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.

The plasma membrane ATPase from red beet (Beta vulgaris L.) storage tissue associated with either native plasma membrane vesicles, a detergent-solubilized enzyme preparation or reconstituted liposomes was subjected to radiation inactivation analysis to determine if changes in target molecular size occurred with modification of its amphipathic environment. For each preparation of the enzyme, the decline in ATP hydrolytic activity with increasing dose of y-ray radiation demonstrated a simple exponential profile indicating the presence of a single target size. Analysis of the radiation inactivation profiles for the plasma membrane associated, solubilized, and reconstituted enzyme revealed target molecular sizes of 225 kilodaltons (kD), 129 kD, and 218 kD, respectively. These results suggest that the plasma membrane associated and reconstituted ATPase preparations consist of enzyme present as a dimer of 100 kD subunits while the solubilized enzyme is present in the monomeric form. These results also indicate that the 100 kD catalytic subunit most likely represents the minimal unit of ATP hydrolytic activity.As with other E1/E2 type ATPases, the catalytic mechanism ofthe plant plasma membrane ATPase involves the formation of a covalent phosphorylated intermediate (7,8,21,25) at an active site aspartyl residue (9, 26). When assayed in the presence of [y-32P]ATP as substrate and analyzed by dodecyl sulfate gel electrophoresis, this phosphorylated intermediate has been shown to be associated with a 100 kD peptide (8,21,25). From this result and the observation that purification of the plasma membrane ATPase results in the enrichment of a 100 kD peptide on stained SDS-gels (2,16,23,24), it has been proposed that this 100 kD peptide represents the catalytic subunit of this transport enzyme (22 and references therein).In a previous study from this laboratory (11), radiation inactivation analysis was used to estimate the holoenzyme mol wt of the red beet plasma membrane ATPase when associated with native plasma membrane vesicles. This method involves irradiating protein samples with high energy radiation such as accelerated electrons, x-rays, or '-rays and then analyzing the decrease in enzyme activity with increasing total radiation dose in terms of target theory (4,17 (20).