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G Inesi

Publications and source records attributed to G Inesi.

At least 37 records · Page 2Linked to original sources
PubMed1997

ATPase gene transfer and mutational analysis of the cation translocation mechanism.

The peptide segment interposed between cation binding and phosphorylation domains retains a high degree of homology in all cation transport ATPases. Mutational analysis and chimeric replacements of Ca2+ ATPase components with corresponding Na+,K(+)-ATPase components indicate that this segment is utilized by various cation ATPases as a common structural device for a long-range functional linkage of enzyme phosphorylation and cation transport. Vectorial displacement of bound cation is rendered possible by a transmembrane channel formed by four clustered helices (M4, M5, M6, and M8). Originating from the four helices, the oxygen functions of Glu309, Glu771, Thr799, Asp800, and Glu908 form a duplex Ca2+ binding site in the middle of the channel, while Lys297 seals the luminal end of the channel with its positively charged side chain. The perturbation triggered by enzyme phosphorylation is apparently transmitted through the linkage segment to produce rotational displacement of the M4 helix with minimal change of secondary structure. The cation binding site is thereby disrupted and the Lys297 side chain removed, permitting Ca2+ to dissociate in exchange for H+ and to flow through the luminal end of the channel.

Amino Acid Sequence↗
PubMed1997

Synthesis of a radioactive azido derivative of thapsigargin and photolabeling of the sarcoplasmic reticulum ATPase.

A thapsigargin C8-derivative (ZTG) was synthesized by acylating debutanoylthapsigargin with 4-azido[carboxyl-14C]benzoic acid. ZTG retains the inhibitory activity of thapsigargin (TG) with respect to the Ca2+ ATPase of sarcoplasmic reticulum (SR). Covalent ATPase labeling was obtained by photoactivation of the ZTG azido moiety under conditions optimized to reduce nonspecific association of ZTG with SR vesicles and to approximate a matching ZTG:ATPase stoichiometry. Specific photolabeling of the Ca2+ ATPase with ZTG was obtained with 30% efficiency and was competitively inhibited by TG. Analysis of the labeled protein and its proteolytic fragments demonstrates that the ZTG label is associated covalently with the membrane-bound portion of tryptic subfragment A1, which spans the sequence between Leu253 and Arg324 and includes segments of S3 and S4 in the stalk, the M3 and M4 transmembrane helices, and the intervening lumenal loop. This finding is in agreement with previous spectroscopic observations and mutational analysis.

Acylation↗
PubMed1997

Lys515-Lys492 cross-linking by DIDS interferes with substrate utilization by the sarcoplasmic reticulum ATPase.

Sarcoplasmic reticulum (SR) Ca2+ ATPase was derivatized with 4,4'-diisothiocyanatostilbene-2,2'-sulfonic acid (DIDS), and complete enzyme inactivation was produced with a molecular stoichiometry of one DIDS per ATPase. It was determined by peptide analysis and sequencing that Lys492 and Lys515 were the ATPase residues derivatized by DIDS. Lack of electrophoretic resolution of the two peptide fragments that result from a single tryptic cut at Arg505 demonstrated that the two derivatized residues were cross-linked. Cross-linking of Lys492 and Lys515 by DIDS interfered with ATPase utilization of both ATP and p-nitrophenyl phosphate substrates, whereas derivatization of only Lys515 with fluorescein isothiocyanate interfered with ATPase utilization of ATP but not of p-nitrophenyl phosphate. Cross-linking with DIDS implies a distance of approximately 13 A between Lys492 and Lys515, which corresponds to the length of ATP bound in an extended configuration. Therefore, within the groove of the nucleotide binding domain, the ATP substrate is positioned with the adenosine moiety near Lys515 and its terminal phosphate near Lys492.

4,4'-Diisothiocyanostilbene-2,2'-Disulfonic Acid↗
PubMed1997

Entropic drive in the sarcoplasmic reticulum ATPase interaction with Mg2+ and Pi.

Thermodynamic quantities for the binding of Mg2+ (in the presence of Ca2+) and Pi (in the presence of Mg2+ and absence of Ca2+) to sarcoplasmic reticulum ATPase were determined from isothermal titration calorimetry measurements at 25 degrees C. Mg2+ and Pi are involved in reversal of the ATPase hydrolytic reaction, and their interactions with the ATPase are conveniently studied under equilibrium conditions. We found that the Mg2+ binding reaction is endothermic with a binding constant (Kb) = 142 +/- 4 M(-1), a binding enthalpy of 180 +/- 3 kJ mol(-1), and an entropy contribution (TdeltaSb) = 192 +/- 3 kJ mol(-1). Similarly, Pi binding is also an endothermic reaction with Kb = 167 +/- 17 M(-1), deltaHb = 65.3 +/- 5.4 kJ mol(-1), and TdeltaSb = 77.9 +/- 5.4 kJ mol(-1). Our measurements demonstrate that the ATPase can absorb heat from the environment upon ligand binding, and emphasize the important role of entropic mechanisms in energy transduction by this enzyme.

Adenosine Triphosphatases↗
PubMed1996

Energy transduction and kinetic regulation by the peptide segment connecting phosphorylation and cation binding domains in transport ATPases.

The sarcoplasmic reticulum ATPase segment (Thr316-Leu356) connecting the extramembranous phosphorylation domain to the preceding transmembrane helix M4 (which is an integral component of the Ca2+ binding domain) retains a high degree of sequence homology with other cation transport ATPases. Single, non conservative mutations of homologous residues in this segment produces enzyme inhibition (Zhang et al., 1995). We have now produced single and multiple mutations of non-homologous residues in this segment of the Ca2+ ATPase to match the corresponding residues of the Na+, K+ ATPase. We find that the main characteristics of the ATPase mechanism (i.e., Ca2+ dependent phosphoenzyme formation and thapsigargin sensitivity) are retained even when the entire 41-amino acid (Thr316-Leu356) segment of the Ca2+ ATPase is rendered identical to the corresponding segment of the Na+, K+ ATPase by sequential mutations of the 14 non-homologous amino acids. However, the phosphoenzyme turnover (likely rate limited by the "Ca2.E1-P-->Ca.E2-P transition") is progressively reduced if four or more Ca2+ ATPase residues are mutated to the corresponding residues of the Na+, K+ ATPase. The time course of enzyme inactivation by EGTA (likely rate limited by the "E1 to E2 transition") is also prolonged. Our findings suggest that an analogous peptide segment provides a functional linkage for energy transduction between phosphorylation and cation binding domains in various cation transport ATPase. However, its kinetic influence on rate-limiting conformational transitions is dependent on matching specific structures in each ATPase.

Adenosine Triphosphate↗
PubMed1996

Short and long range functions of amino acids in the transmembrane region of the sarcoplasmic reticulum ATPase. A mutational study.

Mutational analysis of several amino acids in the transmembrane region of the sarcoplasmic reticulum ATPase was performed by expressing wild type ATPase and 32 site-directed mutants in COS-1 cells followed by functional characterization of the microsomal fraction. Four different phenotype characteristics were observed in the mutants: (a) functions similar to those sustained by the wild type ATPase; (b) Ca2+ transport inhibited to a greater extent than ATPase hydrolytic activity; (c) inhibition of transport and hydrolytic activity in the presence of high levels of phosphorylated enzyme intermediate; and (d) total inhibition of ATP utilization by the enzyme while retaining the ability to form phosphoenzyme by utilization of P(i). Analysis of experimental observations and molecular models revealed short and long range functions of several amino acids within the transmembrane region. Short range functions include: (a) direct involvement of five amino acids in Ca2+ binding within a channel formed by clustered transmembrane helices M4, M5, M6, and M8; (b) roles of several amino acids in structural stabilization of the helical cluster for optimal channel function; and (c) a specific role of Lys297 in sealing the distal end of the channel, suggesting that the M4 helix rotates to allow vectorial flux of Ca2+ upon enzyme phosphorylation. Long range functions are related to the influence of several transmembrane amino acids on phosphorylation reactions with ATP or P(i), transmitted to the extramembranous region of the ATPase in the presence or in the absence of Ca2+.

Amino Acid Sequence↗
PubMed1995

Mutational analysis of the peptide segment linking phosphorylation and Ca(2+)-binding domains in the sarcoplasmic reticulum Ca(2+)-ATPase.

The sarcoplasmic reticulum ATPase segment extending from the phosphorylation site (Asp-351) to the preceding transmembrane helix M4 (which is involved in Ca2+ binding in conjunction with transmembrane helices M5, M6, and M8) retains a marked sequence homology to the corresponding segments of other cation ATPases. We made 26 point mutations in this segment and found that nonconservative mutations of residues that are homologous in various cation ATPases result in strong inhibition of catalytic and transport function. Mutations of nonhomologous residues to match the corresponding residues of other cation ATPases are not inhibitory and, in some cases, produce higher activity. The inhibitory mutations affect the phosphorylated intermediate turnover, which is associated with the vectorial translocation of bound Ca2+. The same mutations do not affect the kinetics of ATPase activation by Ca2+ following enzyme preincubation with EGTA. This suggests that activation of the phosphoryl transfer reaction by Ca2+ binding and vectorial displacement of bound Ca2+ by enzyme phosphorylation do not occur simply as the forward and reverse directions of the same process, but are linked to distinct structural features of the enzyme. The peptide segment extending from the phosphorylation site in the enzyme extramembranous headpiece through the M4 helix in the membrane-bound region sustains a prominent role in transmission of the phosphorylation signal for displacement of bound Ca2+. A critical structural role of this segment is also demonstrated by the interference of specific mutations with membrane assembly of the expressed protein.

Animals↗
PubMed1995

Direct involvement of intracellular Ca2+ transport ATPase in the development of thapsigargin resistance by Chinese hamster lung fibroblasts.

Thapsigargin (TG), a specific inhibitor of intracellular Ca2+ transport ATPases (SERCA), inhibits cell proliferation when added to culture media in the nanomolar concentration range. However, long term exposure to gradually increasing concentrations of TG induces resistance to TG inhibition in both the parental Chinese hamster lung fibroblast DC-3F and a subline derived from it via transfection and stable expression of a full-length cDNA encoding avian SERCA1 ATPase (DC-3F/Ca cells). TG resistance develops in parallel with selection of cells expressing higher levels of the endogenous SERCA2 as well as of the exogenous transfected SERCA1 ATPase, whose Ca2+ transport function can be studied in situ by imaging techniques and following isolation in microsomal fractions. Microsomes isolated from resistant cells contain two functionally distinct populations of ATPases: a population that is inhibited by stoichiometric titration with TG, and a population displaying resistance to inhibition even when TG exceeds the enzyme stoichiometry. It is apparent that resistance to TG develops in parallel with (a) selection of cells expressing high levels of SERCA ATPases, and (b) selection of an ATPase that is resistant to TG.

Animals↗
PubMed1995

Synthesis and interaction of fluorescent thapsigargin derivatives with the sarcoplasmic reticulum ATPase membrane-bound region.

Fluorescent derivatives of thapsigargin (TG) were synthesized by replacing the C8-butanoyl chain with a dansyl (DTG) or eosin (ETG) moiety. DTG and ETG retain the inhibitory effect of TG on the sarcoplasmic reticulum (SR) ATPase, displaying a 2 and 10 microM Ki, respectively. Steady state and lifetime fluorescence measurements are consistent with energy transfer between tryptophanyl residues assigned to the ATPase membrane-bound region and DTG. This phenomenon exhibits saturation behavior, occurs in the presence of DTG concentrations producing ATPase inhibition, and is partially prevented by inhibitory concentrations of TG. Although long range conformational effects of TG binding affect the fluorescence properties of endogenous tryptophans as well as of a fluorescein 5'-isothiocyanate (FITC) label of the ATPase extramembranous region, no significant energy transfer was detected between DTG and the FITC label. It is concluded that the inhibitors partition within the membrane and the binding domain resides within or near the membrane-bound region of the ATPase.

Animals↗
PubMed1995

Variable stoichiometric efficiency of Ca2+ and Sr2+ transport by the sarcoplasmic reticulum ATPase.

In comparative experiments with Ca2+ ATPase in native sarcoplasmic reticulum vesicles and reconstituted proteoliposomes, we find that a variable stoichiometry of Ca2+ or Sr2+ transport per ATPase cycle is observed in the absence of passive leak through independent channels. The observed ratio is commonly lower than the optimal value of 2 and depends on the composition of the reaction mixture. In all cases, a progressive rise in the lumenal concentration of Ca2+ and Sr2+ is accompanied by a parallel reduction of coupling ratios. Significant ATPase activity remains even after asymptotic levels of Ca2+ accumulation are reached. This residual activity subsides if the Ca2+ concentration in the outer medium is reduced below activating levels (as it would following Ca2+ transients in muscle fibers). The reduction of stoichiometric coupling is explained with a reaction scheme, including a branched pathway for hydrolytic cleavage of phosphorylated intermediate before release of Ca2+ into the lumen of the vesicles. Flux through this pathway is favored when net lumenal Ca2+ dissociation from the phosphoenzyme is impeded and results in P(i) production accompanied by lumenal and medium Ca2+ exchange. Occurrence of reactions through branched pathways may have general implications for the stoichiometric efficiency of energy-transducing enzymes.

Adenosine Triphosphatases↗
PubMed1995

Ca2+ binding and translocation by the sarcoplasmic reticulum ATPase: functional and structural considerations.

Three experimental systems are described including sarcoplasmic reticulum (SR) vesicles, reconstituted proteoliposomes, and recombinant protein obtained by gene transfer and expression in foreign cells. It is shown that the Ca(2+) ATPase of sarcoplasmic reticulum (SR) includes an extramembranous globular head which is connected through a stalk to a membrane bound region. Cooperative binding of two calcium ions occurs sequentially, within a channel formed by four clustered helices within the membrane bound region. Destabilization of the helical cluster is produced following enzyme phosphorylation by ATP at the catalytic site in the extramembranous region. The affinity and orientation of the Ca2+ binding site are thereby changed, permitting vectorial dissociation of bound Ca2+ against a concentration gradient. A long range linkage between phosphorylation and Ca2+ binding sites is provided by an intervening peptide segment that retains high homology in cation transport ATPases, and whose function is highly sensitive to mutational perturbations.

Animals↗
PubMed1995

Use of thapsigargin to study Ca2+ homeostasis in cardiac cells.

Several reports have documented that thapsigargin is a potent inhibitor of the SR Ca2+ ATPase isolated from cardiac or skeletal muscle. We have characterized the specificity of this agent in intact rat cardiac myocytes using cells maintained in the whole cell voltage clamp configuration. We have shown that thapsigargin decreases the magnitude of the Ca2+ transient and the twitch by about 80% while it slows the decay rate for these responses. These changes were not accompanied by any alterations in sarcolemmal currents or in the trigger Ca2+ generated by the inward calcium current. Taken together these results reveal that the action of thapsigargin is restricted to the SR Ca2+ ATPase in intact cardiac myocytes. Furthermore, it is demonstrated unambiguously that SR intracellular Ca2+ stores are an absolute requirement for the development of contractile tension in rat heart myocytes. It is shown that thapsigargin is a valuable probe to examine the importance of SR pools of Ca2+ and the role of the Ca2+ ATPase in intact myocytes as well as in genetically altered heart cells.

Animals↗
PubMed1994

A pK change of acidic residues contributes to cation countertransport in the Ca-ATPase of sarcoplasmic reticulum. Role of H+ in Ca(2+)-ATPase countertransport.

Proteoliposomal vesicles reconstituted with sarcoplasmic reticulum ATPase and exogenous lipids sustain ATP-dependent Ca2+ uptake and H+ ejection, as well as net charge displacement by Ca2+. We have studied the effect of lumenal (inner) and medium (extravesicular) pH variations on the countertransport ratios of H+ and Ca2+. We find that the Ca2+/H+ molar ratio is approximately 1 when the lumenal and medium pH is near neutrality, but changes with a specific pattern when the medium pH is varied in the presence of a constant lumenal pH and when the lumenal pH is varied in the presence of a constant medium pH. Empirical analysis of the experimental data shows that the apparent pK of the residue(s) releasing H+ into the medium is approximately 6.1, whereas the apparent pK of the residue(s) binding lumenal H+ is approximately 7.7. Assuming that the same acidic residues are involved in H+ and Ca2+ countertransport, our findings suggest a lower affinity for H+ in their outward orientation (prevalent in the ground state of the enzyme) and a higher affinity for H+ in lumenal orientation (prevalent in the phosphorylated state of the enzyme). Cyclic pK changes, coupled to ATP utilization, promote cation exchange, Ca2+ uptake, and H+ ejection by the vesicles. The stoichiometry of countertransport and net charge displacement is matched by a corresponding electrogenic behavior. A calculation of voltage development related to initial rates of charge transfer (dV/dt = (dQ/dt)/Cm) is given as a corrective replacement of a previous steady state calculation.

Adenosine Triphosphate↗
PubMed1994

Ca2+/H+ countertransport and electrogenicity in proteoliposomes containing erythrocyte plasma membrane Ca-ATPase and exogenous lipids.

A reconstituted proteoliposomal system was obtained with Ca-ATPase purified from human erythrocyte membrane (plasma membrane, PM ATPase), and liposomes prepared by reverse-phase evaporation. The reconstituted PM ATPase behaved as an electrogenic Ca2+/H+ exchanger and, under optimal conditions, utilization of 1 mol of ATP was accompanied by uptake of one Ca2+ by the vesicles, and ejection of one H+ from the lumen of the vesicles. Ca2+ uptake was greatly (5-fold) stimulated by the addition of calmodulin, and by collapsing the H+ gradient with the ionophore carbonyl cyanide p-trifluoromethoxyphenylhydrazone. In the presence of calmodulin and p-trifluoromethoxyphenylhydrazone, the reconstituted system sustained transport rates of 1.00 +/- 0.12 mumol of Ca2+/mg of protein min-1 (30 degrees C), reaching asymptotic levels of 8.05 +/- 0.41 mumol of Ca2+/mg of protein (i.e. 20 mM lumenal Ca2+). The corresponding net charge transfer produced a maximal electrical gradient of 40.5 +/- 1.8 mV at steady state. Demonstration of the electrogenic behavior of the PM ATPase, obtained for the first time with these experiments, was critically dependent on the detergent used in the reconstitution procedure. The lumenal pH rise had a much greater rate-limiting effect on the pump, than the electrical potential developed by the pump.

Adenosine Triphosphate↗
PubMed1994

Intracellular signaling through long-range linked functions in the Ca2+ transport ATPase.

The Ca2+ transport ATPases of intracellular membranes exhibit an intracellular long-range functional linkage which is the basic mechanistic device for Ca2+ transport through ATP utilization. The functional linkage operates between a phosphorylation (catalytic) domain located in the extramembranous region, and a Ca2+ binding domain located in the membrane bound region of the enzyme. The two domains are separated by a distance of approximately 50 A, and are both affected by binding of a single molecule of the highly specific inhibitor, thapsigargin, to the enzyme. Functional and structural features are here described to explain the long-range linkage through the protein structure.

Amino Acid Sequence↗
PubMed1994

Teaching active transport at the turn of the twenty-first century: recent discoveries and conceptual changes.

The conceptual advances introduced by recent discoveries in the field of active transport have triggered a transition from a "black box" approach to a "mechanistic" approach. At present, treating this subject in the graduate setting requires consideration of equilibrium and kinetic experimentation, protein chemistry, mutational analysis and molecular structure, with the aim of defining the "transport machine."

Animals↗