Chang H C, Liao T H
Department of Biochemistry, College of Medicine, National Taiwan University, Taipei, Republic of China.
Arch Biochem Biophys. 1990 Aug 1;280(2):320-4. doi: 10.1016/0003-9861(90)90336-w.
DNase II, bound to the lysosomal membrane of porcine spleen, can be extracted from the membrane with 0.4 M NaCl. Reassociation of DNase II with the salt-extracted lysosomal membrane is readily accomplished in 0.01 M sodium acetate (pH 4.5). The reassociable amount of DNase II is approximately equal to the extractable amount. The capacity of the lysosomal membrane to bind DNase II is unaffected by the subtilisin treatment of the membrane. Phosphatidyl serine can bind DNase II as well, but with a much higher capacity. The erythrocyte plasma membrane on the other hand binds only about 20% of DNase II bound to the lysosomal membrane. The DNase II activity can be eluted from a column of the lysosomal membrane entrapped in 2% agarose and the elution pattern is very similar to that of CM-cellulose chromatography of DNase II, suggesting that electrostatic interactions may play an important role in the binding. The pH-reassociation profile is bell-shaped and is similar to the pH-activity profile of DNase II, having a maximum near pH 5. Under the nondenaturing condition, the dissociated alpha and beta subunits of DNase II cannot be reassociated to regain the enzymatic activity with or without the lysosomal membrane.
与猪脾脏溶酶体膜结合的脱氧核糖核酸酶II（DNase II），可用0.4M氯化钠从膜中提取出来。在0.01M醋酸钠（pH 4.5）中，DNase II能很容易地与经盐提取的溶酶体膜重新结合。可重新结合的DNase II量大约等于可提取量。溶酶体膜结合DNase II的能力不受膜的枯草杆菌蛋白酶处理的影响。磷脂酰丝氨酸也能结合DNase II，但结合能力要高得多。另一方面，红细胞质膜只能结合约20%与溶酶体膜结合的DNase II。DNase II活性可从包埋在2%琼脂糖中的溶酶体膜柱上洗脱下来，其洗脱模式与DNase II的CM-纤维素色谱洗脱模式非常相似，这表明静电相互作用可能在结合中起重要作用。pH值重新结合曲线呈钟形，与DNase II的pH值活性曲线相似，在pH 5附近有一个最大值。在非变性条件下，无论有无溶酶体膜，DNase II解离的α和β亚基都不能重新结合以恢复酶活性。
