A plasma membrane-type Ca[sup 2+]-ATPase of 120 kilodaltons on the endoplasmic reticulum from carrot (Daucus carota) cells
- Univ. of Maryland, College Park (United States)
Cytosolic Ca[sup 2+] levels are regulated in part by Ca[sup 2+]-pumping ATPases that export Ca[sup 2+] from the cytoplasm; The types and properties of Ca[sup 2+] pumps in plants are not well understood. The kinetic properties of a 120-kD phosphoenzyme (PE) intermediate formed during the reaction cycle of a Ca[sup 2+]-ATPase from suspension-cultured carrot (Daucus carota) cells are characterized. Only one Ca[sup 2+]-dependent phosphoprotein was formed when carrot membrane vesicles were incubated with [[gamma]-[sup 32]P]ATP. Formation of this 120-kD phosphoprotein was inhibited by vanadate, enhanced by La[sup 3+], and decreased by hydroxylamine, confirming its identification as an intermediate of a phosphorylated-type Ca[sup 2+]-translocating ATPase. The 120-kD Ca[sup 2+]-ATPase was most abundant in endoplasmic reticulum-enriched fractions, in which the Ca[sup 2+]-ATPase was estimated to be 0.1% of membrane protein. Direct quantitation of Ca[sup 2+]-dependent phosphoprotein was used to examine the kinetics of PE formation. PE formation exhibited a K[sub m] for Ca[sup 2+] of 1 to 2 [mu]m and a K[sub m] for ATP of 67 nm. Relative affinities of substrates, determined by competition experiments, were 0.075 [mu]m for ATP, 1 [mu]m for ADP, 100 [mu]m for ITP, and 250 [mu]m for GTP. Thapsigargin and cyclopiazonic acid, specific inhibitors of animal sarcoplasmic/endoplasmic reticulum Ca[sup 2+]-ATPase, had no effect on PE formation; erythrosin B inhibited with 50% inhibition at <0.1 [mu]m. Calmodulin (1 [mu]m) stimulated PE formation by 25%. The results indicate that the carrot 120-kD Ca[sup 2+]-ATPase is similar but not identical to animal plasma membrane-type Ca[sup 2+]-ATPase and yet is located on endomembranes, such as the endoplasmic reticulum. This type of Ca[sup 2+] pump may reside on the cortical endoplasmic reticulum, thought to play a major role in anchoring the cytoskeleton and in facilitating secretion. 34 refs., 9 figs., 3 tabs.
- DOE Contract Number:
- FG05-86ER13461
- OSTI ID:
- 6247744
- Journal Information:
- Plant Physiology; (United States), Vol. 102:2; ISSN 0032-0889
- Country of Publication:
- United States
- Language:
- English
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Related Subjects
ATP-ASE
BIOLOGICAL FUNCTIONS
CALCIUM
MEMBRANE TRANSPORT
ENDOPLASMIC RETICULUM
PLANTS
CALMODULIN
CARROTS
CYTOPLASM
ECOLOGICAL CONCENTRATION
SECRETION
ACID ANHYDRASES
ALKALINE EARTH METALS
CELL CONSTITUENTS
ELEMENTS
ENZYMES
FOOD
HYDROLASES
MAGNOLIOPHYTA
MAGNOLIOPSIDA
METALS
ORGANIC COMPOUNDS
PHOSPHOHYDROLASES
PROTEINS
VEGETABLES
550200* - Biochemistry
550300 - Cytology