Deletions in the Acidic Lipid-binding Region of the Plasma Membrane Ca2+ Pump
Deletions in the Acidic Lipid-binding Region of the Plasma Membrane Ca2+ Pump
复制标题
质膜 Ca2 泵酸性脂质结合区的缺失
DOI：
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发表时间：
2002
影响因子：
4.8
通讯作者：
H. P. Adamo
中科院分区：
文献类型：
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作者：
F. Pinto;H. P. Adamo
质膜钙泵(PMCA)跨膜螺旋2和3(AL区)之间环的C末端片段在其他P-ATPase中不保守。该区域的一部分位于第三跨膜结构域的上游，与酸性脂类激活PMCA有关。构建了编码钙泵异构体h4xb AL区缺失突变体的cDNAs，并在COS和中国仓鼠卵巢细胞中成功表达。296-349片段缺失的突变体具有完整的钙转运活性，而涉及350-356氨基酸片段的缺失突变体则没有活性，这表明这些残基是功能性PMCA所必需的。在不含钙调素的情况下，突变体d296-349的Vmax与重组野生型泵相似，但其对钙离子的K0.5约低5倍。CaM的加入增加了野生型和D296-349酶的vmax和表观Ca~(2+)亲和力，表明CaM的激活作用不受该缺失的影响。在低浓度的Ca~(2+)和饱和的钙调蛋白存在下，磷脂酸的加入使重组野生型泵的活性增加约2倍。相反，在此条件下，磷脂酸对突变体d296-349的活性没有显著影响。综上所述，这些结果表明：(A)296-349残基的缺失重建了一种类似于酸性脂类结合在AL区产生的PMCA的形式；(B)AL区作为酸性脂结合抑制域，能够调节PMCA对膜脂组成的亲和力；(C)AL区的功能独立于C端钙调素结合区的自身抑制。
The C-terminal segment of the loop between transmembrane helices 2 and 3 (AL region) of the plasma membrane Ca2+ pump (PMCA) is not conserved in other P-ATPases. Part of this region, just upstream from the third transmembrane domain, has been associated with activation of the PMCA by acidic lipids. cDNAs coding for mutants of the Ca2+pump isoform h4xb with deletions in the AL region were constructed, and the proteins were successfully expressed in either COS or Chinese hamster ovary cells. Mutants with deletions in the segment 296–349 had full Ca2+ transport activity, but deletions involving the segment of amino acids 350–356 were inactive suggesting that these residues are required for a functional PMCA. In the absence of calmodulin the V max of mutant d296–349 was similar to that of the recombinant wild type pump, but its K 0.5 for Ca2+ was about 5-fold lower. The addition of calmodulin increased theV max and the apparent Ca2+ affinity of both the wild type and d296–349 enzymes indicating that the activating effects of calmodulin were not affected by the deletion. At low concentrations of Ca2+ and in the presence of saturating amounts of calmodulin, the addition of phosphatidic acid increased about 2-fold the activity of the recombinant wild type pump. In contrast, under these conditions phosphatidic acid did not significantly change the activity of mutant d296–349. Taken together these results suggest that (a) deletion of residues 296–349 recreates a form of PMCA similar to that resulting from the binding of acidic lipids at the AL region; (b) the AL region acts as an acidic lipid-binding inhibitory domain capable of adjusting the Ca2+ affinity of the PMCA to the lipid composition of the membrane; and (c) the function of the AL region is independent of the autoinhibition by the C-terminal calmodulin-binding region.
CTP 的脂质激活：磷酸胆碱胞苷酰转移酶 α：第二个激活域的表征和鉴定。
影响因子：
2.9
作者：
Lykidis,A;Jackson,P;Jackowski,S
通讯作者：
Jackowski,S
通过蛋白水解和单克隆抗体揭示原位红细胞膜钙泵的功能域。
DOI：
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发表时间：
1989
期刊：
The Journal of biological chemistry
影响因子：
--
作者：
Papp,B;Sarkadi,B;Enyedi,A;Caride,AJ;Penniston,JT;Gardos,G
通讯作者：
Gardos,G