The role of Ca2+-ATpase and its hydrophobic component in the release of Ca2+ from skeletal muscle sarcoplasmic reticulum

The Ca2+ permeability of proteoliposomes containing Ca2+-ATPase of sarcoplasmic reticulum and its hydrophobic fragment was investigated, using the method of synthetic penetrant ions and the radioisotopic method. The former method was used to determine the diffusional membrane potential formed by Ca2+ concentration gradient. It was demonstrated that Ca2+-ATPase, whose active center is oriented outside, has and asymmetric conductivity, i. e., it facilitates the rapid efflux of Ca2+ from proteoliposomes. This efflux is stimulated by the membrane potential positive inside. The hydrophobic fragment of Ca2+-ATPase forms a Ca2+-channel with a high conductivity for Ca2+. This channel is responsible for the Ca2+ efflux from sarcoplasmic reticulum.     

A Ca2+-binding domain in RyR1 that interacts with the calmodulin binding site and modulates channel activity

A fragment of RyR1 (amino acids 4064-4210) is predicted to fold to at least one lobe of calmodulin and to bind Ca(2+). This fragment of RyR1 (R4064-4210) was subcloned, expressed, refolded, and purified. Consistent with the predicted folding pattern, R4064-4210 was found to bind two molecules of Ca(2+) and undergo a structural change upon binding Ca(2+) that exposes hydrophobic amino acids. R4064-4210 also binds to RyR1, the L-type Ca(2+) channel (Cav(1.1)), and several synthetic calmodulin binding peptides. Both R4064-4210 and a peptide representing the calmodulin-binding region of RyR1 (R3614-3643) alter the Ca(2+) dependence of ((3)H)ryanodine binding to RyR1, suggesting that they may both be interfering with an intramolecular interaction between amino acids 4064-4210 and amino acids 3614-3643 in the native RyR1 to alter or regulate the response of the channel to changes in Ca(2+) concentration. The finding that a domain within RyR1 binds Ca(2+) and interacts with calmodulin-binding motifs may provide insights into the mechanism for calcium- and calmodulin-dependent regulation of this channel and perhaps for its regulation by the L-type Ca(2+) channel.     

Effect of divalent cations on the ATPase activity of Escherichia coli SecA

It was found that Ca(2+) stimulates the intrinsic SecA ATPase activity in the absence as well as in the presence of liposome. On the other hand, Mg(2+), the general cofactor for ATPase, did not affect the intrinsic SecA ATPase but reduced the portion of ATPase activity enhanced by Ca(2+). The enhancement of SecA ATPase activity correlated well with the increase in 8-anilino-1-naphthalene-sulfonic acid binding of SecA, suggesting that increased exposure of hydrophobic residues stimulates the enzyme activity.     

Calmodulin-sensitive calcium-pumping ATPase of plasma membranes: isolation, reconstitution, and regulation

The calmodulin-sensitive Ca2+ -pumping ATPase was purified to virtual homogeneity from erythrocytes. The purified enzyme exists in two functional states, having low and high Ca2+ affinity. Transition from low to high affinity is induced by 1) calmodulin; 2) acidic phospholipids, long-chain polyunsaturated fatty acids, polyphosphoinositides; and 3) a controlled proteolytic treatment with trypsin or chymotrypsin. The ATPase can be reconstituted into liposomes, where it pumps Ca2+ in exchange for H+ with a stoichiometry to ATP approaching 1. The purified enzyme can be fragmented by trypsin into a number of transient and of limit polypeptides, of which the most interesting from the functional standpoint are the following: 1) a limit polypeptide of Mr 76,000 that contains the active site (i.e., the sequence where the acyl-phosphate is formed); 2) a limit polypeptide of Mr 33,500 that binds the hydrophobic photoactivable label 3-trifluoromethyl-3-(m-(125I-iodophenyl]-diazirine, and is thus presumably the most hydrophobic portion of the molecule; and 3) a transient polypeptide of Mr 90,000 and a limit polypeptide of Mr 25,000-28,000, which specifically bind azido-modified, 125 I-labeled calmodulin. The transient 90,000-dalton calmodulin receptor is rapidly degraded to the 81,000-76,000 limit polypeptide. It can be isolated from the other proteolysis products on calmodulin affinity chromatography columns. The isolated 90,000-dalton fragment is a fully competent, calmodulin-sensitive ATPase that pumps Ca2+ into reconstituted liposomes.     

Stimulation of the ATPase activity of rat brain protein kinase C by phospho acceptor substrates of the enzyme

We recently reported that autophosphorylated rat brain protein kinase C (PKC) catalyzes a Ca2(+)- and phosphatidylserine- (PS-) dependent ATPase reaction. The Ca2(+)- and PS-dependent ATPase and histone kinase reactions of PKC each had a Km app(ATP) of 6 microM. Remarkably, the catalytic fragment of PKC lacked detectable ATPase activity. In this paper, we show that subsaturating concentrations of protein substrates accelerate the ATPase reaction catalyzed by PKC and that protein and peptide substrates of PKC induce ATPase catalysis by the catalytic fragment. At subsaturating concentrations, histone III-S and protamine sulfate each accelerated the ATPase activity of PKC in the presence of Ca2+ and PS by as much as 1.5-fold. At saturating concentrations, the protein substrates were inhibitory. Poly(L-lysine) failed to accelerate the ATPase activity, indicating that the acceleration observed with histone III-S and protamine sulfate was not simply a result of their gross physical properties. Furthermore, histone III-S induced the ATPase activity of the catalytic fragment of PKC, at both subsaturating and saturating histone concentrations. The induction of ATPase activity was also elicited by the peptide substrate Arg-Arg-Lys-Ala-Ser-Gly-Pro-Pro-Val, when the peptide was present at concentrations near its Km app. The induction of the ATPase activity by the nonapeptide provides strong evidence that the binding of phospho acceptor substrates to the active site of PKC can stimulate ATP hydrolysis. Taken together, our results indicate that PKC-catalyzed protein phosphorylation is inefficient, since it is accompanied by Pi production.(ABSTRACT TRUNCATED AT 250 WORDS)     

Tentoxin-induced energy-independent adenine nucleotide exchange and ATPase activity with chloroplast coupling factor 1.

1. The effect of energy transfer inhibitors on energy-dependent exchange of tightly bound adenine nucleotides with washed, broken spinach thylakoids has been studied. Energy transfer inhibitors that inhibit the ATPase activity of soluble chloroplast coupling factor 1 (CF1) (e.g. phloridzin and tentoxin) do not inhibit energy-dependent adenine nucleotide exchange. Energy transfer inhibitors that block proton flux through the hydrophobic protein proton channel (CF0) (e.g. dicyclohexylcarbodiimide and triphenyltin chloride) also block light-dependent adenine nucleotide exchange. 2. Tentoxin, at relatively high concentrations, stimulates an energy-independent exchange of adenosine diphosphate. 3. High concentrations of tentoxin elicit a Ca2+-dependent ATPase activity with soluble CF1, but has no effect on the Ca2+-dependent ATPase activity of membrane-bound CF1. 4. The trypsin-activated, Ca2+-dependent, membrane-bound ATPase is not affected by high concentrations of tentoxin, whereas the dithiothreitol-activated, Mg2+-dependent ATPase is markedly inhibited. 5. The reconstitution of chloroplasts, partially depleted in CF1, with soluble CF1 is correlated with the loss of tentoxin-induced, Ca2+-dependent ATPase activity associated with soluble CF1.     

Evidence against involvement of the human erythrocyte plasma membrane Ca2+-ATPase in Ca2+-dependent K+ transport

Two tests were performed to assess the relationship between the Ca2+-activated K+ channel and the Ca2+-pumping ATPase in human erythrocytes. Antibodies against the purified ATPase inhibited the ATPase in resealed erythrocytes, but had no effect on the K+ channel (as assessed by Rb+ efflux). Reconstituted liposomes containing the purified active Ca2+-pumping ATPase showed no Ca2+-activated Rb+ influx. Both of these results suggest that some molecule other than the Ca2+-ATPase is responsible for the K+ channel.     

Isolation and characterization of tryptic fragments of the adenosine triphosphatase of sarcoplasmic reticulum.

Exposure of sarcoplasmic reticulum to trypsin in the presence of 1 M sucrose results in degradation of the Mr = 102,000 ATPase enzyme to two fragments of Mr = 55,000 and 45,000 with subsequent appearance of fragments of Mr = 30,000 and 20,000. These fragments were purified by column chromatography in sodium dodecyl sulfate. Antibodies were raised against the ATPase and the Mr = 55,000, 45,000, and 20,000 fragments. There was no antigenic cross-reactivity between the Mr = 55,000 and 45,000 fragments, indicating that they were derived from a single linear cleavage of the larger enzyme. There was antigenic cross-reactivity between the Mr = 20,000 and 55,000 fragments, indicating an origin of the Mr = 20,000 fragment in the Mr = 55,000 fragment. None of the antibodies inhibited (Ca2+ + Mg2+)-dependent ATPase or Ca2+ transport. The Mr = 20,000 fragment and the Mr = 55,000 fragment were active in Ca2+ ionophore assays. The active site of ATP hydrolysis was labeled with [gamma-32P]ATP and the site of ATP binding was labeled with tritiated N-ethylmaleimide. In both cases radioactivity was found in the intact ATPase and in the Mr = 55,000 and 30,000 fragments, indicating that the Mr = 30,000 fragment was also derived from the Mr = 55,000 fragment. Amino acid composition data showed that the Mr = 45,000 fragment contained about 60% nonpolar and 40% polar amino acids, while the Mr = 55,000 fragment and the Mr = 20,0000 fragment contained about equal amounts of polar and nonpolar amino acids. Studies of the reaction of various antibodies at the external surface of sarcoplasmic reticulum vesicles showed that the ATPase was exposed, whereas calsequestrin and the high affinity Ca2+-binding protein were not. The use of antibodies against the various fragments indicated that the Mr = 55,000 fragment was in large part exposed, whereas the Mr = 20,000 and the 45,000 fragments were only poorly exposed. It is probable that the site of ATP hydrolysis in the Mr = 55,000 fragment is external, whereas the ionophore site is only partially exposed and the Mr = 45,000 fragment is largely buried within the membrane.     

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+.     

The effect of antibodies against Ca2(+)-ATPase and its hydrophobic fragment on the activity of this enzyme and on the passive transport of Ca2(+)]

It is shown that both Ca(2+)-ATPase and its hydrophobic fragment are immunogenic factors. A region between hydrophobic and hydrophilic parts of Ca(2+)-ATP-ase is immunogenic. These antibodies clearly inhibit inflow and outflow of Ca2+ through a hydrophobic fragment reconstructed into liposomes and do not influence Ca(2+)-ATPase activity.     

Replacement of troponin components in myofibrils.

The Ca(2+)-sensitive ATPase activity of rabbit skeletal myofibrils was desensitized by treatment with excess troponin T and was found to be activated irrespective of the Ca2+ concentrations. A SDS-gel electrophoretic study showed that both troponin C and troponin I were removed from the myofibrils on treatment with troponin T. The Ca(2+)- and Sr(2+)- sensitivities of the ATPase of troponin T-treated myofibrils reconstituted with troponin C. I were the same as in the intact myofibrils. The Ca(2+)-activated ATPase of rabbit skeletal myofibrils was also desensitized on treatment with chicken breast troponin T or its 26K fragment. The SDS-gel electrophoretic study revealed that troponin T, in addition to troponin C and troponin I, was also removed from the myofibrils and, instead, chicken breast troponin T or its 26K fragment was incorporated into the myofibrils. The Ca(2+)- sensitivity of myofibrils treated with chicken breast troponin T or its 26K fragment was then regained on reconstitution with troponin C.I. These findings indicate that the change in composition of myofibrils on treatment with troponin T or its 26K fragment is due to the selective replacement of the troponin C.I.T complex in the myofibrils as a whole with troponin T or its 26K fragment.     

A short hydrophobic segment next to tryptophan-130 in myosin heavy chain is close to the ribose ring of ADP bound in the adenosinetriphosphatase site

The ATPase site of rabbit skeletal myosin was covalently labeled by an ADP analogue that carried a biotin moiety on its adenine ring and a photoreactive phenyl azide group on its ribose ring [Sutoh, K., Yamamoto, K., & Wakabayashi, T. (1986) Proc. Natl. Acad. Sci. U.S.A. 83, 212-216]. The ADP analogue was tightly trapped into the ATPase site in the presence of vanadate ions and then covalently cross-linked to the site by UV irradiation. The N-terminal 23,000-dalton tryptic fragment of the heavy chain was selectively labeled with the analogue. Further mapping of the labeled segment along the 23-kDa fragment was carried out by "end-label fingerprinting" which employed site-directed antibodies against both ends of the N-terminal heavy chain fragment. The mapping revealed that a hydrophobic segment of approximately 10 residues next to Trp-130, which was reported to be in proximity to the adenine ring of ADP bound to the ATPase site [Okamoto, Y., & Yount, R. G. (1985) Proc. Natl. Acad. Sci. U.S.A. 82, 1575-1579], was the site of covalent labeling with the ADP analogue. The result indicates that the hydrophobic segment is close to the ribose ring of ADP bound to the ATPase site.     

Temperature-dependent changes in the profile of the sarcoplasmic reticulum membrane hydrophobic zones]

The dependence of the state of the hydrophobic zone of rabbit sarcoplasmic reticulum (SR) membranes on temperature of the membrane fragment suspension before rapid freezing was studied by the freeze fracturing technique. It was shown that within the temperature range of--15-- +37 degrees C the amount of intramembrane particles and their distribution in the membrane plane and between their convex and concave surfaces do not practically depend on the temperature of the SR membrane suspension. This is indicative of the lack of correlation between the physical state of the phospholipid matrix (gel -- liquid crystal) before freezing and the nature of the profile of the membrane hydrophobic zone revealed after fracturing. The disturbances in the protein -- lipid interactions in the membrane under the effects of mersalyl or aqueous solutions of diethyl ester followed by complete inactivation of Ca2+-dependent ATPase lead to a decrease in the amount of intramembrane particles, which is especially well-pronounced at 37 degrees and -15 degrees C.     

Role of membrane-bound calcium in changes in the ATPase activity, permeability and structural state of human erythrocyte membranes]

A study was conducted on the reconstituted erythrocytes obtained by the method of fast reversible hemolysis. The concentration of free Ca2+ ions in the reconstituted erythrocytes was supported by Ca-EGTA and Ca-nitrate buffers. Oubain-uninhibited ATPase component with a high affinity for Ca2+ (K0.5=4 micron) and alteration of passive and active K+-permeability in the region of free Ca2+ concentration up to 10 micron could be determined only when the content of membrane-bound Ca+ varied. Depletion of the inner side of the membrane of reconstituted erythrocyte is accompanied by alteration of hydrophobic character of the hydrocarbon region of the membrane. It is suggested that Ca+-induced alterations in the structure of the erythrocyte membrane may be a direct cause of the alterations in ATPase activity with a high Ca2+ affinity and permeability for univalent cations.     

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.     

RyR1/SERCA1 cross-talk regulation of calcium transport in heavy sarcoplasmic reticulum vesicles

We investigated the functional interdependence of sarco-endoplasmic reticulum Ca2+ ATPase isoform 1 and ryanodine receptor isoform 1 in heavy sarcoplasmic reticulum membranes by synchronous fluorescence determination of extravesicular Ca2+ transients and catalytic activity. Under conditions of dynamic Ca2+ exchange ATPase catalytic activity was well coordinated to ryanodine receptor activation/inactivation states. Ryanodine-induced activation of Ca2+ release channel leaks also produced marked ATPase activation in the absence of measurable increases in bulk free extravesicular Ca2+. This suggested that Ca2+ pumps are highly sensitive to Ca2+ release channel leak status and potently buffer Ca2+ ions exiting cytoplasmic openings of ryanodine receptors. Conversely, ryanodine receptor activation was dependent on Ca2+-ATPase pump activity. Ryanodine receptor activation by cytosolic Ca2+ was (i) inversely proportional to luminal Ca2+ load and (ii) dependent upon the rate of presentation of cytosolic Ca2+. Progressive Ca2+ filling coincided with progressive loss of Ca2+ sequestration rates and at a threshold loading, ryanodine-induced Ca2+ release produced small transient reversals of catalytic activity. These data indicate that attainment of threshold luminal Ca2+ loads coordinates sensitization of Ca2+ release channels with autogenic inhibition of Ca2+ pumping. This suggests that Ca2+-dependent control of Ca2+ release in intact heavy sarcoplasmic reticulum membranes involves a Ca2+-mediated "cross-talk" between sarco-endoplasmic reticulum Ca2+ ATPase isoform 1 and ryanodine receptor isoform 1.     

ATP steal between cation pumps: a mechanism linking Na+ influx to the onset of necrotic Ca2+ overload

We set out to identify molecular mechanisms underlying the onset of necrotic Ca(2+) overload, triggered in two epithelial cell lines by oxidative stress or metabolic depletion. As reported earlier, the overload was inhibited by extracellular Ca(2+) chelation and the cation channel blocker gadolinium. However, the surface permeability to Ca(2+) was reduced by 60%, thus discarding a role for Ca(2+) channel/carrier activation. Instead, we registered a collapse of the plasma membrane Ca(2+) ATPase (PMCA). Remarkably, inhibition of the Na(+)/K(+) ATPase rescued the PMCA and reverted the Ca(2+) rise. Thermodynamic considerations suggest that the Ca(2+) overload develops when the Na(+)/K(+) ATPase, by virtue of the Na(+) overload, clamps the ATP phosphorylation potential below the minimum required by the PMCA. In addition to providing the mechanism for the onset of Ca(2+) overload, the crosstalk between cation pumps offers a novel explanation for the role of Na(+) in cell death.     

Modulation of erythrocyte Ca2+-ATPase by selective calpain cleavage of the calmodulin-binding domain

The activity of the membrane-bound and the purified erythrocyte Ca2+-ATPase in the absence of calmodulin was stimulated by calpain digestion but could be further increased to maximal levels by calmodulin (CaM). Thus, CaM sensitivity was retained by the digested ATPase, at least at short times of incubation. In membranes digested at higher temperatures and in the purified ATPase digested at higher calpain/ATPase ratios, the ATPase became fully activated. The membrane-bound and the purified 138-kDa ATPase were converted by calpain to a fragment of approximately 124 kDa which still bound CaM and could be isolated on CaM columns when proteolysis occurred slowly but not when it occurred rapidly. Carboxypeptidase digestion of the purified enzyme and of its fragment of about 124 kDa has shown that calpain attacked the CaM-binding domain near the C terminus of the ATPase. This has also been supported by digestion of the purified enzyme and of its fragment of about 124 kDa. A first cut occurred in the middle of the domain producing a fragment of about 14 kDa and a (CaM-binding) fragment of about 124 kDa. A second cut closer to the N terminus of the domain also produced a fragment of about 124 kDa and accounted for the loss of CaM binding at prolonged times of incubation of the ATPase with calpain.     

Rhodopsin and other proteins in artificial lipid membranes.

Some basic aspects of incorporation of hydrophobic peptides and proteins in artificial lipid membranes are discussed. As examples valinomycin as a carrier model and gramicidin A as a channel former in lipid vesicles and in planar lipid membranes are presented. In the second part of the lecture some examples of incorporation of membrane proteins into lipid vesicles and planar lipid membranes are reported. The interaction with artificial lipid membranes of the Ca++ ATPase from the sarcoplasmic reticulum, of Rhodopsin, and of Bacteriorhodopsin is presented.     

Localization of ionophore activity in a 20,000-dalton fragment of the adenosine triphosphatase of Sarcoplasmic reticulum.

The (Ca2+ + Mg2+)-dependent ATPase of sarcoplasmic reticulum has been shown to ast as a Ca2+-dependent and selective ionophore in artificial lipid bilayers. Four fragments of 55,000, 45,000, 30,000, and 20,000 daltons have been purified from tryptic digests of the enzyme and it has been shown that the 55,000- and 45,000-dalton fragments are obtained from a single cleavage of the 100,000-dalton ATPase, while the 30,000- and 20,000-dalton fragments are obtained subsequently by a cleavage of the 55,000-dalton fragment. The 55,000- and 20,000-dalton fragments have ionophore activity inhibited by ruthenium red and by mercuric chloride but not by methylmercuric chloride, an inhibitor of the hydrolytic site of the enzyme. Under standard conditions the 45,000-dalton fragment was not active as an ionophore, while the 30,000-dalton fragment acted as a nonselective ionophore. The 55,000- and 30,000-dalton fragments have been shown to contain the site of phosphorylation and of N-ethyl [2-3H]-maleimide binding indicative of the hydrolytic site in the enzyme, and this site is absent from the 20,000-dalton fragment. Therefore, the ionophoric and hydrolytic sites are localized in separate regions of the ATPase molecule and they have now been physically separated. The 20,000-dalton fragment was degraded with cyanogen bromide and fragments were separated by molecular sieving. Ionophore activity was found in fragments of molecular mass less than 2,000 daltons.