SOLUBILIZATION AND RECONSTITUTION OF THE OAT ROOT VACUOLAR H+/CA-2+ EXCHANGER

被引：45

作者：

SCHUMAKER, KS ^{[1
]}

SZE, H ^{[1
]}

机构：

[1] UNIV MARYLAND,DEPT BOT,COLLEGE PK,MD 20742

来源：

PLANT PHYSIOLOGY | 1990年 / 92卷 / 02期

关键词：

D O I：

10.1104/pp.92.2.340

中图分类号：

Q94 [植物学];

学科分类号：

071001 ;

摘要：

Calcium is sequestered into vacuoles of oat (Avena sativa L.) root cells via a H+/Ca2+ antiporter, and vesicles derived from the vacuolar membrane (tonoplast) catalyze an uptake of calcium which is dependent on protons (pH gradient [ApH] dependent). The first step toward purification and identification of the H+/Ca2+ antiporter is to solubilize and reconstitute the transport activity in liposomes. The vacuolar H+/Ca2+ antiporter was solubilized with octylglucoside in the presence of soybean phospholipids and glycerol. After centrifugation, the soluble proteins were reconstituted into liposomes by detergent dilution. A ΔpH (acid inside) was generated in the proteoliposomes with an NH4Cl gradient (NH4+in ≫ NH4+out) as determined by methylamine uptake. Fundamental properties of ΔpH dependent calcium uptake such as the Km, for calcium (∼15 micromolar) and the sensitivity to inhibitors such as N,N′-dicyclohexylcarbodiimide, ruthenium red, and lanthanum, were similar to those found in membrane vesicles, indicating that the H+/Ca2+ antiporter has been reconstituted in active form.

引用

页码：340 / 345

页数：6

共 17 条

[1]

AMBUDKAR SV, 1986, J BIOL CHEM, V261, P79

[2]

AMBUDKAR SV, 1986, J BIOL CHEM, V261, P5596

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