ATPase activity and Ca2+ transport by reconstituted tryptic fragments of the Ca2+ pump of the erythrocyte plasma membrane

Benaím, Gustavo; Clark, Arthur; Carafoli, Ernesto

doi:10.1016/0143-4160(86)90021-7

Cited by 22 publications

(8 citation statements)

References 12 publications

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“…However, we cannot exclude the possibility that some portion of the (Ca^*-I-Mg'*)-ATPase aetivity was inactivated during isolation. For example, Benaim et al (1986) have demonstrated that the ealmodulin stimulation can be negated by trypsin digestion of purified red cell Ca^*-ATPase. The Ca-*-pumping ATPase of the red cell membrane constitutes only ca.…”

Section: Discussionmentioning

confidence: 99%

Identification of a calmodulin‐stimulated (Ca2++ Mg2+)‐ATPase in a plasma membrane fraction isolated from maize (Zea mays) leaves

Robinson

Larsson

Buckhout

1988

Physiologia Plantarum

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Plasma membranes were isolated from light‐grown, 14‐day‐old maize leaves (Zea mays L. cv. Golden Cross Bantam) using aqueous two‐phase partitioning. The plasma membrane (PM) fraction contained < 0.3% of the total chlorophyll, < 0.2% of the mitochondrial marker enzyme activity, minimal contamination by endomembranes and 34% of the total PM. A calmodulin‐stimulated (Ca2++ Mg2+)‐ATPase was identified in the PM‐enriched fraction. The Ca2++ calmodulin stimulation was dependent on Mg2+, saturated at ca 25 μM total Ca2+, had a pH maximum at 7.2 and was maximally stimulated by 600 nM bovine brain calmodulin. The stimulation was not greatly affected by the anion present and showed a divalent cation specificity of Ca2+ > Sr+2 ± Mn+2 > Co2+± Cu2+ > Ba2+. The napthalenesulfonamide W7, an antagonist of calmodulin action, completely inhibited the calmodulin stimulation at 175 μM, while the less active analogue W5 was ineffective at this concentration. La3+, an inhibitor of PM Ca2+ transport, showed a 50% inhibition of calmodulin‐stimulated ATPase activity at ca 200 μM. Taken as a whole, these data demonstrate the presence of a calmodulinstimulated, (Ca2++ Mg2+)‐ATPase on the cytoplasmic surface of the plasma membrane of maize leaf cells.

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Section: Discussionmentioning

confidence: 99%

Identification of a calmodulin‐stimulated (Ca2++ Mg2+)‐ATPase in a plasma membrane fraction isolated from maize (Zea mays) leaves

Robinson

Larsson

Buckhout

1988

Physiologia Plantarum

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“…Variations in free Ca2+ concentrations were monitored by measuring the changes in the absorbance spectrum of Arsenazo III, using the SLM Aminco DW2000 spectrophotometer at the wavelength pair 675-685 nm [30] to avoid interference by Mg2+ [31]. No free radical formation from Arsenazo III occurred under the conditions used [32][33][34].…”

Section: Determination Of Ca2+ Movementmentioning

confidence: 99%

A calmodulin-activated (Ca2+-Mg2+)-ATPase is involved in Ca2+ transport by plasma membrane vesicles from Trypanosoma cruzi

Benaím

Losada

Gadelha

et al. 1991

Biochemical Journal

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High-affinity Ca(2+)-activated ATPases that do not show any demonstrable dependence on Mg2+ have been reported in the plasma membranes of different trypanosomatids, and it has been suggested [McLaughlin (1985) Mol. Biochem. Parasitol. 15, 189-201; Ghosh, Ray, Sarkar & Bhaduri (1990) J. Biol. Chem. 265, 11345-11351] that these enzymes may have a role in Ca2+ transport by the plasma membrane and in the regulation of intracellular Ca2+ in these parasites. In this report we investigated Ca2+ transport by Trypanosoma cruzi plasma membrane vesicles using Arsenazo III as a Ca2+ indicator. These vesicles accumulated Ca2+ upon addition of ATP only when Mg2+ was present and released it in response to the Ca2+ ionophore A23187, but were insensitive to inositol 1,4,5-trisphosphate. Ca2+ transport was insensitive to antimycin A, oligomycin and carbonyl cyanide p-trifluorophenylhydrazone, ruling out any mitochondrial contamination. Staurosporine and phorbol myristate acetate had no effect on this activity, while low concentrations of vanadate (10 microM) completely inhibited it. In addition, we describe a high-affinity vanadate-sensitive (Ca(2+)-Mg2+)-ATPase in the highly enriched plasma membrane fraction of T. cruzi. Kinetic studies indicated that the apparent Km for free Ca2+ was 0.3 microM. On the other hand, Ca(2+)-ATPase activity and Ca2+ transport were both stimulated by bovine brain calmodulin and by endogenous calmodulin purified from these cells. In addition, trifluoperazine and calmidazolium, at concentrations in the range in which they normally exert anti-calmodulin effects, inhibited the calmodulin-stimulated Ca(2+)-ATPase activity. These observations support the notion that a Mg(2+)-dependent plasma membrane Ca2+ pump is present in these parasites.

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“…Peptides with different affinities for Ca# + could be obtained depending on the conditions used for trypsin proteolysis. One of the peptides (81 kDa) had higher affinity for Ca# + than the native enzyme and was not activated by calmodulin [22,37], but its affinity for Ca# + could be further increased by acidic phospholipids [38]. The 76 kDa tryptic fragment had a higher affinity for Ca# + than either the native enzyme or the 81 kDa fragment and was no longer activated by calmodulin or acidic phospholipids [38].…”

Section: Discussionmentioning

confidence: 99%

Phosphatidylethanol stimulates the plasma-membrane calcium pump from human erythrocytes

Suju

Davila

Poleo

et al. 1996

Biochemical Journal

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Phosphatidylethanol is formed by "transphosphatidylation' of phospholipids with ethanol catalysed by phospholipase D and can be accumulated in the plasma membrane of mammalian cells after treatment of animals with ethanol. In the present work we show that phosphatidylalcohols, such as phosphatidylethanol and phosphatidylbutanol, produced a twofold stimulation of the Ca(2+)-ATPase activity of human erythrocytes. This stimulation occurs with the purified, solubilized enzyme as well as with ghost preparations, where the enzyme is in its natural lipidic environment and is different to that obtained with other acidic phospholipids such as phosphatidylserine. Addition of either phosphatidylserine, phosphatidylethanol or phosphatidylbutanol to the purified Ca(2+)-ATPase, or to ghosts preparations, increased the affinity of the enzyme for Ca2+ and the maximal velocity of the reaction as compared with controls in the absence of acidic phospholipids. However, in contrast with what occurs with phosphatidylserine, simultaneous addition of phosphatidyl-alcohols and calmodulin increased the affinity of the enzyme for Ca2+ to a greater extent than each added separately. When ethanol was added to either the purified erythrocyte Ca(2+)-ATPase or to erythrocyte-ghost preparations in the presence of acidic phospholipids, an additive effect was observed. There was an increase in the affinity for Ca2+ and in the maximal velocity of the reaction, well above the values obtained with ethanol or with the acidic phospholipids tested separately. These findings could have pharmacological importance. It is conceivable that the decrease in the intracellular Ca(2+) concentration that has been reported in erythrocytes as a result of ethanol intoxication could be due to the stimulation of the Ca(2+)-ATPase by the accumulated phosphatidylethanol, to a direct effect of ethanol on the enzyme or to an additive combination of both.

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ATPase activity and Ca2+ transport by reconstituted tryptic fragments of the Ca2+ pump of the erythrocyte plasma membrane

Cited by 22 publications

References 12 publications

Identification of a calmodulin‐stimulated (Ca2++ Mg2+)‐ATPase in a plasma membrane fraction isolated from maize (Zea mays) leaves

Identification of a calmodulin‐stimulated (Ca2++ Mg2+)‐ATPase in a plasma membrane fraction isolated from maize (Zea mays) leaves

A calmodulin-activated (Ca2+-Mg2+)-ATPase is involved in Ca2+ transport by plasma membrane vesicles from Trypanosoma cruzi

Phosphatidylethanol stimulates the plasma-membrane calcium pump from human erythrocytes

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