Purification and characterization of the Ca2+-ATPase of plasma membranes from Ehrlich ascites mammary carcinoma cells

Wetzker, Reinhard; Böhmer, Frank‐D.; Klinger, R; Müller, Elke; Hegewald, H; Scheven, M.; Grosse, Richard

doi:10.1016/0005-2736(86)90071-4

Cited by 5 publications

(4 citation statements)

References 31 publications

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“…Vanadate is a potent inhibitor of tyrosine phosphatases (Swarup et al, 1982;Klarlund, 1985;Leis et aL, 1985), and is also found to stimulate certain kinases (Gordon et al, 1983;Brown and Gordon, 1984). Furthermore, vanadium stimulates DNA synthesis (Carpenter, 1981;Swarup et al, 1982) and adenylate cyclase (Krawietz et aL, 1982), inhibits Ca++/Mg++ATPase (Wetzker et al, 1986), and activates Ca ++ influx in A431 cells in a similar way as EGF (Macara, 1986) Na+/H § exchange which results in pH increase as seen for peptide growth factors (Cassel, 1984), and has been reported to disrupt microtubule-intermediate filament interaction (Lee and Mrotek, 1984). On the other hand, vanadium compounds have not been observed to affect phosphatidyl inositol turnover, or protein kinase C activity (Macara, 1986).…”

Section: Discussionmentioning

confidence: 95%

Vanadium compounds promote the induction of morphological transformation of hamster embryo cells with no effect on gap junctional cell communication

1990

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Vanadium compounds were found to promote the induction of morphological transformation of hamster embryo cells. Exposure of the cells to Na-O-vanadate, vanadin (V) oxide or vanadin (IV) oxide sulfate following pre-exposure to a low concentration of benzo[a]pyrene, potentiated the induction of transformed colonies similar to 12-O-tetradecanoylphorbol-13-acetate. Unlike this phorbol ester, vanadium compounds did not inhibit intercellular communication, or active protein kinase C. Nor did vanadate influence the reoccurrence of communication after removal of a communication blocking phorbol ester. On the other hand, vanadate showed strong synergism with the phorbol ester on induction of transformed morphology in the phorbol ester sensitive cell line BPNi. This suggests that vanadium and tumor promoting phorbol esters mediate their effect on the induction of morphological transformation of hamster embryo cells through different mechanisms.

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Section: Discussionmentioning

confidence: 95%

Vanadium compounds promote the induction of morphological transformation of hamster embryo cells with no effect on gap junctional cell communication

1990

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“…A wide variety of tissues appear to have plasmamembrane Ca2+ pumps which resemble that of the erythrocyte. Both a calmodulin-responsive Ca2+-ATPase (or Ca2+ transport) and a phosphorylated intermediate (or purified enzyme) of Mr 140000 have been demonstrated in brain (Papazian et al, 1984;Hakim et al, 1982), skeletal muscle (Michalak et al, 1984), heart muscle (Caroni & Carafoli, 1981), smooth muscle (De Schutter et al, 1984), intestinal epithelium (De Jonge et al, 1981; Nellans & Popovitch, 1981), kidney (De Smedt et al, 1981, pancreas (Ansah et al, 1984) and Ehrlich ascites cells (Wetzker et al, 1986). In addition, calmodulin-responsiveness of Ca2+-ATPase or Ca2+ transport has been demonstrated in osteosarcoma cells (Shen et al, 1983), thyroid (Kasai & Field, 1982), macrophages (Lew & Stossel, 1980), lymphocytes (Lichtman et al, 1981) and chloroplast envelope (Nguyen & Siegenthaler, 1985).…”

Section: Discussionmentioning

confidence: 99%

Ca2+ uptake by corpus-luteum plasma membranes. Evidence for the presence of both a Ca2+-pumping ATPase and a Ca2+-dependent nucleoside triphosphatase

Minami¹,

Penniston²

1987

Biochemical Journal

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Plasma-membrane vesicles from rat corpus luteum showed an ATP-dependent uptake of Ca2+. Ca2+ was accumulated with a K1/2 (concn. giving half-maximal activity) of 0.2 microM and was released by the bivalent-cation ionophore A23187. A Ca2+-dependent phosphorylated intermediate (Mr 100,000) was detected which showed a low decomposition rate, consistent with it being the phosphorylated intermediate of the transport ATPase responsible for Ca2+ uptake. The Ca2+ uptake and the phosphorylated intermediate (E approximately P) displayed several properties that were different from those of the high-affinity Ca2+-ATPase previously observed in these membranes. Both Ca2+ uptake and E approximately P discriminated against ribonucleoside triphosphates other than ATP, whereas the ATPase split all the ribonucleoside triphosphates equally. Both Ca2+ uptake and E approximately P were sensitive to three different Hg-containing inhibitors, whereas the ATPase was inhibited much less. Ca2+ uptake required added Mg2+ (Km = 2.2 mM), whereas the ATPase required no added Mg2+. The maximum rate of Ca2+ uptake was about 400-fold less than that of ATP splitting; under different conditions, the decomposition rate of E approximately P was 1,000 times too slow to account for the ATPase activity observed. All of these features suggested that Ca2+ uptake was due to an enzyme of low activity, whose ATPase activity was not detected in the presence of the higher-specific-activity Ca2+-dependent ATPase.

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“…Phosphatidylcholine (a sonicated suspension of Sigma type IX-E) and EGTA were added to the supernatant to give final concentrations of 0.3 mg/ ml and I mm respectively. To deplete endogenous calmodulin, the procedure of Wetzker et al [16] was followed, using batch treatment with DEAE-Sephadex ion-exchange resin. After addition to the supernatant of CaCl2 (final concentration 1.5 mM), the protein was applied to a I ml calmodulin-agarose affinity column which had been equilibrated with Buffer B (same as Buffer A but also containing 5 mg of Triton X-100/ml and 0.3 mg of phosphatidylcholine/ml).…”

Section: Calmodulin-affinity Chromatographymentioning

confidence: 99%

“…This has an acid-stable, hydroxylamine-sensitive phospho-aspartyl bond, and its steady-state level is enhanced by the addition of La3", which blocks the E1P -+E2P transition [12]. Demonstration of this phosphoprotein (pp), with a molecular mass of between 130 and 150 kDa and appropriate properties, has led to the identification and subsequent purification of the Ca2+ pumps from the erythrocyte [13], heart sarcolemma [14], brain [15] and Ehrlich ascites carcinoma cells [16]. Unlike these cells, epithelial cells from the intestine transport large amounts of Ca2l from dietary sources and are subject to a variety of physiological controls that might be expected to affect Ca2l-pump properties [17].…”

Section: Introductionmentioning

confidence: 99%

Identification and isolation of the phosphorylated intermediate of the calcium pump in rat intestinal basolateral membranes

Wajsman

Walters

Weiser

1988

Biochemical Journal

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Transport of Ca2+ by the ATP-dependent Ca2+ pump has been demonstrated previously in rat intestinal basolateral-membrane vesicles. To identify the Ca2+-pump protein, duodenal basolateral membranes were phosphorylated with [gamma-32P]ATP in the presence of Ca2+ and La3+, under conditions conducive for maximal formation of the phosphorylated intermediate of the Ca2+ pump. Four major phosphoprotein bands were seen on autoradiograms of acidic SDS/polyacrylamide gels; the properties of a phosphoprotein (pp) at 130 kDa (pp130) were consistent with those expected for the plasma-membrane Ca2+ pump. This phosphoprotein was markedly enhanced by La3+, exhibited the characteristics of an acyl-phosphate bond, was preferentially phosphorylated from ATP and inhibited by micromolar concentrations of vanadate. Another phosphoprotein of 115 kDa possibly represented the endoplasmic reticulum Ca2+ pump or a fragment of pp130. Other phosphoproteins of 75 and 95 kDa were predominantly expressions of alkaline phosphatase. Formation of pp130 was highest in duodenal basolateral-membrane preparations when compared with those of jejunum and ileum or other subcellular fractions. A similar correlation between Ca2+-pump activity and pp130 formation was not found in membranes from villus-tip and crypt cells or in vitamin D-deficient animals. pp130 was isolated as a single phosphoprotein by calmodulin-affinity chromatography. We conclude that pp130 represents the phosphorylated intermediate of the rat intestinal basolateral-membrane Ca2+ pump, which can be separated from other phosphoproteins using its properties as a calmodulin-binding protein.

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Purification and characterization of the Ca2+-ATPase of plasma membranes from Ehrlich ascites mammary carcinoma cells

Cited by 5 publications

References 31 publications

Vanadium compounds promote the induction of morphological transformation of hamster embryo cells with no effect on gap junctional cell communication

Vanadium compounds promote the induction of morphological transformation of hamster embryo cells with no effect on gap junctional cell communication

Ca2+ uptake by corpus-luteum plasma membranes. Evidence for the presence of both a Ca2+-pumping ATPase and a Ca2+-dependent nucleoside triphosphatase

Identification and isolation of the phosphorylated intermediate of the calcium pump in rat intestinal basolateral membranes

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