ATPase Catalytic Cycle Research Articles

Metal Substrate Translocation and Transport Mechanism of the Sinorhizobium melilotiP1B‐5‐type ATPase Nia Revealed by In‐vitro Transport Assays in Proteoliposomes

P1B‐type ATPases are a class of transmembrane transporter proteins responsible for metal substrate translocation, and export across cellular membranes. These ion pumps are classified into seven sub‐families (P1B‐1‐P1B‐7‐type) based on conserved amino acid motifs in their transmembrane helices that appear to control each sub‐family’s substrate selectivity. The P1B‐5‐type sub‐family is widely found in nitrogen fixing bacteria, such as Sinorhizobium meliloti,which form a unique symbiotic relationship with legumes. This symbiotic relationship is highly dependent on the nutrition trafficking between the two species, including a tight control of transition metal homeostasis. The P1B‐5‐type ATPase Nia from S. Meliloti is recognized as a putative iron and nickel transporter, and besides possessing the typical P‐type ATPase domains and topology, it features of a C‐terminal hemerythrin‐like domain containing a di‐iron binding site involved in oxygen sensing, as proposed by in‐vivo studies. However, the substrate transport across lipid bi‐layers, the overall mechanism for cargo translocation and the transport kinetic parameters remain elusive due to lack of molecular tools to study putative substrate transport across membranes in real‐time.In this study, we coupled metal‐stimulated ATPase activity assays with an experimental platform based on multiple fluorescence sensor probes, to study substrate transport mechanism and translocation kinetics in real‐time with Nia reconstituted in a native‐like environment. We expressed and purified Nia in detergent micelles and subsequently incorporated it in artificial small unilamellar lipid bi‐layer vesicles (proteoliposomes) which allow to study the substrate specificity and transport mechanism in real‐time. The proteoliposomes were used as an in‐vitrotool to determine the kinetic parameters for metal transport by encapsulating fluorescent detector probes featuring turn‐on florescence signal upon substrate ion binding and translocation.The study revealed that Fe2+, Ni2+ and Co2+are actively translocated by Nia and the kinetic parameter analysis demonstrated that Fe2+ is the preferential Nia substrate. In parallel, by encapsulating in the proteoliposome lumen pH and membrane sensitive fluorescent probes we are also revealing the chemistry underlying the mechanism of transport and potential ions involved as counter‐transported cargos in P1B‐5‐type ATPase catalytic cycle, with the aim of revealing unique molecular features for this subclass of transmembrane metal pumps.

Switch-1 instability at the active site decouples ATP hydrolysis from force generation in myosin II.

Myosin active site elements (i.e., switch‐1) bind both ATP and a divalent metal to coordinate ATP hydrolysis. ATP hydrolysis at the active site is linked via allosteric communication to the actin polymer binding site and lever arm movement, thus coupling the free energy of ATP hydrolysis to force generation. How active site motifs are functionally linked to actin binding and the power stroke is still poorly understood. We hypothesize that destabilizing switch‐1 movement at the active site will negatively affect the tight coupling of the ATPase catalytic cycle to force production. Using a metal‐switch system, we tested the effect of interfering with switch‐1 coordination of the divalent metal cofactor on force generation. We found that while ATPase activity increased, motility was inhibited. Our results demonstrate that a single atom change that affects the switch‐1 interaction with the divalent metal directly affects actin binding and productive force generation. Even slight modification of the switch‐1 divalent metal coordination can decouple ATP hydrolysis from motility. Switch‐1 movement is therefore critical for both structural communication with the actin binding site, as well as coupling the energy of ATP hydrolysis to force generation.

Substrain-related dependence of Cu(I)-ATPase activity among prion protein-null mice

The prion protein (PrPC) binds copper and affects copper metabolism, albeit among a poorly understood functional landscape. Much of the data on physiological roles of PrPC were obtained in mice of mixed genetic background deficient of the PrPC-coding gene Prnp. This strategy is currently under scrutiny due to the flanking gene problem, in particular related with a polymorphism, typical of both the 129Sv and 129Ola mouse substrains, in the Sirpa gene located in the vicinity of Prnp. Here we report an investigation of biochemical properties of Cu(I)-ATPases as a function of genotype in two strains of PrPC-deficient mice. We found that both the brain and liver of Prnp-null mice of mixed B6;129Sv background had diminished activity, accompanied by increased catalytic phosphorylation of Cu(I)-ATPase, as compared with the respective wild-type animals. However, no such differences were found between Prnp-null and wild-type mice of a B10;129Ola background. Activity of Cu(I)-ATPase was strongly reduced in brain tissue from mice of 129Sv strain, when compared with wild-type either of B6;129Sv, and especially of mice of the B6 strain. No differences between wild-type and Prnp-null brain tissue were noted in the expression of either Atp7a or b genes, and RFLP analysis indicated that the Sirpa129 polymorphism was present in both the B6;129Sv and B10;129Ola Prnp-null mouse colonies used in this study. The results suggest a novel substrain-dependent effect of 129Sv, but not 129Ola, genotype upon the regulation of the Cu(I)-ATPase catalytic cycle in Prnp-null mice, rather than either a Prnp-dependent, or a 129 strain-dependent effect.

ATPase reaction cycle of P4-ATPases affects their transport from the endoplasmic reticulum.

P4-ATPases belonging to the P-type ATPase superfamily mediate active transport of phospholipids across cellular membranes. Most P4-ATPases, except ATP9A and ATP9B proteins, form heteromeric complexes with CDC50 proteins, which are required for transport of P4-ATPases from the endoplasmic reticulum (ER) to their final destinations. P-type ATPases form autophosphorylated intermediates during the ATPase reaction cycle. However, the association of the catalytic cycle of P4-ATPases with their transport from the ER and their cellular localization has not been studied. Here, we show that transport of ATP9 and ATP11 proteins as well as that of ATP10A from the ER depends on the ATPase catalytic cycle, suggesting that conformational changes in P4-ATPases during the catalytic cycle are crucial for their transport from the ER.

Steady-State NTPase Activity of Dengue Virus NS3: Number of Catalytic Sites, Nucleotide Specificity and Activation by ssRNA

Dengue virus nonstructural protein 3 (NS3) unwinds double stranded RNA driven by the free energy derived from the hydrolysis of nucleoside triphosphates. This paper presents the first systematic and quantitative characterization of the steady-state NTPase activity of DENV NS3 and their interaction with ssRNA. Substrate curves for ATP, GTP, CTP and UTP were obtained, and the specificity order for these nucleotides - evaluated as the ratio (kcat/KM)- was GTPATPCTP UTP, which showed that NS3 have poor ability to discriminate between different NTPs. Competition experiments between the four substrates indicated that all of them are hydrolyzed in one and the same catalytic site of the enzyme. The effect of ssRNA on the ATPase activity of NS3 was studied using poly(A) and poly(C). Both RNA molecules produced a 10 fold increase in the turnover rate constant (kcat) and a 100 fold decrease in the apparent affinity (KM) for ATP. When the ratio [RNA bases]/[NS3] was between 0 and 20 the ATPase activity was inhibited by increasing both poly(A) and poly(C). Using the theory of binding of large ligands (NS3) to a one-dimensional homogeneous lattice of infinite length (RNA) we tested the hypothesis that inhibition is the result of crowding of NS3 molecules along the RNA lattices. Finally, we discuss why this hypothesis is consistent with the idea that the ATPase catalytic cycle is tightly coupled to the movement of NS3 helicase along the RNA.

Covalent binding of cisplatin impairs the function of Na+/K+-ATPase by binding to its cytoplasmic part

This study was aimed at verifying the hypothesis that acute kidney failure accompanying cisplatin administration in the cancer therapy could be due to cisplatin interaction with the cytoplasmic part of Na+/K+-ATPase. Our results demonstrated that cisplatin-binding caused inhibition of Na+/K+-ATPase, in contrast to other platinated chemotherapeutics such as carboplatin and oxaliplatin, which are known to be much less nephrotoxic. To acquire more detailed structural information, we performed a series of experiments with the isolated large cytoplasmic segment connecting transmembrane helices 4 and 5 (C45 loop) of Na+/K+-ATPase. Electrochemistry showed that cisplatin is bound to the cysteine residues of the C45 loop, mass spectrometry revealed a modification of the C45 peptide fragment GSHMASLEAVETLGSTSTICSDK, which contains the conserved phosphorylated residue Asp369. Hence, we hypothesize that binding of cisplatin to Cys367 can cause sterical obstruction during the phosphorylation or dephosphorylation step of the Na+/K+-ATPase catalytic cycle.

The Ca2+-ATPase (SERCA1) Is Inhibited by 4-Aminoquinoline Derivatives through Interference with Catalytic Activation by Ca2+, Whereas the ATPase E2 State Remains Functional

Several clotrimazole (CLT) and 4-aminoquinoline derivatives were synthesized and found to exhibit in vitro antiplasmodial activity with IC(50) ranging from nm to μm values. We report here that some of these compounds produce inhibition of rabbit sarcoplasmic reticulum Ca(2+)-ATPase (SERCA1) with IC(50) values in the μm range. The highest affinity for the Ca(2+)-ATPase was observed with NF1442 (N-((3-chlorophenyl)(4-((4-(7-chloroquinolin-4-yl)piperazin-1-yl)methyl)phenyl)methyl)-7-chloro-4-aminoquinoline) and NF1058 (N-((3-chlorophenyl)(4-(pyrrolidin-1-ylmethyl)phenyl)methyl)-7-chloro-4-aminoquinoline),yielding IC(50) values of 1.3 and 8.0 μm as demonstrated by measurements of steady state ATPase activity as well as single cycle charge transfer. Characterization of sequential reactions comprising the ATPase catalytic and transport cycle then demonstrated that NF1058, and similarly CLT, interferes with the mechanism of Ca(2+) binding and Ca(2+)-dependent enzyme activation (E(2) to E(1)·Ca(2) transition) required for formation of phosphorylated intermediate by ATP utilization. On the other hand, Ca(2+) independent phosphoenzyme formation by utilization of P(i) (i.e. reverse of the hydrolytic reaction in the absence of Ca(2+)) was not inhibited by NF1058 or CLT. Comparative experiments showed that the high affinity inhibitor thapsigargin interferes not only with Ca(2+) binding and phosphoenzyme formation with ATP but also with phosphoenzyme formation by utilization of P(i) even though this reaction does not require Ca(2+). It is concluded that NF1058 and CLT inhibit SERCA by stabilization of an E(2) state that, as opposed to that obtained with thapsigargin, retains the functional ability to form E(2)-P by reacting with P(i).

Cuomarin‐6‐sulfonamides May be Useful Photoaffinity‐labeling Reagents for Analyzing the Kinetics of Drug Transport and ATP Hydrolysis in ABC Multidrug Transporters

The photoaffinity reagent [125]‐iodoarylazidoprazosin (IAAP) is used to study both the ATPase catalytic cycle and the behavior of drug‐binding sites in a number of ABC multidrug transporter proteins including those found in fungi and mammalian cells. This reagent has several limitations, however. It is synthesized with a radioactive label and the lack of a cold counterpart makes measuring the Kd at a drug binding site onerous. Furthermore, multidrug transporters have multiple drug binding sites and IAAP labels at best only a minority of these. Both of these limitations are potentially addressed with a set of cuomarin‐6‐sulfonamides that contain a free C4‐azidomethyl group that should be suitable for photoaffinity labeling. We have begun to test some of these reagents using the yeast multidrug transporter Pdr5. We demonstrate that these derivatives are strong Pdr5 drug substrates that don't interfere with the protein's ATPase activity. Using whole cell transport assays, we show that several of these compounds are transported from at least one site that is distinct from the one photoaffinity labeled with IAAP.

Modeling the Membrane Role in Ca2+-ATPase Catalytic Cycle

A deep understanding of the function of membrane proteins requires that we understand the direct and indirect effects of the lipid environment. Deformations of the bilayer to accommodate the protein induce energy penalties and potentially change the free energy between conformational states and thereby change the distribution of protein conformations. The lipid bilayer thus plays a regulatory role for the function of a membrane protein.Structures of the Ca2+-ATPase from sarcoplasmic reticulum, SERCA, have been determined by X-ray crystallography in several different functional states. These structures have provided a unique opportunity to study how the protein interacts with the membrane throughout the functional cycle by all-atom molecular dynamics (MD) simulations.MD simulations have been performed with four different structures of SERCA representing a Ca2+- and ATP-bound state (Ca2E1-ATP); a state with the luminal Ca2+-exit path open and the protein phosphorylated (E2P); and two dephosphorylated occluded states with bound protons, one with inorganic phosphor still bound (E2-Pi) and one without (E2). Our results show how the POPC-membrane and the protein in different functional states undergo mutual adaption (see figure) and how the hydrophobic mismatch and protein area profile change during the functional cycle.View Large Image | View Hi-Res Image | Download PowerPoint Slide

Shedding light on drug transport: structure and function of the P-glycoprotein multidrug transporter (ABCB1)This paper is one of a selection of papers published in this Special Issue, entitled CSBMCB — Membrane Proteins in Health and Disease.

P-glycoprotein (Pgp; ABCB1), a member of the ATP-binding cassette (ABC) superfamily, exports structurally diverse hydrophobic compounds from the cell, driven by ATP hydrolysis. Pgp expression has been linked to the efflux of chemotherapeutic drugs in human cancers, leading to multidrug resistance (MDR). The protein also plays an important physiological role in limiting drug uptake in the gut and entry into the brain. Substrates partition into the lipid bilayer before interacting with Pgp, which has been proposed to function as a hydrophobic vacuum cleaner. Low- and medium-resolution structural models of Pgp suggest that the 2 nucleotide-binding domains are closely associated to form a nucleotide sandwich dimer. Pgp is an outwardly directed flippase for fluorescent phospholipid and glycosphingolipid derivatives, which suggests that it may also translocate drug molecules from the inner to the outer membrane leaflet. The ATPase catalytic cycle of the protein is thought to proceed via an alternating site mechanism, although the details are not understood. The lipid bilayer plays an important role in Pgp function, and may regulate both the binding and transport of drugs. This review focuses on the structure and function of Pgp, and highlights the importance of fluorescence spectroscopic techniques in exploring the molecular details of this enigmatic transporter.

Exploiting Reaction Intermediates of the ATPase Reaction to Elucidate the Mechanism of Transport by P-glycoprotein (ABCB1)

The transport cycle of ABC transporters in general and P-glycoprotein in particular has been extensively studied, but the molecular mechanism remains controversial. We identify stable reaction intermediates in the progression of the P-glycoprotein-mediated ATPase reaction equivalent to the enzyme-substrate (E.S, P-glycoprotein.ATP) and enzyme-product (E.P, P-glycoprotein.ADP.P(i)) reaction intermediates. These have been characterized using the photoaffinity analog 8-azido-[alpha-32P]ATP as well as under equilibrium conditions using [alpha-32P]ATP, in which a cross-linking step is not involved. Similar results were obtained when 8-azido-[alpha-32P]ATP or [alpha-32P]ATP was used. The reaction intermediates were characterized based on their kinetic properties and the nature (triphosphate/diphosphate) of the trapped nucleotide. Using this defined framework and the Walker B E556Q/E1201Q mutant that traps nucleotide in the absence of vanadate or beryllium fluoride, the high to low affinity switch in the transport substrate binding site can be attributed to the formation of the E.S reaction intermediate of the ATPase reaction. Importantly, the posthydrolysis E.P state continues to have low affinity for substrate, suggesting that conformational changes that form the E.S complex are coupled to the conformational change at the transport substrate site to do mechanical work. Thus, the formation of E.S reaction intermediate during a single turnover of the catalytic cycle appears to provide the initial power stroke for movement of drug substrate from inner leaflet to outer leaflet of lipid bilayer. This novel approach applies transition state theory to elucidate the mechanism of P-glycoprotein and other ABC transporters and has wider applications in testing cause-effect hypotheses in coupled systems.

Conformational Changes in Sarcoplasmic Reticulum Ca2+-ATPase Mutants: Effect of Mutations either at Ca2+-Binding Site II or at Tryptophan 552 in the Cytosolic Domain

By analyzing, after expression in yeast and purification, the intrinsic fluorescence properties of point mutants of rabbit Ca(2+)-ATPase (SERCA1a) with alterations to amino acid residues in Ca(2+)-binding site I (E(771)), site II (E(309)), in both sites (D(800)), or in the nucleotide-binding domain (W(552)), we were able to follow the conformational changes associated with various steps in the ATPase catalytic cycle. Whereas Ca(2+) binding to purified wild-type (WT) ATPase in the absence of ATP leads to the rise in Trp fluorescence expected for the so-called E2 --> E1Ca(2) transition, the Ca(2+)-induced fluorescence rise is dramatically reduced for the E(309)Q mutant. As this purified E(309)Q mutant retains the ability to bind Ca(2+) at site I (but not at site II), we tentatively conclude that the protein reorganization induced by Ca(2+) binding at site II makes the major contribution to the overall Trp fluorescence changes observed upon Ca(2+) binding to both sites. Judging from the fluorescence response of W(552)F, similar to that of WT, these changes appear to be primarily due to membranous tryptophans, not to W(552). The same holds for the fluorescence rise observed upon phosphorylation from P(i) (the so-called E2 --> E2P transition). As for WT ATPase, Mg(2+) binding in the absence of Ca(2+) affects the fluorescence of the E(309)Q mutant, suggesting that this Mg(2+)-dependent fluorescence rise does not reflect binding of Mg(2+) to Ca(2+) sites; instead, Mg(2+) probably binds close to the catalytic site, or perhaps near transmembrane span M3, at a location recently revealed by Fe(2+)-catalyzed oxidative cleavage. Mutation of W(552) hardly affects ATP-induced fluorescence changes in the absence of Ca(2+), which are therefore mostly due to membranous Trp residues, demonstrating long-range communication between the nucleotide-binding domain and the membranous domain.

Nucleotides and transported substrates modulate different steps of the ATPase catalytic cycle of MRP1 multidrug transporter.

The human ABC (ATP-binding cassette) transporter MRP1 (human multidrug-resistance-associated protein 1; ABCC1) is involved in the cellular extrusion of conjugated metabolites and causes multidrug resistance in tumour cells. The transport of substrate molecules by ABC proteins is energized by ATP hydrolysis, performed by two co-operating ABC units. Orthovanadate (Vi), a non-covalent inhibitor of the ABC ATPases, was found to catalyse a photo-oxidative cleavage of various ATP-binding proteins. In the present study, we have identified three Vi-cleavage sites within MRP1, and found that the cleavage reactions were variably modulated by the presence of nucleotides and by transported substrates. We concluded that Vi cleavage of MRP1 at Site I detects conformational changes due to the binding of MgATP. In contrast, Site II could be identified as part of the substrate-modulated catalytic cycle, probably containing an MRP1.MgADP.Vi transition-state-like complex. Cleavage at Site III was modulated by both the binding and hydrolysis of MgATP, in a biphasic pattern, which was also affected by the presence of transported substrates. We detected two different allosteric effects and found that they control two consecutive steps of the MRP1 ATPase catalytic cycle. Nucleotide binding to the low-affinity site accelerated the formation of the pre-hydrolytic intermediate in the other catalytic centre. Interaction of the transporter with its transported substrates stimulated a later reaction of the hydrolytic cycle, the formation of the post-hydrolytic intermediate, which could be detected in both catalytic sites by the experimental strategy used.

Why we must move on from the E1E2 model for the reaction cycle of the P-type ATPases.

Recent progress regarding the structure of the Ca(2+)-translocating ATPase of sarcoplasmic reticulum in several conformational states, and a substantial accumulation of biochemical information about this and other P-type ATPases, have put everything in place for the final convergence of biochemistry and structure that will lead to a complete understanding of the molecular mechanism of these membrane transport enzymes. But the common paradigm used to describe the reaction cycle of the P-type ATPases, the E1E2 model, is seriously flawed, and this is hindering our progress toward this goal. In this paper, it is first shown why the E1E2 model must be discarded. This is followed by a description of the P-type ATPase catalytic cycle that is much more consistent with the structural and biochemical information now available for these enzymes, and also brings to light the origin of the forces that drive the key reaction in the active transport cycle where high-affinity ion-binding sites are converted to low-affinity binding sites capable of releasing the transported ions against a considerable concentration gradient. This new model will therefore serve us better as we seek to unravel the final details of the molecular mechanism of active ion transport catalyzed by these enzymes. It is thus time to move on from the traditional E1E2 model.

Molecular mechanism of the P-type ATPases.

The recent determination of the structure of the Ca2+-ATPase of sarcoplasmic reticulum to atomic resolution in the Ca2+-bound state and to near atomic resolution in the Ca2+-free, decavanadate-bound state has paved the way for an ultimate complete understanding of the molecular mechanism of the P-type ATPases. Analysis of this new structure information together with the large amount of biochemical information about these enzymes that preceded it has produced important new revelations about how the P-type ATPases work. Most models propose that these transporters operate by a strictly conformational energy coupling mechanism in which conformational changes in the large cytoplasmic head region mechanically drive the ions to be transported from their binding sites in the transmembrane helix region 50 A away. However, while these enzymes do indeed undergo profound conformational changes, the available evidence suggests that they do not mechanically transduce the chemical energy of ATP hydrolysis into transmembrane ion gradients via these conformational changes. As an alternative, it is proposed that the effects of the chemical events that occur at the phosphorylation/dephosphorylation site in the cytoplasmic region are exerted on the ion-binding sites via two well-defined charge transfer pathways that electronically connect the chemical reaction site with the site of ion binding. The recognition of these charge transfer pathways provides rational explanations of all of the key biochemical features of the P-type ATPase catalytic cycle. Thus, although a few details await elucidation, a nearly complete understanding of the P-type ATPase reaction mechanism may be at hand.

A Remarkably Stable Phosphorylated Form of Ca2+-ATPase Prepared from Ca2+-loaded and Fluorescein Isothiocyanate-labeled Sarcoplasmic Reticulum Vesicles

After the nucleotide binding domain in sarcoplasmic reticulum Ca2+-ATPase has been derivatized with fluorescein isothiocyanate at Lys-515, ATPase phosphorylation in the presence of a calcium gradient, with Ca2+ on the lumenal side but without Ca2+ on the cytosolic side, results in the formation of a species that exhibits exceptionally low probe fluorescence (Pick, U. (1981) FEBS Lett. 123, 131-136). We show here that, as long as the free calcium concentration on the cytosolic side is kept in the nanomolar range, this low fluorescence species is remarkably stable, even when the calcium gradient is subsequently dissipated by ionophore. This species is a Ca2+-free phosphorylated species. The kinetics of Ca2+ binding to it indicates that its transport sites are exposed to the cytosolic side of the membrane and retain a high affinity for Ca2+. Thus, in the ATPase catalytic cycle, an intrinsically transient phosphorylated species with transport sites occupied but not yet occluded must also have been stabilized by fluorescein isothiocyanate (FITC), possibly mimicking ADP. The low fluorescence mainly results from a change in FITC absorption. The Ca2+-free low fluorescence FITC-ATPase species remains stable after addition of thapsigargin in the absence or presence of decavanadate, or after solubilization with dodecylmaltoside. The remarkable stability of this phosphoenzyme species and the changes in FITC spectroscopic properties are discussed in terms of a putative FITC-mediated link between the nucleotide binding domain and the phosphorylation domain in Ca2+-ATPase, and the possible formation of a transition state-like conformation with a compact cytosolic head. These findings might open a path toward structural characterization of a stable phosphorylated form of Ca2+-ATPase for the first time, and thus to further insights into the pump's mechanism.

Clotrimazole, an Antimycotic Drug, Inhibits the Sarcoplasmic Reticulum Calcium Pump and Contractile Function in Heart Muscle

Clotrimazole (CLT), an antimycotic drug, has been shown to inhibit proliferation of normal and cancer cell lines and its systemic use as a new tool in the treatment of proliferative disorders is presently under scrutiny (Benzaquen, L. R., Brugnara, C., Byers, H. R., Gattoni-Celli, S., and Halperin, J. A. (1995) Nature Med. 1, 534-540). The action of CLT is thought to involve depletion of intracellular Ca2+ stores but the underlying mechanism has not been defined. The present study utilized membrane vesicles of rabbit cardiac sarcoplasmic reticulum (SR) to determine the mechanism by which CLT depletes intracellular Ca2+ stores. The results revealed a strong, concentration-dependent inhibitory action of CLT on the ATP-energized Ca2+ uptake activity of SR (50% inhibition with approximately 35 microM CLT). The inhibition was of rapid onset (manifested in <15 s), and was accompanied by a 7-fold decrease in the apparent affinity of the SR Ca2+-ATPase for Ca2+ and a minor decrement in the enzyme's apparent affinity toward ATP. Exposure of SR to CLT in the absence or presence of Ca2+ resulted in irreversible inhibition of Ca2+ uptake demonstrating that the Ca2+-bound and Ca2+-free conformations of the Ca2+-ATPase are CLT-sensitive. Introduction of CLT to the reaction medium subsequent to induction of enzyme turnover with Ca2+ and ATP resulted in instantaneous cessation of Ca2+ transport indicating that an intermediate enzyme species generated during turnover undergoes rapid inactivation by CLT. The inhibition of Ca2+ uptake by CLT was accompanied by inhibition of Ca2+-stimulated ATP hydrolysis and Ca2+-induced phosphoenzyme intermediate formation from ATP in the ATPase catalytic cycle. Phosphorylation of the Ca2+-deprived enzyme with Pi in the reverse direction of catalytic cycle and Ca2+ release from Ca2+-preloaded SR vesicles were unaffected by CLT. It is concluded that CLT depletes intracellular Ca2+ stores by inhibiting Ca2+ sequestration by the Ca2+-ATPase. The mechanism of ATPase inhibition involves a drug-induced alteration in the Ca2+-binding site(s) resulting in paralysis of the enzyme's catalytic and ion transport cycle. CLT (50 microM) caused marked depression of contractile function in isolated perfused, electrically paced rabbit heart preparations. The contractile function recovered gradually following withdrawal of CLT from the perfusate indicating the existence of mechanisms in the intact cell to inactivate, metabolize, or clear CLT from its target site.

Kinetic characterization of the perturbation by dodecylmaltoside of sarcoplasmic reticulum Ca(2+)-ATPase.

We investigated the functional aspects of the interaction between the sarcoplasmic reticulum (SR) membranous Ca(2+)-ATPase and the non-ionic detergent dodecylmaltoside, using detergent concentrations allowing perturbation of the membrane but not its solubilization. At pH 7.5, the effects of dodecylmaltoside on ATPase activity and delipidation had previously been shown to resemble, in some respects, those of octa(ethylene glycol) monododecylether (C12E8), an appropriate detergent for ATPase studies. Our aim here was to explore the specific effects of dodecylmaltoside on the different steps in the ATPase catalytic cycle, which may owe their specificity to the difference between the polar head groups of dodecylmaltoside and C12E8. This was done at 20 degrees C, both at pH 6 in the absence of KCl and at pH 7.5 in the presence of 100 mM KCl, two conditions under which the characteristics of unperturbed ATPase have already been well defined. Preliminary estimation of dodecylmaltoside partition between water and SR membranes at pH 6 yielded a partition coefficient K close to 4 x 10(5) (ratio of the molar fraction of dodecylmaltoside in the lipid to that in the aqueous phase at a low detergent concentration, assuming that most of this detergent was present in the lipid phase). At near saturation of SR membranes, bound dodecylmaltoside was roughly equimolar with the constituent phospholipids. Non-solubilizing concentrations of dodecylmaltoside inhibited SR ATPase activity by up to 65-70% at pH 7.5, but not at pH 6, unlike the results of similar experiments with C12E8. The rates of the four main steps in the ATPase catalytic cycle were measured by fast kinetic techniques; they were similarly modified at both pH. Dodecylmaltoside slowed down both the rate of calcium-saturated ATPase phosphorylation and the rate of ATPase isomerization after phosphorylation, two steps which were not targets of perturbation by C12E8. The slowing down of the isomerization step by dodecylmaltoside might well explain why it inhibited overall ATPase activity at pH 7.5. In contrast to C12E8, dodecylmaltoside did not affect the dephosphorylation step, which was the main target of inhibition by C12E8 and the main rate-limiting step at pH 6. However, like C12E8, dodecylmaltoside accelerated the calcium binding-induced transition of nonphosphorylated ATPase. Another striking feature of the perturbation induced by dodecylmaltoside was that it significantly altered the binding of 45Ca2+ to the ATPase and the corresponding conformational changes. At pCa 5-5.5, it almost halved calcium binding to the ATPase but ATPase phosphorylation was unimpaired.(ABSTRACT TRUNCATED AT 400 WORDS)

Kinetic characterization of the normal and procaine-perturbed reaction cycles of the sarcoplasmic reticulum calcium pump.

We investigated the effect of the local anesthetic procaine on the activity of the calcium pump protein of sarcoplasmic reticulum (SR) vesicles. Procaine slowed down the rate of calcium uptake by SR vesicles without enhancing the vesicles' passive permeability. This slowing of the unidirectional pumping rate was reflected by the inhibition of the maximal rate of the transport-coupled Ca(2+)-ATPase activity. The inhibition was dependent on Mg2+ concentration; at optimal (i.e. low) concentrations of magnesium, half-maximal inhibition occurred with procaine concentrations close to 15-20 mM. Inhibition of ATPase was not mediated by a change in the properties of the bulk lipid phase. Procaine moderately reduced the true affinity of ATPase for ATP, whereas equilibrium binding of calcium to ATPase in the absence of ATP was virtually not modified by procaine. In fast-kinetics studies, we explored the various intermediate steps in the ATPase catalytic cycle, in order to determine which of them were targets for inhibition by procaine. We found that procaine slowed down ATPase dephosphorylation, an effect which is at least partly responsible for the observed inhibition of overall ATPase activity. In contrast, procaine accelerated the calcium-induced transconformation of unphosphorylated ATPase in the absence of ATP, and altered neither the rate of the Ca(2+)-dependent phosphorylation of ATPase, nor the rate of the dissociation of Ca2+ from phosphorylated ATPase towards the SR lumen, a critical step, the rate of which was measured by a novel fast-filtration method. These results are discussed with respect to the possible site(s) of binding of this amphiphile on the ATPase, and in relation to the contribution of individual steps in the catalytic cycle to the rate limitation of unperturbed SR ATPase activity.

The substitution of calcium for magnesium in H+,K+-ATPase catalytic cycle. Evidence for two actions of divalent cations: Evidence for two Actions of Divalent Cations

In order to determine the role of divalent cations in the reaction mechanism of the H+,K+-ATPase, we have substituted calcium for magnesium, which is required by the H+,K+-ATPase for phosphorylation from ATP and from PO4. Calcium was chosen over other divalent cations assayed (barium and manganese) because in the absence of magnesium, calcium activated ATP hydrolysis, generated sufficiently high levels of phosphoenzyme (573 +/- 51 pmol.mg-1) from [gamma-32P]ATP to study dephosphorylation, and inhibited K+-stimulated ATP hydrolysis. The Ca2+-ATPase activity of the H+,K+-ATPase was 40% of the basal Mg2+-ATPase activity. However, the Ca2+,K+-ATPase activity (minus the Ca2+ basal activity) was only 0.7% of the Mg2+,K+-ATPase, indicating that calcium could partially substitute for Mg2+ in activating ATP hydrolysis but not in K+ stimulation of ATP hydrolysis. Approximately 0.1 mM calcium inhibited 50% of the Mg2+-ATPase or Mg2+,K+-ATPase activities. Inhibition of Mg2+,K+-ATPase activity was not competitive with respect to K+. Inhibition by calcium of Mg2+,K+ activity p-nitrophenyl phosphatase activity was competitive with respect to Mg2+ with an apparent Ki of 0.27 mM. Proton transport measured by acridine orange uptake was not detected in the presence of Ca2+ and K+. In the presence of Mg2+ and K+, Ca2+ inhibited proton transport with an apparent affinity similar to the inhibition of the Mg2+, K+-ATPase activity. The site of calcium inhibition was on the exterior of the vesicle. These results suggest that calcium activates basal turnover and inhibits K+ stimulation of the H+,K+-ATPase by binding at a cytosolic divalent cation site. The pseudo-first order rate constant for phosphoenzyme formation from 5 microM [gamma-32P]ATP was at least 22 times slower in the presence of calcium (0.015 s-1) than magnesium (greater than 0.310 s-1). The Ca.EP (phosphoenzyme formed in the presence of Ca2+) formed dephosphorylated four to five times more slowly that the Mg.EP (phosphoenzyme formed in the presence of Mg2+) in the presence of 8 mm trans-1,2-diaminocyclohexane-N,N,N',N'-tetraacetic acid (CDTA) or 250 microM ATP. Approximately 10% of the Ca.EP formed was sensitive to a 100 mM KCl chase compared with greater than 85% of the Mg.EP. By comparing the transient kinetics of the phosphoenzyme formed in the presence of magnesium (Mg.EP) and calcium (Ca.EP), we found two actions of divalent cations on dephosphorylation.(ABSTRACT TRUNCATED AT 400 WORDS)