Szymańska G, Kim H W, Kranias E G
Department of Pharmacology and Cell Biophysics, University of Cincinnati, College of Medicine, OH.
Biochim Biophys Acta. 1991 Jan 31;1091(2):127-34. doi: 10.1016/0167-4889(91)90051-x.
The Ca2(+)-ATPase of skeletal sarcoplasmic reticulum was purified and reconstituted in the presence of phosphatidyl choline using the freeze-thaw sonication technique. The effect of incorporation of negatively charged phospholipids, phosphatidylserine and phosphatidylinositol phosphate, into the phosphatidylcholine proteoliposomes was investigated. Various ratios of phosphatidylserine or phosphatidylinositol phosphate to phosphatidylcholine were used, while the total amount of phospholipid in the reconstituted vesicles was kept constant. Enrichment of phosphatidylcholine proteoliposomes by phosphatidylserine or phosphatidylinositol phosphate was associated with activation of Ca2(+)-uptake and Ca2(+)-ATPase activities. The highest activation was obtained at a 50:50 molar ratio of phosphatidylserine:phosphatidylcholine and at a 10:90 molar ratio of phosphatidylinositol phosphate:phosphatidylcholine. The initial rates of Ca2(+)-uptake obtained at 1 microM Ca2+ were 2.6 +/- 0.1 mumol/min per mg of phosphatidylserine:phosphatidylcholine proteoliposomes and 1.5 +/- 0.1 mumol/min per mg of phosphatidylinositol phosphate:phosphatidylcholine proteoliposomes, compared to 0.9 +/- 0.05 mumol/min per mg of phosphatidylcholine proteoliposomes. These findings suggest that negatively charged phospholipids may be involved in the activation of the reconstituted skeletal muscle sarcoplasmic reticulum Ca2(+)-pump.
利用冻融超声技术，在磷脂酰胆碱存在的情况下对骨骼肌肌浆网的Ca2(+)-ATP酶进行了纯化和重组。研究了将带负电荷的磷脂磷脂酰丝氨酸和磷脂酰肌醇磷酸掺入磷脂酰胆碱蛋白脂质体的效果。使用了磷脂酰丝氨酸或磷脂酰肌醇磷酸与磷脂酰胆碱的各种比例，同时重组囊泡中磷脂的总量保持恒定。磷脂酰丝氨酸或磷脂酰肌醇磷酸对磷脂酰胆碱蛋白脂质体的富集与Ca2(+)-摄取和Ca2(+)-ATP酶活性的激活有关。在磷脂酰丝氨酸:磷脂酰胆碱的摩尔比为50:50和磷脂酰肌醇磷酸:磷脂酰胆碱的摩尔比为10:90时获得了最高的激活效果。在1 microM Ca2+下获得的Ca2(+)-摄取的初始速率，对于每毫克磷脂酰丝氨酸:磷脂酰胆碱蛋白脂质体为2.6±0.1微摩尔/分钟，对于每毫克磷脂酰肌醇磷酸:磷脂酰胆碱蛋白脂质体为1.5±0.1微摩尔/分钟，相比之下，每毫克磷脂酰胆碱蛋白脂质体为0.9±0.05微摩尔/分钟。这些发现表明带负电荷的磷脂可能参与了重组的骨骼肌肌浆网Ca2(+)-泵的激活。
