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Light Modulation of Maize Leaf Phosphoenolpyruvate Carboxylase ¹

Research Institute for Biochemical Regulation, School of Agriculture, Nagoya University, Chikusa, Furo-cho, Nagoya 464, Japan, Department of Agricultural Chemistry, School of Agriculture, Nagoya University, Chikusa, Furo-cho, Nagoya 464, Japan

Phosphoenolpyruvate carboxylase (PEPC) was extracted from maize (Zea mays L. cv Golden Cross Bantam T51) leaves harvested in the dark or light and was partially purified by (NH₄)₂SO₄ fractionation and gel filtration to yield preparations that were 80% homogeneous. Malate sensitivity, PEPC activity, and PEPC protein (measured immunochemically) were monitored during purification. As reported previously, PEPC from dark leaves was more sensitive to malate inhibition compared to enzyme extracted from light leaves. Extraction and purification in the presence of malate stabilized the characteristics of the two forms. During gel filtration on Sephacryl S-300, all of the PEPC activity and PEPC protein emerged in a single high molecular weight peak, indicating that no inactive dissociated forms (dimers, monomers) were present. However, there was a slight difference between the light and dark enzymes in elution volume during gel filtration. In addition, specific activity (units at pH 7/milligram PEPC protein) decreased through the peak for both enzyme samples; because the dark enzyme emerged at a slightly higher elution volume, it contained enzyme with a relatively lower specific activity. The variation in specific activity of the dark enzyme corresponded with changes in malate sensitivity. Immunoblotting of samples with different specific activity and malate sensitivity, obtained from gel filtration, revealed only a single polypeptide with a relative molecular mass of 100,000. When the enzyme was extracted and purified in the absence of malate, characteristic differences of the light and dark enzymes were lost, the enzymes eluted at the same volume during gel filtration, and specific activity was constant through the peak. We conclude that maize leaf PEPC exists in situ as a tetramer of a single polypeptide and that subtle conformation changes can affect both enzymic activity and sensitivity to malate inhibition.

¹ Cooperative investigation with the United States Department of Agriculture, Agricultural Research Service, and the North Carolina Agricultural Research Service, Raleigh, NC 27695-7601. Paper No. 10501 of the Journal Series of the North Carolina Agricultural Research Service, Raleigh, NC 27695-7601. Conducted while S. C. H. was at Nagoya University under the Visiting Professorship Program (1985) supported by the Japanese government (Mombusho). Support was also obtained from the Japanese Ministry of Education, Science and Culture (Grant-in-aid for Special Project Research 59127027) to T. S.

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