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Changes in the patterns of myosin heavy chain (MHC) isoforms, isomyosins, and Ca(2+)-ATPase (SERCA) isoforms were studied in long-term (72 d) stimulated fast-twitch extensor digitorum longus (EDL) and tibialis anterior (TA) muscles of euthyroid and hyperthyroid rabbits. The chronic low-frequency stimulation-induced fast-to-slow transitions in MHC isoforms, isomyosins and SERCA isoforms were pronounced in muscles from euthyroid rabbits, but less pronounced in muscles from hyperthyroid rabbits. Thus, hyperthyroidism counteracted to same extent the stimulation-induced fast-to-slow transition. Analyses of all parameters were performed on the same individual muscles, providing information on the co-ordinated expression of SERCA and myosin isoforms. A high correlation (r = 0.97) was detected between relative concentrations of slow SERCA2a and slow MHCI isoforms. This correlation persisted under all experimental conditions, suggesting a co-ordinated expression of slow myosin and Ca(2+)-ATPase isoforms. Conversely, fast SERCA1a was correlated to fast myosin isoforms as a whole.

We have purified Ca2+-ATPase from synaptosomal membranes (SM)1 from rat cerebellum by calmodulin affinity chromatography. The enzyme was identified as plasma membrane Ca2+-ATPase by its interaction with calmodulin and monoclonal antibodies produced against red blood cell (RBC) Ca2+-ATPase, and by thapsigargin insensitivity. The purpose of the study was to establish whether two regulators of the RBC Ca2+-ATPase, calmodulin and protein kinase C (PKC), affect the Ca2+-ATPase isolated from excitable cells and whether their effects are comparable to those on the RBC Ca2+-ATPase. We found that calmodulin and PKC activated both enzymes. There were significant quantitative differences in the phosphorylation and activation of the SM versus RBC Ca2+-ATPase. The steady-state Ca2+-ATPase activity of SM Ca2+-ATPase was approximately 3 fold lower and significantly less stimulated by calmodulin. The initial rate of PKC catalyzed phosphorylation (in the presence of 12-myristate 13-acetate phorbol) was approximately two times slower for SM enzyme. While phosphorylation of RBC Ca2+-ATPase approached maximum level at around 5 min, comparable level of phosphorylation of SM Ca2+-ATPase was observed only after 30 min. The PKC-catalyzed phosphorylation resulted in a statistically significant increase in Ca2+-ATPase activity of up to 20-40%, higher in the SM Ca2+-ATPase. The differences may be associated with diversities in Ca2+-ATPase function in erythrocytes and neuronal cells and different isoforms composition.

The properties of Ca2+-ATPase purified and reconstituted from rabbit skeletal sarcoplasmic reticulum (SR) has been studied in comparison with the preparations obtained by the commonly used detergent poly(oxyethylene)8-lauryl ether (C12E8) and the bile salt detergents cholate and deoxycholate. 1,2-Diheptanoyl-sn-phosphatidylcholine (DHPC) has been shown to be excellent for solubilizing a wide variety of membrane proteins [Kessi, Poiree, Wehrli, Bachofen, Semenza and Hauser (1994) Biochemistry 33, 10825-10836]. The DHPC method consistently gave higher yields of purified Ca2+-ATPase with a greater specific activity than the methods with C12E8, cholate, or deoxycholate. DHPC and C12E8 were superior to cholate and deoxycholate in active enzyme yields and specific activity. DHPC-solubilized Ca2+-ATPase purified on a density gradient retained the E1Ca-E1(*)Ca conformational transition, whereas the enzyme from the C12E8 purification did not retain this transition. The coupling of Ca2+ transported to ATP hydrolysed in the DHPC-purified enzyme was maximal and matched the values obtained with native SR, whereas the coupling was much lower for the C12E8-purified enzyme. The specific activity of Ca2+-ATPase reconstituted into dioleoylphosphatidylcholine vesicles with DHPC was up to 2-fold greater than that achieved with C12E8, and is comparable to that measured in the native SR. Finally, the dissociation of Ca2+-ATPase into monomers by DHPC preserved the ATPase activity, whereas similar dissociation by C12E8 gave only one-sixth the activity of that obtained with DHPC. These studies show that the Ca2+-ATPase solubilized, purified and reconstituted with DHPC is superior to that obtained with C12E8 in significant ways, making it a preparation suitable for detailed studies on the mechanism of ion transport and the role of protein-lipid interactions in the function of membrane proteins.

The subcellular locations of Ca(2+)-ATPases in the membranes of cauliflower (Brassica oleracea L.) inflorescences were investigated. After continuous sucrose gradient centrifugation a 111-kD calmodulin (CaM)-stimulated and caM-binding Ca(2+)-ATPase (BCA1; P. Askerlund [1996] Plant Physiol 110: 913-922; S. Malmström, P. Askerlund, M.G. Plamgren [1997] FEBS Lett 400: 324-328) comigrated with vacuolar membrane markers, whereas a 116-kD caM-binding Ca(2+)-ATPase co-migrated with a marker for the plasma membrane. The 116 kD Ca(2+)-ATPase was enriched in plasma membranes obtained by aqueous two-phase partitioning, which is in agreement with a plasma membrane location of this Ca(2+)-ATPase. Countercurrent distribution of a low-density intracellular membrane fraction in an aqueous two-phase system resulted in the separation of the endoplasmic reticulum and vacuolar membranes. The 111-kD Ca(2+)-ATPase co-migrated with a vacuolar membrane marker after countercurrent distribution but not with markers for the endoplasmic reticulum. A vacuolar membrane location of the 111-kD Ca(2+)-AtPase was further supported by experiments with isolated vacuoles from cauliflower: (a) Immunoblotting with an antibody against the 111-kD Ca(2+)-ATPase showed that it was associated with the vacuoles, and (b) ATP-dependent Ca2+ uptake by the intact vacuoles was found to be CaM stimulated and partly protonophore insensitive.

In an attempt to better define the parameters governing reconstitution and two-dimensional crystallization of membrane proteins, we have studied Ca2(+)-ATPase from rabbit sarcoplasmic reticulum. This ion pump forms vanadate-induced crystals in its native membrane and has previously been reconstituted at high lipid-to-protein ratios for functional studies. We have characterized the reconstitution of purified Ca2(+)-ATPase at low lipid-to-protein ratios and discovered procedures that produce long, tubular crystals suitable for helical reconstruction. C12E8 (n-dodecyl-octaethylene-glycol monoether) was used to fully solubilize various mixtures of lipid and purified Ca2(+)-ATPase, and BioBeads were then used to remove the C12E8. Slow removal resulted in two populations of vesicles, and the proteoliposome population was separated from the liposome population on a sucrose density gradient. These proteoliposomes had a lipid-to-protein ratio of 1:2, and virtually 100% of molecules faced the outside of vesicles, as determined by fluorescein isothiocyanate labeling. Cycles of freeze-thaw caused considerable aggregation of these proteoliposomes, and, if phosphatidyl ethanolamine and phosphatidic acid were included, or if the bilayers were doped with small amounts of C12E8, vanadate-induced tubular crystals grew from the aggregates. Thus our procedure comprised two steps-reconstitution followed by crystallization-allowing us to consider mechanisms of bilayer formation separately from those of crystallization and tube formation.

PMR1, a P-type ATPase cloned from the yeast Saccharomyces cerevisiae, was previously localized to the Golgi, and shown to be required for normal secretory processes (Antebi, A., and Fink, G.R. (1992) Mol. Biol. Cell 3, 633-654). We provide biochemical evidence that PMR1 is a Ca2+-transporting ATPase in the Golgi, a hitherto unusual location for a Ca2+ pump. As a starting point for structure-function analysis using a mutagenic approach, we used the strong and inducible heat shock promoter to direct high level expression of PMR1 from a multicopy plasmid. Yeast lysates were separated on sucrose density gradients, and fractions assayed for organellar markers. PMR1 is found in fractions containing the Golgi marker guanosine diphosphatase, and is associated with an ATP-dependent, protonophore-insensitive 45Ca2+ uptake activity. This activity is virtually abolished in the absence of the expression plasmid. Furthermore, replacement of the active site aspartate within the phosphorylation domain had the expected effect of abolishing Ca2+ transport activity entirely. Interestingly, the mutant enzymes (Asp-371 --> Glu and Asp-371 --> Asn) demonstrated proper targeting to the Golgi, unlike analogous mutations in the related yeast H+-ATPase. Detailed characterization of calcium transport by PMR1 showed that sensitivity to inhibitors (vanadate, thapsigargin, and cyclopiazonic acid) and affinity for substrates (MgATP and Ca2+) were different from the previously characterized sarco/endoplasmic reticulum and plasma membrane Ca2+-ATPases. PMR1 therefore represents a new and distinct P-type Ca2+-ATPase. Because close homologs of PMR1 have been cloned from rat and other organisms, we suggest that Ca2+-ATPases in the Golgi will form a discrete subgroup that are important for functioning of the secretory pathway.

This work establishes the potential of Bio-Beads as a simple alternative to conventional dialysis for removing detergent and for obtaining 2D crystals of integral membrane proteins useful for structure analysis by electron crystallography. Kinetic and equilibrium aspects of removal of different detergents by adsorption onto hydrophobic Bio-Beads SM2 have been systematically investigated and extended to 2D crystallization of different prototypic membrane proteins, including: (a) Ca2+ ATPase from sarcoplasmic reticulum; (b) melibiose permease from Escherichia coli; (c) cytochrome b6f from Chlamydomonas reinhardtii. Different crystals could be produced from all protein preparations, with optical diffraction down to 20-25 A in negative stain.

The subcellular distribution of plasma-membrane-type Ca2+-transporting ATPases was studied in barley leaves (Hordeum vulgare L.). A highly enriched plasma membrane (PM) fraction was analysed for Ca2+ pumps and compared with several inner-membrane preparations, including the tonoplast, the envelope of the chloroplast, and an endoplasmic reticulum (ER)-enriched fraction. The enzymes were identified and characterised with regard to the phosphointermediate formation, their nucleotide specificity and their binding to calmodulin. A Ca2+-transporting ATPase (molecular mass approximately 130 kDa), which showed high specificity for ATP and high affinity for calmodulin, was localised in the PM. A 116-kDa Ca2+-transporting ATPase, probably located in the ER, showed lower nucleotide specificity and weaker affinity for calmodulin. A comparison of the distribution of the pumps in leaves and roots indicated that the ratio of expression of the two enzymes changed in a tissue-specific manner: the PM pump was dominant in leaves, while the inner-membrane pump was expressed at a higher level in the roots. For the purification of calmodulin-binding proteins (Ca2+ pumps), microsomes isolated from tobacco cell cultures were used. Two active Ca2+ pumps were identified, and the one at 116 kDa was partially sequenced.

The Na+,K(+)-ATPase and the sarcoplasmic/endoplasmic reticulum Ca(2+)-(SERCA-) ATPase belong to a family of P-type ATPases that undergo a cycle of conformational changes between the phosphorylated and dephosphorylated stages in an ion-specific manner. The ouabain-inhibitable Na+,K(+)-ATPase activity requires Na+ and K+. On the other hand, the Ca(2+)-dependent and thapsigargin-inhibitable activity of the SERCA-ATPase does not depend upon Na+ and K+ for its basal activity. However, the SERCA-ATPase and Ca(2+)-transport activities can be further activated either by K+ in a two-step fashion with high (ED50 approximately 20 mM) and low affinity (ED50 approximately 70 mM) or by Na+ in a one-step fashion with an ED50 value of approximately 50 mM. A chimera, in which the carboxy-terminal region (Leu861-COOH) of the Na+,K(+)-ATPase alpha 1 subunit replaced the corresponding region (Ser830-COOH) of the SERCA1-ATPase, lacked the low-affinity K+ activation of the SERCA-ATPase but displayed a higher-affinity (ED50 < 10 mM) activation by K+, similar to that of the Na+,K(+)-ATPase, whereas activation by Na+ was not affected. The replacement of the large cytosolic loop (Gly354-Lys712) and the amino-terminal regions (Met1-Asp162) of the SERCA1-ATPase with the corresponding portions of the Na+,K(+)-ATPase alpha 1 subunit did not affect the sensitivity of the SERCA-ATPase activity to K+. Thus, the carboxy-terminal regions of both the SERCA1 and the Na+,K(+)-ATPase alpha 1 subunit are critical for K+ sensitivity. Analysis of additional (Ca2+/Na+,K+)-ATPase chimeras demonstrated that the carboxy-terminal 102 amino acids (Phe920-Tyr1021) of the Na+/K(+)-ATPase alpha 1 subunit are sufficient to shift the K+ affinity for activation of the SERCA-ATPase without the beta subunit. No change in the two-step activation of SERCA-ATPase by K+ was seen when residues Thr871-Thr898 of the SERCA1-ATPase were replaced with residues Asn894-Ala919 of the Na+,K(+)-ATPase alpha 1 subunit, a region known to bind the Na+,K(+)-ATPase beta subunit [Lemas, M. V., et al. (1994) J. Biol. Chem. 269, 8255-8259]. Thus, the Na+,K(+)-ATPase subunit-assembly domain and the K(+)-sensitive region are distinct within the carboxy-terminal 161 amino acids of the Na+,K(+)-ATPase.

Several lines of evidence are presented to show that the Ca(2+)-ATPase activity of total yeast membranes is due to the reticulum (R) type of Ca(2+)-ATPase: (1) Neither calmodulin nor low concentrations of calmodulin antagonists change Ca2+ uptake; (2) removal of plasma membranes (PM) following Con A treatment of spheroplasts (SP) does not significantly alter Ca2+ uptake by the remaining membranes, but increases its specific activity 3.5-fold; (3) after incubation of membranes with [gamma-32P]ATP, SDS-PAGE shows the formation of acyl phosphate intermediates with molecular masses of around 100, 180-190 and 205 kDa; formation of these acyl phosphates requires Ca2+ and is blocked by cyclopiazonic acid, La3+ ions and in the absence of Ca2+. The data on fractionation of yeast membranes are consistent with the suggestion that both the ER and the Golgi are equipped with Ca(2+)-ATPase(s).

We have used frequency-domain fluorescence resonance energy transfer (FRET) and anisotropy measurements to identify the structural properties of wheat germ calmodulin (CaM) bound to either the plasma membrane Ca-ATPase (PM-Ca-ATPase) in native erythrocyte ghost membranes or a peptide (C25W) that has an identical sequence to the CaM-binding domain on the PM-Ca-ATPase. Cross-linking experiments using benzophenone labeled CaM in conjunction with immunoblots using antibodies specific for either CaM or the PM-Ca-ATPase indicate that one molecule of CaM selectively binds one PM-Ca-ATPase polypeptide chain in native erythrocyte ghost membranes. There are no other proteins in the erythrocyte membrane that bind CaM with high affinity, permitting the measurement of the structural properties of CaM bound to the PM-Ca-ATPase in native erythrocyte ghost membranes. FRET measurements between the fluorophore pyrene maleimide (PMal) located at Cys27 in calcium binding loop I and nitrotyrosine139 in calcium binding loop IV on wheat germ CaM indicate that the average spatial separation and conformational heterogeneity associated with the two opposing globular domains of CaM are virtually identical upon CaM binding to either the PM-Ca-ATPase or C25W. Measurements of the solvent accessibility and segmental rotational dynamics of PMal-CaM bound to either the PM-Ca-ATPase or C25W further indicate that the local environment around the pyrene label located at Cys27 is very similar. However, the overall rotational dynamics of CaM bound to the PM-Ca-ATPase is much slower (phi 2 = 83 +/- 14 ns) than observed when CaM binds C25W (phi 2 = 10.3 +/- 0.5 ns). This implies that CaM is tightly associated with the CaM-binding domain of the PM-Ca-ATPase and that the observed rotational motion of pyrenylmaleimide labeled CaM is characteristic of the global motion of the CaM-binding domain on the PM-Ca-ATPase. The similar conformational heterogeneity and local environment of CaM bound to either the PM-Ca-ATPase or C25W indicates that CaM binds to a contiguous sequence of amino acids on the Ca-ATPase that are analogous to C25W and that there are no significant interactions with other structural elements within the PM-Ca-ATPase. The rate of rotational motion associated with CaM bound to the PM-Ca-ATPase is consistent with hydrodynamic calculation in which the calmodulin-binding domain located at the carboxyl-terminus of the PM-Ca-ATPase has a stable and defined tertiary structure that is independent of the other cytoplasmic domains of the PM-Ca-ATPase.

Regulation of calcium transport by sarcoplasmic reticulum provides increased cardiac contractility in response to beta-adrenergic stimulation. This is due to phosphorylation of phospholamban by cAMP-dependent protein kinase or by calcium/calmodulin-dependent protein kinase, which activates the calcium pump (Ca2+-ATPase). Recently, direct phosphorylation of Ca2+-ATPase by calcium/calmodulin-dependent protein kinase has been proposed to provide additional regulation. To investigate these effects in detail, we have purified Ca2+-ATPase from cardiac sarcoplasmic reticulum using affinity chromatography and reconstituted it with purified, recombinant phospholamban. The resulting proteoliposomes had high rates of calcium transport, which was tightly coupled to ATP hydrolysis (approximately 1.7 calcium ions transported per ATP molecule hydrolyzed). Co-reconstitution with phospholamban suppressed both calcium uptake and ATPase activities by approximately 50%, and this suppression was fully relieved by a phospholamban monoclonal antibody or by phosphorylation either with cAMP-dependent protein kinase or with calcium/calmodulin-dependent protein kinase. These effects were consistent with a change in the apparent calcium affinity of Ca2+-ATPase and not with a change in Vmax. Neither the purified, reconstituted cardiac Ca2+-ATPase nor the Ca2+-ATPase in longitudinal cardiac sarcoplasmic reticulum vesicles was a substrate for calcium/calmodulin-dependent protein kinase, and accordingly, we found no effect of calcium/calmodulin-dependent protein kinase phosphorylation on Vmax for calcium transport.

A method for tethering proteins to solid surfaces has been utilized to form vectorially oriented monolayers of the detergent-solubilized integral membrane protein Ca(2+) -ATPase from the sarcoplasmic reticulum (SR). Bifunctional, organic self-assembled monolayers (SAMs) possessing "headgroup" binding specificity for the substrate and "endgroup" binding specificity for the enzyme were utilized to tether the enzyme to the substrate. Specifically, an amine-terminated 11-siloxyundecaneamine SAM was found to bind the Ca(2+)-ATPase primarily electrostatically. The Ca(2+)-ATPase was labeled with the fluorescent probe 5-(2-[(iodoacetyl)amino]ethyl)aminonaphthalene-1-sulfonic acid before monolayer formation. Consequently, fluorescence measurements performed on amine-terminated SAM/enzyme monolayers formed on quartz substrates served to establish the nature of protein binding. Formation of the monolayers on inorganic multilayer substrates fabricated by molecular beam epitaxy made it possible to use x-ray interferometry to determine the profile structure for the system, which was proved correct by x-ray holography. The profile structures established the vectorial orientation of the Ca(2+)-ATPase within these monolayers, to a spatial resolution of approximately 12 A. Such vectorially oriented monolayers of detergent-solubilized Ca(2+)-ATPase from SR make possible a wide variety of correlative structure/function studies, which would serve to elucidate the mechanism of Ca(2+) transport by this enzyme.

Mutants of the plasma membrane Ca2+ pump (PMCA), in which amino acids in transmembrane domains (TM) 4, 6, and 8 had been replaced, have been expressed in COS-7 cells. They were analyzed functionally by measuring the uptake of Ca2+ in microsomal preparations and by following the formation of the phosphorylated intermediate from ATP and from phosphate. The mutated residues corresponded to amino acids whose mutation in the sarcoplasmic reticulum pump (SERCA) caused loss of Ca2+ transport by the pump protein: however, only four of the six SERCA residues were conserved in the PMCA pump. Mutation of Glu423 (TM4), Asn879 or Asp883 (TM6), or Gln97l (TM8) suppressed Ca2+ transport by the pump and its ability to form the phosphorylated intermediate starting from ATP. By contrast, the ability of these mutants to form the intermediate starting from phosphate was not impaired. In two mutants (Glu423 and Asp883) it was even enhanced. Two conserved Pro residues of TM4 were also mutated, leading to the loss of the ability of the pump to form the Ca2+- and ATP-dependent phosphorylated intermediate. Unexpectedly, two of the mutations (Asn879 and Gln971) led to the mistargeting of the mutated proteins, i.e., to their retention in the endoplasmic reticulum.

The P-type Ca2+-ATPase from Flavobacterium odoratum has been purified to homogeneity and characterized. Inside-out membrane vesicles were extracted with C12E8, followed by ammonium sulfate fractionation, centrifugation through two successive 32-48% glycerol gradients, and DE52 ion exchange chromatography. The purified Ca2+-ATPase consists of a single polypeptide. It migrates electrophoretically with an apparent molecular mass of 60,000 Da, consistent with the phosphorylation pattern originally reported in membrane vesicles. This single polypeptide is functional and capable of calcium-dependent vanadate-sensitive ATP hydrolysis and of forward and reverse phosphorylation. Maximum hydrolysis activity occurs at pH 8.0, with a specific activity of approximately 75 micromol of ATP hydrolyzed min-1 mg-1 protein. The purified Ca2+-ATPase has an apparent Km for calcium of 1.5 microM and for ATP of 90 microM. Vanadate strongly inhibits the activity with an IC50 of 0.6 microM. The prokaryotic Ca2+-ATPase is insensitive to the SR Ca2+-ATPase inhibitors fluorescein isothiocyanate, thapsigargin, and cyclopiazonic acid. It is rapidly phosphorylated by [gamma-32P]ATP in a calcium-dependent vanadate-inhibited manner and can be phosphorylated by Pi in both the presence and absence of calcium.

The kinetics of the Ca(2+)-ATPase purified from sarcoplasmic reticulum have been studied after reconstitution into bilayers of dimyristoleoylphosphatidylcholine [di(C14:1)PC], dioleoylphosphatidylcholine[di(C18:1)PC] and dinervonylphosphatidylcholine [di(C24:1)PC]. In di(C24:1)PC the rate of phosphorylation of the ATPase by ATP was comparable with that in di(C18:1)PC (about 70 s-1), but in di(C14:1)PC the rate was much lower (21 s-1). Fluorescence responses of the ATPase suggest changes in the phosphoryl-transfer step rather than in the preceding conformational change E1Ca2ATP<-->E1'Ca2ATP. The rate of dephosphorylation of the phosphorylated ATPase was found to decrease in the order di(C24:1)PC < di(C14:1)PC < di(C18:1)PC. For the ATPase in di(C24:1)PC the rate of dephosphorylation (3.3 s-1) was slow enough to be the rate-limiting step for ATP hydrolysis; in di(C14:1)PC, it is suggested that both phosphorylation and dephosphorylation contribute to rate limitation. Phosphorylation of the ATPase in di(C24:1)PC by Pi was normal, but no phosphoenzyme could be detected in di(C14:1)PC. The rate of the Ca(2+)-transport step was normal in di(C24:1)PC, suggesting that the single Ca2+ ion bound to the ATPase in di(C24:1)PC could be transported.

The mechanism of inhibition of the Ca(2+)-ATPase from sarcoplasmic reticulum by the sesquiterpene lactones thapsigargin, trilobolide and thapsivillosin A (TvA) has been determined. A decrease in the affinity of the ATPase for Ca2+ is observed in the presence of the inhibitors (I), consistent with a shift in the E1/E2 equilibrium for the ATPase towards E2 forms. Amounts of inhibitor beyond a 1:1 molar ratio with ATPase produce no further decrease in affinity for Ca2+, inconsistent with the formation of a dead-end complex. Measurements of the rate of quenching of the tryptophan fluorescence of the ATPase by TvA are consistent with an association step to give E2I followed by an isomerization to a modified state E2AI. The kinetics of the reversal of the effects of TvA by Ca2+ at sub-stoichiometric amounts of TvA are bi-exponential, with a fast component whose rate is independent of TvA concentration and equal to the rate observed in the absence of TvA, and a slow component whose rate decreases with increasing TvA concentration. These observations are also consistent with the formation of a modified state E2AI following the initial binding of I to E2. The equilibrium constant E2AI/E2I increases in the order TvA < trilobolide < thapsigargin. The results suggest that the effects of the inhibitors on the overall ratio of E2 to E1 forms of the ATPase follow largely from the formation of E2AI from E2I, and that binding constants are very similar for E1Ca2, E1 and E2.

The effectiveness of a phenolic antioxidant as a radical scavenger is determined by its reactivity toward peroxyl radicals and also by the reactivity of the anti-oxidant phenoxyl radical toward oxidation substrate. If the phenoxyl radical efficiently interacts with vitally important biomolecules, this interaction may result in oxidative damage rather than antioxidant protection. In the present work, we studied effects of phenoxyl radicals generated from a phenolic antitumor drug, Etoposide (VP-16), on oxidation of thiols and activity of Ca(2+)-ATPase in sarcoplasmic reticulum (SR) membranes from skeletal muscles. We found that VP-16 is an effective scavenger of peroxyl radicals as judged by its ability to inhibit a water-soluble azo-initiator, 2,2'-azobis(2-amidinopropane)dihydrochloride (AAPH)-induced (i) chemiluminescence (oxidation) of luminol, (ii) fluorescence decay (oxidation) of cis-parinaric acid incorporated in SR membranes, and (iii) peroxidation of SR membrane lipids. VP-16 did not prevent AAPH-induced oxidation of sulfhydryl groups and inhibition of Ca(2+)-ATPase in SR membranes. Electron spin resonance measurements showed that AAPH-induced VP-16 phenoxyl radicals were reduced by interaction with SR thiols. By using tyrosinase to generate VP-16 phenoxyl radicals as the only source of free radicals in the model system, we found that inhibition of Ca(2+)-ATPase was accompanied by oxidation of about 5 mol of Ca(2+)-ATPase SH groups per 1 mol of oxidized VP-16. Secondary products of VP-16 oxidation (including VP-16 o-quinone) were not efficient in inhibiting SR Ca(2+)-ATPase. Reduction of VP-16 phenoxyl radicals by ascorbate protected against AAPH- and tyrosinase-induced thiol oxidation and Ca(2+)-ATPase inhibition. The results suggest that efficient phenolic scavengers of peroxyl radicals such as VP-16--which are commonly considered as potent antioxidants--may themselves produce oxidative stress due to secondary reactions of their phenoxyl radicals with thiols.

The genotoxic carcinogen aflatoxin B1 (AFB1) inhibited the calmodulin-stimulated membrane-bound (Ca2+Mg2+)-ATPase. Using the purified enzyme, 12 nmoles per ml of AFB1 caused maximum inhibition of 28% and 50%, of the acidic phospholipid-stimulated and calmodulin-activated Ca(2+)-ATPase activity respectively. Treatment of red cell ghosts with increasing concentrations of Triton X-100, a non-ionic detergent caused a progressive loss of both the basal and calmodulin-stimulated Ca(2+)-ATPase activity. The activity of the phospholipid-free, detergent-solubilized enzyme was almost fully restored by phosphatidyl serine (PS) and its sensitivity to calmodulin was restored in the presence of phosphatidyl choline (PC). Analysis of the results obtained using varying concentrations of ATP shows that AFB1 did not affect the Km and Vmax of the unstimulated enzyme whereas these parameters were reduced by about 75% and 50%, respectively, in the presence of calmodulin. Using the product of limited proteolysis by trypsin i.e. the 90 kDa fragment which still retains its calmodulin binding-domain and the 76 kDa fragment which has lost this domain, kinetic studies on the enzyme activity revealed that AFB1 inhibited the calmodulin-activated 90 kDa fragment by about 50% while the 76 kDa was not affected at all by the toxin and calmodulin. The toxin had no significant affect on the basal activity of the 90 kDa limited proteolysis fragment of the enzyme. These observations suggest that AFB1 inhibits the activated Ca(2+)-ATPase by binding to an important site in the calmodulin-binding domain of the enzyme. It seems likely that the toxin binds to tryptophan in the calmodulin-binding domain, thus causing a reduction in the rate at which this domain can interact with Ca(2+)-calmodulin or acidic phospholipids. The implication of these observations is that Ca(2+)-extrusion and other calmodulin-activated enzymes and processes may be slowed down during prolonged exposure to AFB1 because of its anticalmodulin effect.

Stable Chinese hamster ovary (CHO) cell lines overexpressing the human plasma membrane Ca(2+)-ATPase (PMCA) were generated, and three independent cell clones were characterized in details. They overexpressed high amounts of active PMCA pump (15-20 times over the amount of endogenous PMCA) as indicated by experiments in which the formation of the phosphoenzyme intermediate and the uptake of Ca2+ by microsomes were measured. Immunocytochemistry experiments coupled to the biotinylation of the pump in the intact cells indicated the correct deliver of the expressed pump to the plasma membrane. The expressed pump was purified by affinity chromatography on calmodulin sepharose. The PMCA of transfected CHO cells promoted an increase of Ca2+ into the medium, after induction of Ca2+ release from the internal stores by activation of a purinergic receptor. An evident decrease of the activity of the endogenous sarcoplasmic reticulum Ca(2+)-ATPase pump was observed, probably related to the down-regulation of its expression. The cells overexpressing the PMCA pump had delayed recovery after trypsinization and plating. Their doubling time was, however, the same as CHO cells.

We have determined directly the effects of the inhibitory peptide phospholamban (PLB) on the rotational dynamics of the calcium pump (Ca-ATPase) of cardiac sarcoplasmic reticulum (SR). This was accomplished by comparing mouse ventricular SR, which has PLB levels similar to those found in other mammals, with mouse atrial SR, which is effectively devoid of PLB and thus has much higher (unregulated) calcium pump activity. To obtain sufficient quantities of atrial SR, we isolated the membranes from atrial tumor cells. We used time-resolved phosphorescence anisotropy of an erythrosin isothiocyanate label attached selectively and rigidly to the Ca-ATPase, to detect the microsecond rotational motion of the Ca-ATPase in the two preparations. The time-resolved phosphorescence anisotropy decays of both preparations at 25 degrees C were multi-exponential, because of the presence of different oligomeric species. The rotational correlation times for the different oligomers were similar for the two preparations, but the total decay amplitude was substantially greater for atrial tumor SR, indicating that a smaller fraction of the Ca-ATPase molecules exists as large aggregates. Phosphorylation of PLB in ventricular SR decreased the population of large-scale Ca-ATPase aggregates to a level similar to that of atrial tumor SR. Lipid chain mobility (fluidity), detected by electron paramagnetic resonance of stearic acid spin labels, was very similar in the two preparations, indicating that the higher protein mobility in atrial tumor SR is not due to higher lipid fluidity. We conclude that PLB inhibits by inducing Ca-ATPase lateral aggregation, which can be relieved either by phosphorylating or removing PLB.

1. Several cell-surface domains of sarcolemma and T-tubule from skeletal-muscle fibre were isolated and characterized. 2. A protocol of subcellular fractionation was set up that involved the sequential low- and high-speed homogenization of rat skeletal muscle followed by KCl washing, Ca2+ loading and sucrose-density-gradient centrifugation. This protocol led to the separation of cell-surface membranes from membranes enriched in sarcoplasmic reticulum and intracellular GLUT4-containing vesicles. 3. Agglutination of cell-surface membranes using wheat-germ agglutinin allowed the isolation of three distinct cell-surface membrane domains: sarcolemmal fraction 1 (SM1), sarcolemmal fraction 2 (SM2) and a T-tubule fraction enriched in protein tt28 and the alpha 2-component of dihydropyridine receptor. 4. Fractions SM1 and SM2 represented distinct sarcolemmal subcompartments based on different compositions of biochemical markers: SM2 was characterized by high levels of beta 1-integrin and dystrophin, and SM1 was enriched in beta 1-integrin but lacked dystrophin. 5. The caveolae-associated molecule caveolin was very abundant in SM1, SM2 and T-tubules, suggesting the presence of caveolae or caveolin-rich domains in these cell-surface membrane domains. In contrast, clathrin heavy chain was abundant in SM1 and T-tubules, but only trace levels were detected in SM2. 6. Immunoadsorption of T-tubule vesicles with antibodies against protein tt28 and against GLUT4 revealed the presence of GLUT4 in T-tubules under basal conditions and it also allowed the identification of two distinct pools of T-tubules showing different contents of tt28 and dihydropyridine receptors. 7. Our data on distribution of clathrin and dystrophin reveal the existence of subcompartments in sarcolemma from muscle fibre, featuring selective mutually exclusive components. T-tubules contain caveolin and clathrin suggesting that they contain caveolin- and clathrin-rich domains. Furthermore, evidence for the heterogeneous distribution of membrane proteins in T-tubules is also presented.

Phospholipid molecules containing a cholate or hemisuccinylcholate moiety at the 2-position were synthesized as possible detergents for solubilization of membrane proteins. 1-Myristoleoyl-2-cholyl-sn-glycero-3- phosphatidylcholine ((C14:1,cholyl)PC) was found to solubilize sarcoplasmic reticulum vesicles at concentrations above its cmc of ca. 4 micrograms/ml. Effects of (C14:1,cholyl)PC on the activity of the Ca(2+)-ATPase of sarcoplasmic reticulum were complex, as for other detergents. High concentrations (0.2 mg/ml) of (C14:1,cholyl)PC were able to displace phospholipids from the lipid/protein interface of the ATPase. Although under these conditions the activity of the ATPase was low, the ATPase was not denatured since activity could be regained by displacement of (C14:1,cholyl)PC with the detergent C12E8. 1-oleoyl-2-cholyl-sn-glycero-3-phosphatidylcholine ((C18:1,cholyl)PC) was found to have a very low water solubility, but this could be increased by the introduction of a hemisuccinyl group to give 1-oleoyl-2-(3 alpha-hemisuccinyl)cholyl-sn-glycero-3-phosphatidylcholine ((C18:1,cholylCOOH)PC). This was able to solubilize and delipidate the Ca(2+)-ATPase; the ATPase was stable in (C18:1,cholylCOOH)PC for long periods of time.

We have determined the primary structure of the rat plasma membrane Ca(2+)-ATPase isoform 4 (PMCA4), and have analysed its mRNA tissue distribution and alternative splicing patterns at splice site A. Rat PMCA4 (rPMCA4) genomic clones were isolated and used to determine the coding sequences and intron/exon organization of the 5'-end of the gene, and the remaining coding sequence was determined from PCR-amplified cDNA fragments. Pairwise comparisons reveal that the amino acid sequence of rPMCA4 has diverged substantially from those of rPMCA isoforms 1, 2 and 3 (73-76% identity) and from that of human PMCA4 (87%). Despite the high degree of sequence divergence between the two species, comparisons of intron and untranslated mRNA sequences with the corresponding human sequences confirm the identity of this rat isoform as PMCA4. Northern blot studies demonstrate that the PMCA4 mRNA is expressed in all rat tissues examined except liver, with the highest levels in uterus and stomach. A combination of PCR analysis of alternative splicing patterns and sequence analysis of the gene demonstrate that a 36 nt exon at site A is included in PMCA4 mRNAs of most tissues but is largely excluded in heart and testis. Alternative splicing of both the 36 nt exon and a previously characterized 175 nt exon at splice site C, each of which can be either included or excluded in a highly tissue-specific manner, leads to the production of four different PMCA4 variants ranging in size from 1157 to 1203 amino acids.

Obtaining large, flat, well ordered crystals represents the key to structure determination by electron crystallography. Multilamellar crystals of Ca(2+)-ATPase are a good candidate for this methodology, and we have optimized methods of crystallization and of preparation for cryoelectron microscopy. In particular, high concentrations of glycerol were found to prevent nucleation and to reduce stacking; thus, by seeding solutions containing 40% glycerol, we obtained thin crystals that were 5-30 microns in diameter and 2-10 unit cells thick. We found that removing vesicles and minimizing concentrations of divalent cations were critical to preparing flat crystals in the frozen-hydrated state. Finally, we developed two methods for determining the number of lamellae composing individual crystals, information that is required for structure determination of this crystal form. The first method, using low magnification images of freeze-dried crystals, is more practical in our case. Nevertheless, the alternative method, involving analysis of Laue zones from electron diffraction patterns of slightly tilted crystals, may be of general use in structure determination from thin, three-dimensional crystals.

Despite previous reports [McLaughlin (1985) Mol. Biochem. Parasitol. 15, 189-201; Ghosh, Ray, Sarkar and Bhaduri (1990) J. Biol. Chem. 265, 11345-11351; Mazumder, Mukherjee, Ghosh, Ray and Bhaduri (1992) J. Biol. Chem. 267, 18440-18446] suggesting that the plasma-membrane Ca(2+)-ATPases of different trypanosomatids differ from the Ca2+ pumps present in mammalian cells, Trypanosoma cruzi plasma-membrane Ca(2+)-ATPase shares several characteristics with the Ca2+ pumps present in other systems. This enzyme could be partially purified from epimastigote plasma-membrane vesicles using calmodulin-agarose affinity chromatography. The activity of the partially purified enzyme was stimulated by T. cruzi or bovine brain calmodulin. In addition, the enzyme cross-reacted with antiserum and monoclonal antibody 5F10 raised against human red-blood-cell Ca(2+)-ATPase, has a molecular mass of 140 kDa and forms Ca(2+)-dependent hydroxylamine-sensitive phosphorylated intermediates. These results, together with its high sensitivity to vanadate, indicate that this enzyme belongs to the P-type class of ionic pumps.

The protein responsible for the (Ca2+ or Mg2+)-ATPase activity in brush-border membranes from pig kidney tubular cells was characterized to distinguish this enzyme from the N-ethylmaleimide-sensitive Mg(2+)-ATPase, also present in renal brush borders. Both enzymes are clearly different in their pH optimum and their sensitivity to divalent cations, nucleoside 5'-triphosphates and inhibitors. Solubilization of the (Ca2+ or Mg2+)-ATPase from brush-border membrane vesicles was accomplished with Nonidet P-40 or dodecylmaltoside. However, simultaneous inactivation of the enzyme was inevitable. A tenfold enrichment of the ATPase activity was obtained by chromatofocusing of Nonidet-P-40-solubilized brush borders. A similar degree of purification was achieved by ion-exchange chromatography of dodecylmaltoside-solubilized preparations. From the SDS/polyacrylamide gels of partially purified (Ca2+ or Mg2+)-ATPase, a few protein bands could still be tentatively identified as responsible for the enzyme activity. Labeling of solubilized brush-border preparations with several radioactive ATP analogues also revealed that a protein band of molecular mass 90 kDa is the most probable candidate for the catalytic peptide of the (Ca2+ or Mg2+)-ATPase. Finally, immunoprecipitation as well as semi-dry blotting with antibodies generated against partially purified enzyme preparations, confirmed that a 90-kDa component is a reasonable candidate for the (Ca2+ or Mg2+)-ATPase in renal brush-border membranes.

The aim of this study was to investigate the process leading to Ca(2+)-activated neutral protease (CANP) activation in vivo. The unautolyzed form of CANP has been targeted to the erythrocyte membrane by increasing, in a controlled way, the Ca2+ concentration in the cells; this was achieved by incubating erythrocytes with the Ca2+ ionophore A23187 and fixed Ca2+ concentrations. After isolation of the CANP-bearing erythrocyte membrane, we could observe that CANP remained bound to the membrane in the 80-kDa unautolyzed form in the presence of low Ca2+ concentrations (1.75 microM); under these conditions, the preferred CANP substrates (the Ca(2+)-ATPase and Band 3) were cleaved. That the cleavage was due to CANP was shown by the finding that the two substrates were not degraded in the presence of a membrane-permeable irreversible CANP inhibitor, Cbz-Leu-Leu-Tyr-CHN2, nor when the free Ca2+ concentration was decreased to sub microM levels with EDTA. The findings suggest an activation mechanism of CANP based on its translocation to the membrane rather than on its autolysis. In this mechanism, CANP would become reversibly activated on the membrane and would return to the quiescent state after dissociating from it when the cell Ca2+ concentration has returned to the physiological, submicromolar level.

We describe here an easy system for the production of mg amounts of the rabbit Ca(2+)-ATPase SERCA 1a in the yeast S. cerevisiae. The protein is present in several membranes, including the plasma membrane of the yeast, in a native conformation. It can be purified by immunoprecipitation and can be phosphorylated from ATP in a Ca(2+)-dependent manner. Using a temperature-sensitive secretion mutant strain, the fully active protein can also be obtained in secretory vesicles.

The plasma membrane Ca(2+)-ATPase from human erythrocytes can be stimulated by different treatments such as addition of calmodulin or acidic phospholipids and controlled proteolysis. In this report we show that short chain alkyl alcohols also stimulated this enzyme. At 5% (v/v) ethanol, the maximal velocity of the enzyme was about 2.4-fold higher than in the control, and thus, was also higher than the maximal velocity obtained in the presence of calmodulin (about 2-fold). When ethanol and calmodulin were present simultaneously, the stimulatory effect was additive (3.4-fold stimulation). On the other hand, the stimulatory effect of ethanol was preserved after treatment of the enzyme with trypsin to stimulate the Ca(2+)-ATPase and render it independent of calmodulin, thus suggesting that the interaction of ethanol and calmodulin with the Ca(2+)-ATPase occurred through a different mechanism. Other short chain alkyl alcohols (methanol, n-propanol and n-butanol) stimulated the Ca(2+)-ATPase activity to the same extent than ethanol but with different efficacy. Thus, the larger the carbon number, the lower the concentration needed to get the same maximal stimulation. Ethanol also increased the affinity of the enzyme for ATP to a larger extent and additively, when compared to calmodulin. All the effects of ethanol mentioned above were identically observed on the membrane-bound enzyme (i.e., erythrocyte ghosts) ruling out any effect of the alcohols attributable to the solubilized purified enzyme. Furthermore, Ca2+ transport by inside-out vesicles was also stimulated by ethanol, showing both the same concentration-dependence as the Ca(2+)-ATPase activity and the additive effect observed when calmodulin was also present. The stimulatory effect of ethanol was significant at pharmacological concentrations, thus suggesting potential implications of toxicological relevance.

The plasma membrane calcium pump is the system that ejects Ca2+ out of eukaryotic cells: this is documented in all animal and plant cells, although knowledge on the latter is only now beginning to be established. Information on lower eukaryotic cells, e.g., yeast, is still scarce, but it also is beginning to develop. The pump shares the catalytic properties of ion-motive ATPases of the P-type family, but has distinctive regulation properties: it is modulated by calmodulin, acidic phospholipids, a number of protein kinases, possibly by the interaction of calcium with its COOH-terminal region, and by aggregation (dimerization) through the calmodulin binding domain. The latter acts as an endogenous inhibitor of pump activity, much as phospholamban does for the sarcoplasmic reticulum pump. The analogy of the regulation mechanisms of the two pumps is heightened by the finding that phosphorylation of the calmodulin binding domain by protein kinase C removes its autoinhibiting function, as other kinases do in the case of phospholamban. The pump is the product of a family of four genes located on different human chromosomes. The isoform diversity is dramatically enhanced by alternative splicing of the transcripts, occurring at "hot spot" A (NH2-terminal) and C (COOH-terminal). At present more than 20 different transcripts with striking tissue and developmental specificity have been detected.

Expression of sarcoplasmic reticulum (SR) Ca(2+)-ATPase was shown to be reduced in failing human myocardium. The functional relevance of this finding, however, is not known. We investigated the relation between myocardial function and protein levels of SR Ca(2+)-ATPase in nonfailing human myocardium (8 muscle strips from 4 hearts) and in myocardium from end-stage failing hearts with dilated (10 muscle strips from 9 hearts) or ischemic (7 muscle strips from 5 hearts) cardiomyopathy. Myocardial function was evaluated by the force-frequency relation in isometrically contracting muscle strip preparations (37 degrees C, 30 to 180 min-1). In nonfailing myocardium, twitch tension rose with increasing rates of stimulation and was 76% higher at 120 min-1 compared with 30 min-1 (P < .02). In failing myocardium, there was no significant increase in average tension at stimulation rates above 30 min-1. At 120 min-1, twitch tension was decreased by 59% (P < .05) in dilated cardiomyopathy and 76% (P < .05) in ischemic cardiomyopathy compared with nonfailing myocardium. Protein levels of SR Ca(2+)-ATPase, normalized per total protein or per myosin, were reduced by 36% (P < .02) or 32% (P < .05), respectively, in failing compared with nonfailing myocardium. SR Ca(2+)-ATPase protein levels were closely related to SR Ca2+ uptake, measured in homogenates from the same hearts (r = .70, n = 16, and P < .005).(ABSTRACT TRUNCATED AT 250 WORDS)

Chronic diabetes due to streptozotocin administration has been shown to induce heart dysfunction characterized by prolonged relaxation time as well as decreased Ca2+ transport and Ca(2+)-ATPase (SERCA2) activities of the cardiac sarcoplasmic reticulum (SR). Rats made diabetic with 65 mg/kg of streptozotocin for 3 and 5 weeks exhibited decreased SR Ca(2+)-pump activities; these were normalized upon treatment with insulin. Northern blot and slot blot analyses did not show statistically significant reduction in the relative level of SERCA2 mRNA expression in diabetic or insulin treated rats. Quantitation of SERCA2 protein by Western blot did not reveal any change in diabetic and insulin treated animals. These results suggest that the defect in SR Ca(2+)-pump may not be due to changes at the transcriptional or translational levels in the diabetic heart.

In the present study, we have identified calmodulin binding sequences in the skeletal muscle ryanodine receptor Ca2+ release channel. Ligand overlays on RYR fusion proteins indicate that the skeletal muscle RYR contains three calmodulin binding regions defined by residues 2937-3225, 3546-3655, and 4425-4621. The RYR fusion protein PC28 (residues 2937-3225) bound calmodulin in the presence of EGTA and Ca2+, while RYR fusion protein PC26 (residues 3546-3655) exhibited strong calmodulin binding at 10 microM Ca2+. The RYR fusion protein PC15 (residues 4425-4621) did not bind calmodulin in the presence of either EGTA or 10-50 microM Ca2+. In the presence of 100-500 microM Ca2+, the RYR fusion protein PC15 exhibited an affinity for calmodulin of approximately 50 nM. Peptides RYR1 PM2 (residues 3610-3629) and RYR1 PM3 (4534-4552) encompassing putative RYR-calmodulin binding sites were synthesized. The synthetic peptides interacted directly with dansylcalmodulin as demonstrated by their capacity to affect the fluorescence emission of dansylcalmodulin. Missense mutation analysis indicates that the Lys and Arg residues are essential for calmodulin binding to the synthetic peptide RYR1 PM3. The RYR calmodulin binding site defined by peptide PM3 lies in the myoplasmic loop 2, a few residues upstream of the putative transmembrane segment M5; the other two calmodulin binding sites are next to the putative transmembrane segments M' and M''. Thus, the effect of calmodulin on Ca2+ release might involve the regulation of the putative transmembrane segments M5, M', and M''.

The existence of a Ca2+ pump in rod outer segment disks of bovine retina is strongly suggested by the isolation on sodium dodecyl sulfate polyacrylamide gel electrophoresis of a hydroxylamine-sensitive phosphorylated intermediate (E-P) of molecular mass of about 100 kDa as well as by measurements of active calcium transport and adenosine 5'-triphosphate (ATP) hydrolysis. Active Ca2+ uptake by disks was dependent on the presence of Mg(2+)-ATP, was inhibited by vanadate or lanthanum and appeared poorly sensitive to calmodulin. ATP hydrolysis by disk membranes was a function of free Ca2+ concentration in the absence of exogenous Mg2+. The presence of a Ca2+ pump on disk membranes is discussed in terms of its possible role in Ca2+ ion buffering during photoreceptor cell functioning.

cDNA encoding an epitope tag was joined to cDNAs encoding the chicken Na,K-ATPase beta 1 and beta 2 and H,K-ATPase beta-subunits to allow recognition of these beta-subunits with the same monoclonal antibody during assembly assays. cDNAs encoding chicken Na,K-ATPase alpha 1, alpha 2, or alpha 3 and Na,K-ATPase beta 1 or beta 2 or H,K-ATPase beta-subunits were transiently coexpressed in mammalian cells. Subunit assembly was assayed by immune precipitation of alpha-isoforms with a monoclonal antibody to the epitope-tagged beta-subunits. Each of the chicken alpha-isoforms assembled with each of the Na,K-ATPase beta-subunits and the H,K-ATPase beta-subunit. Each of the epitope-tagged beta-subunits also assembled with a Na,K-ATPase/Ca-ATPase chimera that retained only 26 amino acids of the Na,K-ATPase alpha-subunit, demonstrating that all three beta-subunits recognize this same alpha-subunit assembly site.

1. Parotid plasma membrane nonpump low-affinity Ca(2+)-ATPase, which possesses high-affinity (Ca2+ + Mg2+)-ATPase activity, was characterized. 2. Purified Ca(2+)-ATPase hydrolyzed the nucleoside triphosphates, GTP, ITP, CTP, UTP, TTP (67-93% of ATP) and nucleoside diphosphates, ADP, GDP, IDP, CDP, TDP (12-40% of ATP) but not AMP and p-NPP. 3. The maximum activities of Ca(2+)- and (Ca2+ + Mg2+)-ATPases were obtained in the presence of 1 mM and 0.13 microM Ca2+, respectively. 4. The Km values for Ca2+ in Ca(2+)- and (Ca2+ + Mg2+)-ATPases were 0.2 mM and 22 nM, respectively. 5. The activities of both Ca(2+)- and (Ca2+ + Mg2+)-ATPases were found in the right-side-out-vesicles obtained from the plasma membrane-rich fraction. 6. These features suggest that Ca(2+)-ATPase is an ecto-Ca(2+)-dependent nucleoside triphosphatase.

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

Intracellular Ca2+ pump expression and Ca2+ pool function are shown to be closely associated with growth and proliferation of DDT1MF-2 hamster smooth muscle cells. The Ca2+ pump blocker thapsigargin induces sustained Ca2+ pool emptying and entry of cells into a quiescent G0-like state (Short, A. D., Bian, J., Ghosh, T. K., Waldron, R. T., Rybak, S. L., and Gill, D. L. (1993) Proc. Natl. Acad. Sci. U.S.A. 90, 4986-4990). Using DDT1MF-2 cells growth-arrested by exposure to 3 microM thapsigargin for 24 h, treatment with 20% serum for 6 h without thapsigargin induced expression of functional Ca2+ pump protein detected as a 110-kDa thapsigargin-sensitive phosphorylated intermediate; 2.5% serum treatment resulted in no functional pump expression. Western analysis revealed only a slight serum-induced increase in total Ca2+ pump protein. New functional Ca2+ pump protein could be detected within 1 h of high serum treatment of thapsigargin-arrested cells, increasing over a 6-h period and correlating with the appearance of new Ca2+ pools. Induction of Ca2+ pools required serum at 10% or higher; no pools appeared with 5% serum or less. Significantly, high serum was required for only a brief but precise period of time. Exposure of thapsigargin-arrested cells to a 45-min pulse of 20% serum followed by continued culture in 2.5% serum was sufficient for full induction of new functional Ca2+ pump protein and Ca2+ pools; in contrast, no pumps or pools were detected after a 30-min serum pulse. A 40-min high serum pulse resulted in arrested cells reentering the cell cycle, synthesizing DNA, and resuming normal proliferation; in contrast, 35 min of serum treatment resulted in cells remaining totally quiescent. The results provide important evidence for the necessity of functional endoplasmic reticulum Ca2+ pumps in serum-induced cell growth and reflect a remarkably precise signaling period during which quiescent cells become committed to a progression of events including Ca2+ pump expression, Ca2+ pool function, reentry into the cell cycle, and cell division.

The structure of Ca(2+)-ATPase has been studied by electron microscopy of two different crystal forms: one tubular form induced by vanadate in native sarcoplasmic reticulum (SR) membranes and another multilamellar form grown from detergent-solubilized SR. To determine the conformation of Ca(2+)-ATPase within each crystal form, the respective effects of Ca2+, thapsigargin, adenosine 5'-(beta, gamma-methylene)triphosphate) (AMP-PCP), and chromium(III) (Cr-ATP) on crystallization have been studied. Vanadate-induced tubes were prevented from forming by micromolar Ca2+, but if preformed in the absence of Ca2&#95;, millimolar Ca2+ was required to disrupt these crystals. Thapsigargin promoted tube formation even in the presence of 10 mM Ca2+. Neither AMP-PCP nor Cr-ATP prevented tube formation, and the Ca2+ sensitivity of tube formation from Cr-ATP-inhibited SR was identical to controls. Multilamellar crystals required at least 0.2 mM Ca2+ and were prevented from forming by thapsigargin, AMP-PCP, or Cr-ATP. It is concluded that helical tubes are composed of the Ca(2+)-free, dephosphorylated conformation (E), and the nucleotide-bound conformation (E-ATP) is also tolerated. In contrast, multilamellar crystals are composed of the Ca(2+)-bound conformation (E.Ca2) and do not tolerate nucleotide binding. Thus, comparison of structures obtained from the two crystal forms should reveal physiologically relevant conformational differences.

A water soluble hydrogenation catalyst (palladium di(sodium alizarine monosulphonate)) in a deuterium-containing environment has been used for the in situ insertion of deuterium atoms into the fatty acyl chains of biological membranes. The thermotropic response of the stretching vibrations of the formed C-D bonds, as detected by Fourier transform IR spectroscopy, was used as a selective probe of biological membrane structure. Partial deuteration of unsaturated fatty acyl chains coupled with IR detection potentially provides a means for detecting specific biological roles of particular lipid classes. In the current study of sarcoplasmic reticulum membranes and purified phospholipid/CaATPase vesicles, it is also shown that vC-D monitors change at particular membrane locations which may remain undetected through the CH2 symmetric stretching frequency, a widely used IR spectral parameter. The latter reflects the average environment of the acyl chains. The approach described here may be suitable for wide applications to the study of biomembranes.

Image analysis has been used to compare the projection structure of three-dimensional crystals of (Na+,K+)-ATPase from pig kidney and the Ca(2+)-ATPase from rabbit muscle sarcoplasmic reticulum. These crystals, formed from detergent-solubilized protein in the presence of 20% glycerol and at a low detergent to protein ratio, crystallize in nearly identical unit cells in the space group C2. Average cell dimensions for the Ca(2+)-ATPase were a = 166.8 +/- 4.5 A, b = 57.7 +/- 4.4 A, gamma = 90 degrees while those for the (Na+,K+)-ATPase were a = 166.2 +/- 3.8A, b = 54.25 +/- 3.5A, gamma = 90 degrees. Their projected structures at the resolution of 25-A resolution are indistinguishable. Thus, the (Na+,K+)-ATPase crystals appear to contain only the alpha chain of the alpha beta heterodimer found in native membranes. We conclude from this that the three-dimensional structure of the alpha chain of the (Na+,K+)-ATPase is very similar to that of the Ca(2+)-ATPase despite their relatively weak overall sequence homology.

We have studied the effect of phospholipid chain length on the activity and molecular dynamics of reconstituted Ca-ATPase from skeletal sarcoplasmic reticulum (SR), using time-resolved phosphorescence anisotropy (TPA) and electron paramagnetic resonance (EPR). We used reconstituted Ca-ATPase in exogenous phosphatidylcholines with monounsaturated chains 14-24 carbons long, to determine their effects on the physical properties of the Ca-ATPase and to correlate these physical changes with changes in the ATPase activity. In agreement with previous studies, we found that the enzyme activity was maximal with a chain length of 18 and decreased substantially with longer or shorter chains. Our TPA results show that chain lengths longer or shorter than the optimal 18 result in a significantly decreased mobility of the Ca-ATPase, indicated by higher residual anisotropy and suggesting extensive protein aggregation. Saturation-transfer EPR data obtained with a spin label bound to a different site also indicates substantial immobilization of the enzyme, supporting the TPA results. There is good agreement between the fractional inhibition of the Ca-ATPase activity and the fraction of the enzyme in large aggregates. Solubilization in the nonionic detergent C12E8 demonstrated that inhibition of enzyme activity is reversible. In contrast to the large effects on protein mobility, these changes in chain length had little or no effect on hydrocarbon chain mobility as detected by conventional EPR at different depths in the membrane. We conclude that the Ca-ATPase has an optimum lipid bilayer thickness, presumably matching the thickness of the hydrophobic transmembrane surface of the enzyme, and that deviation from this optimum thickness produces a hydrophobic mismatch that induces protein aggregation and hence Ca-ATPase inhibition. This is consistent with our proposal that protein dynamics and protein-protein interactions are of primary importance to the Ca-ATPase mechanism.

A high-affinity Ca(2+)-ATPase was solubilized from rat parotid plasma membranes and purified by concanavalin A and DEAE-cellulose chromatography. The properties of the purified high-affinity Ca(2+)-ATPase are very different from those of the parotid plasma membrane ATP-dependent Ca2+ pump but appear to be similar to those of a rat liver cell adhesion protein which exhibits high-affinity ecto-Ca(2+)-ATPase activity.

Excitation-contraction (E-C) coupling is thought to involve close interactions between the calcium release channel (ryanodine receptor; RyR) of the sarcoplasmic reticulum (SR) and the dihydropyridine receptor (DHPR) alpha 1 subunit in the T-tubule membrane. Triadin, a 95-kD protein isolated from heavy SR, binds both the RyR and DHPR and may thus participate in E-C coupling or in interactions responsible for the formation of SR/T-tubule junctions. Immunofluorescence labeling of normal mouse myotubes shows that the RyR and triadin co-aggregate with the DHPR in punctate clusters upon formation of functional junctions. Dysgenic myotubes with a deficiency in the alpha 1 subunit of the DHPR show reduced expression and clustering of RyR and triadin; however, both proteins are still capable of forming clusters and attaining mature cross-striated distributions. Thus, the molecular organization of the RyR and triadin in the terminal cisternae of SR as well as its association with the T-tubules are independent of interactions with the DHPR alpha 1 subunit. Analysis of calcium transients in dysgenic myotubes with fluorescent calcium indicators reveals spontaneous and caffeine-induced calcium release from intracellular stores similar to those of normal muscle; however, depolarization-induced calcium release is absent. Thus, characteristic calcium release properties of the RyR do not require interactions with the DHPR; neither do they require the normal organization of the RyR in the terminal SR cisternae. In hybrids of dysgenic myotubes fused with normal cells, both action potential-induced calcium transients and the normal clustered organization of the RyR are restored in regions expressing the DHPR alpha 1 subunit.

The intestinal basolateral membrane Ca(2+)-transporting adenosinetriphosphatase is the energy-dependent step in the absorption of dietary Ca2+ by the vitamin D-dependent transcellular pathway. Multiple plasma membrane Ca(2+)-pump isoforms are produced from four genes (PMCA1 to 4) and alternative mRNA splicing. We have studied which isoforms are detectable in adult human and rat gastrointestinal tissues by polymerase chain reaction (PCR) amplification, sequencing, and blotting. PMCA1 was the predominant gene product amplified from human small intestinal mucosa, although a minor additional variant lacking the exon at splice site B was detected, which resembled that described for PMCA4. Of the variants described at site C, only the shortest transcript of PMCA1 was amplified; both previously described forms of PMCA4 were found, particularly in colon where PMCA4 predominated. From rat intestinal cDNA, mixed primer PCR amplified PMCA1 and a novel sequence, the rat PMCA4 homologue, which was expressed in many tissues including small intestinal muscle and colon. However, PMCA1 was overwhelmingly predominant in the mucosa of the small intestine, being most abundant in duodenum. These results suggest the involvement of the Ca(2+)-pump isoform PMCA1b in intestinal Ca2+ absorption.

A P-type Ca(2+)-ATPase activity has been identified previously in membranes of the Gram-negative bacteria Flavobacterium odoratum. The reaction mechanism of the prokaryotic Ca(2+)-ATPase was examined by isolation of reaction cycle intermediates formed during reversal of the normal cycle using inorganic P(i). The eukaryotic ATPase can be directly phosphorylated by P(i) only in the absence of calcium, and half-maximal inhibition is observed at 1-2 microM calcium. The F. odoratum Ca(2+)-ATPase is also phosphorylated by P(i) in the absence of calcium, but in contrast to the eukaryotic pump, a 30-35-fold increase in phosphoenzyme (E-P) formation is observed when the enzyme is preincubated in millimolar calcium. Furthermore, the half-maximal activation of E-P formation is observed at approximately 100 microM calcium, similar to the value for low-affinity calcium binding in the sarcoplasmic reticulum Ca(2+)-ATPase. The data suggest that the prokaryotic Ca(2+)-ATPase forward reaction is ordered and that the prokaryotic enzyme does not appear to bind calcium at the high-affinity site until ATP binds. Thus, calcium does not inhibit phosphoenzyme formation by P(i) because the high-affinity Ca2+ binding site is not exposed on the free enzyme (E1).

Activation of four target enzymes by two series of calmodulin Ca2+ binding site mutants has been examined. In each mutant, the conserved bidentate glutamate of one of the Ca2+ binding sites is mutated to glutamine or lysine. The enzymes studied were smooth and skeletal muscle myosin light chain kinases, adenylylcyclase, and plasma membrane Ca(2+)-ATPase. For the first three enzymes, the activation patterns with the two mutant series were very similar: mutation of site 4 was most deleterious, then site 2, site 3, and site 1. This ranking was observed previously in Ca2+ binding and Ca(2+)-induced conformational studies of these mutants. Thus the response of these enzymes is probably determined by the extent to which each mutant's competence to interact with target binding regions has been compromised. In contrast, for Ca(2+)-ATPase, mutants of sites 3 and 4 were much poorer activators than those of sites 1 and 2. Events beyond calmodulin binding and related to enzyme activation probably dictate this unusual activation pattern and also the anomalously poor activation of skeletal muscle myosin light chain kinase by site 1 mutant B1Q. Site 1 mutant B1K showed wild type activation of all four enzymes suggesting that in site 1, the lysine substitution can evoke the conformational changes associated with Ca2+ binding.

Different classes of tryptophan residues in sarcoplasmic reticulum calcium-ATPase were investigated with respect to their exposure to quenchers and sensitivity to high-affinity calcium binding to the ATPase. The charged quenchers, iodide and cesium, produced only slight quenching of ATPase fluorescence, whereas noncharged acrylamide and notably oxygen produced significant quenching. This finding gives support to the proposed location of most of the tryptophan residues of the ATPase in transmembrane domains of this protein (MacLennan et al., 1985, Nature 316r, 696-700). Among the different quenchers tested, oxygen quenching alone was sensitive to calcium binding to the ATPase, indicating that oxygen quenched tryptophan residues located in regions of the ATPase molecule which undergo conformational changes upon calcium binding. Time-resolved oxygen quenching data were analyzed with a recently described model that takes into account the existence of two different classes of emitters in the ATPase (Ferreira and Verjovski-Almeida, 1991, J. Lumin. 48, 430-434): a short-lived blue-shifted exponential component plus a long-lived red-shifted continuous lifetime distribution. Oxygen quenching of the single-exponential lifetime component was found to be insensitive to calcium, whereas quenching of the distributed lifetime component was significantly (ca 25%) enhanced by calcium binding. The different sensitivities of the two tryptophan classes to calcium binding to the ATPase are interpreted in terms of the proposed location of tryptophan residues in relation to the calcium transport sites in the ATPase molecule.