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

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Abstract

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.

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  1. Tsuyoshi Furumoto

    Present address: Department of Biological Sciences, Graduate School of Science, University of Tokyo, Hongo, Bunkyo-ku, Tokyo, 113-0033, Japan

Authors and Affiliations

  1. Division of Applied Biosciences, Graduate School of Agriculture, Kyoto University, Sakyo-ku, Kyoto, 606-8502, Japan

    Shingo Hata & Katsura Izui

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  1. Tsuyoshi Furumoto
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  2. Shingo Hata
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  3. Katsura Izui
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Furumoto, T., Hata, S. & Izui, K. cDNA cloning and characterization of maize phosphoenolpyruvate carboxykinase, a bundle sheath cell-specific enzyme. Plant Mol Biol 41, 301–311 (1999). https://doi.org/10.1023/A:1006317120460

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