Palmgren M G, Sommarin M, Serrano R, Larsson C
Department of Plant Biochemistry, University of Lund, Sweden.
J Biol Chem. 1991 Oct 25;266(30):20470-5.
Proteolytic (trypsin) treatment removes a small terminal segment from the 100-kDa plant plasma membrane H(+)-ATPase. This results in activation of H+ pumping across the plasma membrane, suggesting that an inhibitory domain is located in one of the terminal regions of the enzyme (Palmgren, M.G., Larsson, C., and Sommarin, M. (1990) J. Biol. Chem. 265, 13423-13426). In order to identify the origin of the fragment released by trypsin, polyclonal antibodies were raised against the first 55 amino acids (N-terminal region), the last 99 amino acids (C-terminal region), and a portion of 150 amino acids in the central part of the enzyme as deduced from one of the H(+)-ATPase genes (PMA2) of Arabidopsis thaliana. The native, 100-kDa H(+)-ATPase was recognized by all three antisera in Western blots. By contrast, the approximately 90-kDa polypeptide appearing after trypsin treatment was only recognized by the antisera against the N-terminal and central region, but not by the antiserum against the C-terminal region, suggesting that the inhibitory domain is located in this part of the enzyme. To more closely determine the position of the inhibitory domain, three peptides representing conserved parts of the C-terminal region were synthesized (residues 861-888, 912-943, and 936-949 of the Arabidopsis (PMA2) sequence). Only one of the peptides (residues 861-888) affected H+ pumping by the trypsin-activated (approximately 90-kDa) enzyme. This peptide of 28 amino acids inhibited H+ pumping with an IC50 of about 15 microM, suggesting that the auto-inhibitory domain is located within the corresponding part of the C-terminal region.
蛋白水解（胰蛋白酶）处理会从100 kDa的植物质膜H(+) - ATP酶上移除一小段末端片段。这导致跨质膜的H+ 泵浦活性增强，表明抑制结构域位于该酶的一个末端区域（帕尔姆格伦，M.G.，拉尔松，C.，和索马林，M.（1990）《生物化学杂志》265，13423 - 13426）。为了确定胰蛋白酶释放片段的来源，针对拟南芥H(+) - ATP酶基因（PMA2）之一推导的该酶中部150个氨基酸片段、前55个氨基酸（N末端区域）以及后99个氨基酸（C末端区域）制备了多克隆抗体。在蛋白质免疫印迹中，天然的100 kDa H(+) - ATP酶能被所有三种抗血清识别。相比之下，胰蛋白酶处理后出现的约90 kDa多肽仅被针对N末端和中部区域的抗血清识别，而不被针对C末端区域的抗血清识别，这表明抑制结构域位于该酶的这一部分。为了更精确地确定抑制结构域的位置，合成了代表C末端区域保守部分的三种肽（拟南芥（PMA2）序列的第861 - 888、912 - 943和936 - 949位残基）。只有一种肽（第861 - 888位残基）影响胰蛋白酶激活的（约90 kDa）酶的H+ 泵浦。这种28个氨基酸的肽以约15 microM的IC50抑制H+ 泵浦，表明自抑制结构域位于C末端区域的相应部分内。
