateral membranes of kidney proximal tubules.