Bhattacharyya D, Sen P C
Department of Chemistry, Bose Institute, Calcutta, India.
Eur J Biochem. 1997 Mar 15;244(3):829-34. doi: 10.1111/j.1432-1033.1997.00829.x.
A number of low-molecular mass (12-13 kDa) Na+, K+-ATPase inhibitor proteins have been purified from rat brain cytosol by gel filtration followed by FPLC fractionation on a Mono Q anion-exchange column. Eight peaks were obtained using 0.1 M NaCl eluent of which one peak was found to be the most potent inhibitor of Na+, K+-ATPase. The molcular mass of the inhibitor was about 13 kDa on 16.5% SDS/PAGE. The concentration at which 50% inhibition (I50) was found was in the nanomolar range. The inhibitor seems to bind to Na+, K+-ATPase at a site distal from the ATP-binding site. The binding to the ATPase is non-competitive. The CD analysis suggests an unordered secondary structural element. It also inhibits p-nitrophenyl phosphatase activity from rat brain with comparable I50 value to that for Na+, K+-ATPase. The protein does not contain any Trp as evident from Trp fluorescence and amino acid analysis. Amino acid analysis shows that glycine and serine, derivatives of tyrosine and phenylalanine are the predominant amino acids. The data suggests that it is a negatively charged protein in which the contribution of the hydrophobic part is 27%.
通过凝胶过滤，然后在Mono Q阴离子交换柱上进行快速蛋白质液相色谱（FPLC）分级分离，从大鼠脑细胞质中纯化出了多种低分子量（12 - 13 kDa）的Na⁺,K⁺-ATP酶抑制蛋白。使用0.1 M NaCl洗脱液得到了8个峰，其中一个峰被发现是最有效的Na⁺,K⁺-ATP酶抑制剂。在16.5% SDS/PAGE上，该抑制剂的分子量约为13 kDa。发现50%抑制率（I50）时的浓度处于纳摩尔范围。该抑制剂似乎在远离ATP结合位点的位置与Na⁺,K⁺-ATP酶结合。与ATP酶的结合是非竞争性的。圆二色（CD）分析表明存在无序的二级结构元件。它还以与Na⁺,K⁺-ATP酶相当的I50值抑制大鼠脑的对硝基苯磷酸酶活性。从色氨酸荧光和氨基酸分析可以明显看出，该蛋白质不含任何色氨酸。氨基酸分析表明，甘氨酸、丝氨酸、酪氨酸和苯丙氨酸的衍生物是主要氨基酸。数据表明它是一种带负电荷的蛋白质，其中疏水部分占27%。
