Moriyama Y, Nelson N
Roche Institute of Molecular Biology, Roche Research Center, Nutley, New Jersey 07110.
J Biol Chem. 1988 Jun 15;263(17):8521-7.
A vanadate- and N-ethylmaleimide-sensitive ATPase was purified about 500-fold from chromaffin granule membranes. The purified preparation contained a single major polypeptide with an apparent molecular mass of about 115 kDa, which was copurified with the ATPase activity. Immunological studies revealed that this polypeptide has no relation to subunit I (115 kDa) of the H+-ATPase from chromaffin granules. The ATPase activity of the enzyme is inhibited about 50% by 100 microM N-ethylmaleimide or 5 microM vanadate. The enzyme is not sensitive to dicyclohexylcarbodiimide, ouabain, SCH28080, and omeprazole, which distinguishes it from Na+/K+-ATPase and the gastric K+/H+-ATPase. ATP and 2-deoxy ATP are equally effective substrates for the enzyme. However, the enzyme exhibited only 10% activity with GTP as a substrate. UV illumination of the purified enzyme in the presence of [alpha-32P]ATP exclusively labeled the 115 kDa protein. This labeling was increased by Mg2+ and strongly inhibited by Ca2+ ions. Similarly, the ATPase activity was dependent on Mg2+ and inhibited by the presence of Ca2+ ions. The ATPase activity of the enzyme was largely insensitive to monovalent anions and cations, except for F-, which inhibited the vanadate-sensitive ATPase. Incubation of the enzyme in the presence of [14C]N-ethylmaleimide labeled the 115-kDa polypeptide, and this labeling could be prevented by the addition of ATP during the incubation. A reciprocal experiment showed that preincubation with N-ethylmaleimide inhibited the labeling of the 115-kDa polypeptide by [alpha-32P]ATP by UV illumination. This suggests a close proximity between the ATP-binding site and an essential sulfhydryl group. A possible connection between the isolated ATPase and organelle movement is discussed.
从嗜铬粒细胞膜中纯化出一种对钒酸盐和N - 乙基马来酰亚胺敏感的ATP酶，纯化倍数约为500倍。纯化后的制剂含有一条主要的多肽，表观分子量约为115 kDa，它与ATP酶活性一起被共纯化。免疫学研究表明，该多肽与嗜铬粒H⁺ - ATP酶的亚基I（115 kDa）无关。该酶的ATP酶活性被100 microM N - 乙基马来酰亚胺或5 microM钒酸盐抑制约50%。该酶对二环己基碳二亚胺、哇巴因、SCH28080和奥美拉唑不敏感，这使其与Na⁺/K⁺ - ATP酶和胃K⁺/H⁺ - ATP酶不同。ATP和2 - 脱氧ATP是该酶同样有效的底物。然而，该酶以GTP作为底物时仅表现出10%的活性。在[α - ³²P]ATP存在下对纯化酶进行紫外线照射，仅标记了115 kDa的蛋白质。这种标记在Mg²⁺存在时增加，而被Ca²⁺离子强烈抑制。同样，ATP酶活性依赖于Mg²⁺并被Ca²⁺离子的存在所抑制。该酶的ATP酶活性对单价阴离子和阳离子基本不敏感，除了F⁻，它抑制钒酸盐敏感的ATP酶。在[¹⁴C]N - 乙基马来酰亚胺存在下孵育该酶标记了115 kDa的多肽，并且在孵育期间添加ATP可以阻止这种标记。一个相互对照实验表明，用N - 乙基马来酰亚胺预孵育会抑制紫外线照射下[α - ³²P]ATP对115 kDa多肽的标记。这表明ATP结合位点与一个必需的巯基之间距离很近。文中讨论了分离出的ATP酶与细胞器运动之间可能的联系。
