S23-012 Isolation and characterization of envelope membranes of cyanelles from Cyanophora paradoxa F Yusa, Y Kashino, K Satoh, H Koike Faculty of Science, Himeji Institute of Technology, Harima Science Garden City, Hyogo 678- 1297, Japan E-mail: hkoike@sci.himeji-tech.ac.jp Keywords: cyanelles, Cyanophora paradoxa envelope, plasma membrane, peptidoglycan, Introduction Cyanophora paradoxa, a glaucocystophyte, is one of the three eukaryotic descendants which have aquired photosynthesis by primary symbiosis. The photosynthetic machinery of C. paradoxa, the cyanelle, preserves some aspects which are common to those of cyanobacteria such as phycobilisomes, caroxysomes and peptidoglycan wall between outer and inner envelope membranes (Löfferhardt et al. 1997). Thus, C. paradoxa is considered to be the most primitive photosyntheitc eukaryote. According to the current hypothesis that chloroplasts are acquired by symbiosis of progenitor of cyanobacteria, envelope mebranes of cyanelles should be composed of three membanes; the outermost membranes derived from plasma membranes of the host, middle and innermost membranes from outer and plasma membranes of cyanobacteria, respectively. However, cyanelles of extant C. paradoxa contains only two membranes. Judging from the fact that peptidoglycan wall is present between the two envelope membranes, it is assumed that one of the two outer membranes was disappeared or two membranes were fused during the evolutionary process. In the present study, we have isolated envelope membranes from cyanelles of C. paradoxa and partially characterized them. Materials and Methods Cyanelles of C. paradoxa was prepared as described by Koike et al. (2000). They were suspended in sucrose-containing HEM buffer (0.6 M sucrose, 50 mM HEPES-NaOH (pH7.5), 1 mM EDTA, 2 mM EGTA and 1 mM MgCl 2 ) and treated with lysozyme at 0.1 mg/ml for 15 min at 15 °C and passed through a chilled French Pressure cell twice. The homogenate was centrifuged at 5,000×g for 10 min to remove unbroken cyanelles. The concentration of sucrose of the supernatant was adjusted to 55% (w/v) by 90%-sucrose-containing HEM buffer and placed onto 60%- sucrose-containing buffer. A linear sucrose gradient (53 to 4%) was constructed above the sample. The sample was centrifuged for 16 h at 120,000×g to separate membrane fractions. The separated membranes were fractionated as described by Koike et al. (1998). The outer membranes were separated by solubilizing the thylakoid fraction with 0.5% dodecylmaltoside (DM) at 0.5 mg Chl/ml for 20 min at 0 °C. The homogenate was centrifuged at 39,000×g for 20 min. Precipitate was further treated with 0.5% DM and centrifuged again. The outer envelope membranes obtained as a precipitate was resuspended in HEM buffer. Protein compositions of each fractions were analyzed by SDS-PAGE as described by Kashino et al. (2001). Pigment compositions were analyzed by HPLC as described by Kashino et al. (1998).