Grimaldi M E, Adamo H P, Rega A F, Penniston J T
Instituto de Química y Fisicoquímica Biológicas-Facultad de Farmacia y Bioquímica (Universidad de Buenos Aires), Junin 956, 1113 Capital Federal, Buenos Aires, Argentina.
J Biol Chem. 1996 Oct 25;271(43):26995-7. doi: 10.1074/jbc.271.43.26995.
A mutant of the plasma membrane Ca2+ pump hPMCA4b(d18-75)(ct120) containing a deletion of the N-terminal amino acid residues 18-75 and lacking the C-terminal 120 amino acid residues was expressed in COS-1 cells. The deletion in the N-terminal region did not significantly affect the level of expression of the Ca2+ pump. Tryptic digestion of the hPMCA4b(d18-75)(ct120) mutant resulted in the appearance of the same fragments obtained by proteolysis of the hPMCA4b(ct120) enzyme, suggesting that deletion of residues 18-75 neither impeded the insertion in the membrane nor extensively affected the folding of the mutant protein. The functional competence of the hPMCA4b(d18-75)(ct120) enzyme was examined by measuring the Ca2+ transport and the Ca2+ ATPase activity of COS-1 cell microsomes expressing the mutant protein. Both tests proved the mutant to be inactive. Under conditions in which hPMCA4b(ct120) becomes phosphorylated, hPMCA4b(d18-75)(ct120) was incapable of reacting with ATP and Ca2+ to form the phosphoenzyme. Taken together these results suggest that the segment of amino acids 18-75 is essential for the activity of the plasma membrane Ca2+ pump.
一种质膜Ca2+泵hPMCA4b(d18 - 75)(ct120)突变体在COS - 1细胞中表达，该突变体缺失N端18 - 75个氨基酸残基且缺少C端120个氨基酸残基。N端区域的缺失对Ca2+泵的表达水平没有显著影响。对hPMCA4b(d18 - 75)(ct120)突变体进行胰蛋白酶消化，产生的片段与hPMCA4b(ct120)酶经蛋白水解得到的片段相同，这表明缺失18 - 75个残基既不阻碍其插入膜中，也未广泛影响突变蛋白的折叠。通过测量表达该突变蛋白的COS - 1细胞微粒体的Ca2+转运和Ca2+ ATP酶活性，检测了hPMCA4b(d18 - 75)(ct120)酶的功能活性。两项测试均证明该突变体无活性。在hPMCA4b(ct120)发生磷酸化的条件下，hPMCA4b(d18 - 75)(ct120)无法与ATP和Ca2+反应形成磷酸酶。综合这些结果表明，18 - 75位氨基酸片段对质膜Ca2+泵的活性至关重要。
