THE IN-VITRO EFFECTS OF ATP AND PROTEIN-PHOSPHORYLATION ON THE ACTIVITY OF FERREDOXIN-NADP-CHLOROPLASTS( OXIDOREDUCTASE FROM SPINACH)

When ferredoxin:NADP+ oxidoreductase (FNR) was preincubated with [gamm a-P-32]ATP-Mg and separated by native PAGE it was found to be radioact ive. This was not seen on SDS-PAGE, unless FNR was preincubated with a crude protein kinase extract with FNR kinase activity under phosphory lating conditions. These observations suggest that FNR contains an ATP -binding domain. It was found that ATP caused an inhibition of FNR dia phorase activity, which when analysed by Lineweaver-Burk plots indicat ed that ATP was a non-competitive inhibitor with respect to NADPH. Thi s shows that the ATP site is distinct from the NADP(H) active site. Th e in vitro phosphorylation of FNR on a serine residue(s) by the crude FNR kinase extract led to a modification of ferredoxin (Fd)-dependent FNR activity. An analysis of the data showed that after phosphorylatio n the apparent K(m) for Fd and V(max) both increased. This observation suggests that the Fd-FNR interaction is modified after FNR phosphoryl ation in vitro. Limited proteolysis of phosphorylated FNR followed by SDS-PAGE infers that the phosphorylated amino acid(s) is located near the N-terminus.