NAD turnover and utilisation of metabolites for RNA synthesis in a reaction sensing the redox state of the cytochrome b6f complex in isolated chloroplasts.
NAD is normally regarded as a redox molecule or as the substrate for ADP-ribosylation reactions. In this study, we describe the rapid metabolism of NAD by Percoll-gradient-purified lettuce chloroplasts and show that the adenine moiety can be incorporated into RNA in a dark-activated reaction that senses the redox state of the cytochrome b6f complex. Isolated chloroplasts rapidly metabolised radiolabelled NAD+ to 5'-AMP (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 incorporated into trichloroacetic-acid-insoluble material during this period, with approximately 2-4-fold more incorporation occurring in the dark. Most of this radiolabel was rendered acid-soluble by dilute alkaline digestion at 37 degrees C, yielding an approximately equal mixture of 2'-AMP and 3'-AMP, and by RNase digestion, identifying the acid-insoluble radioactive material as RNA. Protein-bound ADP-ribose would have yielded 5'-AMP and/or oligomeric/polymeric ADP-ribose after alkali digestion. The utilisation of NAD metabolites for RNA synthesis was restricted to the thylakoid compartment of the chloroplast. The use of a variety of electron-transport inhibitors such as 2,5-dibromo-3-methyl-6-isopropyl-p-benzoquinone, bromanil (tetrabromo-1,4-benzoquinone), electron donors (dithiothreitol), electron acceptors (ferricyanide) and an uncoupler showed that the incorporation of radiolabel from NAD into acid-insoluble material was favoured when the cytochrome b6f complex was in the oxidised state (as pertaining to incubations in the dark).
Pearson
C K
CK
Department of Molecular and Cell Biology, University of Aberdeen, Scotland.
Wilson
S B
SB
Schaffer
R
R
Ross
A W
AW
D002736:Chloroplasts / Q000201:enzymology*; D002851:Chromatography, High Pressure Liquid; D003580:Cytochromes / Q000737:chemistry / Q000378:metabolism*; D045348:Cytochromes f; D004579:Electron Transport / Q000187:drug effects; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008027:Light; D009243:NAD / Q000378:metabolism*; D010084:Oxidation-Reduction; D012313:RNA / Q000096:biosynthesis*; D011868:Radioisotopes
D003580:Cytochromes; D011868:Radioisotopes; D009243:NAD; D012313:RNA; D045348:Cytochromes f