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G Inesi

Publications and source records attributed to G Inesi.

At least 55 records · Page 3Linked to original sources
PubMed1993

High sensitivity to site directed mutagenesis of the peptide segment connecting phosphorylation and Ca2+ binding domains in the Ca2+ transport ATPase.

Nine residues (Leu321, Lys329, Asn330, Val333, Arg334, Leu336, Pro337, Val339 and Glu340), within the peptide segment intervening between the catalytic domain and the Ca2+ binding domain of the sarcoplasmic reticulum (SERCA 1) ATPase, were individually mutated to Ala. The mutated proteins were recovered in the microsomal fraction of COS-1 cells following transient expression, and exhibited inhibition of Ca2+ uptake and ATPase hydrolytic activity, while forming discernable levels of phosphorylated intermediate. Mutation of Glu340 to Gln (rather than to Ala) was much less effective, suggesting that the functional consequence of the mutation is related to structural perturbation, rather than loss of the acidic side chain. The high sensitivity of this peptide segment to single mutations suggests that its structural integrity is required for functional linkage of the phosphorylation and Ca2+ binding domains.

Animals↗
PubMed1993

Ca(2+)-dependent and thapsigargin-inhibited phosphorylation of Na+,K(+)-ATPase catalytic domain following chimeric recombination with Ca(2+)-ATPase.

Two chimeric proteins comprising the Na,K-ATPase catalytic domain (large cytosolic loop) and the two flanking regions of the Ca-ATPase were obtained by transient or stable expression in mammalian cells transfected with recombinant DNA. In the first chimera (CpNC), a large portion (containing the nucleotide-binding site) of the cytosolic loop between putative membrane spans M4 and M5 of the sarcoendoplasmic reticulum Ca2+ (SERCA) 1 (fast muscle) ATPase was replaced by the corresponding portion of the Na,K-ATPase alpha 1 subunit. In the second chimera (CNpC), an even larger portion (containing the nucleotide-binding site and the phosphorylation site) of the analogous cytosolic loop of the SERCA2 (cardiac muscle) ATPase was replaced by the corresponding portion of the Na,K-ATPase alpha 1 subunit. Steady state Ca2+ transport and coupled ATP hydrolysis by the chimeric proteins were negligible as compared to those obtained with SERCA enzymes. Nevertheless, the chimeric proteins were able to utilize ATP to form phosphoenzyme levels equal to those formed by SERCA ATPases. Chimeric and SERCA enzymes exhibited an identical Ca2+ requirement for ATP utilization and sensitivity to thapsigargin (TG) which is a specific inhibitor of SERCA ATPase and not of Na,K-ATPase. Furthermore, both SERCA and chimeric enzymes could be phosphorylated with P(i), and this reaction required removal of Ca2+. In comparative experiments, the functional pattern of seemingly unaffected phosphoenzyme formation and inhibited Ca2+ transport was produced in the SERCA ATPase even by single mutation of Pro337 to Ala, evidently due to defective protein conformation. Retention of Ca2+ and TG sensitivity by the chimeric proteins demonstrates that the Ca(2+)- and TG-binding domains do not reside within the cytosolic loop replaced by chimeric substitution and strongly support previous studies suggesting that binding of calcium required for enzyme activation occurs within the membrane-bound region of the SERCA ATPases (Clarke et al., 1989a; Sumbilla et al., 1991).

Amino Acid Sequence↗
PubMed1993

Effects of anions on the Ca2+, H+ and electrical gradients formed by the sarcoplasmic reticulum ATPase in reconstituted proteoliposomes.

The chaotropic character of several ions determines their partition on membrane interfaces with aqueous media as predicted by the Hofmeister series. However, specific characteristics of each individual ion determine its ability to cross the membrane and to influence Ca2+, H+ and electrical gradients produced by the sarcoplasmic reticulum (SR) Ca2+ pump in reconstituted proteoliposomal vesicles. Specific effects of this kind may be relevant to a variety of biological systems, including the excitation-contraction coupling of muscle fibers in which SR plays a prominent role.

Adenosine Triphosphatases↗
PubMed1993

Comparative studies of cardiac and skeletal sarcoplasmic reticulum ATPases. Effect of a phospholamban antibody on enzyme activation by Ca2+.

Vesicular fragments of skeletal (LSR) and cardiac (CSR) sarcoplasmic reticulum were compared with the aim of defining the effect of a monoclonal phospholamban (Pl) antibody (Ab). The Pl Ab has no effect on LSR, while enhancing the Ca2+ transport rates of CSR at Ca2+ concentrations below saturation. We found no direct effect of the Pl Ab on Ca2+ binding by the ATPase in the absence of ATP. Equilibrium measurements of Ca2+ binding yield positively cooperative isotherms which are best fit with a two-interacting sites equation. LSR and CSR display nearly identical affinities for Ca2+, and no effect of the Pl Ab is observed. Taking advantage of a stable CrATP-enzyme complex, we demonstrated that the stoichiometric ratio of occluded Ca2+ to catalytic sites is 2 in either LSR or CSR and that the addition of Pl Ab does not affect the Ca2+ concentration dependence of Ca2+ occluded after equilibration of the system. Interestingly, the cooperative interaction between the two Ca2+ sites is lost in the occluded state, with only one of the two sites acquiring lumenal exposure. The concentration dependence of Ca2+ inhibition of CSR ATPase phosphorylation with Pi is also unaffected by the Pl Ab. Contrary to the lack of Pl Ab effect on reactions measured at equilibrium, enhancement of phosphorylated intermediate formation by the Pl Ab is obtained in kinetic experiments in which nonsaturating Ca2+ and ATP are added to CSR preincubated with EGTA. Therefore, Ab binding to Pl reduces specifically the activation energy for a slow transition triggered by Ca2+ binding, with consequent enhancement of overall kinetics under conditions enhancing the rate-limiting contribution of this transition.

Adenosine Triphosphatases↗
PubMed1993

H+ countertransport and electrogenicity of the sarcoplasmic reticulum Ca2+ pump in reconstituted proteoliposomes.

The Ca2+ transport adenosine triphosphatase of sarcoplasmic reticulum was reconstituted in unilamellar liposomes prepared by reverse-phase evaporation. The size of the resulting proteoliposomes was similar to that of native sarcoplasmic reticulum vesicles, but their protein content was much lower, with a protein/lipid ratio (wt/wt) of 1:40-160, as compared with 1:1 in the native membrane. The proteoliposomes sustained adenosine triphosphate-dependent Ca2+ uptake at rates proportional to the protein content (1-2 mumol Ca2+/mg protein/min), reaching asymptotic levels corresponding to a lumenal calcium concentration of 10-20 mM. The low permeability of the proteoliposomes permitted direct demonstration of Ca2+/H+ countertransport and electrogenicity by parallel measurements in the same experimental system. Countertransport of one H+ per one Ca2+ was demonstrated, and inhibition of the Ca2+ pump by lumenal alkalinization was relieved by the H+ ionophore carbonyl cyanide p-(trifluoromethoxy)phenylhydrazone. Consistent with the countertransport stoichiometry, net positive charge displacement was produced by Ca2+ transport, as revealed by a rapid oxonol VI absorption rise. The initial rise and the following steady-state level of oxonol absorption were highest when SO4(2-) was the prevalent anion and lowest in the presence of the lipophilic anion SCN-. The influence of anions was attributed to potential driven counterion compensation. The absorption rise was rapidly collapsed by addition of valinomycin in the presence of K+. Experimentation with Ca2+ and H+ ionophores was consistent with a primary role of Ca2+ and H+ in net charge displacement. The estimated value of the steady-state electrical potential observed under optimal conditions was approximately 50 mV and was accounted for by the estimated charge transfer associated with Ca2+ and H+ countertransport under the same conditions.

Adenosine Triphosphate↗
PubMed1992

Coupled expression of Ca2+ transport ATPase and a dihydrofolate reductase selectable marker in a mammalian cell system.

Stable expression of a full-length cDNA encoding chicken fast muscle Ca2+ transport ATPase was obtained in a Chinese hamster lung cell line (DC-3F), using a dual-promoter expression vector (pH beta FCaA3) in which the ATPase was cloned downstream of a human beta-actin gene promoter, and a mutant dihydrofolate reductase cDNA (A3/DHFR) was cloned downstream of an SV40 promoter-enhancer. Owing to its essentially normal catalytic activity and modest (20-fold) resistance to the antifolate methotrexate (MTX), the A3/DHFR mutant enzyme served as an efficient dominant selection marker in transfected cell populations challenged with MTX and, within a broad range of drug concentrations, allowed subsequent amplification and overexpression of vector sequences. In stable transfectants, the expressed ATPase was targeted to intracellular membranes, and the microsomal fractions from those cells exhibited high rates of Ca2+ transport. In comparative experiments using transient expression in COS1 cells, the level of ATPase per transfected cell was greater, but less than 5% of the transfected population exhibited ATPase expression. Furthermore, as opposed to the stable lines, the transiently expressing cells could not be propagated. Overall, the yield of ATPase was 12-16 and 4-6 micrograms per milligram of microsomal protein in the stable and the transient expression systems, respectively. The advantages of the stably transfected cell lines therefore lie in the homogeneity of ATPase expression and its distribution in cells and microsomes, in the large yield of microsomes obtained by continuous cell propagation, and in the reproducible functional characteristics of the microsomes. Moreover, the microsomes derived from stably transfected cell lines provide a convenient system for studies of Ca2+ transport and ATPase partial reaction, eliminating the need to conduct repetitive transient transfections to obtain sufficient amounts of enzyme for functional studies.

Animals↗
PubMed1992

Thapsigargin inhibits contraction and Ca2+ transient in cardiac cells by specific inhibition of the sarcoplasmic reticulum Ca2+ pump.

Regulation of the level of ionized calcium, [Ca2+]i, is critical for its use as an important intracellular signal. In cardiac and skeletal muscle the control of fluctuations of [Ca2+]i depend on sarcolemmal and sarcoplasmic reticulum ion channels and transporters. We have investigated the sesquiterpine lactone, thapsigargin (TG), because of its reported action to alter cellular calcium regulation in diverse cell types, including striated muscle cells. We have combined biochemical and physiological methods at the cellular level to determine the site of action of this agent, its specificity, and its cellular effects. Using a patch-clamp method in whole cell configuration while measuring [Ca2+]i with Indo-1 salt, we find that TG (100 nM) largely blocks the contraction and the [Ca2+]i transient in rat ventricular myocytes. Analysis of these data indicate that no sarcolemmal current or transport system is directly altered by TG, although indirect [Ca2+]i-dependent processes are affected. In permeabilized myocytes, TG blocked oxalate-stimulated calcium uptake (half-maximal effect at 10 nM) into the SR. However, TG (100 microM) had no effect on Ca(2+)-induced Ca(2+)-release in purified muscle (ryanodine-receptor enriched) vesicles while clearly blocking Ca(2+)-ATPase activity in purified (longitudinal SR) vesicles. We conclude that in striated muscle TG markedly alters calcium metabolism and thus alters contractile function only by its direct action on the Ca(2+)-ATPase.

Animals↗
PubMed1992

Characterization of the inhibition of intracellular Ca2+ transport ATPases by thapsigargin.

The effects of thapsigargin (TG), a specific inhibitor of intracellular Ca(2+)-ATPases, were studied on vesicular fragments of sarcoplasmic reticulum (SR) membranes. Inhibition of Ca2+ transport and ATPase activity was observed following stoichiometric titration of the membrane bound enzyme with TG. When Ca2+ binding to the enzyme was measured in the absence of ATP, or when one cycle of Ca(2+)-dependent enzyme phosphorylation by ATP was measured under conditions preventing turnover, protection against TG by Ca2+ was observed. The protection by Ca2+ disappeared if the phosphoenzyme was allowed to undergo turnover, indicating that a state reactive to TG is produced during enzyme turnover, whereby a dead end complex with TG is formed. Enzyme phosphorylation with Pi, ATP synthesis, and Ca2+ efflux by the ATPase in its reverse cycling were also inhibited by TG. However, under selected conditions (millimolar Ca2+ in the lumen of the vesicles, and 20% dimethyl sulfoxide in the medium) TG permitted very low rates of enzyme phosphorylation with Pi and ATP synthesis in the presence of ADP. It is concluded that the mechanism of ATPase inhibition by TG involves mutual exclusion of TG and high affinity binding of external Ca2+, as well as strong (but not total) inhibition of other partial reactions of the ATPase cycle. TG reacts selectively with the state acquired by the ATPase in the absence of Ca2+. This state is obtained either by enzyme exposure to EGTA, or by utilization of ATP and consequent displacement of bound Ca2+ during catalytic turnover.

Adenosine Triphosphate↗
PubMed1992

Phosphorylation-dependent changes in the spatial relationship between Ca-ATPase polypeptide chains in sarcoplasmic reticulum membranes.

In order to investigate possible structural changes associated with the coupling mechanisms of the Ca-ATPase in sarcoplasmic reticulum membranes, we have utilized fluorescence resonance energy transfer between spectroscopic probes covalently bound to different domains of the ATPase. Using time-correlated single photon counting, we have directly measured the energy transfer efficiency between 5-[2-[(iodoacetyl)amino]ethyl]aminonaphthalene-1-sulfonic acid (IAEDANS), that is specifically bound to the B trypic fragment at cysteines 670 and 674 and acceptors covalently bound either near the nucleotide binding site, i.e. fluorescein 5-isothiocyanate at lysine 515, also on the B fragment, or maleimide-directed probes specifically located on the A1, tryptic fragment, i.e. 4-dimethylaminoazobenzene-4'-maleimide (DABmal) or fluorescein-5-maleimide (Fmal), probably at cysteines 344 and 364. All of these donor-acceptor pairs exhibit energy transfer both within and between Ca-ATPase molecules allowing us to investigate spatial relationships between the A1 and B domains and between different ATPase polypeptide chains. Differentiation between the intra- and intermolecular components of energy transfer was accomplished in two ways: 1) by comparing the transfer efficiencies in native membranes before and after detergent solubilization and 2) by reconstituting ATPase chains that have already been labeled with either the donor or acceptor chromophores. Using this approach, we find no significant change in the intramolecular transfer efficiency between any of these donor-acceptor pairs either upon binding of calcium to the high affinity sites or upon stabilization of the phosphoenzyme intermediate, indicating that there are no large structural changes within the B tryptic fragment or, alternatively, between the A1 and B fragments. With respect to intermolecular energy transfer, we observe no effect of calcium binding on the unliganded enzyme with either donor-acceptor pair. However, formation of the phosphoenzyme intermediate results in a measurable increase in the transfer efficiency between IAEDANS and DABmal (or Fmal); this increase is reversible upon phosphoenzyme destabilization by subsequent addition of calcium. There is no corresponding change in the intermolecular component of fluorescence resonance energy transfer between IAEDANS and fluorescein 5-isothiocyanate, indicating that the change in fluorescence resonance energy transfer probably occurs as a result of reorientation of associated ATPase polypeptide chains with respect to one another.

Animals↗
PubMed1992

Functional evidence of a transmembrane channel within the Ca2+ transport ATPase of sarcoplasmic reticulum.

Ca2+ efflux can be studied conveniently following dilution of sarcoplasmic reticulum (SR) vesicles preloaded with 45Ca2+ by active transport. The rates of efflux are highly dependent on ATPase substrates and cofactors (Pi, Mg2+, Ca2+ and ADP) in the efflux medium. On the other hand, phenothiazines stimulate efflux through a passive permeability channel with no coupled catalytic events. Efflux activation by manipulation of catalytically active ATPase ligands, as well as by the catalytically inactive phenothiazines, can be prevented by thapsigargin, which is a highly specific inhibitor of the Ca(2+)-ATPase. This demonstrates that the passive channel activated by phenothiazines is an integral part of the ATPase, and can operate either uncoupled or coupled to catalytic events.

Animals↗
PubMed1992

A conformational mechanism for formation of a dead-end complex by the sarcoplasmic reticulum ATPase with thapsigargin.

Thapsigargin (TG), a plant sesquiterpene lactone extract, interacts tightly with the sarcoplasmic reticulum (SR) Ca2+ transport ATPase yielding a 1:1 stoichiometric complex. In addition to inhibiting steady state enzyme activity, TG can be shown to inhibit two individual partial reactions of the ATPase cycle (i.e. Ca2+ binding in the absence of ATP and enzyme phosphorylation by Pi in the absence of Ca2+) even when these reactions are studied separately without interdependence. As the two partial reactions occur at domains relatively distant from each other in the protein structure, it is apparent that the TG induced perturbation involves the entire enzyme. The rate of TG interaction with the ATPase, as estimated by the onset of functional inhibition and by the development of an intrinsic fluorescence signal, is relatively low in the presence of Ca2+. The interaction is much faster when Ca2+ is removed from the medium by the addition of EGTA or is dissociated from the enzyme by utilization of ATP. When the TG interaction with the ATPase is studied in the presence of Ca2+ as a function of temperature (15-35 degrees C) and pH (6.0-8.0), two distinct kinetic components are observed: a fast component which is prevalent at high temperature and low pH, and a slow component which is prevalent at low temperature and high pH. This pattern suggests that the enzyme resides in two states, whose relatively slow equilibration is temperature- and pH-dependent. As only one state is reactive to TG, the enzyme population residing in this state reacts immediately with TG. On the other hand, the enzyme population residing in the alternate state must undergo slow conversion to the reactive state before being affected by TG. It can be also demonstrated that in the presence of Ca2+ TG shifts the ATPase from a refractory state to a state which is able to form bidimensional crystalline arrays stabilized by decavanadate. It is concluded that TG reacts specifically with the ATPase conformation which is prevalent in the absence of Ca2+, thereby forming a catalytically inactive dead-end complex.

Adenosine Triphosphatases↗
PubMed1992

Structural features of cation transport ATPases.

Several cation transport ATPases, sharing the common feature of a phosphorylated intermediate in the process of ATP utilization, are compared with respect to their subunit composition and amino acid sequence. The main component of these enzymes is a polypeptide chain of MW slightly exceeding 100,000, comprising an extramembranous globular head which is connected through a stalk to a membrane-bound region. With reference to the Ca2+ ATPase of sarcoplasmic reticulum, it is proposed that the catalytic (ATP binding and phosphorylation) domain resides in the extramembranous globular head, while cation binding occurs in the membrane region. Therefore, these two functional domains are separated by a distance of approximately 50 A. Alignment of amino acid sequences reveals extensive homology in the isoforms of the same ATPases, but relatively little homology in different cation ATPases. On the other hand, all cation ATPases considered in this analysis retain a consensus sequence of high homology, spanning the distance between the phosphorylation site and the preceding transmembrane helix. It is proposed that this sequence provides long-range functional linkage between catalytic and cation-binding domains. Thereby, translocation of bound cation occurs through a channel formed by transmembrane helices linked to the phosphorylation site. Additional sequences at the carboxyl terminal provide regulatory domains in certain ATPases.

Amino Acid Sequence↗
PubMed1991

Nucleotide sequences of avian cardiac and brain SR/ER Ca(2+)-ATPases and functional comparisons with fast twitch Ca(2+)-ATPase. Calcium affinities and inhibitor effects.

Two similar forms of the cardiac/slow Ca(2+)-ATPase (SERCA2a and SERCA2b), differing in sodium dodecyl sulfate-polyacrylamide gel electrophoresis mobility, are expressed in chicken heart and brain (Kaprielian, Z., Campbell, A. M., and Fambrough, D. M. (1989) Mol. Brain Res. 6, 55-60). In the current study, cDNAs encoding each form were cloned and sequenced. Chicken SERCA2a is 94% identical to its rabbit homologue, while SERCA2b has an extended carboxyl terminus with 38 of 49 amino acids identical to mammalian homologues. SERCA2b mRNA contains the SERCA2a encoding sequence within its 3'-untranslated region. Chicken genomic DNA sequence reveals that the alternate RNA splicing used to produced SERCA2a and SERCA2b subtypes involves a splice site within an exon. Tissue culture cells expressing the avian SERCA2a, SERCA2b, and SERCA1, each targetting to the endoplasmic reticulum, were used to measure Ca2+ affinities and inhibitor effects; no differences among the three pumps were detected.

Amino Acid Sequence↗
PubMed1991

Inhibition of the sarcoplasmic reticulum Ca2+ transport ATPase by thapsigargin at subnanomolar concentrations.

ATP-dependent calcium uptake by isolated sarcoplasmic reticulum vesicles is inhibited by concentrations of free thapsigargin as low as 10(-10) M. This effect is due to primary inhibition of the Ca(2+)-dependent ATPase which is coupled to active transport. When binding of calcium to the activating sites of the enzyme is measured under equilibrium conditions in the absence of ATP, addition of thapsigargin produces strong inhibition. On the other hand, if [tau-32P]ATP is added to ATPase preincubated with Ca2+ under favorable conditions, significant levels of 32P-phosphorylated intermediate are still formed transiently, even in the presence of thapsigargin. The phosphoenzyme, however, decays rapidly as the calcium-enzyme complex is destabilized as a consequence of ATP utilization, and formation of the thapsigargin-enzyme complex is favored. Formation of the thapsigargin-enzyme complex is also favored by Ca2+ chelation with EGTA, with consequent inhibition of the enzyme reactivity to Pi (i.e. reverse of the ATPase hydrolytic reaction). Neither the Ca(2+)- and ATP-induced Ca2+ release from junctional sarcoplasmic reticulum nor the Ca(2+)- and calmodulin-dependent ATPase of plasma membranes (erythrocyte ghosts) were found to be altered by thapsigargin at such low concentrations.

Adenosine Triphosphate↗