Beleznay Z, Zachowski A, Devaux P F, Navazo M P, Ott P
Institut für Biochemie und Molekularbiologie, Bern, Switzerland.
Biochemistry. 1993 Mar 30;32(12):3146-52. doi: 10.1021/bi00063a029.
Vesicles released from human red blood cells by incubation with a suspension of sonicated dimyristoylphosphatidylcholine were purified by gel filtration. Purified vesicles and intact red cells had a very similar composition with respect to phospholipids and integral membrane proteins, but spectrin, the major component of the membrane skeleton, was not found in vesicles. Comparison of red cell and vesicle ATP levels (expressed as micromolar ATP per millimolar hemoglobin) showed a marked difference with a reduced content of only about 30% in vesicles, whatever the initial concentration in the erythrocytes. Spin-labeled aminophospholipids (phosphatidylserine and phosphatidylethanolamine) were translocated to the inner vesicle membrane layer at a comparable rate as in intact red cells provided that vesicles contained enough ATP. The maximum fraction of spin-labeled phospholipids translocated to the inner membrane layer was 84% for phosphatidylserine, 65% for phosphatidylethanolamine, 20-40% for phosphatidylcholine, and below 20% for sphingomyelin. The apparent Km of translocation, expressed as percent of total membrane phospholipid, was 0.14% for spin-labeled phosphatidylserine and 1.19% for spin-labeled phosphatidylethanolamine. This compares well to values established earlier for intact red blood cells. The fact that no ATP was synthesized in vesicles allowed determination of ATP consumption by aminophospholipid transport. The basic ATP hydrolysis rate was increased upon the addition of labeled aminophospholipids but not of labeled phosphatidylcholine or sphingomyelin. The stoichiometry between lipid translocation and ATP consumption, calculated from the respective initial velocities, was 1.13 +/- 0.2 for phosphatidylserine and 1.11 +/- 0.16 for phosphatidylethanolamine.
通过与超声处理的二肉豆蔻酰磷脂酰胆碱悬浮液孵育从人红细胞释放的囊泡经凝胶过滤纯化。纯化的囊泡和完整红细胞在磷脂和整合膜蛋白方面具有非常相似的组成，但膜骨架的主要成分血影蛋白在囊泡中未被发现。红细胞和囊泡ATP水平（以每毫摩尔血红蛋白中微摩尔ATP表示）的比较显示出显著差异，无论红细胞中的初始浓度如何，囊泡中的含量仅降低约30%。只要囊泡含有足够的ATP，自旋标记的氨基磷脂（磷脂酰丝氨酸和磷脂酰乙醇胺）以与完整红细胞相当的速率转运到囊泡内膜层。转运到内膜层的自旋标记磷脂的最大比例，磷脂酰丝氨酸为84%，磷脂酰乙醇胺为65%，磷脂酰胆碱为20 - 40%，鞘磷脂低于20%。以总膜磷脂百分比表示的转运表观Km，自旋标记的磷脂酰丝氨酸为0.14%，自旋标记的磷脂酰乙醇胺为1.19%。这与早期为完整红细胞确定的值相当吻合。囊泡中不合成ATP这一事实使得能够确定氨基磷脂转运消耗的ATP。添加标记的氨基磷脂而非标记的磷脂酰胆碱或鞘磷脂后，基础ATP水解速率增加。根据各自的初始速度计算，脂质转运与ATP消耗之间的化学计量比，磷脂酰丝氨酸为1.13±0.2，磷脂酰乙醇胺为1.11±0.16。
