The plasma membrane calcium-transport ATPase plays a major role in maintaining the low cytosolic calcium concentrations required
for normal cellular function. Calcium, magnesium, calmodulin
and lanthanum have been shown to alter the activity of the calcium-stimulated, magnesium-dependent ATPase activity in human erythrocytes. In an attempt to examine the reaction sequence of the (Ca²⁺ + Mg²⁺)-ATPase, the effects of these agents on the kinetics of calcium dependent phosphoprotein formation, the first step in the partial reaction sequence, were examined. Calmo-dulin-depleted erythrocyte membranes were prepared by hypotonic lysis in the presence of EDTA, according to the method of Carafoli et al (1980).
Calcium-dependent formation of the phosphorylated intermediate
was biphasic; the high calcium-affinity component was associated with low levels of E.Ca.P and a shallow response to changing calcium concentrations, whereas in the region of the low calcium-affinity component, E.Ca.P rose sharply in response to increasing calcium concentrations. The low affinity component of E.Ca.P lies in the range of calcium concentrations which inhibit (Ca²⁺ + Mg²⁺)-ATPase activity. When analyzed on LiDS acid PAGE, both components of calcium-dependent phosphoprotein formation were due to hydroxylamine-sensitive phosphorylation of a 135,000-145,000 dalton protein. Hence, the low calcium-affinity component of phosphoprotein formation and calcium-dependent inhibition of (Ca²⁺ + Mg²⁺)-ATPase activity were likely due to calcium-inhibition of dephosphorylation. Kinetic studies of calcium-dependent phosphoprotein formation, at two different calcium concentrations (1.0 μM, 0.4 mM), indicated that a steady-state was reached much sooner at higher calcium concentrations. Lanthanum, which is known to block dephosphorylation of the intermediate
complex, increased both the apparent rate of formation and the steady-state level of the phosphorylated intermediate.
Calmodulin, which has previously been shown to increase both the maximum velocity and the calcium affinity of the (Ca²⁺ + Mg²⁺)-ATPase, did not affect either calcium-dependent inhibition of (Ca²⁺ + Mg²⁺ )-ATPase activity or the biphasic nature of calcium-dependent phosphoprotein formation. At low calcium concentrations, calmodulin increased the apparent rate of phosphoprotein
formation, whereas at higher calcium concentrations (0.4 mM) calmodulin reduced the steady-state level of the phosphoprotein; the apparent rate of formation was unaffected. In the presence of lanthanum, calmodulin increased both the apparent rate of formation and steady-state level of the phosphoprotein, suggesting that the true rate of formation was increased by calmodulin
at higher calcium concentrations, but this was normally hidden by a simultaneous increase in the rate of dephosphorylation.
Removal of endogenous magnesium, using trans-1,2-diamino-cyclohexane tetraacetic acid (CDTA) did not alter the calcium sensitivity or rate of formation of the phosphorylated intermediate,
however turnover of the intermediate was markedly reduced. In the absence of free magnesium, both the velocity and calcium sensitivity of the (Ca²⁺ + Mg²⁺)-ATPase were also found to be lower.
The low calcium-affinity component of calcium-dependent phosphoprotein formation, which Schatzmann (1982) has attributed to an action of calcium at a "magnesium-specific" site, was not affected by magnesium concentrations as high as 1 mM. Furthermore, this phosphoprotein could be dephosphorylated along either the forward or reverse pathways. These results indicate that the transformation from E₁.Ca.P to E₂.Ca.P may not be the site of the calcium-dependent inhibition of dephosphorylation.
Calmodulin-depleted membrane fragments were prepared from the erythrocytes of cystic fibrosis patients as well as age- and sex-matched controls. Under conditions in which dephosphoryla-tion is inhibited, phosphoprotein formation and (Ca²⁺ + Mg²⁺)-ATPase activities were determined. Both (Ca²⁺ + Mg²⁺)-ATPase activity and phoshoprotein formation were found to be significantly
reduced in the preparations derived from patients with cystic fibrosis. Turnover of the phosphorylated intermediate did not differ significantly between the two groups. A reduction in (Ca²⁺ + Mg²⁺)-ATPase activity and phosphoprotein formation suggests that there may be fewer active calcium-pumping sites in the erythrocyte membranes of cystic fibrosis patients compared to normal subjects. / Pharmaceutical Sciences, Faculty of / Graduate
| Identifer | oai:union.ndltd.org:UBC/oai:circle.library.ubc.ca:2429/24458 |
| Date | January 1985 |
| Creators | Allen, Bruce Gordon |
| Publisher | University of British Columbia |
| Source Sets | University of British Columbia |
| Language | English |
| Detected Language | English |
| Type | Text, Thesis/Dissertation |
| Rights | For non-commercial purposes only, such as research, private study and education. Additional conditions apply, see Terms of Use https://open.library.ubc.ca/terms_of_use. |
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