Biochemical characterization of a calcium-sensitive protein kinase LeCPK2 from tomato

LeCPK2 (GenBank GQ205414), a versatile calcium-dependent protein kinase (CDPK or CPK) gene was isolated from tomato in our previous study. In this study, the biochemical properties of LeCPK2 were further investigated. To examine the role of the C-terminal calmodulin-like domain (CLD) of LeCPK2 with respect to Ca2+ activation, the kinase activities of recombinant full-length and truncated LeCPK2 were measured by Kinase-Glo Luminescent kinase assay (Promega). The results showed that LeCPK2 activity was Ca(2+)-dependent and the C-terminal CLD of 161 residues was essential for the activation of LeCPK2. The activity of LeCPK2 was sharply stimulated by Ca2+ with K0.5 (concentration of Ca2+ for half-maximal activity) of 48.8 and 45.5 nM with substrate histone IIIs and syntide 2, respectively. The optimal concentration of Mg2+ for LeCPK2 activity was 20 and 10 mM for substrate histone IIIs and syntide 2, respectively. The K(m) value of LeCPK2 towards histone IIIs and syntide 2 was 44.9 microg/ml and 89.52 microM, respectively. The determination of biochemical properties of LeCPK2 would provide some clues on how its activity was regulated in vivo.     

茉莉酸甲酯处理对绿豆下胚轴质膜H+-ATPase水解活力及磷酸化水平的影响

运用γ-32P示踪、蛋白激酶和磷酸酶抑制剂药理实验探讨茉莉酸甲酯(MeJA)对质膜H -ATP酶水解活力及磷酸化水平的影响.结果如下:MeJA可促进H -ATP酶水解活力30%;斑蝥素和岗田酸促进了MeJA对质膜H -ATP酶的刺激作用;星形孢菌素和白屈菜红碱削弱了MeJA对质膜H -ATP酶的刺激作用.H -ATP酶活力变化同时,其上的γ-32P标记量发生变化.Ca2 对H -ATP酶水解活力有很大的刺激作用,但对MeJA促进H -ATP酶活力的作用没有进一步的影响.根据这些结果可以得出结论:MeJA刺激质膜H -ATP酶水解活力的变化与H -ATP酶磷酸化水平呈正相关,并且催化这一作用的蛋白激酶可能不依赖于Ca2 ,而蛋白磷酸酶依赖于Ca2 .     

Ca2+-ATPase activity in pancreatic acinar plasma membranes. Regulation by calmodulin and acidic phospholipids

A high degree of ATP hydrolytic activity present in purified rat pancreatic acinar cells was localized to plasma membranes. This activity was stimulated almost equally by Mg2+ or Ca2+. Kinetic analysis revealed that the enzyme had a higher affinity for Ca2+ (Kd = 1.73 microM) than Mg2+ (Kd = 2.98 microM) but a similar maximal rate of activity. A comparison of substrate requirements revealed very similar profiles for the Mg2+- and Ca2+-stimulated activities. Combinations of saturating concentrations of Mg2+ or Ca2+ produced the same degree of maximal activity. Investigation of the partial reactions of the ATPase activity revealed two phosphoprotein intermediates (Mr = 115,000 and 130,000) in the presence of Ca2+ and Mg2+. A significant stimulation of the Ca2+-ATPase activity by calmodulin was observed (Kd = 0.7 microM). Calmodulin increased the Ca2+-sensitivity of this enzyme system; Mg2+ appeared to be required for this effect. The Ca2+-ATPase activity was also stimulated by acidic phospholipids. Using an 125I-labeled calmodulin gel overlay technique, calmodulin was shown to bind in a Ca2+-dependent fashion to 133,000- and 230,000-dalton proteins present in the plasma membrane-enriched fraction. Under conditions that favor Ca2+-dependent kinase activity, calmodulin enhanced the phosphorylation of a 30,000- and 19,000-dalton protein. The major ATP hydrolytic activity in pancreatic acinar plasma membranes was present as an ectoenzyme.     

Modulation of plasma membrane H+-ATPase activity differentially activates wound and pathogen defense responses in tomato plants.

Systemin is an important mediator of wound-induced defense gene activation in tomato plants, and it elicits a rapid alkalinization of the growth medium of cultured Lycopersicon peruvianum cells. A possible mechanistic link between proton fluxes across the plasma membrane and the induction of defense genes was investigated by modulating plasma membrane H+-ATPase activity. Inhibitors of H+-ATPase (erythrosin B, diethyl stilbestrol, and vanadate) were found to alkalinize the growth medium of L. peruvianum cell cultures and to induce wound response genes in whole tomato plants. Conversely, an activator of the H+-ATPase (fusicoccin) acidified the growth medium of L. peruvianum cell cultures and suppressed systemin-induced medium alkalinization. Likewise, in fusicoccin-treated tomato plants, the wound- and systemin-triggered accumulation of wound-responsive mRNAs was found to be suppressed. However, fusicoccin treatment of tomato plants led to the accumulation of salicylic acid and the expression of pathogenesis-related genes. Apparently, the wound and pathogen defense signaling pathways are differentially regulated by changes in the proton electrochemical gradient across the plasma membrane. In addition, alkalinization of the L. peruvianum cell culture medium was found to depend on the influx of Ca2+ and the activity of a protein kinase. Reversible protein phosphorylation was also shown to be involved in the induction of wound response genes. The plasma membrane H+-ATPase as a possible target of a Ca2+-activated protein kinase and its role in defense signaling are discussed.     

Regulation of Plant Defense Response to Fungal Pathogens: Two Types of Protein Kinases in the Reversible Phosphorylation of the Host Plasma Membrane H+-ATPase

The role of reversible phosphorylation of the host plasma membrane H+-ATPase in signal transduction during the incompatible interaction between tomato cells and the fungal pathogen Cladosporium fulvum was investigated. Tomato cells (with the Cf-5 resistance gene) or isolated plasma membranes from Cf-5 cells treated with elicitor preparations from race 2.3 or 4 of C. fulvum (containing the avr5 gene product) showed a marked dephosphorylation of plasma membrane H+-ATPase. Similar treatment with elicitor preparations from races 5 and 2.4.5.9.11 (lacking the avr5 gene product) showed no change in dephosphorylation. Elicitor (race 4) treatment of cells, but not of isolated plasma membranes, for 2 hr resulted in rephosphorylation of the ATPase via Ca2+-dependent protein kinases. The initial (first hour) rephosphorylation was enhanced by protein kinase C (PKC) activators and was prevented by PKC inhibitors. Activity of a second kinase appeared after 1 hr and was responsible for the continuing phosphorylation of the H+-ATPase. This latter Ca2+-dependent kinase was inhibited by a calmodulin (CaM) antagonist and by an inhibitor of Ca2+/CaM-dependent protein kinase II. The activation of the Ca2+/CaM-dependent protein kinase depended on the prior activation of the PKC-like kinase.     

Ca2+ transport by rat liver plasma membranes: the transporter and the previously reported Ca2+-ATPase are different enzymes

A rat liver plasma membrane fraction showed an ATP-dependent uptake of Ca2+ which was released by the ionophore A23187. This activity represents a plasma membrane component and is not due to microsomal contamination. The Ca2+ transport displayed several properties which were different from those of the high-affinity Ca2+-ATPase previously observed in these membranes (Lotersztajn et al. (1981) J. Biol. Chem. 256, 11209-11215; Birch-Machin, M.A. and Dawson, A.P. (1986) Biochim. Biophys. Acta 855, 277-285). These observations have shown that Ca2+-ATPase does not require added Mg2+ whereas we have demonstrated that, in the same membrane preparation, Ca2+ uptake required millimolar concentrations of added Mg2+. The Ca2+-ATPase has a broad specificity for the nucleotides ATP, GTP, UTP and ITP while Ca2+ uptake remains specific for ATP. Ca2+ uptake also displayed different affinities for free Ca2+ and MgATP compared to Ca2+-ATPase activity, with apparent Km values of 0.25 microM Ca2+, 0.15 mM MgATP and 1.0 microM Ca2+, 4 microM MgATP respectively. The apparent maximum rate of Ca2+ uptake was about 150-fold less than Ca2+-ATPase activity. These features suggest that the high-affinity Ca2+-ATPase is not the enzymic expression of the ATP-dependent Ca2+ transport mechanism.     

Molecular dissection of the C-terminal regulatory domain of the plant plasma membrane H+-ATPase AHA2: mapping of residues that when altered give rise to an activated enzyme.

The plasma membrane H+-ATPase is a proton pump belonging to the P-type ATPase superfamily and is important for nutrient acquisition in plants. The H+-ATPase is controlled by an autoinhibitory C-terminal regulatory domain and is activated by 14-3-3 proteins which bind to this part of the enzyme. Alanine-scanning mutagenesis through 87 consecutive amino acid residues was used to evaluate the role of the C-terminus in autoinhibition of the plasma membrane H+-ATPase AHA2 from Arabidopsis thaliana. Mutant enzymes were expressed in a strain of Saccharomyces cerevisiae with a defective endogenous H+-ATPase. The enzymes were characterized by their ability to promote growth in acidic conditions and to promote H+ extrusion from intact cells, both of which are measures of plasma membrane H+-ATPase activity, and were also characterized with respect to kinetic properties such as affinity for H+ and ATP. Residues that when altered lead to increased pump activity group together in two regions of the C-terminus. One region stretches from K863 to L885 and includes two residues (Q879 and R880) that are conserved between plant and fungal H+-ATPases. The other region, incorporating S904 to L919, is situated in an extension of the C-terminus unique to plant H+-ATPases. Alteration of residues in both regions led to increased binding of yeast 14-3-3 protein to the plasma membrane of transformed cells. Taken together, our data suggest that modification of residues in two regions of the C-terminal regulatory domain exposes a latent binding site for activatory 14-3-3 proteins.     

Characterization of a putative Ca2(+)-transporting Ca2(+)-ATPase in the pellicles of Paramecium tetraurelia

In Paramecium, no Ca2(+)-ATPases with the properties of Ca2+ pumps have been identified. Here we report a pellicle associated Ca2(+)-ATPase activity and a corresponding phosphoprotein intermediate characteristic of a pump. The Ca2(+)-ATPase activity requires 3 mM Mg for optimal Ca2+ stimulation (KCa = 90 nM) and is specific for ATP as substrate (Km = 75 microM). Vanadate and calmidazolium inhibit Ca2(+)-stimulated activity with an EC50 of about 2 microM and 0.5 microM, respectively. Likewise, 10 microM trifluoperazine inhibits 80% of Ca2(+)-ATPase activity, but bovine calmodulin fails to stimulate. The Ca2(+)-ATPase is not inhibited by sodium azide (10 mM), oligomycin (10 micrograms/ml) or ouabain (0.2 mM). Incubation of pellicles with [gamma-32P]ATP specifically labels a 133 kDa protein in a Ca2(+)-dependent, hydroxylamine-sensitive manner, and the level of phosphorylation is increased by 100 microM La3+. Phosphorylation of an endoplasmic reticulum-enriched fraction labels a Ca2(+)-dependent protein different from the pellicle protein, being lower in molecular mass and unaffected by La3+. Ca2+ uptake by the alveolar sacs, integral components of the pellicle membrane complex, is poorly coupled to Ca2(+)-stimulated ATP hydrolysis (Ca2+ transported/ATP hydrolysed less than 0.2) and is much less sensitive to vanadate inhibition (EC50 approx. 20 microM) compared to the total Ca2(+)-ATPase activity. Therefore, the majority of the Ca2(+)-ATPase activity is likely to be plasma membrane associated.     

The effect of thyroid hormone on the high affinity Ca2+-ATPase in rat liver plasma membrane

The effect of thyroid hormone on the high affinity Ca2+-ATPase activity in rat liver plasma membrane was studied. The high affinity Ca2+-ATPase activity in plasma membrane was activated by 10(-7)-10(-5) M of Ca2+ and was inhibited by 70 microM trifluoperazine. Thyroidectomy of rats was associated with an increase in the activity of high affinity Ca2+-ATPase. The increased enzyme activity was normalized by T4 administration to the animals. On the other hand, Na+-K+-ATPase activity in the membrane was decreased by thyroidectomy and the decreased enzyme activity was normalized by T4 administration. The results suggest that thyroid hormone inhibits the Ca2+ extrusion system by inhibiting calmodulin-independent high affinity Ca2+-ATPase in liver plasma membrane.     

The binding site for regulatory 14-3-3 protein in plant plasma membrane H+-ATPase: involvement of a region promoting phosphorylation-independent interaction in addition to the phosphorylation-dependent C-terminal end

14-3-3 proteins constitute a family of well conserved proteins interacting with a large number of phosphorylated binding partners in eukaryotic cells. The plant plasma membrane H+-ATPase is an unusual target in that a unique phosphothreonine motif (946YpTV, where pT represents phosphothreonine) in the extreme C-terminal end of the H+-ATPase interacts with the binding cleft of 14-3-3 protein (Wurtele, M., Jelich-Ottmann, C., Wittinghofer, A., and Oecking, C. (2003) EMBO J. 22, 987-994). We report binding of 14-3-3 protein to a nonphosphorylated peptide representing the 34 C-terminal residues of the Arabidopsis plasma membrane H+-ATPase isoform 2 (AHA2). Following site-directed mutagenesis within the 45 C-terminal residues of AHA2, we conclude that, in addition to the 946YpTV motif, a number of residues located further upstream are required for phosphorylation-independent binding of 14-3-3. Among these, Thr-924 is important for interaction with 14-3-3 protein even when Thr-947 is phosphorylated. We suggest that the role of phosphorylation, which is accentuated by fusicoccin, is to stabilize protein-protein interaction between 14-3-3 protein and several residues of the H+-ATPase C-terminal domain.     

Purification, characterization, and reconstitution of the Ca2+-transport system (high-affinity Ca2+, Mg2+-ATPase) of the human erythrocyte membrane

The Ca2+-transport system of human erythrocyte membranes was solubilized by deoxycholate in the presence of the nonionic detergent Tween 20 and was purified by calmodulin affinity chromatography. The method yields a functional enzyme, which as compared with the erythrocyte membrane was purified 207-fold based on specific activity, and about 330-fold based on protein content. The activity of the isolated enzyme can be increased about 9-fold by the addition of calmodulin, resulting in a specific activity of 10.1 mumoles/mg . min at 37 degrees C. Triton X-100 and deoxycholate stimulate the calmodulin-deficient Ca2+-ATPase in a concentration dependent manner, which results in a loss of the calmodulin-sensitivity. The Ca2+-transport ATPase could be reconstituted after solubilization of the ATPase by deoxycholate and controlled dialysis near room temperature. The system was reconstituted to form membraneous vesicles capable of energized Ca2+ accumulation. The membrane vesicles showed a protein to lipid ratio (approx. 60% protein and 40% lipid) similar to that of the original erythrocyte membrane. The stimulation by calmodulin of the calmodulin-depleted membrane-bound and partially purified Ca2+-ATPase is strongly time dependent. At a Ca2+-concentration of 40 microM and low calmodulin concentrations, approx. 120 min are required to regain full activity. This time period is decreased to about 15 min in the presence of a high excess of calmodulin. Vice versa, at fixed concentrations of calmodulin, the time necessary for regain of full activity is decreased as the Ca2+ concentrations is increased. The dependence of the Ca2+-ATPase activity on the calmodulin concentration shows strong deviation from Michaelis-Menten kinetics at Ca2+ concentrations below (4--10 microM) and above (200 microM) the optimum concentration of 40 microM. Mathematical analysis of the results at 200 microM Ca2+ leads to the assumption that 4 calmodulin molecules interact with one oligomer of Ca2+-ATPase consisting of 4 identical subunits.     

A Ca2(+)-activated, Mg2(+)-dependent ATPase with high affinities for both Ca2+ and Mg2+ in vascular smooth muscle microsomes: comparison with plasma membrane Ca2(+)-pump ATPase

A Ca2(+)-ATPase with a high affinity for free Ca2+ (apparent Km of 0.13 microM) was found and characterized in membrane fractions from porcine aortic and coronary artery smooth muscles in comparison with the plasma membrane Ca2(+)-pump ATPase purified from porcine aorta by calmodulin affinity chromatography. The activity of the high-affinity Ca2(+)-ATPase became enriched in a plasma membrane-enriched fraction, suggesting its localization in the plasma membrane. The enzyme was fully active in the absence of exogenously added Mg2+, but required a minute amount of Mg2+ for its activity as evidenced by the findings that it was fully active in the presence of 0.1 microM free Mg2+ but lost the activity in a reaction mixture containing trans-cyclohexane-1,2-diamine-N,N,N',N'-tetraacetic acid as a divalent cation chelator which has, unlike EGTA, high affinities for both Ca2+ and Mg2+. It was able to utilize a variety of nucleoside di- and triphosphates as substrates, such as ADP, GDP, ATP, GTP, CTP, and UTP, showing a broad substrate specificity. The activity of the enzyme was not modified by calmodulin (5, 10 micrograms/ml). Trifluoperazine, a calmodulin antagonist, had a partial inhibitory effect on the activity at 30 to 240 microM, but this inhibition could not be reproduced by a more specific calmodulin antagonist, W-7, indicating that this inhibition by trifluoperazine was not specific. Furthermore, the high-affinity Ca2(+)-ATPase activity was not modified either by low concentrations (0.5-9 microM) of vanadate or by 1-100 microM p-chloromercuribenzoic acid. Cyclic GMP, nitroglycerin, and nicorandil did not have any effect on the enzyme activity.(ABSTRACT TRUNCATED AT 250 WORDS)     

Protein kinase C phosphorylates the carboxyl terminus of the plasma membrane Ca(2+)-ATPase from human erythrocytes.

Purified Ca(2+)-stimulated, Mg(2+)-dependent ATPase (Ca(2+)-ATPase) from human erythrocytes was phosphorylated with a stoichiometry of about 1 mol of phosphate/mol of ATPase at both threonine and serine residues by purified rat brain type III protein kinase C. In the presence of calmodulin, the phosphorylation was markedly reduced. Labeled phosphate from [gamma-32P]ATP was retained on an 86-kDa calmodulin-binding tryptic fragment of Ca(2+)-ATPase but not on 82- and 77-kDa non-calmodulin-binding fragments. Similarly, fragmentation of the phosphorylated Ca(2+)-ATPase by calpain I revealed that calmodulin-binding fragments (127 and 125 kDa) retained phosphate label whereas a non-calmodulin-binding fragment (124 kDa) did not. The calmodulin-binding domain, located about 12 kDa from the carboxyl terminus of the Ca(2+)-ATPase, was thus located as a site of protein kinase C phosphorylation. A synthetic peptide corresponding to a segment of the calmodulin-binding domain (H2 N-R-G-L-N-R-I-Q-T-Q-I-K-V-V-N-COOH) was indeed phosphorylated at the single threonine residue within this sequence. The additional serine phosphorylation site was carboxyl terminal to the calmodulin domain. Phosphorylation by purified type III protein kinase C (canine heart) antagonized the calmodulin activation of the Ca(2+)-ATPase, particularly at lower Ca2+ concentrations (0.2-1.0 microM). By contrast, a purified but unresolved protein kinase C isoenzyme mixture from rat brain stimulated the activity of Ca(2+)-ATPase prepared in asolectin, but not glycerol, by more than 2-fold in the presence of the ionophore A23187, without increasing its Ca2+ sensitivity. The results clearly indicate that human erythrocyte Ca(2+)-ATPase is a substrate of protein kinase C, but the effect of phosphorylation on the activity of the enzyme depends on the isoenzyme form of protein kinase C used and on the lipid associated with the Ca(2+)-ATPase.     

Increase of (Ca2++Mg2+)-ATPase activity of renal basolateral membrane by parathyroid hormone via cyclic AMP-dependent membrane phosphorylation

Studies were made on the mechanism of the effect of parathyroid hormone (PTH) on the activity of (Ca2++Mg2+)-ATPase, a membrane bound Ca2+-extrusion pump enzyme from the basolateral membranes (BLM) of canine kidney (Km for free Ca2+ = 1.3 X 10(-7) M, Vmax = 200 nmol Pi/mg/min). At 1 X 10(-7) M free Ca2+, both PTH (10(-7)-10(-6) M) and cAMP (10(-6)-10(-4) M) stimulated (Ca2++Mg2+)-ATPase activity dose-dependent and their stimulatory effects were inhibited completely by 5 microM H-8, an inhibitor of cAMP-dependent protein kinase. PTH (10(-7) M) also caused 40% increase in 32P incorporation into the BLM and 5 microM H-8 inhibited this increase too. PTH (10(-7) M) was found to stimulate phosphorylation of a protein of Mr 9000 by cAMP dependent protein kinase and 5 microM H-8 was found to block this stimulation also. From these results, it is proposed that PTH stimulates (Ca2++Mg2+)-ATPase activity by enhancing its affinity for free Ca2+ via cAMP-dependent phosphorylation of a BLM protein of Mr 9000.     

Identification of Ca2+-pump-related phosphoprotein in plasma membrane vesicles of Ehrlich ascites carcinoma cells

Plasma membrane vesicles of Ehrlich ascites carcinoma cells have been isolated to a high degree of purity. In the presence of Mg2+, the plasma membrane preparation exhibits a Ca2+-dependent ATPase activity of 2 mumol Pi per h per mg protein. It is suggested that this (Ca2+ + Mg2+)-ATPase activity is related to the measured Ca2+ transport which was characterized by Km values for ATP and Ca2+ of 44 +/- 9 microM and 0.25 +/- 0.10 microM, respectively. Phosphorylation of plasma membranes with [gamma-32P]ATP and analysis of the radioactive species by polyacrylamide gel electrophoresis revealed a Ca2+-dependent hydroxylamine-sensitive phosphoprotein with a molecular mass of 135 kDa. Molecular mass and other data differentiate this phosphoprotein from the catalytic subunit of (Na+ + K+)-ATPase and from the catalytic subunit of (Ca2+ + Mg2+)-ATPase of endoplasmic reticulum. It is suggested that the 135 kDa phosphoprotein represents the phosphorylated catalytic subunit of the (Ca2+ + Mg2+)-ATPase of the plasma membrane of Ehrlich ascites carcinoma cells. This finding is discussed in relation to previous attempts to identify a Ca2+-pump in plasma membranes isolated from nucleated cells.     

Effects of C-terminal truncations on trafficking of the yeast plasma membrane H+-ATPase

Within the large family of P-type cation-transporting ATPases, members differ in the number of C-terminal transmembrane helices, ranging from two in Cu2+-ATPases to six in H+-, Na+,K+-, Mg2+-, and Ca2+-ATPases. In this study, yeast Pma1 H+-ATPase has served as a model to examine the role of the C-terminal membrane domain in ATPase stability and targeting to the plasma membrane. Successive truncations were constructed from the middle of the major cytoplasmic loop to the middle of the extended cytoplasmic tail, adding back the C-terminal membrane-spanning helices one at a time. When the resulting constructs were expressed transiently in yeast, there was a steady increase in half-life from 70 min in Pma1 delta452 to 348 min in Pma1 delta901, but even the longest construct was considerably less stable than wild-type ATPase (t(1/2) = 11 h). Confocal immunofluorescence microscopy showed that 11 of 12 constructs were arrested in the endoplasmic reticulum and degraded in the proteasome. The only truncated ATPase that escaped the ER, Pma1 delta901, traveled slowly to the plasma membrane, where it hydrolyzed ATP and supported growth. Limited trypsinolysis showed Pma1 delta901 to be misfolded, however, resulting in premature delivery to the vacuole for degradation. As model substrates, this series of truncations affirms the importance of the entire C-terminal domain to yeast H+-ATPase biogenesis and defines a sequence element of 20 amino acids in the carboxyl tail that is critical to ER escape and trafficking to the plasma membrane.     

Membrane adenosine triphosphatase activities in rat pancreas

The membrane ATPase activities present in rat pancreas were studied to investigate the possible role of ATPase enzymes in HCO3(-) secretion in the pancreas. It was found that all the HCO3(-)-sensitive (anion-sensitive) ATPase activity was accountable as pancreatic mitochondrial ATPase, thus supporting the view that a distinct plasma membrane 'bicarbonate-ATPase' is not involved in HCO3(-) secretion in pancreas. A remarkably high Mg+- and CA2+-requiring ATPase activity (30 mumol ATP hydrolysed/min per mg) was found in the plasma membrane fraction (rho = 1.10-1.13). This activity has been characterized in some detail. It is inhibited by p-fluorosulfonylbenzoyladenosine, an affinity label analogue of ATP and the analogue appears to label covalently a protein of Mr approximately 35 000. The (Ca2+ + Mg2+)-ATPase activity did not form a 'phosphorylated-intermediate' and was vanadate-insensitive. These and other tests have served to demonstrate that the (Ca2+ + Mg2+)-ATPase activity is different in properties from (Na+ + K+)-ATPase, Ca2+-ATPase, (H+ + K+)-ATPase or mitochondrial H+-ATPase. Apart from the (Ca2+ + Mg2+)-ATPase of plasma membrane and mitochondrial ATPase, the only other membrane ATPase activities noted were (Na+ + K+)-ATPase, which occurred in the same fractions as the (Ca2+ + Mg2+)-AtPase at rho = 1.10-1.13 and was of surprisingly low activity, and an ATPase activity in light membrane fractions (rho - 1.08-1.09) derived from zymogen granule membranes. At this time, therefore, there is no obvious candidate for an ATPase activity at the luminal surface of pancreatic cells which is directly involved in ion transport, but the results presented here direct attention to the high activity (Ca2+ + Mg2+)-ATPase in the plasma membrane fraction.     

A calcium pump in plasma membrane vesicles from Leishmania braziliensis

A subcellular fraction highly enriched in plasma membrane vesicles was prepared from Leishmania promastigotes. This fraction showed (Ca2+ + Mg2+)-ATPase activity. This, however, represented a small fraction (about 25%) of the overall ATPase activity. The Ca2(+)-ATPase showed general characteristics common to plasma membrane ATPases involved in Ca2+ transport. Thus, the Ca2(+)-ATPase was activated by Ca2+ with a high affinity (Km about 0.7 microM), saturating at about 5 microM Ca2+. Furthermore, it was stimulated by calmodulin (about 70-80% with 5 micrograms/ml) and almost fully inhibited by trifluoperazine (100 microM). The above vesicles accumulated Ca2+ against a concentration gradient and released it after the addition of A23187, as shown independently by 45Ca2+ and Arsenazo III studies. The transport mechanism showed the same kinetics parameters as described for the enzyme, indicating a single molecular entity. In addition, Ca2(+)-ATPase activity and Ca2+ uptake were completely inhibited by vanadate (20 microM), indicating that an E1-E2 type mechanism is involved. The results clearly demonstrate the presence of a Ca2+ pump in the plasma membrane of Leishmania which is capable of maintaining a low cytoplasmic Ca2+ concentration.     

Inhibition by quercetin of thyroid hormone stimulation in vitro of human red blood cell Ca2+-ATPase activity

Human red blood cell membrane Ca2+-ATPase activity is stimulated in vitro by physiological concentrations of thyroid hormone. Quercetin, a flavonoid that inhibits several membrane-linked ATPases, suppressed thyroid hormone action on red cell Ca2+-ATPase activity and also interfered with binding of the hormone by red cell membranes. These effects of quercetin were dose-dependent over a range of concentrations (1-50 microM). In contrast, in the absence of thyroid hormone, quercetin at low concentrations stimulated Ca2+-ATPase activity and at 50 microM inhibited the enzyme. The effects of quercetin at low concentrations (1-10 microM), namely, stimulation of Ca2+-ATPase and inhibition of membrane-binding of thyroid hormone, mimic those of thyroid hormone and are consistent with the thyronine-like structure of quercetin. At high concentrations, quercetin is generally inhibitory of Ca2+-ATPase activity. Chalcone, fisetin, hesperetin and tangeretin are other flavonoids shown to reduce susceptibility of membrane Ca2+-ATPase to hormonal stimulation.     

Expression in yeast and purification of a membrane protein, SERCA1a, using a biotinylated acceptor domain

We have recently described the final steps leading to the crystallization of a mammalian membrane protein, the rabbit sarcoplasmic reticulum Ca2+-ATPase, after heterologous expression. Here, we detail the initial steps leading to this new purification method. A biotin acceptor domain was fused at the C-terminal part of Ca2+-ATPase and a thrombin site was inserted between both coding regions. The recombinant protein was expressed under the control of a galactose-inducible promoter in the yeast Saccharomyces cerevisiae. The biotinylation reaction of the protein was performed directly in vivo in yeast. After solubilization of the yeast light membrane fraction, the biotinylated protein was retained specifically using the strong biotin-avidin interaction. Finally, digestion by the protease thrombin allowed the separation of the Ca2+-ATPase from the biotinylated domain. At this step, Ca2+-ATPase is in a relatively purified form (about 40%). After a size-exclusion HPLC step, the purity of the protein is about 70%, and evaluation of the conformational changes during the catalytic cycle by monitoring the intrinsic fluorescence is demonstrated. The major advantage of this avidin procedure is the particularly good specific ATPase activity as compared with that of a purified His-tagged Ca2+-ATPase.