Isolation and characterization of a thylakoid membrane module showing partial light and dark reactions

A functional thylakoid membrane module of photosynthesis was isolated from cell free extracts of Anacystis nidulans by stepwise sequential ultracentrifugation. The thylakoid membrane fractions sedimenting at 40,000×g, followed by 90,000×g and finally at 150,000×g were collected. These fractions had all the components of electron transport chain, ATP synthase, phycobiliproteins, ferredoxin-NADP reductase but no ferredoxin. Five sequential enzymes of Calvin cycle viz phosphoriboisomerase, phosphoribulokinase, RuBP carboxylase, 3-PGA kinase and glyceraldehyde-3-phosphate dehydrogenase were found to be associated with thylakoid membranes. Among the three different thylakoid fractions, the 150,000×g fraction showed highest activities of these enzymes and also higher rate of whole chain electron transport activity on chlorophyll basis. An important finding was that the 150,000×g fraction showed appreciably higher rate of R-5-P+ADP+Pi dependent CO2 fixation in light compared to the other two fractions, indicating the efficiency of this fraction in utilizing ATP for Calvin cycle. This thylakoid membrane fraction represents a fully functional module exhibiting a synchronized system of light and dark reactions of photosynthesis. Most of the components of this module remained together even after sucrose density gradient centrifugation. This is the first report on the isolation of a photosynthetic module involving membrane and soluble proteins.