A Cl(-)-translocating adenosinetriphosphatase in Acetabularia acetabulum. 2. Reconstitution of the enzyme into liposomes and effect of net charges of liposomes on chloride permeability and reconstitution.
A Cl(-)-translocating adenosinetriphosphatase in Acetabularia acetabulum. 2. Reconstitution of the enzyme into liposomes and effect of net charges of liposomes on chloride permeability and reconstitution.
复制标题
髋臼中的 Cl(-)-转位腺苷三磷酸酶。
作者：
M. Ikeda;D. Oesterhelt
将从髋臼分离的Mono Q-III组分（一种Mg 2（+）-ATP酶）重组到通过反相方法制备的各种净电荷的脂质体中，并检测Cl（-）-转运活性。来自卵磷脂、磷酸双十六烷基酯和胆固醇的混合物（63：18：9摩尔比，阴性脂质体）以及来自卵磷脂和胆固醇的混合物（63：9摩尔比，中性脂质体）的脂质体比来自asolectin的阳性脂质体以及来自卵磷脂、硬脂胺和胆固醇的混合物（63：18：9摩尔比）的脂质体的泄漏更少。通过短期透析掺入酶后，在缬氨霉素存在下加入ATP，观察到36 Cl-从阴性和中性脂质体流出的显著增加。ATP驱动的36 Cl-外排被抑制的叠氮化物，ATP酶的抑制剂。预孵育的酶与苯丙酮醛，一种甘氨酸修饰试剂，灭活ATP介导的36 Cl-流出，但ATP酶活性的制备不受影响。当氯化物被35 SO 4（2）-取代时，从脂蛋白体中检测不到ATP依赖的35 SO 4（2）-流出。还测试了酶的质子移位活性，并且没有观察到9-ACMA的荧光猝灭。
The Mono Q-III fraction, a Mg2(+)-ATPase, isolated from Acetabularia acetabulum was reconstituted into liposomes of various net charges prepared by the reversed-phase method and tested for a Cl(-)-translocating activity. The liposomes from a mixture of egg lecithin, dicetyl phosphate, and cholesterol (63:18:9 mole ratio, negative liposomes) and from a mixture of egg lecithin and cholesterol (63:9 mole ratio, neutral liposomes) were less leaky than positive liposomes from asolectin, and from a mixture of egg lecithin, stearylamine, and cholesterol (63:18:9 mole ratio). A significant increase in 36Cl- efflux from the negative and neutral liposomes was observed by addition of ATP in the presence of valinomycin after incorporation of the enzyme by short-term dialysis. The ATP-driven 36Cl- efflux was inhibited by addition of azide, an inhibitor of the ATPase. The preincubation of the enzyme with phenylglyoxal, an arginine-modifying reagent, inactivated ATP-mediated 36Cl- efflux, but the ATPase activity of the preparation was not affected. When chloride was replaced by 35SO4(2)-, no ATP-dependent 35SO4(2)- efflux was detectable from the proteoliposomes. Proton-translocating activity of the enzyme was also tested, and no fluorescent quenching of 9-ACMA was observed.