Further characterization and reconstitution of the purified Ca2+-pumping ATPase of heart sarcolemma.

Caroni, Pico; Zurini, Mauro; Clark, Arthur; Carafoli, Ernesto

doi:10.1016/s0021-9258(18)32178-1

Cited by 69 publications

(5 citation statements)

References 18 publications

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“…Among the individual PL head groups tested, substitution of increasing parts of egg PC with bovine brain PS led to a consistent increase in ATP-dependent Ca2+ transport. This is consistent with similar findings with the Ca2+ pump from erythrocytes (Carafoli & Zurini, 1982) and with the cardiac Na+-Ca2+ exchanger in the heart (Luciani, 1984;Carroni et al, 1983; Vemuri & Philipson, 1987). One possible explanation of the effects of increasing amounts of PS in the PCcontaining membrane could be linked to its well-known effects in promoting Ca2+ binding.…”

Section: Discussionsupporting

confidence: 89%

Role of the phospholipid environment in modulating the activity of the rat brain synaptic plasma membrane calcium ATPase

Hermoni-Levine¹,

Rahamimoff²

1990

Biochemistry

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The role of the phospholipid environment in modulating the activity of the rat brain synaptic plasma membrane (SPM) Ca2(+)-ATPase was investigated by its reconstitution into different phospholipids. Retention of activity of the solubilized Ca2(+)-ATPase depended on addition of exogenous phospholipids. As the cholate concentration used for solubilization of native SPM increased, a larger excess of exogeneous phospholipids, relative to membrane protein, had to be added to maintain optimal activity. Highest ATP-dependent Ca2+ transport activity was obtained when reconstitution was carried out in calf brain phospholipids (BPLs) followed by soybean phospholipids (SPLs) and the lowest in egg PC; reconstitution at a 40:1 weight ratio of exogenous phospholipids to native SPM protein resulted in ATP-dependent Ca2+ transport of 40.0 +/- 4.16, 23.4 +/- 8.48, and 11.54 +/- 2.31 nmol of Ca2+ (mg of protein)-1 (5 min)-1, respectively. Partial substitution of egg PC with BPLs led to an increase in the activity of the reconstituted Ca2+ pump. The highest ATP-dependent Ca2+ uptake was obtained when ratios of 15:25 or 10:30 egg PC to BPLs were used. Testing the individual phospholipids participating in the BPL mixture showed that addition of PS to egg PC led to a consistent increase in Ca2+ pump activity. Substitution of 50% of the PC with PS resulted in a 3.8-fold higher ATP-dependent Ca2+ uptake than that obtained in egg PC alone. No other phospholipid tested--PE, SM, or PI--had a similar effect. Increasing the proportion of PS within the BPL mixture above its original content led to a gradual decrease in the reconstituted SPM Ca2+ pump activity. Enrichment of asolectin with PS led first to increased Ca2+ pump activity; then, as the proportion of PS increased, Ca2+ transport of the reconstituted pump decreased. An increased proportion of PE, SM, or PI within the BPLs or asolectin, above their original contents, resulted in decreased Ca2+ transport. These results indicate that optimal SPM Ca2+ pump activity requires the combined presence of a critical amount of PC and PS within the reconstituted membrane.

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Section: Discussionsupporting

confidence: 89%

Role of the phospholipid environment in modulating the activity of the rat brain synaptic plasma membrane calcium ATPase

Hermoni-Levine¹,

Rahamimoff²

1990

Biochemistry

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“…Pig erythrocyte ghosts were prepared according to the method of Rega et al (18). PMCA was solubilized and purified by a Sepharose-4B-calmodulin affinity chromatography, as described by Caroni et al (19) and modified by Pasa et al (20). Protein concentration was determined according to the method of Lowry et al (21) (ghosts) or Peterson (22) (purified PMCA).…”

Section: Methodsmentioning

confidence: 99%

“…Ghosts (18 mL at 4 mg/ mL) were incubated in a medium containing 20 µM FITC, 10 mM BTP-Cl (pH 7.4), and no added Ca 2+ , in a dark shell at room temperature. After 40 min, the sample was pelleted for 10 min at 20000g, resuspended to 18 mL with 100 mM Tris-Cl (pH 7.4), repelleted, and resuspended again two times, and the last pellet was dissolved in the solubilization medium described by Caroni et al (19), with 0.2 µM lysine added to further deplete residual FITC. The rest of the purification procedure was as stated by those authors.…”

Section: Methodsmentioning

confidence: 99%

Conformational Changes of the Nucleotide Site of the Plasma Membrane Ca²⁺-ATPase Probed by Fluorescence Quenching

et al. 2002

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Fluorescence quenching by the water-soluble ions I(-) and Cs(+) was used to probe solvent accessibility and polarity of the nucleotide/fluorescein isothiocyanate binding pocket of the purified soluble Ca(2+)-ATPase from plasma membranes. The E(1).Ca.CaM conformer was the least accessible state studied, presenting the lowest suppression constant (K(q)) for both I(-) (K(q) = 6.7 M(-)(1)) and Cs(+) (K(q) = 0.7 M(-)(1)). Accessibility to I(-) was similar for the E(2).VO(4) and E(1).Ca states (K(q) = 7.13 and 7.5 M(-)(1), respectively), whereas E(2) was slightly more accessible (K(q) = 9.1 M(-)(1)). The phosphorylated state E(2)-P presented the highest accessibility, with a K(q) of 16.5 M(-)(1), very near the K(q) of 20.3 M(-)(1) for free FITC. I(-) was unequivocally a better fluorescence quencher, being usually nearly 3-fold as efficient as Cs(+), as indicated by the K(q)(I(-))/K(q)(Cs(+)) ratio (R(q)). The advent of a positive charge cluster on the nucleotide/fluorescein binding pocket in different states was suggested by the increase in R(q), which reached a value as high as 9.5 for the E(1).Ca.CaM conformer. These results indicate (i) a very high water accessibility of the nucleotide/fluorescein pocket for E(2)-P that (ii) is more restricted on the free E(2) state and (iii) becomes rather lower for the E(1).Ca states. Additionally, a positive charge effect of amino acids on the nucleotide site, possibly related to ATP binding and phosphoryl transfer, appears in these E(1).Ca states, being absent in the phosphorylated and nonphosphorylated E(2) states.

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“…Purification of the Ca 2+ -ATPase. The Ca 2+ -ATPase was purified from red cell membrane ghosts by solubilization with polydocanol and affinity chromatography through a CaM-Sepharose 4B column, according to the method of Caroni et al (35) as modified by Pasa et al (36). On SDS-PAGE (37), silver-staining (38) of this preparation reveals a single band of 135-145 kDa.…”

Section: Methodsmentioning

confidence: 99%

A Novel Role of 4,4‘-Diisothiocyanatostilbene-2,2‘-disulfonic Acid as an Activator of the Phosphatase Activity Catalyzed by Plasma Membrane Ca²⁺-ATPase

et al. 1999

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The hydrolysis of p-nitrophenyl phosphate catalyzed by the erythrocyte membrane Ca2+-ATPase is stimulated by low concentrations of the compound 4,4'-diisothiocyanatostilbene-2,2'-disulfonic acid (DIDS), a classic inhibitor of anion transport. Enhancement of the phosphatase activity varies from 2- to 6-fold, depending on the Ca2+ and calmodulin concentrations used. Maximum stimulation of the pNPPase activity in ghosts is reached at 4-5 microM DIDS. Under the same conditions, but with ATP rather than pNPP as the substrate, the Ca2+-ATPase activity is strongly inhibited. Activation of pNPP hydrolysis by DIDS is equally effective for both ghosts and purified enzyme, and therefore is independent of its effect as an anion transport inhibitor. Binding of the activator does not change the Ca2+ dependence of the pNPPase activity. Stimulation is partially additive to the activation of the pNPPase activity elicited by calmodulin and appears to involve a strong affinity binding or covalent binding to sulfhydryl groups of the enzyme, since activation is reversed by addition of dithiothreitol but not by washing. The degree of activation of pNPP hydrolysis is greater at alkaline pH values. DIDS decreases the apparent affinity of the enzyme for pNPP whether in the presence of Ca2+ alone or Ca2+ and calmodulin or in the absence of Ca2+ (with 5 microM DIDS the observed Km shifts from 4.8 +/- 1.4 to 10.1 +/- 2.6, from 3.8 +/- 0.4 to 7.0 +/- 0.8, and from 9.3 +/- 0.7 to 15.5 +/- 1.1 mM, respectively). However, the pNPPase rate is always increased (as above, from 3.6 +/- 0.6 to 11.2 +/- 1.7, from 4.4 +/- 0.5 to 11.4 +/- 0.9, and from 2.6 +/- 0.6 to 18.6 +/- 3.9 nmol mg-1 min-1, in the presence of Ca2+ alone or Ca2+ and calmodulin or in the absence of Ca2+, respectively). ATP inhibits the pNPPase activity in the absence of Ca2+, both in the presence and in the absence of DIDS. Therefore, kinetic evidence indicates that DIDS does more than shift the enzyme to the E2 conformation. We propose that the transition from E2 to E1 is decreased and a new enzyme conformer, denoted E2*, is accumulated in the presence of DIDS.

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Further characterization and reconstitution of the purified Ca2+-pumping ATPase of heart sarcolemma.

Cited by 69 publications

References 18 publications

Role of the phospholipid environment in modulating the activity of the rat brain synaptic plasma membrane calcium ATPase

Role of the phospholipid environment in modulating the activity of the rat brain synaptic plasma membrane calcium ATPase

Conformational Changes of the Nucleotide Site of the Plasma Membrane Ca²⁺-ATPase Probed by Fluorescence Quenching

A Novel Role of 4,4‘-Diisothiocyanatostilbene-2,2‘-disulfonic Acid as an Activator of the Phosphatase Activity Catalyzed by Plasma Membrane Ca²⁺-ATPase

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Further characterization and reconstitution of the purified Ca2+-pumping ATPase of heart sarcolemma.

Cited by 69 publications

References 18 publications

Role of the phospholipid environment in modulating the activity of the rat brain synaptic plasma membrane calcium ATPase

Role of the phospholipid environment in modulating the activity of the rat brain synaptic plasma membrane calcium ATPase

Conformational Changes of the Nucleotide Site of the Plasma Membrane Ca2+-ATPase Probed by Fluorescence Quenching

A Novel Role of 4,4‘-Diisothiocyanatostilbene-2,2‘-disulfonic Acid as an Activator of the Phosphatase Activity Catalyzed by Plasma Membrane Ca2+-ATPase

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Product

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Conformational Changes of the Nucleotide Site of the Plasma Membrane Ca²⁺-ATPase Probed by Fluorescence Quenching

A Novel Role of 4,4‘-Diisothiocyanatostilbene-2,2‘-disulfonic Acid as an Activator of the Phosphatase Activity Catalyzed by Plasma Membrane Ca²⁺-ATPase