Chlamydomonas reinhardtii cell preparation with altered permeability toward substrates of organellar reactions.

Chlamydomonas reinhardtii cells disrupted under low pressure in a Yeda press yielded a preparation ("presate") with high permeability toward substrates for Class A chloroplasts and intact mitochondria. The stoichiometric rates of CO2 uptake and O2 photoevolution by the wild-type cell pressate were severely suppressed by 10 mM exogenous phosphate, and this suppression could be reversed by the addition of either 3-phosphoglycerate or dihydroxyacetone phosphate. A mutant, F60, which lacks phosphoribulokinase activity and hence CO2-dependent O2 photoevolution, was studied by using intact cells, pressate, and sonicated pressate. In the pressate, the rate of 3-phosphoglycerate-dependent O2 photoevolution was high, whereas that dependent on K3Fe(CN)6 was low; the opposite was true of the sonicated pressate. p-Benzoquinone supported high rates of O2 evolution in both the pressate and the sonicated pressate. The slow O2 uptake in the dark by the dark-adapted wild-type pressate could be increased by the addition of succinate and further stimulated by ADP. Addition of KCN resulted in rapid but only partial suppression of this activity. Dark O2 uptake by the unpressed preparation did not show similar responses. The procedure described here opens the possibility of in situ analysis of Class A chloroplasts from wild-type and mutant strains of C. reinhardtii.