Monesterolo N E, Amaiden M R, Campetelli A N, Santander V S, Arce C A, Pié J, Casale C H
Departamento de Biología Molecular, Universidad Nacional de Río Cuarto, Argentina.
Biochim Biophys Acta. 2012 Mar;1818(3):601-8. doi: 10.1016/j.bbamem.2011.11.022. Epub 2011 Dec 3.
We demonstrated previously that acetylated tubulin inhibits plasma membrane Ca(2+)-ATPase (PMCA) activity in plasma membrane vesicles (PMVs) of rat brain through a reversible interaction. Dissociation of the PMCA/tubulin complex leads to restoration of ATPase activity. We now report that, when the enzyme is reconstituted in phosphatidylcholine vesicles containing acidic or neutral lipids, tubulin not only loses its inhibitory effect but is also capable of activating PMCA. This alteration of the PMCA-inhibitory effect of tubulin was dependent on concentrations of both lipids and tubulin. Tubulin (300μg/ml) in combination with acidic lipids at concentrations >10%, increased PMCA activity up to 27-fold. The neutral lipid diacylglycerol (DAG), in combination with 50μg/ml tubulin, increased PMCA activity >12-fold, whereas tubulin alone at high concentration (≥300μg/ml) produced only 80% increase. When DAG was generated in situ by phospholipase C incubation of PMVs pre-treated with exogenous tubulin, the inhibitory effect of tubulin on PMCA activity (ATP hydrolysis, and Ca(2+) transport within vesicles) was reversed. These findings indicate that PMCA is activated independently of surrounding lipid composition at low tubulin concentrations (<50μg/ml), whereas PMCA is activated mainly by reconstitution in acidic lipids at high tubulin concentrations. Regulation of PMCA activity by tubulin is thus dependent on both membrane lipid composition and tubulin concentration.
我们之前证明，乙酰化微管蛋白通过可逆相互作用抑制大鼠脑细胞膜囊泡（PMV）中的质膜Ca(2+)-ATP酶（PMCA）活性。PMCA/微管蛋白复合物的解离导致ATP酶活性恢复。我们现在报告，当该酶在含有酸性或中性脂质的磷脂酰胆碱囊泡中重组时，微管蛋白不仅失去其抑制作用，而且还能够激活PMCA。微管蛋白对PMCA抑制作用的这种改变取决于脂质和微管蛋白的浓度。浓度>10%的酸性脂质与300μg/ml微管蛋白结合，可使PMCA活性增加高达27倍。中性脂质二酰甘油（DAG）与50μg/ml微管蛋白结合，可使PMCA活性增加>12倍，而单独的高浓度（≥300μg/ml）微管蛋白仅使活性增加80%。当通过用外源微管蛋白预处理的PMV经磷脂酶C孵育原位生成DAG时，微管蛋白对PMCA活性（ATP水解以及囊泡内Ca(2+)转运）的抑制作用被逆转。这些发现表明，在微管蛋白浓度较低（<50μg/ml）时，PMCA的激活与周围脂质组成无关，而在微管蛋白浓度较高时，PMCA主要通过在酸性脂质中重组而被激活。因此，微管蛋白对PMCA活性的调节取决于膜脂质组成和微管蛋白浓度。
