Britten C J, Zhen R G, Kim E J, Rea P A
Department of Biology, University of Pennsylvania, Philadelphia 19104-6018.
J Biol Chem. 1992 Oct 25;267(30):21850-5.
A procedure for reconstitution of the transport function of the vacuolar H(+)-translocating inorganic pyrophosphatase (H(+)-PPase; EC 3.6.1.1) prepared from etiolated hypocotyls of Vigna radiata (mung bean) is described. The method entails sequential extraction of isolated vacuolar membrane (tonoplast) vesicles with deoxycholate and CHAPS (3-[(3-cholamidopropyl)-dimethylammonio]-1-propanesulfonate), combination of CHAPS-solubilized protein with phospholipid-cholesterol mixtures, dialysis, and glycerol density gradient centrifugation. The final proteoliposome preparation is 9-fold enriched for PPase activity and active in pyrophosphate (PPi)-energized electrogenic H(+)-translocation. Since both PPi hydrolysis and PPi-dependent H(+)-translocation by the proteoliposomes are indistinguishable from the corresponding activities of native tonoplast vesicles, the functional integrity of the H(+)-PPase appears to be conserved during solubilization and reconstitution. The high transport capacity and amenability of the reconstituted enzyme to both radiometric membrane filtration and fluorimetric H(+)-translocation assays, on the other hand, demonstrate its applicability to a broad range of transport studies. SDS-polyacrylamide gel electrophoresis of the proteoliposomes reveals selective enrichment of the M(r) 66,000, substrate-binding subunit of the H(+)-PPase and two additional polypeptides of M(r) 21,000 and 20,000. Although the M(r) 21,000 and 20,000 polypeptides have not been described previously, all attempts to reconstitute H(+)-PPase lacking these components were unsuccessful. It is therefore tentatively proposed that the M(r) 21,000 and 20,000 polypeptides, as well as the M(r) 66,000 subunit, are required for the productive reconstitution of PPi-dependent H(+)-translocation.
本文描述了一种用于重建从绿豆黄化下胚轴制备的液泡H⁺转运无机焦磷酸酶（H⁺-PPase；EC 3.6.1.1）转运功能的方法。该方法包括用脱氧胆酸盐和CHAPS（3-[(3-胆酰胺丙基)-二甲基铵]-1-丙烷磺酸盐）依次提取分离的液泡膜（液泡膜）囊泡，将CHAPS溶解的蛋白质与磷脂-胆固醇混合物混合，透析，以及甘油密度梯度离心。最终的蛋白脂质体制剂中PPase活性富集了9倍，并且在焦磷酸（PPi）驱动的电致H⁺转运中具有活性。由于蛋白脂质体的PPi水解和PPi依赖的H⁺转运与天然液泡膜囊泡的相应活性没有区别，因此H⁺-PPase的功能完整性在溶解和重建过程中似乎得以保留。另一方面，重建酶对放射性膜过滤和荧光H⁺转运测定具有高转运能力且适用性良好，这表明其适用于广泛的转运研究。蛋白脂质体的SDS-聚丙烯酰胺凝胶电泳显示，H⁺-PPase的M(r) 66,000底物结合亚基以及另外两种M(r) 21,000和20,000的多肽有选择性富集。尽管之前尚未描述M(r) 21,000和20,000的多肽，但所有重建缺乏这些成分的H⁺-PPase的尝试均未成功。因此，初步推测M(r) 21,000和20,000的多肽以及M(r) 66,000亚基是PPi依赖的H⁺转运有效重建所必需的。
