NAD TURNOVER AND UTILIZATION OF METABOLITES FOR RNA-SYNTHESIS IN A REACTION SENSING THE REDOX STATE OF THE CYTOCHROME-B(6)F COMPLEX IN ISOLATED-CHLOROPLASTS

NAD is normally regarded as a redox molecule or as the substrate for A DP-ribosylation reactions. In this study, we describe the rapid metabo lism of NAD by Percoll-gradient-purified lettuce chloroplasts and show that the adenine moiety can be incorporated into RNA in a dark-activa ted reaction that senses the redox state of the cytochrome b6f complex . Isolated chloroplasts rapidly metabolised radiolabelled NAD+ to 5'-A MP (within seconds) and adenosine during a 60-min incubation in vitro; the products were analysed by high-performance liquid chromatography. No radiolabelled ADP-ribose was detected. Radioactivity was incorpora ted into trichloroacetic-acid-insoluble material during this period, w ith approximately 2-4-fold more incorporation occurring in the dark. M ost of this radiolabel was rendered acid-soluble by dilute alkaline di gestion at 37-degrees-C, yielding an approximately equal mixture of 2' -AMP and 3'-AMP, and by RNase digestion, identifying the acid-insolubl e radioactive material as RNA. Protein-bound ADP-ribose would have yie lded 5'-AMP and/or oligomeric/polymeric ADP-ribose after alkali digest ion. The utilisation of NAD metabolites for RNA synthesis was restrict ed to the thylakoid compartment of the chloroplast. The use of a varie ty of electron-transport inhibitors such as 2,5-dibromo-3-methyl-6-iso propyl-p-benzoquinone, bromanil (tetrabromo-1,4-benzoquinone), electro n donors (dithiothreitol), electron acceptors (ferricyanide) and an un coupler showed that the incorporation of radiolabel from NAD into acid -insoluble material was favoured when the cytochrome b6f complex was i n the oxidised state (as pertaining to incubations in the dark).