两相分配法制备南极红酵母质膜

Isolation of Plasma Membrane of Antarctic Yeast by Two-phase Partition

摘要:

用葡聚糖T-500(Dextran T-500)和聚乙二醇(PEG-3350)两相体系制备南极红酵母(Rhodotorula sp.)菌株 NJ298的质膜。首先在2 mmol/L KCl浓度下, 选用5种不同的聚合物浓度(5.6%、5.8%、6.0%、6.2%、6.4 %, W/W), 研究了NJ298质膜在两相体系中的分配情况, 在此基础上进一步研究了KCl浓度(2 mmol/L、4 mmol/L、6 mmol/L、8 mmol/L、10 mmol/L)对NJ298质膜的纯度及得率的影响。结果表明, 选用6.0%聚合物浓度, 4 mmol/L KCl的两相分配体系, 分离3次可得到相对纯度在78.2%的南极红酵母质膜组分, 标志酶鉴定及磷钨酸染色电镜检测均表明获得了高纯度密实的正向型的质膜囊泡。这为进一步研究该菌株的南极极端环境适应机制奠定了基础。

Abstract:

Plasma Membrane of Antarctic yeast Rhodotorula sp. NJ298 was purified from microsome in a two-phase aqueous system including Dextran T 500 and PEG 3350. The effects of the polymer and KCl concentration in the system on the plasma membrane partition were studied. The result indicated that the system with 6.0% (W/W) Dextran T 500 and PEG 3350, 4 mmol/L KCl was suitable for the plasma membrane isolation in Rhodotorula sp. NJ298. The electron micrograph stained with phosphotungstic acid and the purity identification analyzed by marker enzyme activities proved that the above two-phase system could obtain sealed positive plasma membrane vesicles with the purity of up to 78.2%. This paper was a basic about studying adaptation mechanism of Rhodotorula sp. NJ298 by isolation the plasma membrane.