cDNA cloning and characterization of maize phosphoenolpyruvate carboxykinase, a bundle sheath cell-specific enzyme.
We isolated a full-length cDNA that encodes ATP-dependent phosphoenolpyruvate carboxykinase (EC 4.1.1.49, PCK) from leaves of maize, an NADP-malic enzyme type C4 plant. The mRNA was specifically and rather abundantly expressed in bundle sheath cells in accordance with the recent finding of cell-type-specific localization of PCK protein in maize, which has been detected with antibodies against cucumber PCK protein. The predicted protein had an N-terminal extension, which is characteristic of plant PCKs. The transcript level 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 maize PCK was expressed in Escherichia coli with the pET32 plasmid and purified to homogeneity. Through digestion with enterokinase, two types of enzyme 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 protein had about 2-fold higher specific activity than the intact one, and was inhibited by 3-phosphoglycerate (3-PGA) with an I0.5 of 17.5 mM. In contrast, the intact protein was almost insensitive to 3-PGA. These results strongly suggest that the intact N-terminal extension may be involved in the regulation of PCK activity in vivo through some modification such as reversible phosphorylation.
Furumoto
T
T
Division of Applied Biosciences, Graduate School of Agriculture, Kyoto University, Japan.
Hata
S
S
Izui
K
K
D000595:Amino Acid Sequence; D002940:Circadian Rhythm; D003001:Cloning, Molecular; D017868:Cyclic AMP-Dependent Protein Kinases; D018076:DNA, Complementary; D005988:Glyceric Acids / Q000378:metabolism; D008291:Malate Dehydrogenase; D008956:Models, Chemical; D008969:Molecular Sequence Data; D019764:Phosphoenolpyruvate Carboxykinase (ATP) / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism; D018515:Plant Leaves / Q000201:enzymology; D012333:RNA, Messenger / Q000302:isolation & purification; D018749:RNA, Plant / Q000302:isolation & purification; D011994:Recombinant Proteins / Q000378:metabolism; D017422:Sequence Analysis, DNA; D017386:Sequence Homology, Amino Acid; D014018:Tissue Distribution; D003313:Zea mays / Q000201:enzymology / Q000235:genetics*
D018076:DNA, Complementary; D005988:Glyceric Acids; D012333:RNA, Messenger; D018749:RNA, Plant; D011994:Recombinant Proteins; C005156:3-phosphoglycerate; D008291:Malate Dehydrogenase; C031917:malate dehydrogenase (oxaloacetate-decarboxylating) (NADP+); D017868:Cyclic AMP-Dependent Protein Kinases; D019764:Phosphoenolpyruvate Carboxykinase (ATP)