Isolation and characterization of a specific endogenous Na+,K+-ATPase inhibitor from bovine adrenal.
Isolation and characterization of a specific endogenous Na+,K+-ATPase inhibitor from bovine adrenal.
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从牛肾上腺中分离和表征特定的内源性 Na ,K -ATP 酶抑制剂。
DOI：
10.1021/bi00412a007
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发表时间：
1988
期刊：
影响因子：
2.9
通讯作者：
Inagami,T
中科院分区：
文献类型：
--
作者：
Tamura,M;Lam,TT;Inagami,T
材料和方法化学品。[12 a-3 H]地高辛（19.0 Ci/mmol）、[G-3 H]-哇巴因（20.0 Ci/mmol）、45 CaCl 2（21.92 mCi/mg）和86-RbCl（2.72-4.82 mCi/mg）购自新英格兰核公司。在绵羊体内培养的地高辛与牛血清白蛋白结合的抗血清是Dr.马萨诸塞州总医院的埃德加哈伯。Rh（-）O型人红细胞由范德比尔特医院血库提供。腺苷S ′-三磷酸（无钒）、地高辛、哇巴因、蛋白酶（XIV型）、胰蛋白酶（III型）、脱氧核糖核酸酶I（II型）、核糖核酸酶A（III-A型）和磷脂酶C（XIV型）从Sigma Chemical Co.获得。氧化氘（99.96原子% D）从Aldrich Chemical Co.购买。有机溶剂为1级高效液相色谱（HPLC）。其他化学品为分析级。Na+，K*-ATP酶活性通过改进Jorgensen的快速方法（Jorgensen，1974 a），从犬肾脏外髓制备膜结合Na+，K+-AT-Pase。除另有说明外，在37 ℃下，用P ~+-ATP酶抑制率估算抑制Na ~+，K ~+-ATP酶活性的能力。在含有77 mM NaCl、17 mM NaHCO 3、0.5 mM KCl、2.7 mM MgCl 2、0.77 mM Tris-EGTA、23 mM咪唑、11.5 mM Hepes、77 gg脱脂牛血清白蛋白（BSA）和6.8 µg纯化酶蛋白。孵育30 min后，加入0.5 mL 5% SDS终止反应。释放的P °根据
Materials and MethodsChemicals.[12a-3H] Digoxin (19.0 Ci/mmol),[G-3H]-ouabain (20.0 Ci/mmol), 45CaCl2 (21.92 mCi/mg), and 86-RbCl (2.72-4.82 mCi/mg) were purchased from New England Nuclear. Antisera against digoxin conjugated to bovine serum albumin raised in sheep were the generous gift ofDr. Edgar Haber of Massachusetts General Hospital. Human red blood cells, type Rh (-) O, were supplied by the blood bank of Vanderbilt Hospital. Adenosine S'-triphosphate (vanadium free), digoxin, ouabain, protease (type XIV), trypsin (type III), deoxyribonuclease I (type II), ribonuclease A (type III-A), and phospholipase C (type XIV) were obtained from Sigma Chemical Co. Deuterium oxide (99.96 atom% D) was pur-chased from Aldrich Chemical Co. Organic solvents were of high-performance liquid chromatography (HPLC) 1 grade. Other chemicals were of analytical grade. Na+, K*-ATPase Activity. Membrane-bound Na+, K+-AT-Pase was prepared from canine kidney outer medulla by a modification of Jorgensen’s rapid method (Jorgensen, 1974a). Unless otherwise stated, the capacity to inhibit Na+, K+-AT-Pase activity was estimated at 37 C by the rate of P¡ release from 3.1 mM Na2ATP at pH 7.4 in a 1.3-mL reaction mixture containing 77 mM NaCl, 17 mM NaHC03, 0.5 mM KC1, 2.7 mM MgCl2, 0.77 mM Tris-EGTA, 23 mM imidazole, 11.5 mM Hepes, 77 gg of fat-free bovine serum albumin (BSA), and 6.8 µg of the purified enzyme protein. After incubation for 30 min, the reaction was terminated by addition of 0.5 mL of 5% SDS. The P¡ released was measured according to the