Mimura K, Matsui H, Takagi T, Hayashi Y
1st Department of Biochemistry, Kyorin University School of Medicine, Tokyo, Japan.
Biochim Biophys Acta. 1993 Jan 18;1145(1):63-74. doi: 10.1016/0005-2736(93)90382-a.
Membrane-bound Na+/K(+)-ATPase purified from dog kidney was solubilized with octaethylene glycol dodecyl ether (C12E8), and the resultant solubilized enzyme was chromatographed on a TSKgel G4000SWXL or G3000SWXL column equilibrated with elution buffers containing various ligands affecting oligomerization of the enzyme. Weight-averaged molecular weight (Mw) values for the main protein components eluted were estimated by low-angle laser light-scattering photometry. With increasing concentration of C12E8 included in the elution buffer from 0.1 to 5 mg/ml, the Mw decreased from 230,000 to 153,000, indicating that C12E8 induced dissociation of the enzyme. In contrast, the Mw of the protein component increased up to 1.44.10(6) as the concentration of phosphatidylserine (PS) added to the elution buffer containing a fixed concentration of 0.3 mg/ml C12E8 was increased to 120 micrograms/ml. The association and/or aggregation were reversible by removal of the PS by rechromatography. Addition of PS to the elution buffer also allowed the solubilized enzyme to exhibit ATPase activity comparable to that of the membrane-bound enzyme during passage through the column. This was also the case with phosphatidylglycerol (PG) and phosphatidylinositol, but not with phosphatidylcholine or phosphatidylethanolamine. The specific refractive index increment (dn/dcp) of the solubilized enzyme was increased by addition of exogenous PG or PS, strongly suggesting that the phospholipid became bound to the enzyme, and that it induced association of the enzyme. The association induced by PS was inhibited by ATP and ADP, but not AMP. The concentrations for half-maximal inhibition were 0.44 mM for ATP and 0.88 mM for ADP. The PS-induced associated enzyme isolated by chromatography in the presence of 120 micrograms/ml PS was dissociated by ATP with K0.5 of 0.16 mM. The dissociating effect of C12E8, ATP and ADP and the associating effect of PS on the solubilized enzyme are consistent with the reports that C12E8 mimics the effect of regulatory ATP at the low-affinity site on the conformational transition from E2 to E1, and that phospholipids are essential for the reverse transition from E1 to E2. The results can be explained by assuming that the enzyme takes the form of a loosely associated diprotomer in the E1 state and a tightly associated one in the E2 state.
从狗肾中纯化得到的膜结合型钠钾ATP酶用八甘醇十二烷基醚（C12E8）进行增溶处理，然后将所得的增溶酶在TSKgel G4000SWXL或G3000SWXL柱上进行色谱分离，该柱用含有影响酶寡聚化的各种配体的洗脱缓冲液平衡。通过低角度激光散射光度法估算洗脱的主要蛋白质组分的重均分子量（Mw）值。随着洗脱缓冲液中C12E8浓度从0.1mg/ml增加到5mg/ml，Mw从230,000降至153,000，表明C12E8诱导酶解离。相反，当添加到含有固定浓度0.3mg/ml C12E8的洗脱缓冲液中的磷脂酰丝氨酸（PS）浓度增加到120μg/ml时，蛋白质组分的Mw增加到1.44×10⁶。通过再色谱法去除PS可使缔合和/或聚集作用逆转。向洗脱缓冲液中添加PS还使增溶酶在通过柱子的过程中表现出与膜结合酶相当的ATP酶活性。磷脂酰甘油（PG）和磷脂酰肌醇也有同样的情况，但磷脂酰胆碱或磷脂酰乙醇胺则不然。添加外源PG或PS可增加增溶酶的比折光率增量（dn/dcp），强烈表明磷脂与酶结合，并诱导酶缔合。PS诱导的缔合作用受到ATP和ADP的抑制，但不受AMP的抑制。半最大抑制浓度对于ATP为0.44mM，对于ADP为0.88mM。在120μg/ml PS存在下通过色谱法分离得到的PS诱导的缔合酶被ATP解离，K0.5为0.16mM。C12E8、ATP和ADP的解离作用以及PS对增溶酶的缔合作用与以下报道一致：C12E8在低亲和力位点模拟调节性ATP对从E2到E1构象转变的作用，并且磷脂对于从E1到E2的反向转变至关重要。这些结果可以通过假设酶在E1状态下以松散缔合的双聚体形式存在，在E2状态下以紧密缔合的形式存在来解释。
