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.     

Studies on the structure of the calcium-dependent adenosine triphosphatase from rabbit skeletal muscle sarcoplasmic reticulum

The linear arrangement of the three fragments of Ca²⁺-ATPase from rabbit skeletal muscle sarcoplasmic reticulum with molecular weights of 20,000, 30,000, and 45,000 obtained by limited tryptic hydrolysis was determined by locating the NH₂-terminal acetylated methionyl residue of the original peptide in the M_r = 20,000 fragment. Since both the M_r = 20,000 and 30,000 polypeptides originate from a M_r = 55,000 fragment which is distinct from the M_r = 45,000 polypeptide, the sequence of these three fragments was determined to be 20,000, 30,000, and 45,000. The M_r = 20,000 fragment was further cleaved by cyanogen bromide to yield a M_r = 7,000 COOH-terminal fragment which is relatively hydrophilic. The NH₂-terminal portion is rich in glutamyl residues. The COOH-terminus of the M_r = 30,000 fragment was determined by both digestion with carboxypeptidases and cyanogen bromide cleavage. Using the partial amino acid sequence of the Ca²⁺-ATPase, it was deduced that the active site phosphoaspartyl residue is 154 amino acids from the COOH-terminus of the M_r = 30,000 fragment and hence approximately 35,000 M_r from the NH₂-terminus of the original Ca²⁺-ATPase molecule. Furthermore, it was shown that the two tryptic cleavages of the Ca²⁺-ATPase generating these three large fragments were both single hydrolyses of arginylalanine peptide bonds.     

Restoration of calcium transport in the trypsin-treated (Ca+ + Mg2+)-dependent adenosine triphosphatase of sarcoplasmic reticulum exposed th sodium dodecyl sulfate.

When sarcoplasmic reticulum vesicles are exposed to trypsin for 1 min the adenosine triphosphatase (Mr = 102,000) is cleaved to fragments of Mr = 45,000 and 55,000. The purified ATPase, containing both fragments, transports Ca2+ when incorporated into vesicles containing excess phospholipid. The two fragments can only be dissociated in solutions containing 1% sodium dodecyl sulfate (SDS). Ca2+ transport activity is restored in SDS-dissociated preparations in a series of steps involving dilution with 5 volumes of 5% phospholipids in 0.75% sodium cholate, incubation in ice for 30 min, and passage through an anion exchange column. Vesicles formed in this procedure regain high Ca2+ transport activity if they are incubated in SDS solution at 24 degrees for less than 20 min. However, the extent of renaturation diminishes if the vesicles are incubated for longer periods and little acitivity is recovered in vesicles incubated longer than 60 min at 24 degrees.     

Purification and characterization of the 45,000-Dalton fragment from tryptic digestion of (Ca2+ + Mg2+)-adenosine triphosphatase of sarcoplasmic reticulum.

Tryptic digestion of (Ca2+ + Mg2+)-ATPase from sarcoplasmic reticulum of rabbit skeletal muscle has previously been shown to cleave the enzyme initially into a 55,000-dalton fragment and a 45,000-dalton fragment. In the present study the two fragments are solubilized in sodium dodecyl sulfate (SDS) and separated by preparative polyacrylamide gel electrophoresis. The 45,000-dalton fragment is found to be a relatively nonselective, divalent cation-dependent ionophore when incorporated into an oxidized cholesterol membrane (BLM). Ionophoric activity of this fragment is inhibited by low concentrations of LaCl3, HgCl2, and various reducing agents. There appears to be one or two relatively inaccessible disulfide bonds in the 45,000-dalton fragment that are essential for transport. Addition of reducing agents inhibits the ionophoric activity of the succinylated undigested enzyme and the 45,000-dalton fragment, but has no effect on the 55,000-dalton fragment. These experiments imply that the 45,000-dalton fragment and the 55,000-dalton fragment are in a series arrangement in the membrane.     

Purification and characterization of the 45,000-dalton fragment from tryptic digestion of (Ca2++Mg2+)-adenosine triphosphatase of sarcoplasmic reticulum

Summary Tryptic digestion of (Ca²⁺+Mg²⁺)-ATPase from sarcoplasmic reticulum of rabbit skeletal muscle has previously been shown to cleave the enzyme initially into a 55,000-dalton fragment and a 45,000-dalton fragment. In the present study the two fragments are solubilized in sodium dodecyl sulfate (SDS) and separated by preparative polyacrylamide gel electrophoresis. The 45,000-dalton fragment is found to be a relatively nonselective, divalent cation-dependent ionophore when incorporated into an oxidized cholesterol membrane (BLM). Ionophoric activity of this fragment is inhibited by low concentrations of LaCl₃, HgCl₂, and various reducing agents. There appears to be one or two relatively inaccessible disulfide bonds in the 45,000-dalton fragment that are essential for transport. Addition of reducing agents inhibits the ionophoric activity of the succinylated undigested enzyme and the 45,000-dalton fragment, but has no effect on the 55,000-dalton fragment. These experiments imply that the 45,000-dalton fragment and the 55,000-dalton fragment are in a series arrangement in the membrane.     

Ca2+-activated ATPase in microsomes from human liver

Human liver microsomal fractions exhibit ATP-supported Ca2+ uptake which is half-maximal at 7 X 10(-7) M free Ca2+ in the presence of oxalate. Ca2+ uptake is coupled to a Ca2+-stimulated ATPase activity, which is half-maximal at 4 X 10(-7) M free Ca2+. Catalysis involves formation of an Mr = 116,000 phosphoprotein with stability characteristics of an acylphosphate compound suggested to represent a phosphoryl protein intermediate of the Ca2+-ATPase. Phosphorylation is half-maximal at about 10(-6) M free Ca2+. The Mr = 116,000 protein is highly susceptible to proteolysis with trypsin. The phosphorylated active site was localized in an Mr = 58,000 primary tryptic fragment and in an Mr = 34,000 subfragment. Analyses on the mechanism of the Ca2+-ATPase suggest the following reaction sequence: formation of an ADP-reactive phosphoenzyme (Mr = 116,000) with bound Ca2+, which can transphosphorylate its Pi to ADP, giving rise to synthesis of ATP; reversible transformation of the ADP-reactive phosphoenzyme into an isomer without bound Ca2+, which cannot further react with ADP; hydrolytical cleavage, probably catalyzed by Mg2+, of the ADP-unreactive phosphoenzyme with liberation of Pi. Comparison with the Ca2+-transport ATPase in sarcoplasmic reticulum of skeletal muscle led us to suggest that the Mr = 116,000 Ca2+-ATPase belongs to the class of E1P . E2P-ATPases and might be operative as a Ca2+-transport ATPase at the level of the endoplasmic reticulum in human liver.     

Properties of a reconstituted calcium pump.

1. The translocation of 45Ca2+ in vesicles reconstituted with purified Ca2+ ATPase of sarcoplasmic reticulum and phospholipids was dependent on ATP and varied greatly with the composition of the phospholipids. 2. In contrast to sarcoplasmic reticulum fragments, the reconstituted vesicles were impermeable to 14C-labeled oxalate, 3H- or 32P-labeled ATP, or 32P-i. There was no translocation of phosphate from gamma-labeled ATP during Ca2+ uptake. These results are inconsistent with some current formulations of the mechanism of pump action. 3. Reversal of the Ca2+ pump and generation of ATP and ADP and P-i was observed when vesicles loaded with Ca2+ were exposed to ethylene glycol bis(beta-aminoethyl ether)-N,N'-tetraacetic acid. 4. Experiments on the formation of phosphoenzyme with 32P-labeled ATP showed that most if not all functional ATPase molecules in the reconstituted vesicles were oriented in the same direction, as in the case of sarcoplasmic reticulum fragments.     

Hydrophobic photolabelling of sarcoplasmic reticulum with [125I]TID

[125I]TID, a small photoreactive lipophylic reagent, was used to label intrinsic proteins of rabbit and rat sarcoplasmic reticulum membranes. A 160,000 glycoprotein, the Ca2+-ATPase and polypeptides of mol. wt 53-55,000, 30,000, 20,000 and 6000 dalton were labelled suggesting that these proteins are integral membrane components.     

Isolation and characterization of staphylocoagulase chymotryptic fragment. Localization of the procoagulant- and prothrombin-binding domain of this protein

Several strains of Staphylococcus aureus secrete a protein, staphylocoagulase, that binds stoichiometrically to human prothrombin, resulting in a coagulant complex designated staphylothrombin. In the present study, staphylocoagulase was digested with alpha-chymotrypsin and the resulting fragments were isolated by gel filtration. One fragment (Mr 43,000) exhibited a high affinity for human prothrombin (Kd = 1.7 X 10(-9) M), which is comparable to the affinity observed using intact staphylocoagulase (Kd = 4.6 X 10(-10) M). A complex of the Mr 43,000 fragment and prothrombin possessed both clotting and amidase activity essentially identical to that observed in a complex of intact staphylocoagulase and prothrombin. A second fragment (Mr 30,000) exhibited weaker affinity for prothrombin (Kd = 1.2 X 10(-7) M). While clotting activity was not observed with a complex of this fragment and prothrombin, it nonetheless possessed a weak amidase activity. A third fragment (Mr 20,000) was found to bind to prothrombin, but the resultant complex did not exhibit clotting or amidase activity. Amino-terminal sequence analyses of these staphylocoagulase fragments revealed that the Mr 43,000 fragment constitutes the amino-terminal portion of staphylocoagulase and also contains the Mr 30,000 and 20,000 fragments. Moreover, the amino-terminal sequence of the Mr 20,000 fragment was identical to that observed for the Mr 30,000 fragment. From these results, we conclude that the functional region of staphylocoagulase for binding and activation of human prothrombin is localized in the amino-terminal region of the intact bacterial protein.     

Photolabeling of the integral proteins of skeletal muscle sarcoplasmic reticulum: comparison of junctional and nonjunctional membrane fractions

Rabbit skeletal muscle sarcoplasmic reticulum (SR) was fractionated by isopycnic density gradient centrifugation into longitudinal tubules (LSR) and terminal cisternae (TC). Junctional face membranes (JFM) were obtained by Triton X-100 treatment of the TC fraction (Costello, B., Chadwick, C., Saito, A., Chu, A., Maurer, A. and Fleischer, S. (1986) J. Cell Biol. 103, 741-753). Photoactivatable phospholipid analogs were introduced into LSR, TC, and JFM fractions to specifically label integral membrane proteins. Remarkably different labeling patterns were observed. Proteins of the following Mr were labeled and identified in the junctional sarcoplasmic reticulum (JFM): 350,000, 325,000, 80,000, 49,000, 37,000, 32,000, 30,000, and 6000. Polypeptides of Mr 105,000 (Ca2+-dependent ATPase), 77,000, 55,000, 41,000, 22,000, and 9000 (proteolipid) were labeled and found to be selectively localized in the nonjunctional sarcoplasmic reticulum (LSR). Calsequestrin, a key protein responsible for Ca2+ storage within the SR lumen, was never labeled, whether 1 mM CaCl2 was present or absent, and is termed a nonintegral membrane protein.     

Evidence that coated vesicles isolated from brain are calcium- sequestering organelles resembling sarcoplasmic reticulum

Coated vesicles from the brain have been purified to near morphological homogeneity by a modification of the method of Pearse. These vesicles resemble sarcoplasmic reticulum fragments isolated from skeletal muscle. They contain proteins with 100,000- and 55,000-dalton mol wt which co-migrate on polyacrylamide gels, in the presence of sodium dodecyl sulfate, with the two major proteins of the sarcoplasmic reticulum fragment. These vesicles contain adenosine triphosphatase (ATPase) activity which is stimulated by calcium ions in the presence of Triton X-100 (Rohm & Haas Co., Philadelphia, Pa.), displaying maximal activity at 8 x 10(-7) M Ca ++. They take up calcium ions from the medium, and this uptake is stimulated by ATP and by potassium oxalate, a calcium-trapping agent. The 100,000-dalton protein of the coated vesicles displays immunological reactivity with an antiserum directed against the 100,000-dalton, calcium-stimulated ATPase of the sarcoplasmic reticulum. As with the sarcoplasmic reticulum fragment, this protein becomes radiolabeled when coated vesicles are briefly incubated with gamma-labeled [32P]ATP. The possible functions of coated vesicles as calcium-sequestering organelles are discussed.     

Definition of an N-terminal actin-binding domain and a C-terminal Ca2+ regulatory domain in human brevin

Brevin is a Ca2+-modulated actin-associated protein that will sever F-actin and cap barbed filament ends. Limited proteolysis with chymotrypsin or subtilisin cleaves the molecule approximately in half. Cleavage is approximately 10-fold more rapid in Ca2+ than in EGTA. The two fragments are readily separated from each other and from undigested brevin by high pressure liquid chromatography on a DEAE resin. A 40,000-mol-wt fragment from the N-terminal is not retained by DEAE, while a 45,000-mol-wt C-terminal fragment binds more tightly than brevin. The N-terminal fragment retains approximately 10% of the nucleation activity, caps barbed ends, and retains 50% of the total severing activity defined by dilution induced depolymerization of pyrenyl actin, but, in contrast to brevin, none of these functions are affected by Ca2+. Fluorescent actin binding studies and gel-filtration demonstrate that the 40,000-mol-wt fragment binds two actin monomers. The 45,000-mol-wt C-terminal fragment has no severing, nucleating, or capping activity. Cross-reaction with two monoclonal antibodies against two specific Ca2+-induced conformations of human platelet gelsolin suggest that both Ca2+ binding sites are located on the carboxyl half of the brevin molecule. One epitope, defined as the rapidly exchanging Ca2+ binding site in the gelsolin-actin complex, is lost when a 20,000-mol-wt fragment is cleaved from the carboxyl terminal. The second epitope, related to the poorly exchanging Ca2+ binding site in the complex, is nearer the middle of the brevin molecule.     

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.     

The binding of monoclonal and polyclonal antibodies to the Ca2(+)-ATPase of sarcoplasmic reticulum: effects on interactions between ATPase molecules.

We analyzed the interaction of 14 monoclonal and 5 polyclonal anti-ATPase antibodies with the Ca2(+)-ATPase of rabbit sarcoplasmic reticulum and correlated the location of their epitopes with their effects on ATPase-ATPase interactions and Ca2+ transport activity. All antibodies were found to bind with high affinity to the denatured Ca2(+)-ATPase, but the binding to the native enzyme showed significant differences, depending on the location of antigenic sites within the ATPase molecule. Of the seven monoclonal antibodies directed against epitopes on the B tryptic fragment of the Ca2(+)-ATPase, all except one (VIE8) reacted with the enzyme in native sarcoplasmic reticulum vesicles in both the E1 and E2V conformations. Therefore these regions of the Ca2(+)-ATPase molecule are freely accessible in the native enzyme. The monoclonal antibody VIE8 bound with high affinity to the Ca2(+)-ATPase only in the E1 conformation stabilized by 0.5 mM Ca2+ but not in the E2V conformation stabilized by 0.5 mM EGTA and 5 mM vanadate. Several antibodies that reacted with the B fragment interfered with the crystallization of Ca2(+)-ATPase in the presence of EGTA and vanadate and at least two of them destabilized preformed Ca2(+)-ATPase crystals, suggesting inhibition of interactions between ATPase molecules. Of five monoclonal antibodies with epitopes on the A1 tryptic fragment of the Ca2(+)-ATPase only one gave strong reaction with the native enzyme, and none interfered with ATPase-ATPase interactions as measured by the polarization of fluorescence of FITC-labeled Ca2(+)-ATPase. Therefore the regions of the molecule containing these epitopes are relatively inaccessible in the native structure. Partial tryptic cleavage of the Ca2(+)-ATPase into the A1, A2 and B fragments did not promote the reaction of anti-A1 antibodies with sarcoplasmic reticulum vesicles, but solubilization of the membrane with C12E8 rendered the antigenic site fully accessible to several of them, suggesting that their epitopes are located in areas of contacts between ATPase molecules. Two monoclonal anti-B antibodies that interfered with ATPase-ATPase interactions, produced close to 50% inhibition of the rate of ATP-dependent Ca2+ transport, with significant inhibition of ATPase; this may suggest a role for ATPase oligomers in the regulation of Ca2+ transport. The other antibodies that interact with the native Ca2(+)-ATPase produced no significant inhibition of ATPase activity even at saturating concentrations; therefore their antigenic sites do not undergo major movements during Ca2+ transport.     

Differences in domain structure between human fibronectins isolated from plasma and from culture supernatants of normal and transformed fibroblasts. Studies with domain-specific antibodies

The domain structure of human fibronectins isolated from plasma and from the conditioned medium of normal and transformed fibroblasts was analyzed by limited proteolysis and S-cyanylation followed by immunostaining of released fragments with five kinds of antibodies, each specific for one functional domain. The results indicate that all three human fibronectins are composed of the same set of functional domains aligned in the same topological order. However, the following clear differences were found in specific fragments released from plasma fibronectin (pFN) and those released from fibronectin of normal (N-cFN) and transformed fibroblasts (T-cFN). Two fragments (Mr = 70,000 and 60,000) were released from the COOH-terminal region of pFN by cathepsin D. These fragments represent the COOH-terminal heparin-binding (Hep-2) and fibrin-binding (Fib-2) domains. The corresponding fragments released from both N-cFN and T-cFN by cathepsin D had much larger molecular weights (Mr = 100,000 and 83,000-74,000) than those from pFN. The fragments from the Fib-2 domain alone, however, did not show any difference among all three FNs. The internal region, from the gelatin-binding (Gel) domain through the Hep-2 domain, of N-cFN and T-cFN was released as a Mr = 210,000 fragment upon mild trypsin digestion. The corresponding fragment from pFN was released as a Mr = 185,000 fragment. The COOH-terminal half, including the Hep-2 domain, of both N-cFN and T-cFN was released by S-cyanylation as Mr = 160,000-145,000 fragments, which are 25,000-20,000 larger than the corresponding fragments of pFN. These results clearly indicate that the Hep-2 domain of N-cFN and T-cFN is 30,000-20,000 daltons larger than the same domain of pFN. Although various fragments released from N-cFN and T-cFN showed a similar pattern, there were minor differences. Thermolysin fragments derived from the Hep-2 domain of N-cFN were clearly distinguishable from those from T-cFN. Three groups of fragments with Mr = 40,000, 35,000-32,000, and 30,000 were released from N-cFN, while only the 35,000-32,000 fragment was released from T-cFN. The Mr = 44,000/60,000 thermolysin fragments representing the Gel domain and the Mr = 210,000/165,000 tryptic fragments representing the internal domains of T-cFN were slightly, but consistently, larger than those of N-cFN.(ABSTRACT TRUNCATED AT 400 WORDS)     

Calmodulin Affinity for Brain Coated Vesicle Proteins

A systematic characterization of the affinity of calmodulin for brain coated vesicles was undertaken. Binding of 125I-labeled calmodulin to coated vesicles was saturable and competed with unlabeled calmodulin, but not with troponin-C. Scatchard analysis revealed one high-affinity, low-capacity binding site, KD = 3.9 +/- 0.6 nM, Bmax = 16.3 +/- 2.4 pmol/mg, and one low-affinity, high-capacity binding site, KD = 102 +/- 15.0 nM, Bmax = 151 +/- 23.0 pmol/mg. Radioimmunoassay revealed that coated vesicles contain 1.05 microgram calmodulin/mg protein. Because this value remained constant even after removal of clathrin, the major coat protein, from the coated vesicle, it is apparent that calmodulin is associated with the vesicle per se rather than with its clathrin lattice. When a Triton X-100-treated extract of coated vesicles was passed through a Sepharose 4B-calmodulin affinity column, polypeptides with Mrs (molecular weights) of 100,000, 55,000, and 30,000 bound in a Ca2+-dependent manner. A 30,000 Mr protein doublet purified from coated vesicles was completely eluted by EGTA from the calmodulin affinity column, confirming that this protein doublet represents one of the coated vesicle calmodulin binding sites. Because calmodulin stimulated [Ca2+-Mg2+]-ATPase activity as well as Ca2+ uptake in coated vesicles, it is postulated that the 100,000 and 55,000 Mr calmodulin binding proteins represent the [Ca2+-Mg2+]-ATPase complex, the other coated vesicle calmodulin binding site.     

Separation and characterisation of tryptic fragments from the adenosine triphosphatase of sarcoplasmic reticulum.

The two halves of the ATPase, M, 115,000, from sarcoplasmic reticulum produ-ed by limited trypsin treatment have been purified in sodium dodecylsulphate. The fragment of Mr60,000 has been purified by electrophoresis on cellulose acetate slabs and that of Mr 55,000 by gel filtration. The two halves of the 60,000 Mr fragment (Mr33,000 and 24,000) produced by more extensive trypsin treatment have also been purified by gel filtration in sodium dodecylsulphate. The sum of the amino acid analyses of the constituent tryptic fragments is in good agreement with that for the whole ATPase. The amino acid compositions of the two halves of the ATPase were strikingly similar. N-terminal analysis shows that the ATPase and its constituent tryptic polypeptides all possess a single N-terminal alanine implying no further cleavage of the polypeptide by trypsin. Attempts to solubilize selectively the tryptic fragments from the membrane by a variety of denaturing and solubilising agents under a variety of conditions have proved unsuccessful, suggesting that the interaction between the tryptic polypeptides is stronger than between the lipid and the protein. The possibility that the interaction between the tryptic polypeptides includes disulphide bonding has been eliminated.     

Modification of the (Ca2+ + Mg2+)-ATPase protein of sarcoplasmic reticulum with 7-chloro-4-nitrobenzo-2-oxa-1,3-diazole

The (Ca2+ + Mg2+)-ATPase (ATP phosphohydrolase (Ca2+-transporting), EC 3.6.1.38) protein of rabbit skeletal sarcoplasmic reticulum (SR) rapidly incorporated 2 mol of 7-chloro-4-nitrobenzo-2-oxa-1,3-diazole (NBD-Cl) per 10(5) g of protein with little change in the Ca2+-dependent ATPase activity. When 2 additional mol of the reagent were bound the Ca2+-ATPase, activity was inhibited. The same pattern was found for modified intact SR and the Ca2+ uptake ability was inhibited. MgATP, CaATP and MgADP protected the Ca2+-ATPase activity concurrent with a decrease of about 1 mol of the NBD group per 10(5) g protein, but the Ca2+ uptake ability was not protected. Calcium alone had no effect on the modification. The modified ATPase protein or SR formed non-serial oligomers or aggregates, but the ATPase protein remained the predominant species present. In the presence of MgATP, oligomer formation was reduced partially but the major changes in the Ca2+-ATPase activity were due to the modification of the ATPase monomer. Thiolysis of the NBD-ATPase protein with dithiothreitol did not restore the Ca2+-ATPase activity, although more than 1 mol of the NBD group was removed from cysteine residues. Cysteine residues were modified in the NBD-ATPase protein or SR when the enzyme activity was inhibited. Trypsin digestion of NBD-SR or its ATPase protein released the A, B, A1, and A2 fragments. The A fragment and its subfragment A2 contained most of the label. Substrate MgATP protection studies showed that the A1 and A2 fragments were involved in maintaining the Ca2+-ATPase activity. Reagent-induced conformational changes of these fragments rather than direct active site group labeling accounted for the loss of ATPase activity.     

Smooth muscle expresses a cardiac/slow muscle isoform of the Ca2+-transport ATPase in its endoplasmic reticulum.

Smooth muscle expresses in its endoplasmic reticulum an isoform of the Ca2+-transport ATPase that is very similar to or identical with that of the cardiac-muscle/slow-twitch skeletal-muscle form. However, this enzyme differs from that found in fast-twitch skeletal muscle. This conclusion is based on two independent sets of observations, namely immunological observations and phosphorylation experiments. Immunoblot experiments show that two different antibody preparations against the Ca2+-transport ATPase of cardiac-muscle sarcoplasmic reticulum also recognize the endoplasmic-reticulum/sarcoplasmic-reticulum enzyme of the smooth muscle and the slow-twitch skeletal muscle whereas they bind very weakly or not at all to the sarcoplasmic-reticulum Ca2+-transport ATPase of the fast-twitch skeletal muscle. Conversely antibodies directed against the fast-twitch skeletal-muscle isoform of the sarcoplasmic-reticulum Ca2+-transport ATPase do not bind to the cardiac-muscle, smooth-muscle or slow-twitch skeletal-muscle enzymes. The phosphorylated tryptic fragments A and A1 of the sarcoplasmic-reticulum Ca2+-transport ATPases have the same apparent Mr values in cardiac muscle, slow-twitch skeletal muscle and smooth muscle, whereas the corresponding fragments in fast-twitch skeletal muscle have lower apparent Mr values. This analytical procedure is a new and easy technique for discrimination between the isoforms of endoplasmic-reticulum/sarcoplasmic-reticulum Ca2+-transport ATPases.     

Separate effects of mercurial compounds on the ionophoric and hydrolytic functions of the (Ca++ +Mg++)-ATPase of sarcoplasmic reticulum.

We have shown that a Ca++-ionophore activity is present in the (Ca++ +Mg++)-ATPase of rabbit skeletal muscle sarcoplasmic reticulum (A. E. Shamoo & D. H. MacLennan, 1974. Proc. Nat. Acad. Sci. USA 71:3522). Methylmercuric chloride inhibited the (Ca++ +Mg++)-ATPase and Ca++ transport, but had no effect on the activity of the Ca++ ionophore. Mercuric chloride inhibited ATPase, transport and ionophore activity. The ATPase and transport functions were more sensitive to methylmercuric chloride than to mercuric chloride. The two functions were inhibited concomitantly by methylmercuric chloride but slightly lower concentrations of mercuric chloride were required to inhibit Ca++ transport than were required to inhibit ATPase. Methylmercuric chloride and mercuric chloride probably inhibited ATPase and Ca++ transport by blocking essential -SH groups. However, it appears that there are no essential -SH groups in the Ca++ ionophore and that mercuric chloride inhibited the Ca++ ionophore activity by competition with Ca++ for the ionophoric site. Blockage of Ca++ transport by mercuric chloride probably occurs both at sites of essential -SH groups and at sites of ionophoric activity. These data suggest the separate identity of the sites of ATP hydrolysis and of Ca++ ionophoric activity.