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Characterization of Nigericin-Stimulated ATPase from Sealed Microsomal Vesicles of Tobacco Callus ¹

Department of Botany, University of Kansas, Lawrence, Kansas 66045, Department of Biochemistry, University of Kansas, Lawrence, Kansas 66045

To understand the function and membrane origin of ionophore-stimulated ATPases, the activity of nigericin-stimulated ATPase was characterized from a low-density microsomal fraction containing sealed vesicles of autonomous tobacco (Nicotiana tabacum Linnaeous cv. Wisconsin no. 38) callus. The properties of KCl-stimulated, Mg-requiring ATPases (KCl-Mg,ATPase) were similar in the absence or presence of nigericin. Nigericin (or gramicidin) stimulation of a KCl-Mg,ATPase activity was optimum at pH 6.5 to 7.0. The enzyme was inhibited completely by N,N'-dicyclohexylcarbodiimide (10 µM), tributyltin (5 µM), and partially by vanadate (200 µM), but it was insensitive to fusicoccin and mitochondrial ATPase inhibitors, such as azide (1 mM) and oligomycin (5 µg/ml). The ATPase was more sensitive to anions than cations. Cations stimulated ATPase activity with a selectivity sequence of NH₄⁺ > K⁺, Rb⁺, Cs⁺, Na⁺, Li⁺ > Tris⁺. Anions stimulated Mg, ATPase activity with a decreasing sequence of Cl^– = acetate > SO₄^2– > benzene sulfonate > NO₃^–. The anion stimulation was caused partly by dissipation of the electrical potential (interior positive) by permeant anions and partly by a specific ionic effect. Plant membranes had at least two classes of nigericin-stimulated ATPases: one sensitive and one insensitive to vanadate. Many of the properties of the nigericin-sensitive, salt-stimulated Mg,ATPase were similar to a vanadate-sensitive plasma membrane ATPase of plant tissues, yet other properties (anion stimulation and vanadate insensitivity) resembled those of a tonoplast ATPase. These results support the idea that nigericin-stimulated ATPases are mainly electrogenic H⁺ pumps originated in part from the plasma membrane and in part from other nonmitochondrial membranes, such as the tonoplast.

¹ Supported in part by the General Research Fund of the University of Kansas and National Science Foundation Grant PCM 80-16808.