Localization of a Proton-Translocating ATPase on Sucrose Gradients

Abstract

lonophore-stimulated ATPase activity and ATP-dependent quinacrine quench were enriched in parallel when microsomal vesicles were prepared from corn (Crow Single Cross Hybrid WF9-Mo17) roots and collected on a cushion of 10% dextran. Activities were highest in the apical 1.5 centimeters of the roots. Vesicles collected on the dextran cushion also contained NADH cytochrome c reductase (enriched in the apical 0.5 cm of the root) and nucleoside diphosphatase (distributed throughout the first four cm). On continuous sucrose gradients, ATP-dependent proton transport and ionophore-stimulated ATPase In a previous paper (5) we described a proton-translocating ATPase in membranes from corn roots. It was stimulated by C1-and by ionophores, did not appear to be of mitochondrial origin, as it was not inhibited by oligomycin, and differed from the previously described plasma membrane ATPase of corn roots in that it was unaffected by monovalent cations, and was not inhibited by vanadate. Sze (23,24) suggested that similar vesicles obtained from tobacco callus originated from the plasma membrane and that the ionophore-stimulated ATPase, ATP-dependent methylamine uptake, and ATP-dependent SCN-uptake are the function of a proton translocating ATPase located on the plasma membrane. In this paper we describe preliminary attempts to determine the origin of the vesicles. We present evidence that the proton-translocating ATPase of corn root microsomal membranes is not of plasma membrane origin, and present evidence that the enzyme is likely to be of tonoplast origin. Similar findings have recently been reported for corn coleoptiles (8,9,15,17 Preparation of Vesicles. Sealed microsomal vesicles were prepared by a modification of the method of Sze (23). Corn seeds were germinated between damp paper towels in plastic trays. Root tips (approximately 15 mm in length) of primary and secondary roots of 4-to 5-day-old seedlings were excised into aerated 0.1 mM CaCl2 at room temperature. Roots were ground with a chilled mortar and pestle. The homogenization buffer consisted of 0.25 M sorbitol, 2 mm EGTA, 0.1% f8-mercaptoethanol, 0.1% BSA, and 25 mm Bis-Tris-propane adjusted to pH 7.5 with solid Mes. Approximately 10 ml of buffer/g fresh weight of root was used. The homogenate was centrifuged at 10,000g for 10 min, the mitochondrial pellet discarded, the supernatant centrifuged again at 10,000g for 10 min, the pellet discarded, and the supernatant centrifuged at 80,000g for 30 min to prepare the microsomal pellet. The microsomal pellet was suspended in suspension buffer consisting of 0.25 M sorbitol and 2.5 mm Bis-Tris-propane/Mes (pH 7.2), underlain with 8 ml of 10%o dextran T-70 in the same buffer, and centrifuged at 80,000g for 1 h. The cloudy interface (approximately 50%o of the protein applied to the gradient) was collected from the dextran cushion, and vesicles were stored on ice for 1 to 6 h before use. Vesicles showed little loss of activity for up to 10 h, and activity was preserved for several days if vesicles were frozen i...

Proton Transport in Isolated Vacuoles from Corn Coleoptiles

“…In some systems, two populations of proton 21,22). This also seems to be the case for other groups working with corn (12,26), although recent work in our laboratory using a different variety of corn and a modified membrane isolation procedure has resulted in the identification of a second proton pump attributed to the Golgi (10). Our previous work with microsomal vesicles from corn coleoptiles demonstrated that the characteristics of the proton pump and the light density ofthe active vesicles were inconsistent with a plasma membrane, ER, Golgi, or mitochondrial origin (18,21,22).…”

Separation of the Mg2+-ATPases from the Ca2+-Phosphatase Activity of Microsomal Membranes Prepared from Barley Roots

“…Seeds of barley (Hordeum vulgare L. cv California Mariout 72) were sown on moist cheesecloth above aerated 0.5 mM CaSO4 and grown at 22°C in the dark for 7 …”

“…We compared the substrate specificity of three areas of the gradient and the I00,OOOg supernatant (Table II). The [6][7]. The activity of the fraction presumed to be rich in plasma membrane vesicles (fractions 10-13) was highly specific for ATP We compared the distribution of marker enzymes in 18-h gradients with the results from 2-h gradients (Figs.…”

ADP Is a Competitive Inhibitor of ATP-Dependent H+ Transport in Microsomal Membranes from Zea mays L. Coleoptiles

“…The following criteria strongly suggest a tonoplast origin of Recently, evidence has accumulated for the presence of two microsomal H+-ATPases in plants involved in the production of electrogenic H+ gradients (4,6,8,10, 11,18,19). One is associated with high density membranes (about 1.16-1.17 g/cm3 inhibited by vanadate and correlated with marker enzymes for plasma membrane (4,7,8,19).…”