2 RESIDUES THAT MAY LIGATE CA2+ IN TRANSMEMBRANE DOMAIN 6 OF THE PLASMA-MEMBRANE CA2+-ATPASE
2 RESIDUES THAT MAY LIGATE CA2+ IN TRANSMEMBRANE DOMAIN 6 OF THE PLASMA-MEMBRANE CA2+-ATPASE
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DOI：
10.1074/jbc.270.46.27812
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发表时间：
1995-11-17
影响因子：
4.8
通讯作者：
PENNISTON, JT
中科院分区：
文献类型：
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作者：
ADEBAYO, AO;ENYEDI, A;PENNISTON, JT
为了鉴定质膜Ca 2+泵的推定跨膜结构域6中的Ca 2+配体，单独改变氨基酸Asn（879）、Met（882）、Asp（883）和Ser（887），Asn（879）、Met（882）、和Asp（883），因为已经提出了相应的氨基酸作为肌浆网Ca 2+泵中的Ca 2+配体（Clarke，D，M，，Loo，T，W，和MacLennan，D，H，（1990）J，Biol，Chem，265，6262-6267）。对于改变，使用了完全活性的截短形式的泵，因为可以在不受钙调蛋白结合干扰的情况下研究Ca 2+与泵的相互作用。Asn和Asp的突变体不进行ATP支持的Ca 2+摄取，并且不从[γ-P-32]ATP形成酰基磷酸，这表明，像肌浆网Ca 2+泵中的相应氨基酸一样，这两个是Ca 2+配体，然而，在Met（882）位置的所有突变体都显示出一些活性。实际上，Met（882）→ Ile突变体在饱和Ca 2+浓度下是完全活性的，并且仅对于Ca 2+活化的K-1/2稍微向上移动，将Met转换为Thr（其是肌浆网Ca 2+泵中的相应残基）将活性降低至野生型的20%，进一步强调了两种Ca 2+泵之间的差异，突变体Ser（887）-> Ala的表达量大于野生型，并且比活性比野生型高约50%，这表明该丝氨酸也不能是Ca 2+配体，并且不能取代Met（882）位置上缺失的Thr。
In order to identify Ca2+ ligands in the putative transmembrane domain 6 of the plasma membrane Ca2+ pump, amino acids Asn(879), Met(882), Asp(883), and Ser(887) were singly altered, Asn(879), Met(882), and Asp(883) were chosen because the corresponding amino acids have been proposed as Ca2+ ligands in the sarcoplasmic reticulum Ca2+ pump (Clarke, D, M,, Loo, T, W,, and MacLennan, D, H, (1990) J, Biol, Chem, 265, 6262-6267). For the alterations, a fully active truncated version of the pump was used, because the interaction of Ca2+ with the pump could be studied without interference from calmodulin binding, The mutants at Asn and Asp did not carry out ATP-supported Ca2+ uptake and formed no acylphosphate from [gamma-P-32]ATP, suggesting that, like the corresponding amino acids in the sarcoplasmic reticulum Ca2+ pump, these two are Ca2+ ligands, However, all the mutants at the position of Met(882) showed some activity, Indeed, the Met(882) --> Ile mutant was fully active at a saturating Ca2+ concentration and only the K-1/2, for Ca2+ activation was shifted slightly upward, Converting the Met to Thr (which is the corresponding residue in the sarcoplasmic reticulum Ca2+ pump) reduced the activity to 20% of the wild type, further emphasizing the differences between the two Ca2+ pumps, The mutant Ser(887) --> Ala was expressed in greater amounts than, and had a specific activity about 50% higher than, the wild type, indicating that this serine also could not be a Ca2+ ligand and could not replace the missing Thr at position Met(882).