Cytosolic CaZ+ levels are regulated in part by Caz+-pumping ATPases that export Caa+ from the cytoplasm; however, the types and properties of Caz+ pumps in plants are not well understood. We have characterized the kinetic properties of a 120-kD phosphoenzyme (PE) to examine the kinetics of PE formation. PE formation exhibited a K,,, for Ca" of 1 to 2 p~ and a K,,, for ATP of 67 nM. Relative affinities of substrates, determined by competition experiments, were 0.075 NM for ATP, 1 p~ for ADP, 100 p~ for ITP, and 250 p~ for CTP. Thapsigargin and cyclopiazonic acid, specific inhibitors of animal sarcoplasmic/endoplasmic reticulum Ca'+-ATPase, had no effect on PE formation; erythrosin B inhibited with 50% inhibition at <0.1 KM. Calmodulin (1 p~) stimulated PE formation by 25%. The results indicate that the carrot 120-kD CaZ+-ATPase i s similar but not identical to animal plasma membrane-type Ca'+-ATPase and yet is located on endomembranes, such as the endoplasmic reticulum. This type of Caz+ pump may reside on the cortical endoplasmic reticulum, which is thought to play a major role in anchoring the cytoskeleton and in facilitating secretion.

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Diclofop-methyl (methyl ester of 2-[4-(2',4'-dichlorophenoxy)phenoxy]propionate; 100 micromolar) and diclofop (100 micromolar) inhibited both ATP-and PPi-dependent formation of H+ gradients by tonoplast vesicles isolated from oat (Avena sativa L., cv Dal) roots. Diclofop-methyl (1 micromolar) significantly reduced the steady-state H+ gradient generated in the presence of ATP. The ester (diclofop-methyl) was more inhibitory than the free acid (diclofop) at pH 7.4, but this relative activity was reversed at pH 5.7. Neither compound affected the rate of ATP or PPi hydrolysis by the proton-pumping enzymes. Diclofop-methyl (50, 100 micromolar), but not diclofop (100 micromolar), accelerated the decay of nonmetabolic H gradients established across vesicle membranes. Diclofop-methyl (100 micromolar) did not collapse K+ gradients across vesicle membranes. Both the (+)-and (-)-enantiomers of diclofop-methyl dissipated nonmetabolic H+ gradients established across vesicle membranes. Diclofopmethyl, but not diclofop (each 100 micromolar), accelerated the decay of H+ gradients imposed across liposomal membranes. These results show that diclofop-methyl causes a specific increase in the H permeability of tonoplast.The herbicide DM3 and its free acid form, DA, inhibit shoot and root growth and cause chlorosis and necrosis of oat and wild oat tissues (7,14,23,28). Ultrastructural damage observed in treated tissues includes breakdown of cellular membranes and destruction of cytoplasmic structure (5). Evidence indicates that they interfere with membrane-associated functions causing inhibition of mineral ion absorption (1), leakage of electrolytes from cells (7) cides is not known, presently two hypothetical mechanisms are being evaluated experimentally (10). In one model, the basis for structural and functional effects on cellular membranes is a direct interaction of the herbicide with the membrane or membrane components. A direct effect of DA on membrane function is indicated by the rapidity of depolarization of electrical potentials by DA (within 10 min [21,36]) and repolarization after removal of DA (40 s, with IAA addition [32]). The nature of the DA-membrane interaction or of the membrane components required for DA activity has not yet been determined. The second model suggests that membrane disruption is an indirect effect of herbicidal action resulting from an inhibition of the biosynthesis of membrane lipids. De novo fatty acid biosynthesis has been shown to be a sensitive site of action for DA (15,17). The I50 values for the inhibition by DA of ['4C]acetate incorporation by maize and oat chloroplasts were 0.2 and 0.1 M, respectively (15, 17). DA was more inhibitory than was DM (Iso = 10 uM) in these assays. Recent evidence indicates that the enzyme acetylCoA carboxylase is the site of inhibition by DA in this biosynthetic pathway ( 17).We have used an in vitro system of tonoplast vesicles isolated from oat root to study the possible direct action of DM and DA on membranes. With isolated membranes, effects of the h...

Two biochemical types of Ca2+-pumping ATPases were distinguished in membranes that were isolated from carrot (Daucus carota) suspension-cultured cells. One type hydrolyzed GTP nearly as well as ATP, was stimulated by calmodulin, and was resistant to cyclopiazonic acid. This plasma membrane (PM)-type pump was associated with PMs and endomembranes, including vacuolar membranes and the endoplasmic reticulum (ER). Another pump (“ER-type”) that was associated mainly with the ER hydrolyzed ATP preferentially, was insensitive to calmodulin, and was inhibited partially by cyclopiazonic acid, a blocker of the animal sarcoplasmic/ER Ca2+ pump. Oxalate stimulation of Ca2+ accumulation by ER-type, but not PM-type, pump(s) indicated a separation of the two types on distinct compartments. An endomembrane 120-kD Ca2+ pump was partially purified by calmodulin-affinity chromatography. The purified polypeptide bound calmodulin reacted with antibodies to a calmodulin-stimulated Ca2+ pump from cauliflower and displayed [32P]phosphoenzyme properties that are characteristic of PM-type Ca2+ pumps. The purified ATPase corresponded to a phosphoenzyme and a 120-kD calmodulin-binding protein on endomembranes. Another PM-type pump was suggested by a 127-kD PM-associated protein that bound calmodulin. Thus, both ER- and PM-type Ca2+ pumps coexist in most plant tissues, and each type can be distinguished from another by a set of traits, even in partially purified membranes.

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