Investigation of the Calcium-Transporting ATPases at the Endoplasmic Reticulum and Plasma Membrane of Red Beet (Beta vulgaris)

Abstract

Calcium-transporting ATPases were compared in endoplasmic reticulum (ER)-and plasma membrane-enriched fractions of red beet (Beta vulgaris 1.) storage tissue by measuring "Ca uptake and calcium-dependent phosphoenzyme formation. l h e plasma membrane fraction was prepared by aqueous two-phase partitioning of a microsomal fraction and collecting the upper phase. l h e ERenriched fraction was obtained by submitting a sucrose-gradient ER-enriched fradion to aqueous two-phase partitioning and collecting the lower phase; this reduced contaminating plasma membrane, which partitioned into the upper phase. l h e ATP-dependent calcium uptake observed in both fractions was released by the calcium ionophore A23187. Calcium uptake showed saturation kinetics for calcium with K, values of 0.92 mmol m-3 for the ER fraction and 1.24 mmol m-3 for the plasma membrane fraction. Uptake into both fractions was inhibited by vanadate and erythrosin 6, although the plasma membrane system was slightly more sensitive to both inhibitors. Cyclopiazonic acid and thapsigargin, at low concentrations, had no marked effect on uptake. The plasma membrane system was less substrate-specific for ATP than the ER system, since it was able to use CTP and ITP to drive calcium transport at up to 50% of the leve1 obtained with ATP. Following phosphorylation with [-p3'P]ATP, two high molecular mass, calcium-dependent phosphoproteins (119 and 124 kD) and a low molecular mass, calcium-independent phosphoprotein (1 7 kD) were observed in the plasma membrane fraction. The ER fraction showed one high molecular mass phosphoprotein (119 kD) in the presence of calcium and two low molecular mass phosphoproteins (17 and 20 kD) that showed no calcium dependence. l h e low molecular mass phosphoproteins were insensitive to hydroxylamine, but they did show turnover. l h e identity of these proteins is unknown, but they do not have the properties of phosphorylated intermediates of calcium-ATPases. In contrast, the high molecular mass phosphoproteins displayed properties consistent with their representing phosphorylated intermediates of E,Ez-type ATPases; they were hydroxylamine-sensitive, showed rapid turnover, and were inhibited by vanadate. Because they showed calcium-dependent phosphorylation and were sensitive to erythrosin B, the 119-and 124-kD phosphoproteins may be phosphorylated intermediates of the ER and plasma membrane calcium ATPases. These phosphoproteins were characterized further with respect to inhibitor sensitivity, responses to ions, and substrate specificity.

Identification of the Plasma Membrane Ca2+-ATPase and of Its Autoinhibitory Domain

“…These values are in the same range as those previously determined under similar conditions for the PM Ca2+-ATPase from other plant materials (Williams et al, 1990;Askerlund and Evans, 1993;Thomson et al, 1993). However, quite different values are obtained from standard (Laemmli, 1970) SDS-PAGE: 133 and 118 kD, respeciively, for the entire and proteolyzed PM Ca2+-ATPase.…”

“…Vol. 108, 1995 fairly similar biochemical characteristics and their reported values of molecular mass fall in the same range (Evans et al, 1989;Williams et al, 1990;Hsieh et al, 1991;Askrdund andEvans, 1992, 1993;Chen et al, 1993;Thomson et al, 1993Thomson et al, , 1994; therefore, they are not easily discerned.…”

“…Molecular masses of the two phosphoproteins estimated from different acidic gels ranged from 109 to 124 kD for that of higher molecular mass and from 94 to 108 kD for that of lower molecular mass. The variability of molecular mass estimated in different experiments but not in adjacent lanes of the same gel, probably depending on scarce solubility of SDS at acidic pHs and incorrect negative charging of proteins, does not allow a clear-cut estimation of the mo1 wt of the PM Ca2+-ATPase and is likely to be a source of variability of mo1 wt values reported in the literature for this enzyme (Williams et al, 1990;Askerlund and Evans, 1993;Thomson et al, 1993).…”

“…The formation of 32P-phosphorylated intermediate was assayed according to the method of Thomson et al (1993) with some modifications. The reaction mixture (0.2 mL final volume) contained 12 p~ MgSO,, 50 mM KC1,lO mM Hepes-KOH, pH 7,50 p~ CaC1, or 0.5 mM EGTA, and 0.1 p~ [y-32P]GTP (250 pCi/nmol); 100 p~ LaCI,, 20 pg/mL CaM, and 1 p~ eosin Y were included when indicated in the text.…”

“…Identification of the PM Ca2+-ATPase lon gel electrophoresis has been accomplished up to now by utilizing the characteristic of the enzyme to form a phosphorylated intermediate during the catalytic process (Williams et al, 1990;Askerlund and Evans, 1993;Thomson et al, 1993). In this work, to study the effect of controlled proteolysis on the PM Ca2+-ATPase at the molecular le\rel, besides following the migration of its phosphorylated intermediate in acidic gels, we have developed methods suitable also for SDS-PAGE according to Laemmli (1970), exploiting some characteristics of the enzyme.…”