Inhibitory effect platinum and palladium complexes as indicator of conformational changes in sarcoplasmic reticulum membranes]

Inhibition of Ca2+-dependent ATPase of sarcoplasmic reticulum membranes (SRM) by platinum and palladium complexes is considerable enhanced during the incubation of these compunds with SRM preparations in the presence of small (10(-5) M) concentrations of ATP or ADP. AMP and nucleotides with non-adenine bases do not have inhibitory effect. To increase the sensitivity of Ca2+-dependent ATPase to platinum and palladium complexes under the action of ATP (but not ADP), the presence of free Ca2+-ions in the medium is required. In the absence of ATP Ca2+-ions do not affect the inhibiting effect of the complexes. The increase in pH of the medium up to 8.5 and the increase of temperature up to 45degree C sharply decrease the ATP ability to enchance the sensitivity of Ca2+-dependent ATPase to platinum and palladium compunds. It is assumed that the ATP ability to enhance Ca2+-dependent ATPase inhibition by platinum and palladium complexes is due to ATP-dependent structural changes in SRM, which increase the availability of certain groups of the enzyme to those compounds.     

Spectrofluorometric topography of the ATPase center of Ca2+-Mg2+-dependent ATPase of sarcoplasmic reticulum by means of platinum and palladium compounds

The fluorescence of tryptophan residues in Ca2+--Mg2+-ATPase was studied in the presence of K2PtCl4, K2PdCl4 and 5-sulpho-8-mercaptochinolinate platinum and palladium. It has been shown that both first two compounds quenched the fluorescence dye to bonding with SH-groups in ATPase active centre, but the last two compounds influence the fluorescence by bonding with tryptophan residues. The distance between the SH-groups and tryptophan in the active centre was determined by Foerster--Galanin equation and was equal to 14 +/- 3 A.     

Localization of tryptophan residues in Ca2+-ATPase from sarcoplasmic reticulum

By the methods of spectroscopy, fluorimetry and chemical modification of tryptophane residues with N-bromsuccinimide, the sarcoplasmic reticulum of rabbit sceletal muscle was shown to contain 18 +/- 1 tryptophane residues per Ca2+-ATPase molecule, 6 of which were, probably, inside the protein globule, in its hydrophobic region, and thus unavailable for modifier, while the rest 12 +/- 1 were easily transformed to the 6-oxyindole chromophore being the main source of the intrinsic fluorescence of the enzyme. The quantum yield for the rest four residues was equal to 0.015. Four tryptophane residues are located at the distance of less than 14 A from the ATP-binding site of the enzyme. The quantum yields of fluorescence for 8 of the tryptophane residues of Ca2+-ATPase were similar and equal to 0.03.     

Differential regulation of calcium-dependent and calcium-independent cyclic nucleotide phosphodiesterases from heart by palmitoylcarnitine and palmitoyl coenzyme A

Regulation of Ca2+-dependent (peak I) and Ca2+-independent (peak II) phosphodiesterases from the heart by various fatty acyl esters and phospholipids were studied. DL-Palmitoylcarnitine stimulated the basal activity (in the absence of Ca2+) of peak I enzyme, while non-competitively inhibiting peak II enzyme with respect to cyclic AMP. It had no effect on other species of Ca2+-independent phosphodiesterases, including cyclic AMP- and cyclic GMP-specific enzymes from the lung, and cyclic CMP enzyme from the liver Palmitoyl-CoA and phosphatidylserine also stimulated the basal activity of peak I enzyme, but they were without effect on peak II enzyme. In comparison, DL-palmitoylcarnitine inhibited Ca2+-dependent activity of cardiac myosin light chain kinase, whereas phosphatidylserine was without effect. It is conceivable that differential regulation of phosphodiesterases by these lipids could profoundly alter the levels or effects, or both, of cyclic nucleotides and Ca2+ in the myocardium.     

Calcium-dependent protein-protein interactions induce changes in proximity relationships of Cys48 and Cys64 in chicken skeletal troponin I.

The goal of this study was to relate conformational changes in the N-terminal domain of chicken troponin I (TnI) to Ca2+ activation of the actin-myosin interaction. The two cysteine residues in this region (Cys48 and Cys64) were labeled with two sulfhydryl-reactive pyrene-containing fluorophores [N-(1-pyrene)maleimide, and N-(1-pyrene)iodoacetamide]. The labeled TnI showed a typical fluorescence spectrum: two sharp peaks of monomer fluorescence and a broad peak of excimer fluorescence arising from the formation of an excited dimer (excimer). Results obtained show that forming a binary complex of labeled TnI with skeletal TnC (sTnC) in the absence of Ca2+ decreases the excimer fluorescence, indicating a separation of the two residues. This reduction in excimer fluorescence does not occur when labeled TnI is complexed with cardiac TnC (cTnC). The latter causes only partial activation of the Ca2+-dependent myofibrillar ATPase. The binding of Ca2+ to the two N-terminal sites of sTnC causes a significant decrease in excimer fluorescence and an increase in monomer fluorescence in complexes of labeled TnI with skeletal TnC or TnC/TnT, while Ca2+ binding to site II of cTnC only causes an increase in monomer fluorescence but no change in excimer fluorescence. Thus a conformational change in the N-terminal region of TnI may be necessary for full activation of muscle contraction.     

Proton pump-linked Mg2+-ATPase activity in isolated rat liver lysosomes

ATPase activity in highly purified rat liver lysosome preparations was evaluated in the presence of other membrane cellular ATPase inhibitors, and compared with lysosome ATP-driven proton translocating activity. Replacement of 5 mM Mg2+ with equimolar Ca2+ brought about a 50% inhibition in divalent cation-dependent ATPase activity, and an 80% inactivation of ATP-linked lysosomal H+ pump activity. In the presence of optimal concentrations of Ca2+ and Mg2+, ATPase activity was similar to that seen in an Mg2+ medium. Mg2+-dependent ATPase activity was greatly inhibited (from 70 to 80%) by the platinum complexes; cis-didimethylsulfoxide dichloroplatinum(II) (CDDP) at approximately 90 microM and cis-diaminedichloroplatinum(II) at twofold higher concentrations. Less inhibition, about 30 and 45%, was obtained with N,N'-dicyclohexylcarbodiimide and N-ethylmaleimide, and the maximal effect occurred in the 50-100 microM and 0.1-1.5 mM ranges, respectively. The concentration dependence of inhibition by the above drugs was determined for both proton pumping and ATPase activities, and half-maximal inhibition concentration of each activity was found at nearly similar values. A micromolar concentration of carbonylcyanide p-trifluoromethoxyphenylhydrazone (FCCP) prevented ATP from setting up a pH gradient across the lysosomal membranes, but stimulated Mg2+-ATPase activity significantly. ATPase activity in Ca2+ medium was also inhibited by CDDP and stimulated by FCCP, but both effects were two- to threefold less than those observed in Mg2+ medium. FCCP failed to stimulate ATPase activity in a CDDP-supplemented medium, thus suggesting that the same ATPase activity fraction was sensitive to both CDDP and FCCP. Mg2+-ATPase activity, like the proton pump, was anion dependent. The lowest activity was recorded in a F-medium, and increased in the order of F- less than SO2-4 less than Cl- approximately equal to Br-. The CDDP-sensitive ATPase activity observed, supported by Mg2+ and less so by Ca2+, may be related to lysosome proton pump activity.     

Effects of N-acetylimidazole on human erythrocyte ATPase activity. Evidence for a tyrosyl residue at the ATP binding site of the (Na+ plus K+)-dependent ATPase.

1. Acetylation of human erythrocytes by N-acetylimidazole alters the structure of stroma prepared from these cells and the degree of alteration appears to be dependent upon the level of the initial treatment. These changes do not occur when stroma are acetylated. 2. Deacetylation by hydroxylamine or mild alkaline treatment causes a complete recovery of the (Na+ plus K+)-dependent and the Ca2+ -stimulated ATPase activities and indicates that the inhibition is due to the acetylation of a tyrosyl residue. There is only partial recovery of the Mg2+ -dependent ATPase after deacetylation. 3. ATP or Mg-ATP completely protect the (Na+ plus K+)-dependent ATPase, but not the Ca2+ -stimulated system. 4. The results indicate that the (Na+ plus K+)-dependent and the Ca2+ -stimulated ATPase activities have separate substrate binding sites and most likely are separate enzyme systems. 5. Acetylation of human erythrocytes has no effect on D-glucose transport.     

Determination and functional significance of low affinity nucleotide sites of Ca2+ + Mg2+ -dependent ATPase of sarcoplasmic reticulum.

The protective effect of ATP, ADP and GTP against the inactivation of Ca2+ + Mg2+ -dependent ATPase by the thiol reagent NBD-chloride is used to calculate the apparent dissociation constants (K'D) of nucleotide enzyme complexes on the basis of a simple kinetic model. The K'D-values of the complexes with Mg-ATP (80 micrometer) and Mg-GTP (500 micrometer) are found to be rather close to their Km-values in the high concentration range supporting maximum activity. The requirement of the occupancy of the low affinity site by Mg ATP for a high rate of the Ca2+ transport system is explained in terms of the flip-flop mechanism established earlier for the analogous Na+ + K+-transporting ATPase system.     

Involvement of conserved, acidic residues in the N-terminal domain of troponin C in calcium-dependent regulation

We have mutated eight conserved, charged amino acid residues in the N-terminal, regulatory domain of troponin C (TnC) so we could investigate their role in troponin-linked Ca2+ regulation of muscle contraction. These residues surround a hydrophobic pocket in the N-terminal domain of TnC which, when Ca2+ binds to regulatory sites in this domain, is exposed and interacts with the inhibitory region of troponin I (TnI). We constructed three double mutants (E53A/E54A, E60A/E61A, and E85A/D86A) and two single mutants (R44A and R81A) of rabbit fast skeletal muscle troponin C (TnC) in which the charged residues were replaced with neutral alanines. All five of these mutants retained TnC's ability to bind TnI in a Ca2+-dependent manner, to neutralize TnI's inhibition of actomyosin S1 ATPase activity, and to form a ternary complex with TnI and troponin T (TnT). Ternary complexes formed with TnC(R44A) or TnC(R81A) regulated actomyosin S1 ATPase activity normally, with TnI-based inhibition in the absence of Ca2+ and TnT-based activation in the presence of Ca2+. TnC(E53A/E54A) and TnC(E85A/D86A) interacted weakly with TnT, as judged by native gel electrophoresis. Ternary complexes formed with these mutants inhibited actomyosin S1 ATPase activity in both the presence and absence of Ca2+, and did not undergo Ca2+-dependent structural changes in TnI which can be detected by limited chymotryptic digestion. TnC(E60A/E61A) interacted normally with TnT. Its ternary complex showed Ca2+-dependent structural changes in TnI, inhibited actomyosin S1 ATPase in the absence of Ca2+, but did not activate ATPase in the presence of Ca2+. This is the first demonstration that selective mutation of TnC can abolish the activating effect of troponin while its inhibitory function is retained. Our results suggest the existence of an elaborate network of protein-protein interactions formed by TnI, TnT, and the N-terminal domain of TnC, all of which are important in the Ca2+-dependent regulation of muscle contraction.     

Interaction of fluorescein isothiocyanate with the (H+ + K+)-ATPase

Fluorescein isothiocyanate was used to covalently label the gastric (H+ + K+)-ATPase. FITC treatment of the enzyme inhibited the ATPase activity while largely sparing partial reactions such as the associated p-nitrophenylphosphatase activity. ATP protected against inhibition suggesting the ligand binds at or near an ATP binding site. At 100% inhibition the stoichiometry of binding was 1.5 nmol FITC per mg Lowry protein a value corresponding to maximal phosphoenzyme formation. Binding occurred largely to a peptide of 6.2 isoelectric point, although minor labelling of a peptide of pI 5.6 was also noted. Fluorescence was quenched by K+, Rb+ and Tl+ in a dose-dependent manner, and the K0.5 values of 0.28, 0.83 and 0.025 mM correspond rather well to the values required for dephosphorylation at a luminal site. Vanadate, a known inhibitor of the gastric ATPase produced a slow Mg2+-dependent fluorescent quench. Ca2+ reversed the K+-dependent loss of fluorescence and inhibited it when added prior to K+. This may relate to the slow phosphorylation in the presence of ATP found when Ca2+ was substituted for Mg2+ and the absence of K+-dependent dephosphorylation. The results with FITC-modified gastric ATPase provide evidence for a conformational change with K+ binding to the enzyme.     

Purification and characterization of two wheat-embryo protein phosphatases.

Two protein phosphatases (enzymes I and II) were extensively purified from wheat embryo by a procedure involving chromatography on DEAE-cellulose, phenyl-Sepharose CL-4B, DEAE-Sephacel and Ultrogel AcA 44. Preparations of enzyme I (Mr 197,000) are heterogeneous. Preparations of enzyme II (Mr 35,000) contain only one major polypeptide (Mr 17,500), which exactly co-purifies with protein phosphatase II on gel filtration and is not present in preparations of enzyme I. However, this major polypeptide has been identified as calmodulin. Calmodulin and protein phosphatase II can be separated by further chromatography on phenyl-Sepharose CL-4B. Protein phosphatases I and II do not require Mg2+ or Ca2+ for activity. Both enzymes catalyse the dephosphorylation of phosphohistone H1 (phosphorylated by wheat-germ Ca2+-dependent protein kinase) and of phosphocasein (phosphorylated by wheat-germ Ca2+-independent casein kinase), but neither enzyme dephosphorylates a range of non-protein phosphomonoesters tested. Both enzymes are inhibited by Zn2+, Hg2+, vanadate, molybdate, F-, pyrophosphate and ATP.     

ADP-ribosylation of Ca2+-dependent ATPase in vitro suppresses the enzyme activity

We investigated the effect on the Ca2+-dependent ATPase activity of ADP-ribosylation of the enzyme from the rabbit skeletal muscle sarcoplasmic reticulum. A reconstituted ADP-ribosylation system of Ca2+-dependent ATPase in which the enzyme and ADP-ribosyltransferase, both were partially purified from the vesicles, and poly L-lysine were contained, was preincubated with 1 mM NAD, and the Ca2+-dependent ATPase activity was assayed. The NAD-dependent suppression of the enzyme activity depended on both the concentration of NAD and preincubation-time for the ADP-ribosylation, and was reversed by adding 20 mM arginine during the preincubation. These results taken together with the findings that Ca2+-dependent ATPase is a major acceptor protein for the modification in rabbit skeletal muscle sarcoplasmic reticulum [Hara et al. (1987) Biochem. Biophys. Res. Commun. 144; 856-862] suggest that Ca2+-transport in the sarcoplasmic reticulum may be regulated through changes in the rate of ADP-ribosylation of Ca2+-dependent ATPase.     

Dicarboxylate platinum(II) complexes as inhibitors of plasma membrane H(+)-ATPase in the yeast Saccharomyces cerevisiae

A series of cytotoxic neutral dicarboxylatoplatinum(II) complexes containing D(+), L(-) or DL-malate dianion and ethylenediamine or 1-ethylimidazole as ligands were examined using ATPase activity assays and the proton extrusion test. ATPase activity assays in vitro on plasma membrane H+-ATPase and on mitochondrial ATPase were carried out. The concentrations of compounds inhibiting enzyme activity to 50 per cent (J50) was determined. The new platinum complexes showed a stronger level of inhibition of both ATPases than the reference carboplatin; this inhibitory activity is related to a stereoisomeric form of anionic platinum ligands. ATPase inhibition in vivo was tested by glucose-stimulated proton extrusion and the influence of platinum compounds on this process in yeast cells was determined. Significant differences in activity levels were observed between those complexes with 1-ethylimidazole and those with ethylenediamine.     

Circular dichroism and fluorescence studies on interaction of calmodulin (CaM) with purified (Ca2(+)-Mg2+)ATPase of erythrocyte ghosts

It was found, using circular dichroism spectroscopy, that CaM, in the presence of Ca2+, decreases the alpha-helix content of (Ca2(+)-Mg2+)ATPase of porcine erythrocytes from 66% to 55%. In the absence of Ca2+ the enzyme showed 46% of alpha-helix. Moreover, quenching of the ATPase intrinsic fluorescence by acrylamide indicated that, depending on the enzyme conformational status, the accessibility of its tryptophan residues is influenced by direct interaction with CaM at micromolar Ca2+ concentration. This was also confirmed by the observation that fluorescence energy transfer occurred from tryptophan residues of (Ca2(+)-Mg2+)ATPase to dansylated CaM. The presented results may indicate that binding of CaM gives rise to a novel conformational state of the enzyme, distinct from E1 and E2 forms of the Ca2+ pump.     

Interaction between free fatty acids and Ca2+-dependent ATPase from sarcoplasmic reticulum membranes

The interaction between free fatty acids and Ca2+-dependent ATPase, an intrinsic protein of sarcoplasmic reticulum membranes, was studied with relevance to the changes in membrane permeability induced by free fatty acids. It was found that only unsaturated fatty acids increase the permeability of reticulum membranes for Ca2+, this effect being completely reversible. The increase in the membrane permeability by fatty acids is coupled to a generation of a channel for Ca2+ efflux under effect of Ca2+-dependent ATPase. The interaction between fatty acids and Ca2+-dependent ATPase was demonstrated by the protein fluorescence and electron paramagnetic resonance methods, using spin-labelled fatty acid derivatives. A model demonstrating the increase of sarcoplasmic reticulum membrane permeability for Ca2+ in the presence of the fatty acid-Ca2+-dependent ATPase complex is proposed.     

Temperature-induced change in the Ca2+-dependent ATPase activity and in the state of the ATPase protein of sarcoplasmic reticulum membrane.

The temperature dependence of the Ca2+-dependent ATPase activity and of the conformational fluctuation of the ATPase molecule has been measured for four kinds of preparations: fragmented sarcoplasmic reticulum, MacLennan's enzyme (purified ATPase preparation), and DOL and egg PC-ATPase (purified ATPase preparations in which lipids are replaced with dioleoyllecithin and egg yolk lecithin, respectively). It has been found that Arrhenius plots of the Ca2+-dependent ATPase activity show a break at about 18 degrees C for all the preparations. Hydrogen--deuterium exchange kinetics of the peptide NH protons were used to measure the conformational fluctuation of the protein molecules. Van't Hoff plots of the conformational fluctuation amplitude of a region near the surface of the ATPase molecule also show a break at about 18 degrees C for all the preparations. It is concluded that the break at around 18 degrees C is not related to a gel-liquid crystalline transition of lipids but to a change in the conformation of the ATPase molecule existing in fluid lipids.     

Structural perturbation of the transmembrane region interferes with calcium binding by the Ca2+ transport ATPase

The Ca(2+)-ATPase of sarcoplasmic reticulum reacts with N-cyclohexyl-N'-(4-dimethylamino-1-naphthyl) carbodiimide (NCD4) yielding a fluorescence labeling that interferes with calcium binding to activating and transport sites of the enzyme and, thereby, with Ca(2+)-dependent ATPase activity. On the other hand, the catalytic site does not appear altered, as revealed by the normal occurrence of Ca(2+)-independent reactions, such as enzyme phosphorylation with Pi in the reverse direction of the catalytic cycle. This reaction is not inhibited by Ca2+ in the labeled enzyme, while it is inhibited in the native enzyme. The NCD4 reaction which is involved in functional inactivation occurs in the membrane-bound portion of the ATPase. Sodium dodecyl sulfate solubilization of hydrophobic peptides, electrophoresis, and microsequencing of transblotted electrophoretic bands revealed that the fluorescent NCD4 label resides in a segment of tryptic fragment A1, intervening between Glu231 and Glu309. This segment includes two transmembrane helices, and does not include the domain involved in the phosphoryl transfer reaction during catalytic activity. This specific labeling does not occur when the NCD4 derivatization procedure is carried out in the presence of Ca2+ concentrations that also prevent functional inactivation. Fluorescence characterization by steady state and intensity decay measurements shows only negligible energy transfer between the NCD4 label and fluorescein isothiocyanate label of Lys515, indicating that the NCD4 label is unlikely to reside within the extramembranous region of the ATPase. On the other hand, the fluorescence emission of intrinsic tryptophan residues clustered within or near the transmembrane region of the ATPase, is distinctly affected by NCD4 label specifically bound to the ATPase, and NCD4 label nonspecifically bound to the sarcoplasmic reticulum membrane. The combined sequencing and spectroscopic observations indicate that derivatization with NCD4 induces a perturbation within or near the transmembrane region of the ATPase (at a relatively large distance from the catalytic site) that interferes with specific calcium binding. This is in agreement with experiments (Clarke et al., 1989) demonstrating that mutations of any of six amino acids within the transmembrane region of the ATPase interfere with enzyme activation by Ca2+.     

ATPase and phosphatase activities from human red cell membranes: I. The effects of N-ethylmaleimide.

In human red cell membranes the sensitivity to N-ethylmaleimide of Ca2+-dependent ATPase and phosphatase activities is at least ten times larger than the sensitivity to N-ethylmaleimide of (Na+ + K+)-ATPase and K+-activated phosphatase activities. All activities are partially protected against N-ethylmaleimide by ATP but not by inorganic phosphate or by p-nitrophenylphosphate. (ii) Protection by ATP of (Na+ + K+)-ATPase is impeded by either Na+ or K+ whereas only K+ impedes protection by ATP of K+-activated phosphatase. On the other hand, Na+ or K+ slightly protects Ca2+-dependent activities against N-ethylmaleimide, this effect being independent of ATP. (iii) The sensitivity to N-ethylmaleimide of Ca2+-dependent ATPase and phosphatase activities is markedly enhanced by low concentrations of Ca2+. This effect is half-maximal at less than 1 micron Ca2+ and does not require ATP, which suggests that sites with high affinity for Ca2+ exist in the Ca2+-ATPase in the absence of ATP. (IV) Under all conditions tested the response to N-ethylmaleimide of the ATPase and phosphatase activities stimulated by K+ or Na+ in the presence of Ca2+ parallels that of the Ca2+-dependent activities, suggesting that the Ca2+-ATPase system possesses sites at which monovalent cations bind to increase its activity.     

Studies of gastric Ca2+-stimulated adenosine triphosphatase. I. characterization and general properties

Gastric microsomes do not contain any significant Ca2+-stimulated ATPase activity. Trypsinization of pig gastric microsomes in presence of ATP results in significant (2-3 fold) increase in the basal (with Mg2+ as the only cation) ATPase activity, with virtual elimination of the K+-stimulated component. Such treatment causes unmasking of latent Mg2+-dependent Ca2+-stimulation ATPase. Other divalent cations such as Sr2+, Ba2+, Zn2+, and Mn2+ were found ineffective as a substitute for Ca2+. Moreover, those divalent cations acted as inhibitors of the Ca2+-stimulated ATPase activity. The pH optimum of the enzyme is around 6.8. The enzyme has a Km of 70 microM for ATP and the Ka values for Mg2+ and Ca2+ are about 4 x 10(-4) and 10(-7) M, respectively. Studies with inhibitors suggest the involvement of sulfhydryl and primary amino groups in the operation of the enzyme. Possible roles of the enzyme in gastric H+ transport have been discussed.     

Mg2+ and ATP effects on K+ activation of the Ca2+-transport ATPase of cardiac sarcoplasmic reticulum.

ATP and the divalent cations Mg2+ and Ca2+ regulated K+ stimulation of the Ca2+-transport ATPase of cardiac sarcoplasmic reticulum vesicles. Millimolar concentrations of total ATP increased the K+-stimulated ATPase activity of the Ca2+ pump by two mechanisms. First, ATP chelated free Mg2+ and, at low ionized Mg2+ concentrations, K+ was shown to be a potent activator of ATP hydrolysis. In the absence of K+ ionized Mg2+ activated the enzyme half-maximally at approximately 1 mM, whereas in the presence of K+ the concentration of ionized Mg2+ required for half-maximal activation was reduced at least 20-fold. Second MgATP apparently interacted directly with the enzyme at a low affinity nucleotide site to facilitate K+-stimulation. With a saturating concentration of ionized Mg2+, stimulation by K+ was 2-fold, but only when the MgATP concentration was greater than 2 mM. Hill plots showed that K+ increased the concentration of MgATP required for half-maximal enzymic activation approx. 3-fold. Activation of K+-stimulated ATPase activity by Ca2+ was maximal at an ionized Ca2+ concentration of approx. 1 microM. At very high concentrations of either Ca2+ or Mg2+, basal Ca2+-dependent ATPase activity persisted, but the enzymic response to K+ was completely inhibited. The results provide further evidence that the Ca2+-transport ATPase of cardiac sarcoplasmic reticulum has distinct sites for monovalent cations, which in turn interact allosterically with other regulatory sites on the enzyme.