A kinetic method for the study of solvent environments of thiol groups in proteins involving the use of a pair of isomeric reactivity probes and a differential solvent effect. Investigation of the active centre of ficin by using 2,2''- and 4,4''- dipyridyl disulphides as reactivity probes.

1. Whereas the second-order rate constants for the reaction of the thiolate ion of 2-mercaptoethanol with 4,4'-dipyridyl disulphide (k4PDS) and with 5,5'-dithiobis-2-nitrobenzoate dianion increase with decreasing dielectric constant of the solvent, or remain unchanged, the rate constant for the analogous reaction with 2,2'-dipyridyl disulphide (k2PDS) decreases. This anomalous solvent effect and other unusual physicochemical properties of 2,2'-dipyridyl disulphide are discussed. 2. The differential effect of solvent on the reactions of thiolate ion with the 2,2'- and 4,4'-dipyridyl disulphides is shown to provide a method of characterizing solvent environments of thiol groups in proteins by a reactivity-probe method that should not suffer from the usual drawback associated with the existence of steric or binding effects of unknown magnitude. Application of the method to ficin (EC 3.4.22.3) suggests that its active-centre thiol group resides in a relatively hydrophobic environment. 3. The pH-k profile for the reaction of ficin with 4,4'-dipyridyl disulphide is reported.     

Transmembranous organization of (Ca2(+)-Mg2+)-ATPase from sarcoplasmic reticulum. Evidence for lumenal location of residues 877-888

An antipeptide antibody was produced against a peptide corresponding to residues 877-888 of fast twitch rabbit sarcoplasmic reticulum ATPase. This antipeptide antibody bound strongly to the ATPase in sarcoplasmic reticulum vesicles only after the vesicles had been solubilized with the detergent C12E8 indicating that its epitope was located in the lumen of the sarcoplasmic reticulum. Digestion of sarcoplasmic reticulum or purified (Ca2(+)-MG2+)-ATPase by proteinase K for up to 1 h resulted in a stable ATPase fragment of 30 kDa containing the epitope for the above antibody and the epitope for an antibody directed against the C terminus. Further proteolysis revealed smaller fragments (Mr 19,000 and 13,000) containing both epitopes. By contrast, small fragments of the ATPase (less than 29 kDa) containing the N-terminal epitope were not observed even after short exposures to proteinase K. These data support the view that the (Ca2(+)-MG2+)-ATPase has 10 transmembranous helices.     

Calcium-induced dissociation of human plasma factor XIII and the appearance of catalytic activity

1. The Ca(2+) dependence of the activity of plasma Factor XIII(a) was studied by using the continuous assay based on the incorporation of dansylcadaverine into dephosphorylated acetylated beta-casein (beta-substrate). The K(m) for Ca(2+) is about 0.170mm. 2. At low concentrations of Ca(2+) there was a lag in attaining the steady-state rate. The size of the lag was decreased and eventually abolished if the enzyme was preincubated with a high concentration of Ca(2+) before assay. The concentration of Ca(2+) required to decrease the lag phase by 50% in 10min depended on the protein concentration: at 0.87mg of protein/ml it required 17mm-Ca(2+) and at 0.44mg/ml it needed 10mm-Ca(2+). 3. The concentrations of Ca(2+) required either to abolish the lag phase in the appearance of enzyme activity or to activate the essential thiol for reaction with 5,5'-dithiobis-(2-nitrobenzoate) in 10min incubation were similar at the same protein concentration. This indicated that Ca(2+) induces a conformation change that is responsible for both phenomena. A model is proposed that links this conformation change to the dissociation of the tetrameric enzyme. 4. This was supported by the observation that the addition of excess of b chains to the Factor XIII(a) (a'(2)b(2)) increased the concentration of Ca(2+) required to expose the reactive thiol, and inhibited the Ca(2+)-dependent aggregation of a' chains. 5. Platelet Factor XIII(a) (a'(2)) was inhibited by 5,5'-dithiobis-(2-nitrobenzoate) in the absence of Ca(2+), and no lag phases were observed in attaining the steady-state rate at low Ca(2+) concentrations, thus confirming the model for the activation of the plasma enzyme. 6. The Ca(2+) dependence of platelet Factor XIII(a) indicated that Ca(2+) has an additional role in the enzyme mechanism of the plasma enzyme, perhaps being involved in substrate binding. 7. The dependence of the stability of plasma Factor XIII(a) on Ca(2+) and protein concentration indicates that the decay in activity is related to the tetramer dissociation. 8. beta-Substrate decreased the Ca(2+) concentration required for (1) abolition of the lag phase and (2) enzyme inhibition by thiol reagents. The effect on the former is greater than on the latter. 9. The role of the b chains of the plasma Factor and the evolutionary significance of the plasma and platelet Factors are considered.     

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.     

A study of the subunit structure and the thiol reactivity of bovine liver uridine diphosphate glucose dehydrogenase

1. The amino acid analysis of UDP-glucose dehydrogenase is reported. 2. N-Terminal-group analysis indicates only one type of N-terminal amino acid, methionine, to be present. 3. Peptide ;mapping' in conjunction with the amino acid analysis indicates that the subunits of the enzyme are similar if not identical. 4. The various kinetic classes of thiol group were investigated by reaction with 5,5'-dithiobis-(2-nitrobenzoate). 5. NAD(+), UDP-glucose and UDP-xylose protect the two rapidly reacting thiol groups of the hexameric enzyme. 6. Inactivation of the enzyme with 5,5'-dithiobis-(2-nitrobenzoate) indicates the involvement of six thiol groups in the maintenance of enzymic activity. 7. The pH-dependence of UDP-xylose inhibition of the enzyme was investigated. 8. The group involved in the binding of UDP-xylose to the protein has a heat of ionization of about 33kJ/mol and a pK of 8.4-8.6. 9. It is suggested that UDP-xylose has a cooperative homotropic effect on the enzyme.     

The soluble carnitine palmitoyltransferase from bovine liver. A comparison with the enzymes from peroxisomes and from the mitochondrial inner membrane.

The properties of two carnitine acyltransferases (CPT) purified from bovine liver are compared to confirm that they are different proteins. The soluble CPT and the inner CPT from mitochondria differ in subunit Mr, native Mr, pI and reactivity with thiol reagents. All eight free thiol groups in soluble CPT react with 5,5'-dithiobis-(2-nitrobenzoate) in the absence of any unfolding reagent, and activity is gradually lost. The inner CPT activity is completely stable in the presence of 5,5'-dithiobis-(2-nitrobenzoate), and only one thiol group per molecule of subunit is modified in the native enzyme. Antisera to each enzyme inhibit that enzyme, but do not cross-react. CPT activity in subcellular fractions can now be identified by titration with these antibodies. The soluble CPT from bovine liver is probably peroxisomal in origin, but, although antigenically similar, it differs from the peroxisomal carnitine octanoyltransferase found in rat and mouse liver in its specificity for the longer-chain acyl-CoA substrates.     

The reaction of papain with Ellman''s reagent [5,5′-dithiobis-(2-nitrobenzoate) dianion]

1. The report by Robyt et al. (1971) that the 2-nitro-5-mercaptobenzoate dianion (Nbs(2-)) produced by reaction of papain with the 5,5'-dithiobis-(2-nitrobenzoate) dianion (Nbs(2) (2-); Ellman's reagent) cleaves the three disulphide bonds in papain is shown to be incorrect. 2. When partially active papain containing approx. 0.4 mol of thiol/mol of protein is incubated with excess of Nbs(2) (2-) at pH8, Nbs(2) (2-) reacts with the protein in an amount stoicheiometric with the cysteinyl thiol group of papain to produce Nbs(2-) in an amount stoicheiometric with the original papain cysteinyl thiol group, and the catalytically inactive mixed disulphide, papain-Nbs(-). 3. Papain catalyses the hydrolysis of Nbs(2) (2-) at pH10.5 probably by nucleophilic catalysis involving the enzyme's thiol group. 4. These results cast very serious doubts on the claim by Robyt et al. (1971) to have established a new general method for the determination of cystinyl disulphide residues in proteins.     

The location of the thiol groups of myosin that are protected by pyrophosphate against reaction with 2,4-dinitrophenyl β-hydroxyethyl disulphide

Myosin modified in the presence or in the absence of pyrophosphate by 2,4-dinitrophenyl beta-hydroxyethyl disulphide was treated with iodo[1-(14)C]acetamide. The residual Ca(2+)-stimulated adenosine triphosphatase (ATPase) activity of the modified myosin was different depending on the presence or absence of PP(i) during modification and the number of 2,4-dinitrophenyl beta-hydroxyethyl disulphide-modified thiol groups. The radioactivity incorporated into the light components of myosin correlated with the Ca(2+)-stimulated ATPase activity of the modified myosin and decreased with decreasing residual Ca(2+)-stimulated ATPase activity of the modified myosin. When native myosin was treated with low concentrations of iodo[1-(14)C]acetamide the residual Ca(2+)-stimulated ATPase activity of carboxyamidomethylated myosin was high and the radioactivity incorporated into the light components of myosin was negligible. The thiol groups of the light components of myosin are essential to preserve the ATPase activity of the protein and are close to the pyrophosphate-binding sites.     

The presence of sarcolipin results in increased heat production by Ca(2+)-ATPase

Skeletal muscle sarcoplasmic reticulum of large mammals such as rabbit contains sarcolipin (SLN), a small peptide with a single transmembrane alpha-helix. When reconstituted with the Ca(2+)-ATPase from skeletal muscle sarcoplasmic reticulum into sealed vesicles, the presence of SLN leads to a reduced level of accumulation of Ca(2+). Heats of reaction of the reconstituted Ca(2+)-ATPase with ATP were measured using isothermal calorimetry. The heat released increased linearly with time over 30 min and increased with increasing SLN content. Rates ATP hydrolysis by the reconstituted Ca(2+)-ATPase were constant over a 30-min time period and were the same when measured in the presence or absence of an ATP-regenerating system. The calculated values of heat released per mol of ATP hydrolyzed increased with increasing SLN content and fitted to a simple binding equation with a dissociation constant for the SLN.ATPase complex of 6.9 x 10(-4) +/- 2.9 x 10(-4) in units of mol fraction per monolayer. It is suggested that the interaction between Ca(2+)-ATPase and SLN in the sarcoplasmic reticulum could be important in thermogenesis by the sarcoplasmic reticulum.     

Interaction of amphipols with sarcoplasmic reticulum Ca2+-ATPase

Amphipols are short-chain amphipathic polymers designed to keep membrane proteins soluble in aqueous solutions. We have evaluated the effects of the interaction of amphipols with sarcoplasmic reticulum Ca(2+)-ATPase either in a membrane-bound or a soluble form. If the addition of amphipols to detergent-solubilized ATPase was followed by removal of detergent, soluble complexes formed, but these complexes retained poor ATPase activity, were not very stable upon long incubation periods, and at high concentrations they experienced aggregation. Nevertheless, adding excess detergent to diluted detergent-free ATPase-amphipol complexes incubated for short periods immediately restored full activity to these complexes, showing that amphipols had protected solubilized ATPase from the rapid and irreversible inactivation that otherwise follows detergent removal. Amphipols also protected solubilized ATPase from the rapid and irreversible inactivation observed in detergent solutions if the ATPase Ca(2+) binding sites remain vacant. Moreover, in the presence of Ca(2+), amphipol/detergent mixtures stabilized concentrated ATPase against inactivation and aggregation, whether in the presence or absence of lipids, for much longer periods of time (days) than detergent alone. Our observations suggest that mixtures of amphipols and detergents are promising media for handling solubilized Ca(2+)-ATPase under conditions that would otherwise lead to its irreversible denaturation and/or aggregation.     

Immunological relatedness of the sarcoplasmic reticulum Ca(2+)-ATPase and the Na+,K(+)-ATPase.

The effect of anti-ATPase antibodies with epitopes near Asp-351 (PR-8), Lys-515 (PR-11) and the ATP binding domain (D12) of the Ca(2+)-ATPase of sarcoplasmic reticulum (EC 3.6.1.38) was analyzed. The PR-8 and D12 antibodies reacted freely with the Ca(2+)-ATPase in the native membrane, indicating that their epitopes are exposed on the cytoplasmic surface. Both PR-8 and D12 interfered with the crystallization of the Ca(2+)-ATPase, suggesting that their binding sites are at interfaces between ATPase molecules. PR-11 had no effect on ATPase-ATPase interactions or on the ATPase activity of sarcoplasmic reticulum. The epitope of PR-11 is suggested to be the VIDRC sequence at residues 520-525, while that of D12 at residues 670-720 of the Ca(2+)-ATPase. The use of predictive algorithms of antigenicity for identification of potential antigenic determinants in the Ca(2+)-ATPase is analyzed.     

Myeloperoxidase-derived oxidants inhibit sarco/endoplasmic reticulum Ca2+-ATPase activity and perturb Ca2+ homeostasis in human coronary artery endothelial cells

The sarco/endoplasmic reticulum Ca(2+)-ATPase (SERCA) plays a critical role in Ca(2+) homeostasis via sequestration of this ion in the sarco/endoplasmic reticulum. The activity of this pump is inhibited by oxidants and impaired in aging tissues and cardiovascular disease. We have shown previously that the myeloperoxidase (MPO)-derived oxidants HOCl and HOSCN target thiols and mediate cellular dysfunction. As SERCA contains Cys residues critical to ATPase activity, we hypothesized that HOCl and HOSCN might inhibit SERCA activity, via thiol oxidation, and increase cytosolic Ca(2+) levels in human coronary artery endothelial cells (HCAEC). Exposure of sarcoplasmic reticulum vesicles to preformed or enzymatically generated HOCl and HOSCN resulted in a concentration-dependent decrease in ATPase activity; this was also inhibited by the SERCA inhibitor thapsigargin. Decomposed HOSCN and incomplete MPO enzyme systems did not decrease activity. Loss of ATPase activity occurred concurrent with oxidation of SERCA Cys residues and protein modification. Exposure of HCAEC, with or without external Ca(2+), to HOSCN or HOCl resulted in a time- and concentration-dependent increase in intracellular Ca(2+) under conditions that did not result in immediate loss of cell viability. Thapsigargin, but not inhibitors of plasma membrane or mitochondrial Ca(2+) pumps/channels, completely attenuated the increase in intracellular Ca(2+) consistent with a critical role for SERCA in maintaining endothelial cell Ca(2+) homeostasis. Angiotensin II pretreatment potentiated the effect of HOSCN at low concentrations. MPO-mediated modulation of intracellular Ca(2+) levels may exacerbate endothelial dysfunction, a key early event in atherosclerosis, and be more marked in smokers because of their higher SCN(-) levels.     

Inhibition by 4-hydroxynonenal (HNE) of Ca2+ transport by SERCA1a: low concentrations of HNE open protein-mediated leaks in the membrane

Exposure of sarcoplasmic reticulum membranes to 4-hydroxy-2-nonenal (HNE) resulted in inhibition of the maximal ATPase activity and Ca(2+) transport ability of SERCA1a, the Ca(2+) pump in these membranes. The concomitant presence of ATP significantly protected SERCA1a ATPase activity from inhibition. ATP binding and phosphoenzyme formation from ATP were reduced after treatment with HNE, whereas Ca(2+) binding to the high-affinity sites was altered to a lower extent. HNE reacted with SH groups, some of which were identified by MALDI-TOF mass spectrometry, and competition studies with FITC indicated that HNE also reacted with Lys(515) within the nucleotide binding pocket of SERCA1a. A remarkable fact was that both the steady-state ability of SR vesicles to sequester Ca(2+) and the ATPase activity of SR membranes in the absence of added ionophore or detergent were sensitive to concentrations of HNE much smaller than those that affected the maximal ATPase activity of SERCA1a. This was due to an increase in the passive permeability of HNE-treated SR vesicles to Ca(2+), an increase in permeability that did not arise from alteration of the lipid component of these vesicles. Judging from immunodetection with an anti-HNE antibody, this HNE-dependent increase in permeability probably arose from modification of proteins of about 150-160kDa, present in very low abundance in longitudinal SR membranes (and in slightly larger abundance in SR terminal cisternae). HNE-induced promotion, via these proteins, of Ca(2+) leakage pathways might be involved in the general toxic effects of HNE.     

Comparison of the (Ca2+ + Mg2+)-ATPase proteins from normal and dystrophic chicken sarcoplasmic reticulum.

The involvement of membrane protein in dystrophic chicken fragmented sarcoplasmic reticulum alterations has been examined. A purified preparation of the (Ca2+ + Mg2+)-ATPase protein from dystrophic fragmented sarcoplasmic reticulum was found to have a reduced calcium-sensitive ATPase activity and phosphoenzyme level, in agreement with alterations found in dystrophic chicken fragmented sarcoplasmic reticulum. An amino acid analysis of the ATPase preparations showed no difference in the normal and dystrophic (Ca2+ + Mg2+)-ATPase. The (Ca2+ + Mg2+)-ATPase was investigated further by isoelectric focusing and proteolytic digestion of the fragmented sarcoplasmic reticulum. Neither of these methods indicated any alteration in the composition of the dystrophic (Ca2+ + Mg2+)-ATPase. We have concluded that the alterations observed in dystrophic fragmented sarcoplasmic reticulum are not due to increased amounts of non-(Ca2+ + Mg2+)-ATPase protein, and that the normal and dystrophic (Ca2+ + Mg2+)-ATPase protein are not detectably different.     

Calcium binding and allosteric signaling mechanisms for the sarcoplasmic reticulum Ca(2+) ATPase

The sarcoplasmic reticulum Ca(2+) ATPase (SERCA) is a membrane-bound pump that utilizes ATP to drive calcium ions from the myocyte cytosol against the higher calcium concentration in the sarcoplasmic reticulum. Conformational transitions associated with Ca(2+) -binding are important to its catalytic function. We have identified collective motions that partition SERCA crystallographic structures into multiple catalytically-distinct states using principal component analysis. Using Brownian dynamics simulations, we demonstrate the important contribution of surface-exposed, polar residues in the diffusional encounter of Ca(2+) . Molecular dynamics simulations indicate the role of Glu309 gating in binding Ca(2+) , as well as subsequent changes in the dynamics of SERCA's cytosolic domains. Together these data provide structural and dynamical insights into a multistep process involving Ca(2+) binding and catalytic transitions.     

A disulfide crosslink between Cys98 of troponin-C and Cys133 of troponin-I abolishes the activity of rabbit skeletal troponin.

Various thio-reactive bifunctional crosslinkers as well as 5,5'-dithiobis(2-nitrobenzoate)-mediated disulfide bond formation were used to crosslink troponin-C and troponin-I, the Ca(2+)-binding and inhibitory subunits of troponin, respectively. In all cases, substantial crosslinking was obtained when the reactions were carried out in the absence of Ca2+. No disulfide crosslinking occurred if either Cys98 of TnC, or Cys133 of TnI were blocked, indicating that these thiols are involved in the crosslinking. Troponin containing the disulfide crosslink is no longer capable of regulating actomyosin ATPase activity in a Ca(2+)-dependent manner. Our results suggest that the relative movement between the Cys98 region of TnC and the Cys133 region of TnI is required for the Ca(2+)-regulatory process in skeletal muscle.     

Effect of calcium on the interactions between Ca2+-ATPase molecules in sarcoplasmic reticulum

The interaction between Ca2+-ATPase molecules in the native sarcoplasmic reticulum membrane and in detergent solutions was analyzed by chemical crosslinking, high performance liquid chromatography (HPLC), and by the polarization of fluorescence of fluorescein 5'-isothiocyanate (FITC) covalently attached to the Ca2+-ATPase. Reaction of sarcoplasmic reticulum vesicles with glutaraldehyde causes the crosslinking of Ca2+-ATPase molecules with the formation of dimers, tetramers and higher oligomers. At moderate concentrations of glutaraldehyde solubilization of sarcoplasmic reticulum by C12 E8 or Brij 36T (approximately equal to 4 mg/mg protein) decreased the formation of higher oligomers without significant interference with the appearance of crosslinked ATPase dimers. These observations are consistent with the existence of Ca2+-ATPase dimers in detergent-solubilized sarcoplasmic reticulum. Ca2+ (2-20 mM) and glycerol (10-20%) increased the degree of crosslinking at pH 6.0 both in vesicular and in solubilized sarcoplasmic reticulum, presumably by promoting interactions between ATPase molecules; at pH 7.5 the effect of Ca2+ was less pronounced. In agreement with these observations, high performance liquid chromatography of sarcoplasmic reticulum proteins solubilized by Brij 36T or C12 E10 revealed the presence of components with the expected elution characteristics of Ca2+-ATPase oligomers. The polarization of fluorescence of FITC covalently attached to the Ca2+-ATPase is low in the native sarcoplasmic reticulum due to energy transfer, consistent with the existence of ATPase oligomers (Highsmith, S. and Cohen, J.A. (1987) Biochemistry 26, 154-161); upon solubilization of the sarcoplasmic reticulum by detergents, the polarization of fluorescence increased due to dissociation of ATPase oligomers. Based on its effects on the fluorescence of FITC-ATPase, Ca2+ promoted the interaction between ATPase molecules, both in the native membrane and in detergent solutions.     

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.     

Modulation of the sarcoplasmic reticulum (Ca2+ + Mg2+)-ATPase by pentobarbital

The dependence of the (Ca2+ + Mg2+)-ATPase activity of sarcoplasmic reticulum vesicles upon the concentration of pentobarbital shows a biphasic pattern. Concentrations of pentobarbital ranging from 2 to 8 mM produce a slight stimulation, approximately 20-30%, of the ATPase activity of sarcoplasmic reticulum vesicles made leaky to Ca2+, whereas pentobarbital concentrations above 10 mM strongly inhibit the activity. The purified ATPase shows a higher sensitivity to pentobarbital, namely 3-4-fold shift towards lower values of the K0.5 value of inhibition by this drug. These effects of pentobarbital are observed over a wide range of ATP concentrations. In addition, this drug shifts the Ca2+ dependence of the (Ca2+ + Mg2+)-ATPase activity towards higher values of free Ca2+ concentrations and increases several-fold the passive permeability to Ca2+ of the sarcoplasmic reticulum membranes. At the concentrations of pentobarbital that inhibit this enzyme in the sarcoplasmic reticulum membrane, pentobarbital does not significantly alter the order parameter of these membranes as monitored with diphenylhexatriene, whereas the temperature of denaturation of the (Ca2+ + Mg2+)-ATPase is decreased by 4-5 C degrees, thus, indicating that the conformation of the ATPase is altered. The effects of pentobarbital on the intensity of the fluorescence of fluorescein-labeled (Ca2+ + Mg2+)-ATPase in sarcoplasmic reticulum also support the hypothesis of a conformational change in the enzyme induced by millimolar concentrations of this drug. It is concluded that the inhibition of the sarcoplasmic reticulum ATPase by pentobarbital is a consequence of its binding to hydrophobic binding sites in this enzyme.     

Deduced amino acid sequence and E1-E2 equilibrium of the sarcoplasmic reticulum Ca(2+)-ATPase of frog skeletal muscle. Comparison with the Ca(2+)-ATPase of rabbit fast twitch muscle.

The cDNA encoding a Ca(2+)-transport ATPase of frog (Rana esculenta) skeletal muscle was isolated and characterized. The deduced amino acid sequence, consisting of 994 residues, showed 89% identity to the fast twitch muscle sarcoplasmic reticulum Ca(2+)-ATPases of chicken and rabbit. Northern blot analysis using a fragment of this cDNA as probe detected a 5.0 kb message in frog skeletal muscle but did not detect any mRNA encoding sarcoplasmic reticulum Ca(2+)-ATPase in frog cardiac muscle. The enzymatic properties of the amphibian skeletal muscle Ca(2+)-ATPase were compared with those of the rabbit fast twitch muscle Ca(2+)-ATPase by functional expression of the cDNAs in COS-1 cells. The amphibian Ca(2+)-ATPase displayed a reduced apparent affinity for Ca2+ and an increased apparent affinity for the inhibitors, vanadate and thapsigargin, relative to the mammalian enzyme. This may be explained by a mechanism in which relatively more of the E2 conformation accumulated in the frog Ca(2+)-ATPase than in the mammalian enzyme.