Antibodies against erythrocyte Ca2+-transport ATPase specifically inhibit the calmodulin-dependent fraction of the enzyme's activity

Antibodies against purified Ca2+-transport ATPase from human erythrocytes were raised in rabbits. Immunodiffusion experiments revealed that precipitating antibodies had been developed. The immunoglobulin fraction inhibited solely the calmodulin-dependent fraction of erythrocyte Ca2+-transport ATPase activity, whereas the basal (in the absence of added calmodulin) activity of the enzyme was not significantly affected by the antibodies. The antibodies produced similar doseresponse curves for the calmodulin- and the oleic acid-stimulated enzyme. However, the immunoglobulin fraction was considerably less effective in inhibiting Ca2+-transport ATPase activated by limited proteolysis. The results obtained with our antibodies are compatible with the interpretation that at least one subpopulation of the antibodies attacks the enzyme at or close to the calmodulin-binding site of the ATPase. The antibodies also inhibited the calmodulin-regulated Ca2+-transport ATPase from pig smooth-muscle plasma membrane, though with lower potency. However, the immunoglobulin fraction failed to suppress pig cardiac sarcoplasmicreticulum Ca2+-transport ATPase activity in the concentration range investigated. In addition, the activity of phosphodiesterase from rat brain, another enzyme modulated by calmodulin, was not at all affected by the immunoglobulin fraction.

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Inhibitory antibodies to plasmalemmal Ca2+-transporting ATPases. Their use in subcellular localization of (Ca2+ + Mg2+)-dependent ATPase activity in smooth muscle

Biochemical Journal ◽

10.1042/bj2310737 ◽

1985 ◽

Vol 231 (3) ◽

pp. 737-742 ◽

Cited By ~ 7

Author(s):

J Verbist ◽

F Wuytack ◽

L Raeymaekers ◽

R Casteels

Keyword(s):

Endoplasmic Reticulum ◽

Smooth Muscle ◽

Plasma Membrane ◽

Atpase Activity ◽

Membrane Fraction ◽

Transport Atpase ◽

Calmodulin Binding ◽

Dependent Atpase ◽

Inhibitory Antibodies ◽

Stimulation Of

Antibodies directed against the purified calmodulin-binding (Ca2+ + Mg2+)-ATPase [(Ca2+ + Mg2+)-dependent ATPase] from pig erythrocytes and from smooth muscle of pig stomach (antral part) were raised in rabbits. Both the IgGs against the erythrocyte (Ca2+ + Mg2+)-ATPase and against the smooth-muscle (Ca2+ + Mg2+)-ATPase inhibited the activity of the purified calmodulin-binding (Ca2+ + Mg2+)-ATPase from smooth muscle. Up to 85% of the total (Ca2+ + Mg2+)-ATPase activity in a preparation of KCl-extracted smooth-muscle membranes was inhibited by these antibodies. The (Ca2+ + Mg2+)-ATPase activity and the Ca2+ uptake in a plasma-membrane-enriched fraction from this smooth muscle were inhibited to the same extent, whereas in an endoplasmic-reticulum-enriched membrane fraction the (Ca2+ + Mg2+)-ATPase activity was inhibited by only 25% and no effect was observed on the oxalate-stimulated Ca2+ uptake. This supports the hypothesis that, in pig stomach smooth muscle, two separate types of Ca2+-transport ATPase exist: a calmodulin-binding ATPase located in the plasma membrane and a calmodulin-independent one present in the endoplasmic reticulum. The antibodies did not affect the stimulation of the (Ca2+ + Mg2+)-ATPase activity by calmodulin.

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AIF4-induced inhibition of the ATPase activity, the Ca2+-transport activity and the phosphoprotein-intermediate formation of plasma-membrane and endo(sarco)plasmic-reticulum Ca2+-transport ATPases in different tissues. Evidence for a tissue-dependent functional difference

Biochemical Journal ◽

10.1042/bj2610655 ◽

1989 ◽

Vol 261 (2) ◽

pp. 655-660 ◽

Cited By ~ 22

Author(s):

L Missiaen ◽

F Wuytack ◽

H De Smedt ◽

F Amant ◽

R Casteels

Keyword(s):

Smooth Muscle ◽

Plasma Membrane ◽

Sarcoplasmic Reticulum ◽

Atpase Activity ◽

Intermediate Formation ◽

Functional Difference ◽

Transport Activity ◽

Fast Skeletal Muscle ◽

Transport Atpase ◽

Plasmic Reticulum

AIF4- inhibits the (Ca2+ + Mg2+)-ATPase activity of the plasma-membrane and the sarcoplasmic-reticulum Ca2+-transport ATPase [Missiaen, Wuytack, De Smedt, Vrolix & Casteels (1988) Biochem. J. 253, 827-833]. The aim of the present work was to investigate this inhibition further. We now report that AIF4- inhibits not only the (Ca2+ + Mg2+)-ATPase activity, but also the ATP-dependent 45Ca2+ transport, and the formation of the phosphoprotein intermediate by these pumps. Mg2+ potentiated the effect of AIF4-, whereas K+ had no such effect. The plasma-membrane Ca2+-transport ATPase from erythrocytes was 20 times less sensitive to inhibition by AIF4- as compared with the Ca2+-transport ATPase from smooth muscle. The endoplasmic-reticulum Ca2+-transport ATPase from smooth muscle was inhibited to a greater extent than the sarcoplasmic-reticulum Ca2+-transport ATPase of slow and fast skeletal muscle.

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Conformational changes during the reaction cycle of Plasma Membrane Ca2+-ATPase in the autoinhibited and activated states

Biochemical Journal ◽

10.1042/bcj20210036 ◽

2021 ◽

Author(s):

Nicolás A Saffioti ◽

Marilina de Sautu ◽

Ana Sol Riesco ◽

Mariela Soledad Ferreira-Gomes ◽

Juan Pablo F.C. Rossi ◽

...

Keyword(s):

Plasma Membrane ◽

Atpase Activity ◽

Binding Site ◽

Conformational Changes ◽

Ground State ◽

Binding Pocket ◽

Product State ◽

Extracellular Medium ◽

Reaction Cycle ◽

Calmodulin Binding

Plasma membrane Ca2+-ATPase (PMCA) transports Ca2+ by a reaction cycle including phosphorylated intermediates. Calmodulin binding to the C-terminal tail disrupts autoinhibitory interactions, activating the pump. To assess the conformational changes during the reaction cycle, we studied the structure of different PMCA states using a fluorescent probe, hydrophobic photolabeling, controlled proteolysis and Ca2+-ATPase activity.  Our results show that calmodulin binds to E2P-like states, and during dephosphorylation, the hydrophobicity in the nucleotide-binding pocket decreases and the Ca2+ binding site becomes inaccessible to the extracellular medium. Autoinhibitory interactions are disrupted in E1Ca and in the E2P ground state whereas they are stabilized in the E2∙Pi product state. Finally, we propose a model that describes the conformational changes during the Ca2+ transport of PMCA.

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A monoclonal antibody to the calmodulin-binding (Ca2+ + Mg2+)-dependent ATPase from pig stomach smooth muscle inhibits plasmalemmal (Ca2+ + Mg2+)-dependent ATPase activity

Biochemical Journal ◽

10.1042/bj2400633 ◽

1986 ◽

Vol 240 (3) ◽

pp. 633-640 ◽

Cited By ~ 7

Author(s):

J Verbist ◽

F Wuytack ◽

L Raeymaekers ◽

F Van Leuven ◽

J J Cassiman ◽

...

Keyword(s):

Monoclonal Antibody ◽

Endoplasmic Reticulum ◽

Smooth Muscle ◽

Atpase Activity ◽

Plasma Membranes ◽

Polyacrylamide Gel Electrophoresis ◽

Competitive Inhibitor ◽

Transport Atpase ◽

Calmodulin Binding ◽

Dependent Atpase

A monoclonal antibody (2B3) directed against the calmodulin-binding (Ca2+ + Mg2+)-dependent ATPase from pig stomach smooth muscle was prepared. This antibody reacts with a 130,000-Mr protein that co-migrates on SDS/polyacrylamide-gel electrophoresis with the calmodulin-binding (Ca2+ + Mg2+)-ATPase purified from smooth muscle by calmodulin affinity chromatography. The antibody causes partial inhibition of the (Ca2+ + Mg2+)-ATPase activity in plasma membranes from pig stomach smooth muscle, in pig erythrocytes and human erythrocytes. It appears to be directed against a specific functionally important site of the plasmalemmal Ca2+-transport ATPase and acts as a competitive inhibitor of ATP binding. Binding of the antibody does not change the Km of the ATPase for Ca2+ and its inhibitory effect is not altered by the presence of calmodulin. No inhibition of (Ca2+ + Mg2+)-ATPase activity or of the oxalate-stimulated Ca2+ uptake was observed in a pig smooth-muscle vesicle preparation enriched in endoplasmic reticulum. These results confirm the existence in smooth muscle of two different types of Ca2+-transport ATPase: a calmodulin-binding (Ca2+ + Mg2+)-ATPase located in the plasma membrane and a second one confined to the endoplasmic reticulum.

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Phosphorylation of Membrane Proteins by Protein Kinase in a Smooth Muscle Plasma Membrane Fraction

Experimental Biology and Medicine ◽

10.3181/00379727-170-41426 ◽

1982 ◽

Vol 170 (2) ◽

pp. 245-250 ◽

Cited By ~ 1

Author(s):

J. F. Krall ◽

S. G. Korenman

Keyword(s):

Smooth Muscle ◽

Plasma Membrane ◽

Protein Kinase ◽

Membrane Proteins ◽

Membrane Fraction ◽

Plasma Membrane Fraction ◽

Muscle Plasma Membrane

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The effects of acetylcholine and atropine on the 22Na+ permeability of intestinal smooth muscle vesicles

Canadian Journal of Physiology and Pharmacology ◽

10.1139/y78-146 ◽

1978 ◽

Vol 56 (6) ◽

pp. 921-925

Author(s):

L. Spero

Keyword(s):

Smooth Muscle ◽

Plasma Membrane ◽

Membrane Vesicles ◽

Intestinal Smooth Muscle ◽

Erythrocyte Ghosts ◽

Muscarinic Agonists ◽

Plasma Membrane Vesicles ◽

Muscle Plasma Membrane ◽

Whole Muscle ◽

Kinetics Of

A technique is described which has enabled us to measure changes in 22Na+ efflux from smooth muscle plasma membrane vesicles. The resting 22Na+ efflux from these sealed vesicles showed a concentration-dependent increase in response to acetylcholine and other muscarinic agonists, in similar concentrations to those which increased 42K+ efflux in whole muscle. The kinetics of this efflux were complex and could not be described by less than three exponential processes. The response to agonists has, therefore, been characterized by measurement of the half-life of 22Na+ efflux (t1/2). The acetylcholine effect was inhibited by atropine, but unlike the situation in the whole muscle, this inhibition was noncompetitive. Tubocuraine (a nicotinic antagonist) had no effect on this acetylcholine response. Atropine has no effect by itself on the resting 22Na+ efflux, neither did tetrodotoxin or ouabain. 22Na+ efflux from erythrocyte ghosts and liposomes, prepared from lipid extracts of the smooth muscle plasma membrane, was not modified by acetylcholine or atropine.

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Calcium pump, high-affinity Ca2+-ATPase, and other ATPases in dog antrum smooth muscle plasma membrane

AJP Cell Physiology ◽

10.1152/ajpcell.1985.248.5.c449 ◽

1985 ◽

Vol 248 (5) ◽

pp. C449-C456 ◽

Cited By ~ 36

Author(s):

A. K. Grover ◽

C. Y. Kwan ◽

P. J. Oakes

Keyword(s):

Smooth Muscle ◽

Plasma Membrane ◽

Maximal Activity ◽

Calcium Pump ◽

Disulfonic Acid ◽

Calmodulin Antagonists ◽

High Affinity ◽

Chelex 100 ◽

Muscle Plasma Membrane ◽

Absolute Requirement

The plasma membrane-enriched fraction from dog antrum smooth muscle is enriched in ATP-dependent azide-insensitive Ca2+ uptake (0.3-0.4 microM Ca2+ required for half-maximal activity), a high-affinity Ca2+-ATPase (Km of 0.3-0.8 microM for Ca2+), a low-affinity Ca2+-ATPase (Km for 250-400 microM for Ca2+), and a Mg2+-ATPase. Studies using membranes washed with EDTA and assay media treated with Chelex 100 showed that the high-affinity Ca2+-ATPase did not depend on contaminating Mg2+. Thus, whereas the ATP-dependent Ca2+ uptake had an absolute requirement for Mg2+, the Ca2+-ATPases did not. Studies using gamma-irradiation showed that the protein responsible for the ATP-dependent Ca2+ uptake was inactivated at significantly lower doses of radiation than the three ATPases. The Ca2+ uptake and the high-affinity Ca2+-ATPase also differed in their inhibition by calmodulin antagonists and 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid. Thus it is unlikely that the high-affinity Ca2+-ATPase by itself is responsible for the ATP-dependent Ca2+ uptake.

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