Perrone J R, Hackney J F, Dixon J F, Hokin L E
J Biol Chem. 1975 Jun 10;250(11):4178-84.
The chemical properties of two highly purified preparations of (sodium + potassium)-activated adenosine triphosphatase (NaK ATPase) and their subunits have been compared. One preparation is derived from the rectal gland of the spiny dogfish shark, Squalus acanthias and the other preparation is derived from the electric organ of the electric eel, Electrophorus electricus. Ouabain binding and phosphorylation from [gamma-32-P]ATP for both enzymes ranged from 4000 to 4300 pmol per mg of protein. This gives a stoichiometry for ouabain binding and phosphorylation of 1:1 for both enzymes. The molar ratios of catalytic subunit to glycoprotein was 2:1 for both enzymes, suggesting a minimum molecular weight of 250, 000, which agrees with the molecular weight obtained by radiation inactivation. Assuming that only one of the two catalytic subunits is phosphorylated and binds ouabain per (sodium + potassium)-activated adenosine triphosphatase molecule the data on phosphorylation and ouabain binding also give a molecular weight of 250, 000. The data on phosphorylatiion, ouabain binding, subunit composition, and molecular weight based on radiaion inactivation are thus all internally consistent. A technique has been developed for isolation of pure catalytic subunit and glycoprotein in good yields by preparative sodium dodecyl sulfate-polyacrylamide gel electrophoresis. A variety of chemical studies have been carried out with the purified subunits. The amino acid composition of the catalytic subunit was different from that of the glycoprotein, but the amino acid composition of each of the two subunits was essentially the same for both species. However, the NH2-terminal amino acid for the catalytic subunit was alanine for the rectal gland enzyme and serine for the electric organ enzyme, suggesting some differencesin amino acid sequences for the two species. The NH2-terminal amino acid for the glycoprotein was alanine for the two species. The glycoproteins from both species contained the same carbohydrates but in quite differing amounts. The carbohydrates were glucosamine, sialic acid, fucose, galactose, mannose, and glucose. The release of all the sialic acid from the electric organ enzyme and the release of 40% of the sialic acid from the rectal gland enzyme did not affect (sodium + potassium)-activated adenosine triphosphatase activity. Both enzymes contained the following phospholipids, which accounted for 98 to 100% of the total phospholipid phosphorus: sphingomyelin, lecithin, phosphatidylserine, phosphatidylethanolamine, and phosphatidylinositol. With the exception of phosphatidylethanolamine, and phosphatidylinositol. With the exception of phosphatidylserine, the amount of any phospholipid per mg of enzyme as well as the total phospholipid content were quite different for the two enzymes.
对两种高度纯化的（钠+钾）激活的三磷酸腺苷酶（NaK ATPase）制剂及其亚基的化学性质进行了比较。一种制剂来源于棘鲨（Squalus acanthias）的直肠腺，另一种制剂来源于电鳗（Electrophorus electricus）的电器官。两种酶的哇巴因结合以及由[γ-32-P]ATP进行的磷酸化反应，每毫克蛋白质的范围为4000至4300皮摩尔。这使得两种酶的哇巴因结合与磷酸化的化学计量比为1:1。两种酶的催化亚基与糖蛋白的摩尔比均为2:1，表明最小分子量为250,000，这与通过辐射失活获得的分子量一致。假设每个（钠+钾）激活的三磷酸腺苷酶分子中只有两个催化亚基中的一个被磷酸化并结合哇巴因，那么关于磷酸化和哇巴因结合的数据也得出分子量为250,000。因此，基于辐射失活的磷酸化、哇巴因结合、亚基组成和分子量的数据在内部都是一致的。已经开发出一种通过制备性十二烷基硫酸钠-聚丙烯酰胺凝胶电泳以高产率分离纯催化亚基和糖蛋白的技术。对纯化的亚基进行了各种化学研究。催化亚基的氨基酸组成与糖蛋白不同，但两种物种的两个亚基各自的氨基酸组成基本相同。然而，直肠腺酶催化亚基的NH2末端氨基酸是丙氨酸，而电器官酶的是丝氨酸，这表明两种物种在氨基酸序列上存在一些差异。两种物种糖蛋白的NH2末端氨基酸都是丙氨酸。两种物种的糖蛋白都含有相同的碳水化合物，但含量差异很大。这些碳水化合物是氨基葡萄糖、唾液酸、岩藻糖、半乳糖、甘露糖和葡萄糖。从电器官酶中释放所有唾液酸以及从直肠腺酶中释放40%的唾液酸并不影响（钠+钾）激活的三磷酸腺苷酶活性。两种酶都含有以下磷脂，它们占总磷脂磷的98%至100%：鞘磷脂、卵磷脂、磷脂酰丝氨酸、磷脂酰乙醇胺和磷脂酰肌醇。除了磷脂酰乙醇胺和磷脂酰肌醇外。除了磷脂酰丝氨酸外，每毫克酶中任何一种磷脂的量以及总磷脂含量在两种酶中都有很大差异。
