Therien A G, Karlish S J, Blostein R
Department of Biochemistry, McGill University, Montreal, Quebec H3G 1A4, Canada.
J Biol Chem. 1999 Apr 30;274(18):12252-6. doi: 10.1074/jbc.274.18.12252.
The functional role of the gamma subunit of the Na,K-ATPase was studied using rat gamma cDNA-transfected HEK-293 cells and an antiserum (gammaC33) specific for gamma. Although the sequence for gamma was verified and shown to be larger (7237 Da) than first reported, it still comprises a single initiator methionine despite the expression of a gammaC33-reactive doublet on immunoblots. Kinetic analysis of the enzyme of transfected compared with control cells and of gammaC33-treated kidney pumps shows that gamma regulates the apparent affinity for ATP. Thus, gamma-transfected cells have a decreased K'ATP as shown in measurements of (i) K'ATP of Na,K-ATPase activity and (ii) K+ inhibition of Na-ATPase at 1 microM ATP. Consistent with the behavior of gamma-transfected cells, gammaC33 pretreatment increases K'ATP of the kidney enzyme and K+ inhibition (1 microM ATP) of both kidney and gamma-transfected cells. These results are consistent with previous findings that an antiserum raised against the pig gamma subunit stabilizes the E2(K) form of the enzyme (Therien, A. G., Goldshleger, R., Karlish, S. J., and Blostein, R. (1997) J. Biol. Chem. 272, 32628-32634). Overall, our data demonstrate that gamma is a tissue (kidney)-specific regulator of the Na,K-ATPase that can increase the apparent affinity of the enzyme for ATP in a manner that is reversible by anti-gamma antiserum.
利用大鼠γ亚基cDNA转染的HEK - 293细胞和针对γ亚基的特异性抗血清（γC33）研究了钠钾ATP酶γ亚基的功能作用。尽管γ亚基的序列已得到验证，且显示其分子量比最初报道的更大（7237 Da），但尽管在免疫印迹上出现了γC33反应性双峰，它仍包含一个起始甲硫氨酸。与对照细胞相比，对转染细胞的酶以及经γC33处理的肾泵进行动力学分析表明，γ亚基调节酶对ATP的表观亲和力。因此，γ亚基转染的细胞具有降低的K'ATP，如在以下测量中所示：（i）钠钾ATP酶活性的K'ATP，以及（ii）在1μM ATP时钾对钠ATP酶的抑制作用。与γ亚基转染细胞的行为一致，γC33预处理增加了肾酶的K'ATP以及肾细胞和γ亚基转染细胞的钾抑制作用（1μM ATP）。这些结果与先前的发现一致，即针对猪γ亚基产生的抗血清可稳定酶的E2（K）形式（Therien, A. G., Goldshleger, R., Karlish, S. J., and Blostein, R. (1997) J. Biol. Chem. 272, 32628 - 32634）。总体而言，我们的数据表明，γ亚基是钠钾ATP酶的组织（肾脏）特异性调节因子，它可以增加酶对ATP的表观亲和力，且这种作用可被抗γ亚基抗血清逆转。
