玉米根V-ATPase的A亚基磷酸化位点的鉴定

以玉米 (Zea mays L.) 根的高纯度液泡膜为材料进行的磷酸化反应表明,液泡膜蛋白的磷酸化可明显提高V型H -ATPase (V-ATPase) 的ATP水解活性和H 转运活性。进一步研究表明,纯化的液泡膜蛋白能被硫代磷酸化,用V-ATPase的A亚基抗体将一条约69 kD的条带鉴定为A亚基。为了测定V-ATPase的A亚基的磷酸化位点,从硫代磷酸化的凝胶中切下A亚基条带并用胰蛋白酶彻底消化。用RP-HPLC分离纯化酶解片断,收集纯化的硫代磷酸化肽段进行质谱分析所测定的分子量为573.83 Da。A亚基胰蛋白酶彻底消化后能产生61个肽段,只有F56肽段的分子量573.66 Da与573.83 Da最接近,而且F56肽段上只有第525位的丝氨酸可以被磷酸化。因此可以确定,玉米根V-AT-Pase A亚基的潜在磷酸化位点为Ser525。就我们所知,这是首次确定植物V-ATPase A亚基的磷酸化位点。

Identification of the Phosphorylation Site of the V-ATPase Subunit A in Maize Roots

In the present study our investigation shows that phosphorylation of tonoplast proteinspurified from maize (Zea mays L.) roots increases obviously V-type H -ATPase (V-ATPase) activities of ATPhydrolysis and H transport. Further research indicates that some of the purified tonoplast proteins can bethiophosphorylated and one band about 69 kD is identified as subunit A with antibody against subunit A of V-ATPase. In order to determine the phosphorylation site(s) in subunit A of V-ATPase, the subunit A band at69 kD was isolated from thiophosphorylated gel and then completely digested by trypsin. After purificationof these enzymatic lysis fragments with RP-HPLC, the molecular weight of phosphorylated peptidefragment was determined as 573.83 Da with mass spectrometry. Data search indicates that subunit A cangenerate 61 peptide fragments after tryptic digestion, of which only F56 with molecular weight of 573.66Da is close to that of the identified fragment, and F56 can only be phosphorylated at Ser525. Therefore ourresearch suggests that Ser525 is the potential phosphorylation site of V-ATPase subunit A in maize roots.To our knowledge, this is the first time to determine the phosphorylation site of V-ATPase subunit A inplants.