van Hunnik E, Livne A, Pogenberg V, Spijkerman E, van den Ende H, Mendoza E G, Sültemeyer D, de Leeuw J W
Institute for Molecular Cell Biology, BioCentrum Amsterdam, University of Amsterdam, The Netherlands.
Planta. 2001 Feb;212(3):454-9. doi: 10.1007/s004250000418.
In order to broaden our understanding of the eukaryotic CO2-concentrating mechanism the occurrence and localization of a thylakoid-associated carbonic anhydrase (EC 4.2.1.1) were studied in the green algae Tetraedron minimum and Chlamydomonas noctigama. Both algae induce a CO2-concentrating mechanism when grown under limiting CO2 conditions. Using mass-spectrometric measurements of 18O exchange from doubly labelled CO2, the presence of a thylakoid-associated carbonic anhydrase was confirmed for both species. From purified thylakoid membranes, photosystem I (PSI), photosystem II (PSII) and the light-harvesting complex of the photosynthetic apparatus were isolated by mild detergent gel. The protein fractions were identified by 77 K fluorescence spectroscopy and immunological studies. A polypeptide was found to immunoreact with an antibody raised against thylakoid carbonic anhydrase (CAH3) from Chlamydomonas reinhardtii. It was found that this polypeptide was mainly associated with PSII, although a certain proportion was also connected to light harvesting complex II. This was confirmed by activity measurements of carbonic anhydrase in isolated bands extracted from the mild detergent gel. The thylakoid carbonic anhydrase isolated from T. minimum had an isoelectric point between 5.4 and 4.8. Together the results are consistent with the hypothesis that thylakoid carbonic anhydrase resides within the lumen where it is associated with the PSII complex.
为了拓宽我们对真核生物二氧化碳浓缩机制的理解，我们研究了绿藻小四角藻（Tetraedron minimum）和夜生衣藻（Chlamydomonas noctigama）中类囊体相关碳酸酐酶（EC 4.2.1.1）的存在和定位。这两种藻类在低二氧化碳条件下生长时都会诱导出一种二氧化碳浓缩机制。通过对双标记二氧化碳中18O交换的质谱测量，证实了这两个物种中都存在类囊体相关碳酸酐酶。从纯化的类囊体膜中，通过温和去污剂凝胶分离出光合系统I（PSI）、光合系统II（PSII）和光合装置的捕光复合体。通过77K荧光光谱和免疫学研究对蛋白质组分进行了鉴定。发现一种多肽与针对莱茵衣藻（Chlamydomonas reinhardtii）类囊体碳酸酐酶（CAH3）产生的抗体发生免疫反应。发现该多肽主要与PSII相关，尽管也有一定比例与捕光复合体II相连。从温和去污剂凝胶中提取的分离条带中的碳酸酐酶活性测量证实了这一点。从小四角藻中分离出的类囊体碳酸酐酶的等电点在5.4至4.8之间。这些结果共同支持了类囊体碳酸酐酶存在于类囊体腔中并与PSII复合体相关的假说。
