Calcium Transport in Sealed Vesicles from Red Beet (Beta vulgaris L.) Storage Tissue

Abstract

Calcium transport was examined in microsomal membrane vesicles from red beet (Beta vulgaris L.) storage tissue using chlorotetracycline as a fluorescent probe. This probe demonstrates an increase in fluorescence corresponding to calcium accumulation within the vesicles which can be collapsed by the addition of the calcium ionophore A23187. Calcium uptake in the microsomal vesicles was ATP dependent and completely inhibited by orthovanadate. Centrifugation of the microsomal membrane fraction on a linear 15 to 45% (w/w) sucrose density gradient revealed the presence of a single peak of calcium uptake which comigrated with the marker for endoplasmic reticulum. The calcium transport system associated with endoplasmic reticulum vesicles was then further characterized in fractions produced by centrifugation on discontinous sucrose density gradients. Calcium transport was insensitive to carbonylcyanide m-chlorophenylhydrazone indicating the presence of a prmary transport system directly linked to ATP utilization. The endoplasmic reticulum vesicles contained an ATPase activity that was calcium dependent and further stimulated by A23187 (Ca2, A23187 stimulatedATPase). Both calcium uptake and Ca2+, A23187 stimulated ATPase demonstrated similar properties with respect to pH optimum, inhibitor sensitivity, substrate specificity, and substrate kinetics. Treatment of the red beet endoplasmic reticulum vesicles with [fr-32P-ATP over short time intervals revealed the presence of a rapidly turning over 96 kilodalton radioactive peptide possibly representing a phosphorylated intermediate of this endoplasmic reticulum associated ATPase. It is proposed that this ATPase activity may represent the enzymic machinery responsible for mediating primary calcium transport in the endoplasmic reticulum linked to ATP utilization.

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

“…However, although previous phosphorylation studies in red beet were conducted in the presence of calcium (Giannini et al, 1987a;Williams et al, 1990), the calcium-dependence of the phosphoenzymes observed in the Suc-gradient-enriched ER fractions (Giannini et al, 1987a) and plasma membrane fractions (Williams et al, 1990) was not established, since the phosphoenzymes were not characterized in detail. In addition, assigning a Plant Physiol.…”

“…In most reports characterizing calcium transport at particular cell membranes, the membrane fractions were isolated by discontinuous Suc gradients (Evans et al, 1991); this includes previous studies of the transporters at the ER and plasma membrane of red beet (Beta vulgaris) (Giannini et al, 1987a(Giannini et al, , 1987bWilliams et al, 1990). However, a comparison of the properties of the calcium transporters at particular cell membranes is complicated because these fractions show some degree of cross-contamination (Giannini et al, 1987a).…”

“…However, a comparison of the properties of the calcium transporters at particular cell membranes is complicated because these fractions show some degree of cross-contamination (Giannini et al, 1987a). This is especially important when comparing the ER and plasma membrane transporters, which operate by similar mechanisms .…”

Regulation of High-Affinity Nitrate Uptake in Roots of Arabidopsis Depends Predominantly on Posttranscriptional Control of the NRT2.1/NAR2.1 Transport System

“…To obtain microsomes the pellet was resuspended in a minimal volume of conservation buffer (10 mM Tris, 10 mM borate, 300 mM Suc, 9 mM KCl, 5 mM EDTA, 5 mM EGTA, 50 mM NaF pH 8.3, 4.2 mM leupeptine, 1 mM phenylmethylsulfonyl fluoride, 5 mM dithiothreitol) and frozen at 280°C. For plants grown in hydroponic, microsomes were purified as described by Giannini et al (1987).…”

Calcium release from the endoplasmic reticulum of higher plants elicited by the NADP metabolite nicotinic acid adenine dinucleotide phosphate

“…The presence of highly active Ca 2ϩ sequestration mechanisms (21)(22)(23)(24) and Ca 2ϩ channels, including ligand-gated channels (3,4,25,26), underlines the importance of Ca 2ϩ metabolism in these experimental systems.…”