From the Instituto de Química y Fisicoquímica Biológicas, Facultad de Farmacia y Bioquímica, Universidad de Buenos Aires, 1113 Ciudad Autónoma de Buenos Aires, Argentina.
J Biol Chem. 2012 Aug 31;287(36):30477-84. doi: 10.1074/jbc.M112.363465. Epub 2012 Jun 28.
The P5-ATPases are important components of eukaryotic cells. They have been shown to influence protein biogenesis, folding, and transport. The knowledge of their biochemical properties is, however, limited, and the transported ions are still unknown. We expressed in Saccharomyces cerevisiae the yeast Spf1 P5A-ATPase containing the GFP fused at the N-terminal end. The GFP-Spf1 protein was localized in the yeast endoplasmic reticulum. Purified preparations of GFP-Spf1 hydrolyzed ATP at a rate of 0.3-1 μmol of P(i)/mg/min and formed a phosphoenzyme in a simple reaction medium containing no added metal ions except Mg(2+). No significant differences were found between the ATPase activity of GFP-Spf1 and recombinant Spf1. Omission of protease inhibitors from the purification buffers resulted in a high level of endogenous proteolysis at the C-terminal portion of the GFP-Spf1 molecule that abolished phosphoenzyme formation. The Mg(2+) dependence of the GFP-Spf1 ATPase was similar to that of other P-ATPases where Mg(2+) acts as a cofactor. The addition of Mn(2+) to the reaction medium decreased the ATPase activity. The enzyme manifested optimal activity at a near neutral pH. When chased by the addition of cold ATP, 90% of the phosphoenzyme remained stable after 5 s. In contrast, the phosphoenzyme rapidly decayed to less than 20% when chased for 3 s by the addition of ADP. The greater effect of ADP accelerating the disappearance of EP suggests that GFP-Spf1 accumulated the E1P phosphoenzyme. This behavior may reflect a limiting countertransported substrate needed to promote turnover or a missing regulatory factor.
P5-ATPases 是真核细胞的重要组成部分。它们已被证明影响蛋白质的生物发生、折叠和运输。然而，人们对它们的生化特性知之甚少，其转运的离子仍不清楚。我们在酿酒酵母中表达了酵母 Spf1 P5A-ATPase，该酶的 N 端融合了 GFP。GFP-Spf1 蛋白定位于酵母内质网。在不含添加金属离子（仅含 Mg2+）的简单反应介质中，纯化的 GFP-Spf1 水解 ATP 的速度约为 0.3-1 μmol Pi/mg/min，并形成磷酰酶。GFP-Spf1 的 ATP 酶活性与重组 Spf1 没有显著差异。从纯化缓冲液中去除蛋白酶抑制剂会导致 GFP-Spf1 分子的 C 端发生高水平的内源性蛋白水解，从而阻止磷酰酶的形成。GFP-Spf1 ATP 酶对 Mg2+的依赖性与其他 P-ATPases 相似，其中 Mg2+ 作为辅因子发挥作用。在反应介质中添加 Mn2+会降低 ATP 酶的活性。该酶在近中性 pH 下表现出最佳活性。当用冷 ATP 追踪时，90%的磷酰酶在 5 秒后保持稳定。相比之下，当用 ADP 追踪 3 秒时，磷酰酶迅速衰减至低于 20%。ADP 加速 EP 消失的更大影响表明 GFP-Spf1 积累了 E1~P 磷酰酶。这种行为可能反映了促进周转率所需的有限反向转运底物或缺失的调节因子。
