Subcellular Localization and Identification of Hydrogenase Isolated from the Marine Green Alga Platymonas subcordiformis Using Immunoprecipitation and MALDI-TOF MS
应用免疫和质谱法对亚心形扁藻氢酶蛋白进行亚细胞定位和分类

A marine unicellular green alga,Platymonas subcordiformis,was demonstrated to photobiologically produce hydrogen gas from seawater.The objective of this study was to localize and identify the hydrogenase isolated from P.subcordifo rmis.Adaptation in the presence of inhibitors of protein biosynthesis indicat ed that the hydrogenase was much more inhibited by cycloheximide than that by ch loramphenicol.The result suggested that the hydrogenase isolated from P.subc ordiformis is probably synthesized in cytoplasmic ribosomes.Both Western blot analysis and immunogold electron microscopy demonstrate that the P.subcordif ormis hydrogenase is mainly located in the chloroplast stroma.The proteins th at reacted specifically with the antibodies against the iron hydrogenase isolate d from Chlamydomonas reinhardtii were concentrated by immunoprecipitation.T he separated protein bands were cut out of the SDS-PAGE gel,in-gel digested b y trypsin,and analyzed by Matrix-Assisted Laser Desorption Ionization Time-of-Flight Mass Spectrometry(MALDI-TOF MS).Mascot was employed for analysis of the MALDI data using the public databases NCBInr.The hydrogenase isolated from P.subcordiformis was identified to be the Fe-hydrogenase.