cDNA cloning and characterization of maize phosphoenolpyruvate carboxykinase, a bundle sheath cell-specific enzyme

We isolated a full-length cDNA that encodes ATP-dependent phosphoenolpyruva te carboxykinase (EC 4.1.1.49, PCK) from leaves of maize, an NADP-malic enz yme type C-4 plant. The mRNA was specifically and rather abundantly express ed in bundle sheath cells in accordance with the recent finding of cell-typ e-specific localization of PCK protein in maize, which has been detected wi th antibodies against cucumber PCK protein. The predicted protein had an N- terminal extension, which is characteristic of plant PCKs. The transcript l evel was much higher in the daytime than at night in 14-day old seedlings. However, in 42-day old plants the extent of diurnal change decreased. The m aize PCK was expressed in Escherichia coli with the pET32 plasmid and purif ied to homogeneity. Through digestion with enterokinase, two types of enzym e were prepared; one with an intact N-terminus and the other lacking its N- terminal 77 amino acid residues due to over-digestion. The truncated protei n had about 2-fold higher specific activity than the intact one, and was in hibited by 3-phosphoglycerate (3-PGA) with an I-0.5 of 17.5 mM. In contrast , the intact protein was almost insensitive to 3-PGA. These results strongl y suggest that the intact N-terminal extension may be involved in the regul ation of PCK activity in vivo through some modification such as reversible phosphorylation.