Triton X-100加KI对燕麦根细胞质膜ATP酶的溶解

Solubilization of the Plasma Membrane ATPase of Oat Roots by Triton X-100 and KI

在酸性条件下,1％ Triton X—100加 0.25mol/L KI能有效地溶解燕麦根细胞质膜ATP酶。溶解的ATP酶水解ATP的最适pH在6.5左右,酶活性受到Na_3VO_4和DES的强烈抑制,而不受Na_2MoO_4和NaN_3的抑制。溶解的酶液经透析后,K^+—ATP酶活性占Mg^(2+),KCl—ATP酶活性的85％。

The plasma membrane ATPase was succes-sfully solubilized with a two-step procedure. Asthe first step, the plasma membrane preparationwas washed with 0. 1% Triton X-100 and 0.2mol/L KC1. This removed approxima?ely 60%of the total proteins of the membrane prepara-tion giving about a 2-fold activity of K^+, Mg^(2+)-ATPase (Table 1). As the second step, TritonX-100 and KI were used to treat the washedplasma membranes, and produced an unexpec-ted result. Then the system of Triton X-100and KI of solubilization of the plasma mem-brane ATPase was studied in detail. The optimum condition of the solubiliza-tion of K^+, Mg^(2+)-ATPase was 1% Triton X-100and 0.25 mol/L KI at pH 4.5 (Fig. 1 and Fig.2). The low pH of the system may be importantin stabilizing the activity of tbe solubilized ATP-ase. The optimum pH of the solubilized ATPasewas about 6. 5 (Fig. 3). The activity of the enzyme was inhibited by Na_3VO_4. and DES, but notby NaN_3, and Na_2MoO_4. (Table 3). When thesolubilized ATPase was dialyed, higher Octivityof K^+-ATPase (312 units) was obtained, and thepercentage of the activity of K^+-APTase in thatof K^+,Mg^(2+)-ATPase was 85% (Table 4), whichis much higher than what others obtained (Hod-ges and Leonard 1974, Vara and Serrano1982). This result is important for further studyof the relationship between the plasma membr-ane ATPase and the transport of K^+ through theplasma membrane. Several other detergents were also testedin an attempt to solubilize the plasma mem-brane ATPase. But the results were not as goodas with Triton X-100 and KI (Table 2).