Abstract
Higher plant cells have one or more vacuoles important for maintaining cell turgor and for the transport and storage of ions and metabolites. One driving force for solute transport across the vacuolar membrane (tonoplast) is provided by an ATP-dependent electrogenic H+ pump. The tonoplast H+-pumping ATPase from oat roots has been solubilized with Triton X-100 and purified 16-fold by Sepharose 4B chromatography. The partially purified enzyme was sensitive to the same inhibitors (N-ethylmaleimide, N,N'-dicyclohexylcarbodiimide (DCCD), 7-chloro-4-nitrobenzo-2-oxa-1,3-diazole, 4,4'-diisothiocyano-2,2'-stilbene disulfonic acid, and NO-3) as the native membrane-bound enzyme. The partially purified enzyme was stimulated by Cl- (Km(app) = 1.0 mM) and hydrolyzed ATP with a Km(app) of 0.25 mM. Thus, the partially purified tonoplast ATPase has retained the properties of the native membrane-bound enzyme. [14C]DCCD labeled a single polypeptide (14-18 kDa) in the purified tonoplast ATPase preparation. Two major polypeptides, 72 and 60 kDa, that copurified with the ATPase activity and the 14-18-kDa DCCD-binding peptide are postulated to be subunits of a holoenzyme of 300-600 kDa (estimated by gel filtration). Despite several catalytic similarities with the mitochondrial H+-ATPase, the major polypeptides of the tonoplast ATPase differed in mass from the alpha and beta subunits (58 and 55 kDa) and the [14C] DCCD-binding proteolipid (8 kDa) of the oat F1F0-ATPase.
Highlights
(tonoplast) is provided by an ATP-dependent electro- nitrate, NEM,’ NBD-C1, and DCCD [9,10,11]
(1.5 x 65 cm)or a Sepharose CL-GBcolumn (3 X 100 cm) equilibrated solubilized at 3% (v/v) TritonX-100 (Fig. 1). about 75% of the membrane protein an5d0%of the ATPase activity in 2 mM BTP-Hepes, 0.15 mM Na2ATP, 0.1 mM DTT, 30% was recovered in the supernatant with 5% Triton in Fig. 1, glycerol, and 0.2% Triton X-100, and 0.2 mg/ml phospholipids and we subsequently found that over 90% of the ATPase and eluted with the same buffer at 4 "C
We have shown that NEM and NBD-Cl inhibition of the Properties of the Partially Purified Tonoplast ATPase-The tonoplast H+-ATPasefrom oat roots has been solubilized by Triton X-100 and purified 16-fold by gel filtration using a Sepharose 4B (Table I) oCr L-GB column
Summary
Mated bygel filtration).Despite several catalytic similarities withthe mitochondrial H+-ATPase,the major polypeptides of the tonoplast ATPase differed in mass from the a and j3 subunits (58and 55 kDa) and the [14C] DCCD-binding proteolipid (8 kDa) of the oat FIFoATPase. The brei was strainedthrough cheesecloth, andthe homogenate was Vacuoles of higher plant cells play a fundamental role in centrifuged for 10 min a t 480 X g. The pellet was resuspended in a resuspension buffer An electrogenic H+-ATPase has been found on vtahceuolar dextran (79 kDa) cushion, and centrifuged a t 70,000 (rmaxfo)r 2 h membrane (tonoplast)of several higher plant tissues, includ-. The pelleted membranes, enriched in tonoplast ATPase, oatroots [4,5,6,7,8] This H’ pump is thoughtto provide the were resuspended in resuspension buffer a t approximately 4 mg of proton motive force for active transport of solutes across the protein/ml. Density microsomal vesicles indicatethatthe low-density V) glycerol, 0.3 mM Na,ATP, pH 7.4), was added to an equal volume membranesareenrichedintonoplast vesicles [4,5, 7,8,9,10]
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