Reactive sulfhydryl groups of sarcoplasmic reticulum ATPase. III. Identification of cysteine residues whose modification with N-ethylmaleimide leads to loss of the Ca2+-transporting activity

The reactive sulfhydryl group (SHD) (Kawakita et al. (1980) J. Biochem. 87, 609-617) which is essential for the decomposition of the E-P intermediate of Ca2+-transporting ATPase of the rabbit skeletal muscle sarcoplasmic reticulum has been identified. One sample of sarcoplasmic reticulum membranes was reacted for 3 min with 0.4 mM N-[3H]ethylmaleimide at pH 7.0 at 30 degrees C to a labeling density of 1 mol/mol ATPase without loss of the Ca2+-transporting activity. Another sample of the membranes was treated similarly with non-radioactive N-ethylmaleimide and then labeled with 0.4 mM N-ethyl[14C]maleimide for 17 min. An extensive loss of the Ca2+-transporting activity occurred during the period of this radio-labeling, thus substantiating the 14C-labeling of SHD. The labeled membranes were digested by thermolysin, and the labeled peptides were fractionated by gel filtration and reversed-phase HPLC. Two major radioactive peptides were present in both 3H- and 14C-labeled thermolytic digests, and each of the major components of 14C-labeled peptides had a counterpart in the major components of 3H-labeled peptides which behaved identically on HPLC. The major 14C-labeled peptides were purified and found to be identical with the two SHN peptides, TL-I and TL-II (Saito-Nakatsuka et al. (1987) J. Biochem. 101, 365-376), and 0.5 mol/mol ATPase each of Cys344 and Cys364 was assigned as SHD. It seems that the Ca2+-transport system retains its activity while either of the two Cys residues is unoccupied, but loses it when both of them are modified with N-ethylmaleimide.     

Studies on conformational transitions of Ca2+, Mg2+-adenosine triphosphatase of sarcoplasmic reticulum. I. Selective labeling of functionally distinct sulfhydryl groups with conformational probes and evidence for a Ca2+-dependent conformational change

Several maleimide derivatives of potential usefulness as conformational probes were tested for reactivity toward SH groups of Ca2+, Mg2+-ATPase of sarcoplasmic reticulum. These include three fluorescent labels, N-(1-anilinonaphthyl-4)maleimide (ANM), N-(p-(2-benzimidazolyl)phenyl)maleimide (BIPM), and N-(7-dimethylamino-4-methyl-3-coumarinyl)maleimide (DACM), and a spin label, 4-maleimido-2,2,6,6-tetramethylpiperidinooxyl (MSL). These reagents also exhibit a selective reactivity toward SH groups which is similar to that of N-ethylmaleimide, although these conformational probes were somewhat more reactive than N-ethylmaleimide. Based on the above finding, procedures were devised to specifically label either one of two reactive SH groups of the ATPase, namely one highly reactive but functionally nonessential (SHN) and the other, essential for the decomposition of the E-P intermediate (SHD) [Kawakita, M., et al. (1980) J. Biochem. 87, 609-617], with any one of these conformational probes. Sarcoplasmic reticulum membranes labeled with ANM at either SHN or SHD showed a characteristic fluorescence whose intensity reversibly changed in response to the removal and readdition of Ca2+ ions in the range of 10(-6) to 10(-7) M. The change could be ascribed to a conformational change of the ATPase in response to dissociation and association of Ca2+ ions at the transport site. The Ca2+-dependent fluorescence change was quantitatively different, depending on whether the ATPase was labeled at SHN or SHD. Moreover, it was probe-specific in that BIPM and DACM fluorescence did not change in response to Ca2+. The possible significance of these observations is discussed.     

Studies on conformational transitions of Ca2+,Mg2+-adenosine triphosphatase of sarcoplasmic reticulum. II. Conformational characteristics of stabilized reaction intermediates as revealed by fluorescent and paramagnetic probes

Various reaction intermediates of sarcoplasmic reticulum Ca2+,Mg2+-ATPase were stabilized and accumulated by modifying a specific SH group or by using nucleotide analogs. Conformational changes of the Ca2+,Mg2+-ATPase during the catalytic cycle were studied in the stabilized intermediates by the use of fluorescent and spin probes, which were introduced at specific SH groups of ATPase, namely one highly reactive but functionally nonessential (SHN) and one essential for the decomposition of the E-P intermediate (SHD) [Kawakita, M., et al. (1980) J. Biochem. 87, 609-617]. The fluorescence intensity of N-(7-dimethylamino-4-methyl-3-coumarinyl)maleimide attached to SHD decreased by 2.5% upon addition of 10 microM AMP-P(NH)P provided that Ca2+ was also present. The AMP-P(NH)P-induced fluorescence change could also be detected by using other fluorescent probes such as N-[p-(2-benzimidazolyl)phenyl]maleimide and N-(1-anilinonaphthyl-4)maleimide. Moreover, labeling at SHN gave similar results. When SHN was labeled with N-[p-(2-benzimidazolyl)phenyl]maleimide, the fluorescence intensity also decreased by 2.5% upon addition of ATP only in the presence of Ca2+, where E-P formation took place. A conformational difference between ECa1-P X ADP and ECa1-P was suggested from saturation transfer ESR measurement of spin-labeled ATPase by using ADP beta S as an ADP analog to cause accumulation of ECa1-P X ADP beta S complex. Possible structural similarities among some of the intermediates are discussed based on these findings.     

Determination of the primary structure of intermolecular cross-linking sites on the Ca2(+)-ATPase of sarcoplasmic reticulum using 14C-labeled N,N'-(1,4-phenylene)bismaleimide or N-ethylmaleimide

To determine the intermolecular cross-linking site on the primary structure sarcoplasmic reticulum (SR) Ca-ATPase, the conditions for the specific binding of 14C-labeled 1,4-phenylene bis maleimide (PBM) or 14C-labeled N-ethylmaleimide (NEM) to the ATPase were explored. SR vesicles were preincubated with nonradioactive PBM in the presence of 1 mM vanadate for 1 h, then washed by centrifugation to remove free PBM and vanadate. When the pretreated SR vesicles were allowed to react with 1 mM [14C]PBM in the presence of 1 mM AMPPNP, the amount of [14C]PBM incorporated into the ATPase increased with time in parallel with the formation of dimeric ATPase and reached the maximum labeling density of 1 mol of [14C]PBM per mol of dimeric ATPase at 40 min after the start of the reaction. When the pretreated SR vesicles were allowed to react with 2 mM [14C]NEM in the absence of AMPPNP, a maximum of about 2 mol of NEM was bound per mol of the ATPase monomer. The labeling density of [14C]NEM decreased from 2 to 1 mol per mol of the ATPase when the SR vesicles were allowed to react with [14C]NEM in the presence of AMPPNP. From the analysis of the amino acid composition of the two major [14C]NEM-labeled peptides isolated from the thermolytic digest of the enzyme after the reaction of SR with [14C]NEM in the absence of AMPPNP, we deduced that [14C]NEM was incorporated into Cys377 and Cys614. On the other hand, the labeling of SR in the presence of AMPPNP resulted in inhibition of the [14C]NEM binding to Cys614, leaving Cys377 unaltered.(ABSTRACT TRUNCATED AT 250 WORDS)     

Reactive sulfhydryl groups of sarcoplasmic reticulum ATPase. II. Site of labeling with iodoacetamide and its fluorescent derivative

Iodoacetamide (IAA) and its fluorescent derivative, 5-(2-iodoacetamidoethyl) amino-naphthalene-1-sulfonate (IAEDANS) specifically bind to a site on the C-terminal half of sarcoplasmic reticulum (SR) Ca2+,Mg2+-ATPase. The location of this specific binding site was identified. SR membranes were treated with 150 microM [14C]IAA at pH 7.0 and 30 degrees C. One mole of IAA per mole of ATPase was bound in 6 h without affecting the Ca2+-transport activity. [14C]IAA-labeled SR membranes were cleaved with BrCN, and 14C-labeled peptide fragments were separated by Sephadex LH-60 chromatography and then digested further with trypsin. A radioactive peptide (Ala-Cys 674-Cys-Phe-Ala-Arg) was purified by Sephadex LH-20 chromatography and C18 reversed phase HPLC (Cys denotes the [14C]IAA-binding site). IAEDANS-labeling was carried out by reacting SR membranes with 50 microM IAEDANS for 5 h, at pH 7.0 and 30 degrees C. A fluorescent peptide was successfully purified by the same procedures as for the IAA-labeled peptide, and the amino acid sequence analysis of this peptide revealed that the IAEDANS labeling site was identical with the IAA binding site.     

Chemical modification of sarcoplasmic reticulum. Stimulation of Ca2+ release.

Pretreatment of sarcoplasmic membranes with acetic or maleic anhydrides, which interact principally with amino groups, resulted in an inhibition of Ca2+ accumulation and ATPase activity. The presence of ATP, ADP or adenosine 5'-[beta, gamma-imido]triphosphate in the modification medium selectively protected against the inactivation of ATPase activity by the anhydride but did not protect against the inhibition of Ca2+ accumulation. Acetic anhydride modification in the presence of ATP appeared to increase specifically the permeability of the sarcoplasmic reticulum membrane to Ca2+ but not to sucrose, Tris, Na+ or Pi. The chemical modification stimulated a rapid release of Ca2+ from sarcoplasmic reticulum vesicles passively or actively loaded with calcium, from liposomes reconstituted with the partially purified ATPase fraction but not from those reconstituted with the purified ATPase. The inactivation of Ca2+ accumulation by acetic anhydride (in the presence of ATP) was rapid and strongly pH-dependent with an estimated pK value above 8.3 for the reactive group(s). The negatively charged reagents pyridoxal 5-phosphate and trinitrobenzene-sulphonate, which also interact with amino groups, did not stimulate Ca2+ release. Since these reagents do not penetrate the sarcoplasmic reticulum membranes, it is proposed that Ca2+ release is promoted by modification of internally located, positively charged amino group(s).     

Oxidation of reactive sulfhydryl groups of sarcoplasmic reticulum ATPase

The role of reactive sulfhydryl groups of sarcoplasmic reticulum ATPase has been investigated. Incubation of ATPase with 17 mol o-iodosobenzoic acid per mol ATPase results in a 15% inhibition of Ca2+ uptake with only a 5% loss of ATPase activity. When ATPase is treated with 15 mol KMnO4 per mol ATPase, Ca2+ uptake is completely inhibited. From the measurement of remaining SH groups using 5,5'-dithiobis-(2-nitrobenzoic acid), it is found that the oxidation of approximately four SH groups per ATPase molecule with KMnO4 leads to a complete loss of Ca2+ uptake, while the oxidation of five SH groups per ATPase with o-iodosobenzoic acid results in only 15% inhibition of Ca2+ uptake. The results of amino acid analysis indicate that KMnO4 oxidizes the reactive SH groups to sulfonic acid groups. Among the five o-iodosobenzoic acid-reactive SH groups, at least one shows a distinct Ca2+ dependence. Addition of o-iodosobenzoic acid to the reaction medium containing KMnO4 does not increase the number of oxidized SH groups, indicating that both o-iodosobenzoic acid and KMnO4 oxidize the same SH groups of the enzyme. The different effects of two oxidizing agents on sarcoplasmic reticulum ATPase eliminate the possibility of direct involvement of SH group(s) in the ATPase reaction.     

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

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

Chemical modification of an arginine residue in the ATP-binding site of Ca2+-transporting ATPase of sarcoplasmic reticulum by phenylglyoxal

Phenylglyoxal (PGO) was used as a reagent for chemical modification of the ATP-binding site of Ca2+-transporting ATPase of rabbit skeletal muscle sarcoplasmic reticulum (SR-ATPase). When 1 mM PGO was reacted with SR-ATPase at 30°C at pH 8.5, PGO was bound to the ATPase molecule in two-to-one stoichiometry with concomitant loss of activity of the ATPase to form the phosphorylated intermediate (E-P). ATP and ADP prevented the binding of PGO and thereby protected the enzyme from inactivation. The SR membranes were labeled with [14C]PGO and then digested with pepsin to identify the attachment site of PGO. A 14C-labeled peptide (402lle-Arg*-Ser-Gly-Gln406) was purified to homogeneity by C18-reversed phase HPLC (Arg* denotes the binding site of [14C]PGO). These results indicate that Arg403 is located in the ATP binding site of the SR-ATPase.     

The NH2 terminus of the (Ca2+ + Mg2+)-adenosine triphosphatase is located on the cytoplasmic surface of the sarcoplasmic reticulum membrane

The (Ca2+ + Mg2+)-adenosine triphosphatase (ATPase) of sarcoplasmic reticulum contains a cysteine residue at position 12 of its sequence. This sulfhydryl group was 1 out of a total of 10-11 that were labeled by treatment of sarcoplasmic reticulum vesicles with N-[3H]ethylmaleimide under saturating conditions. This was shown by isolating a 31-residue NH2-terminal peptide from a tryptic digest of the succinylated ATPase, prepared from N-[3H]ethylmaleimide-labeled vesicles. Reaction of the vesicles with glutathione maleimide, parachloromercuribenzoic acid, or parachloromercuriphenyl sulfonic acid, membrane-impermeant reagents, prevented further reaction of sulfhydryl groups with N-ethylmaleimide. This result indicates that all sulfhydryl groups that are reactive with N-ethylmaleimide are on the outside of the vesicles. Since Cys12 is located in a hydrophilic NH2-terminal portion of the ATPase, the labeling results suggest that the NH2 terminus of the ATPase is on the cytoplasmic side of the membrane. These results are consistent with earlier observations (Reithmeier, R. A. F., de Leon, S., and MacLennan, D. H. (1980) J. Biol. Chem. 255, 11839-11846) that the (Ca2+ + Mg2+)-ATPase is synthesized without an NH2-terminal signal sequence.     

Effects of sulfhydryl reagents and other inhibitors on Ca2+ transport and inositol trisphosphate-induced Ca2+ release from human platelet membranes

The effects of modifiers of Ca2+ uptake and release in sarcoplasmic reticulum were studied in human platelet membranes. AgNO3,p-chloromercuribenzoate (pClHgBzO), N-ethylmaleimide (MalNEt), quercetin, vanadate, A23187, and caffeine all had the same effects on Ca2+ uptake in platelet membranes as had been observed for sarcoplasmic reticulum. These results strengthen our earlier conclusion that the Ca2+-pump proteins from internal human platelet membranes and muscle sarcoplasmic reticulum are very similar in functional properties. The sulfhydryl reagents Ag+ and pClHgBzO elicited rapid release of Ca2+ from platelet membranes in the presence of ATP, whereas MalNEt induced slow release. Quercetin also caused slow release of Ca2+ from platelet membranes in the presence of ATP. The effects of all three sulfhydryl reagents could be reversed by dithiothreitol, and Ag+-induced release was also reversed by ruthenium red. These effects are similar to those observed in sarcoplasmic reticulum, but in contrast caffeine did not induce Ca2+ release. In the absence of ATP, passively loaded platelet membranes did not release Ca2+ when exposed to sulfhydryl reagents. However, AgCl and pClHgBzO inhibited inositol trisphosphate (InsP3)-induced Ca2+ release from platelet membranes and this effect was reversed by dithiothreitol. Ruthenium red also inhibited InsP3-induced release, but ATP was found not to be required for InsP3-mediated release. LiCl enhanced Ca2+ release from platelet membranes. These results demonstrate that the InsP3-gated Ca2+ release channel is a separate entity from the Ca2+-pump and that essential protein sulfhydryls are involved in the release process.     

A fluorescence probe study of the phosphorylation reaction of the calcium ATPase of sarcoplasmic reticulum

The fluorescent thiol reagent N-(1-anilinonaphthyl-4)maleimide (ANM) reacts covalently with the Ca2+ ATPase moiety of fragmented sarcoplasmic reticulum in two phases as determined by the increase of fluorescence intensity and optical density at 350 nm. In the rapid phase, 5.5 nmol of ANM reacts with 1 mg of fragmented sarcoplasmic reticulum protein. Assuming that 55% of the total membrane protein is the Ca2+ ATPase, this is equivalent to 1 mol of SH/10(5) g of ATPase, designated as SH1-ANM. ANM reacts with the second SH (SH2-ANM) at a much slower rate. Reaction of ANM with both SH1-ANM and SH2-ANM produces no inhibition of phosphoenzyme (EP) formation. Upon addition of Mg . ATP in the micromolar range, at [Ca2+] = 1 microM there is an increase in the fluorescence intensity of ANM attached to SH2-ANM, while the ANM attached to SH1-ANM does not respond to Mg . ATP. Under conditions in which there is no EP formation, there is no fluorescence change. Furthermore, the enhancement of ANM fluorescence produced by Mg . ATP is reversed by ADP as it reacts with EP to form ATP. Thus, it appears that the Mg . ATP-induced fluorescence increase reflects changes of enzyme conformation produced by EP formation.     

The base-exchange enzyme activities of sarcolemma and sarcoplasmic reticulum from rat heart

The Ca2+ dependent incorporation of [14C]ethanolamine, L-[14C]serine and [14C]choline into phosphatidylethanolamine, phosphatidylserine and phosphatidylcholine, respectively, were investigated in membrane preparations from rat heart. The ethanolamine and serine base-exchange enzyme-catalyzed reactions were associated with the sarcolemma and sarcoplasmic reticulum. There was a 17.2-fold and 6.8-fold enrichment, respectively, of the serine and the ethanolamine base-exchange enzyme activities in the sarcolemma compared to the starting whole homogenate. The sarcoplasmic reticulum was enriched in the ethanolamine and serine base-exchange enzyme activities. The choline base-exchange enzyme activity of all membranes fractions was negligible compared to the ethanolamine or serine base-exchange enzyme activities. The apparent Km for the ethanolamine and serine base-exchange enzyme in sarcolemma was 14 microM and 25 microM, respectively. The pH optimum for these base-exchange activities was 7.5-8.0. There was a dependence upon Ca2+ for these reactions with a 1 or 4 mM concentration required for maximal activity. The properties of the sarcoplasmic reticulum base-exchange enzymes were similar to the sarcolemmal base-exchange enzymes.     

Sulfhydryl group modification of sarcoplasmic reticulum membranes.

Modification of calcium-translocating sarcoplasmic reticulum membranes (SR) with 5,5'-dithiobis(2-nitrobenzoate) (Nbs2) reveals four classes (kinetic sets) of sulfhydryl groups. Of the 25 mol/1.5 X 10(5) G OF SR protein (i.e., containing 1 mol of ATPase protein) estimated in the presence of sodium dodecyl sulfate, 8 mol are unreactive, while 7, 8, and 2 mol display pseudo-first-order rate constants (k1) of 0.16, 0.68, and 8.3 min(-1), respectively (25 decrees C, pH 7.8, 4 MM Nbs2). Under these conditions, the Ca-ATPase activity is lost with k1 = 0.73 min(-1), whereas the Ca-independent ATPase activity is essentially unchanged. These results are little changed by the presence of Mg2+ or Ba2+ in the modification mixture, while Ca2+ or Sr2+ causes all 16-17 reactable sulfhydryls to be modified with k1 = 0.50 and 0.53 min(-1), respectively. The corresponding values for the loss of Ca-ATPase activity are 0.53 and 0.67 min(-1); this suggests that blocking of only one of the 16-17 SH groups inactivates the enzyme, i.e., that there is a single "essential" SH group. The midpoint of the transition between the Ca2+-free and Ca2+-modification patterns occurs at a free Ca2+ concentration of about 0.9 muM, implying that it is Ca2+ binding at the active sites (KD = 0.1 muM), rather than at the low-affinity nonspecific sites, that effects a conformation change in the ATPase protein (which contains greater than 90% of the cysteines). A calcium-induced conformation change is also suggested by increased ultraviolet absorbance spectrum of the purified ATPase protein upon calcium binding. If protein-lipid interaction is disrupted with deoxycholate or Triton X-100 (which does not destroy the Ca-ATPase activity and hence presumably leaves the tertiary structure of the ATPase protein largely intact), 95% of the sulfhydryls react with Nbs2 considerably faster; thus, at 2 mg/ml o- deoxycholate, 14 groups react with k1 greater than 20, 5 with k1 = 2.3, and 5 with k1 = 0.4 min(-1). These results suggest that the inaccessibility of SH groups in the absence of detergents is due to extensive interaction of the bilayer phospholipids with the ATPase protein.     

Affinity labeling of 3 alpha-hydroxysteroid dehydrogenase with 3 alpha-bromoacetoxyandrosterone and 11 alpha-bromoacetoxyprogesterone. Isolation and sequence of active site peptides containing reactive cysteines; sequence confirmation using nucleotide sequence from a cDNA clone

Homogeneous 3 alpha-hydroxysteroid dehydrogenase (3 alpha-HSD, EC 1.1.1.50) of rat liver cytosol is potently inhibited at its active site by nonsteroidal anti-inflammatory drugs (NSAIDs). Using 3 alpha-bromoacetoxy-5 alpha-androstan-17-one (BrAnd, a substrate analog) and 11 alpha-bromoacetoxyprogesterone (Br11P, a glucocorticoid analog) as affinity-labeling agents, kinetic evidence was obtained that these agents alkylate this site. Inactivation of 3 alpha-HSD with either [14C]BrAnd or [14C] Br11P led to the incorporation of 1 mol of affinity-labeling agent per enzyme monomer. Complete acid hydrolysis of 3 alpha-HSD radiolabeled with either agent followed by amino acid analysis led to the identification of [14C]carboxymethylcysteine indicating that [14C]BrAnd and [14C]Br11P covalently tag discrete reactive cysteine(s) at the enzyme active site. Trypsin digestion of [14C]BrAnd-inactivated 3 alpha-HSD followed by peptide mapping led to the purification of a single radiolabeled peptide (3A1) which gave the following sequence: H2N-Ser-Ile-Gly-Val-Ser-Asn-Phe-Asn-X-Arg-CO2H. Identical experiments on [14C] Br11P-inactivated 3 alpha-HSD led to the purification of three radiolabeled peptides (11P1-11P3). The major radiolabeled peptide (11P1) had an identical sequence to 3A1 which was tagged with [14C]BrAnd. The minor radiolabeled peptides had the following sequences: H2N-Ser-Lys-Asp-Ile-Ile-Leu-Val-Ser-Tyr-X-Thr-Leu-Gly-Ser-Ser-Arg-CO2H (11P2) and H2N-Ser-Pro-Val-Leu-Leu-Asp-Asp-Pro-Val-Leu-X-Ala-Ile-Ala-Lys-CO2H (11P3). In each peptide group X was identified as carboxymethylcysteine. Alignment of the peptide sequences with the primary structure of 3 alpha-HSD, deduced from its cDNA clone, assigned peptide 11P1 to residues 162-171, peptide 11P2 to residues 208-223, and peptide 11P3 to residues 232-246 of the amino acid sequence. The reactive cysteines correspond to Cys170, Cys217, and Cys242. We propose that Cys170 labeled by BrAnd may lie within the catalytic pocket of the enzyme. By contrast the 11 alpha-bromoacetoxy group in Br11P labeled several reactive cysteines which may be involved in the binding of glucocorticoids and NSAIDs.     

Interactions between the oligomycin sensitivity conferring protein (OSCP) and beef heart mitochondrial F1-ATPase. 1. Study of the binding parameters with a chemically radiolabeled OSCP

Upon treatment of beef heart mitochondrial oligomycin sensitivity conferring protein (OSCP) with [14C]-N-ethylmaleimide ( [14C]NEM) or dithiobis(nitro[14C] benzoate), 1 mol of either SH reagent was incorporated per mol of OSCP. Radiolabeling occurred at the level of the only cysteine residue, Cys-118, present in the OSCP sequence reported by Ovchinnikov et al. [Ovchinnikov, Y. A., Modyanov, N. N., Grinkevich, V. A., Aldanova, N. A., Trubetskaya, O. E., Nazimov, I. V., Hundal, T., & Ernster, L. (1984) FEBS Lett. 166, 19-22]; it did not alter the biological activity of OSCP tested in a reconstituted F0-F1 system that catalyzed oligomycin-sensitive ATPase activity or ATP-Pi exchange. The parameters of [14C]NEM-OSCP binding to isolated beef heart mitochondrial F1 were assessed by equilibrium dialysis. Addition of trace amounts of Tween 20 prevented unspecific adsorption of OSCP. The binding curves showed that each F1 possesses a high-affinity OSCP binding site (Kd = 0.08 microM) and two low-affinity OSCP binding sites (Kd = 6-8 microM). Binding of OSCP to the high-affinity site on F1 is probably responsible for the ability of OSCP to confer oligomycin sensitivity to F1 in the ATPase complex.     

A coupling factor from sarcoplasmic reticulum required for the translocation of Ca2+ ions in a reconstituted Ca2+ATPase pump.

1. During purification of the Ca2+ATPase from sarcoplasmic reticulum of rabbit muscle, different fractions with similar Ca2+ATPase activity were found to vary greatly in their ability to catalyze 45Ca2+ translocation in reconstituted liposomal systems. 2. A heat-stable fraction isolated from the fraction most active in Ca2+ translocation enhanced several-fold the Ca2+ translocation rate of the least active fraction. It also increased the ratio of Ca2+ translocation to ATP hydrolysis over 5-fold. The properties of the coupling factor resemble those of the proteolipid previously described by MacLennan et al. (MACLENNAN, D.H., YIP, C. C., ILES, G. H., and SEAMAN, P. (1972) Cold Spring Harbor Symp. Quant. Biol. 37, 469-478). 3. When the heat-stable factor was added to either sarcoplasmic reticulum fragments or to liposomes after, rather than before, reconstitution, it acted as an ionophore abolishing Ca2+ translocation.     

Effect of the purified (Mg2+ + Ca2+)-activated ATPase of sarcoplasmic reticulum upon the passive Ca2+ permeability and ultrastructure of phospholipid vesicles.

The passive Ca2+ permeability of fragmented sarcoplasmic reticulum membranes is 10(4) to 10(61 times greater than that of liposomes prepared from natural or synthetic phospholipids. The contribution of membrane proteins to the Ca2+ permeability was studied by incorporating the purified [Ca2+ + Mg2+]-activated ATPase into bilayer membranes prepared from different phospholipids. The incorporation of the Ca2+ transport ATPase into the lipid phase increased its Ca2+ permeability to levels approaching that of sarcoplasmic reticulum membranes. The permeability change may arise from a reordering of the structure of the lipid phase in the environment of the protein or could represent a specific property of the protein itself. The calcium-binding protein of sarcoplasmic reticulum did not produce a similar effect. The increased rate of Ca2+ release from reconstituted ATPase vesicles is not a carrier-mediated process as indicated by the linear dependence of the Ca2+ efflux upon the gradient of Ca2+ concentration and by the absence of competition and countertransport between Ca2+ and other divalent metal ions. The increased Ca2+ permeability upon incorporation of the transport ATPase into the lipid phase is accompanied by similar increase in the permeability of the vesicles for sucrose, Na+, choline, and SO42- indicating that the transport ATPase does not act as a specific Ca2+ channel. Native sarcoplasmic reticulum membranes are asymmetric structures and the 75-A particles seen by freeze-etch electron microscopy are located primarily in the outer fracture face. In reconstituted ATPase vesicles the distribution of the particles between the two fracture faces is even, indicating that complete structural reconstitution was not achieved. The Ca2+ transport activity of reconstituted ATPase vesicles is also much less than that of fragmented sarcoplasmic reticulum. The density of the 40-A surface particles visible after negative staining of native or reconstituted vesicles is greater than that of the intramembranous particles and the relationship between these two structures remains to be established.     

Cyclopiazonic acid is a specific inhibitor of the Ca2+-ATPase of sarcoplasmic reticulum

The mycotoxin, cyclopiazonic acid (CPA), inhibits the Ca2+-stimulated ATPase (EC 3.6.1.38) and Ca2+ transport activity of sarcoplasmic reticulum (Goeger, D. E., Riley, R. T., Dorner, J. W., and Cole, R. J. (1988) Biochem. Pharmacol. 37, 978-981). We found that at low ATP concentrations (0.5-2 microM) the inhibition of ATPase activity was essentially complete at a CPA concentration of 6-8 nmol/mg protein, indicating stoichiometric reaction of CPA with the Ca2+-ATPase. Cyclopiazonic acid caused similar inhibition of the Ca2+-stimulated ATP hydrolysis in intact sarcoplasmic reticulum and in a purified preparation of Ca2+-ATPase. Cyclopiazonic acid also inhibited the Ca2+-dependent acetylphosphate, p-nitrophenylphosphate and carbamylphosphate hydrolysis by sarcoplasmic reticulum. ATP protected the enzyme in a competitive manner against inhibition by CPA, while a 10(5)-fold change in free Ca2+ concentration had only moderate effect on the extent of inhibition. CPA did not influence the crystallization of Ca2+-ATPase by vanadate or the reaction of fluorescein-5'-isothiocyanate with the Ca2+-ATPase, but it completely blocked at concentrations as low as 1-2 mol of CPA/mol of ATPase the fluorescence changes induced by Ca2+ and [ethylenebis(oxyethylenenitrilo)]tetraacetic acid (EGTA) in FITC-labeled sarcoplasmic reticulum and inhibited the cleavage of Ca2+-ATPase by trypsin at the T2 cleavage site in the presence of EGTA. These observations suggest that CPA interferes with the ATP-induced conformational changes related to Ca2+ transport. The effect of CPA on the sarcoplasmic reticulum Ca2+-ATPase appears to be fairly specific, since the kidney and brain Na+,K+-ATPase (EC 3.6.1.37), the gastric H+,K+-ATPase (EC 3.6.1.36), the mitochondrial F1-ATPase (EC 3.6.1.34), the Ca2+-ATPase of erythrocytes, and the Mg2+-activated ATPase of T-tubules and surface membranes of rat skeletal muscle were not inhibited by CPA, even at concentrations as high as 1000 nmol/mg protein.     

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.