Adenylate effects on protein phosphorylation in the interenvelope lumen of pea chloroplasts.

A 64-kilodalton (kDa) protein, situated in the lumen between the inner and outer envelopes of pea (Pisum sativum L.) chloroplasts (Soll and Bennett 1988, Eur. J. Biochem., 175, 301-307) is shown to undergo reversible phosphorylation in isolated mixed envelope vesicles. It is the most conspicuously labelled protein after incubation of envelopes with 33 nmol·1(-1) [γ-(32)P]ATP whereas incubation with 50 μmol·1(-1) [γ-(32)P]ATP labels most prominently two outer envelope proteins (86 and 23 kDa). Half-maximum velocity for phosphorylation of the 64-kDa protein occurs with 200 nmol·1(-1) ATP, and around 40 μmol·1(-1) ATP for phosphorylation of the 86- and 23-kDa proteins, indicating the operation of two distinct kinases. GGuanosine-, uridine-, cytidine 5'-triphosphate and AMP are poor inhibitors of the labelling of the 64-kDa protein with [γ-(32)P]ATP. On the other hand, ADP has a potent influence on the extent of labelling (half-maximal inhibition at 1-5 μmol·1(-1)). The ADP-dependent appearance of (32)P in ATP indicates that ADP acts by reversal of kinase activity and not as a competitive inhibitor. However, the most rapid loss of (32)P from pre-labelled 64-kDa protein occurs when envelope vesicles are incubated with ATP t1/2=15 s at 20 μmolsd1(-1) ATP). This induced turnover of phosphate appears to be responsible for the rapid phosphoryl turnover seen in situ.