Suzuki H, Kamakura M, Morii M, Takeguchi N
Faculty of Pharmaceutical Sciences, Toyama Medical and Pharmaceutical University, 2630 Sugitani, Toyama 930-01, Japan.
J Biol Chem. 1997 Apr 18;272(16):10429-34. doi: 10.1074/jbc.272.16.10429.
We found that isolated gastric vesicles contain a novel Mg2+-ATP-dependent phospholipid translocation (flippase) activity. Fluorescence analogue of phosphatidylcholine, 2-(12-(7-nitrobenz-2-oxa-1, 3-diazol-4-yl)amino)dodecanoyl-1-hexadecanoyl-sn-glycero-3- phosphocholine, was ATP-dependently translocated from the outer (cytosolic) to inner (luminal) leaflet of the lipid membrane bilayer of hog gastric vesicles. The translocation was saturable and depended on time and the ATP concentration (Km = 3.1 microM). The basal Mg2+-ATPase activity of gastric vesicles in the absence of K+ showed high (Km = 1.6 microM) and low (Km = 80 microM) affinities for ATP, indicating that the present flippase activity is driven mostly by the high affinity Mg2+-ATPase activity. It required Mg2+ but not K+. Verapamil, which is an inhibitor of mouse mdr2 phosphatidylcholine flippase, did not inhibit the present flippase activity. Isolated sarcoplasmic reticulum vesicles that contain Ca2+-ATPase did not show any flippase activity. Fluorescence analogues of phosphatidylserine and phosphatidylethanolamine were similarly translocated by the gastric flippase. These phospholipid flippase activities were inhibited by 2-methyl-8-(phenylmethoxy)imidazo[1,2-a]pyridine-3-acetonitrile (SCH 28080) (IC50 = 0.14-0.25 microM), a specific K+-ATPase inhibitor of gastric H+,K+-ATPase rich in gastric vesicles. IC50 value for the SCH 28080-inhibitable Mg2+-ATPase activity was about 0.13 microM, indicating that the phospholipid translocation was driven mostly by the SCH 28080-sensitive Mg2+-ATPase activity. Possible physiological roles of flippases were discussed in relation with the gastric acid secretory and cytoprotective mechanisms.
我们发现，分离出的胃小泡含有一种新型的Mg2+依赖的ATP磷脂转运（翻转酶）活性。磷脂酰胆碱的荧光类似物，2-（12-（7-硝基苯并-2-恶唑-1,3-二氮杂环丁烷-4-基）氨基）十二烷酰-1-十六烷酰-sn-甘油-3-磷酸胆碱，在ATP的作用下从猪胃小泡脂质膜双层的外侧（胞质侧）转运至内侧（腔侧）小叶。这种转运是可饱和的，并且依赖于时间和ATP浓度（Km = 3.1 microM）。在无K+的情况下，胃小泡的基础Mg2+依赖的ATP酶活性对ATP表现出高亲和力（Km = 1.6 microM）和低亲和力（Km = 80 microM），这表明目前的翻转酶活性主要由高亲和力的Mg2+依赖的ATP酶活性驱动。它需要Mg2+但不需要K+。维拉帕米是小鼠mdr2磷脂酰胆碱翻转酶的抑制剂，它并不抑制目前的翻转酶活性。含有Ca2+依赖的ATP酶的分离出的肌浆网小泡没有显示出任何翻转酶活性。磷脂酰丝氨酸和磷脂酰乙醇胺的荧光类似物也同样被胃翻转酶转运。这些磷脂翻转酶活性被2-甲基-8-（苄氧基）咪唑并[1,2-a]吡啶-3-乙腈（SCH 28080）（IC50 = 0.14 - 0.25 microM）抑制，SCH 28080是富含胃小泡的胃H+，K+依赖的ATP酶的特异性K+依赖的ATP酶抑制剂。SCH 28080可抑制的Mg2+依赖的ATP酶活性的IC50值约为0.13 microM，这表明磷脂转运主要由SCH 28080敏感的Mg2+依赖的ATP酶活性驱动。文中讨论了翻转酶可能的生理作用与胃酸分泌和细胞保护机制的关系。
