Activation and Inactivation of Phosphoenolpyruvate Carboxylase in Leaf Extracts From C4 Species

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Abstract
The stability of phosphoenolpyruvate carboxylase (EC 4.1.1.31) was examined following extraction of the enzyme from leaves of several C4 plants. Extracts were rapidly processed on small Sephadex G-25 columns to free protein of small-molecular-weight compounds. With most of the species examined, activity was rapidly lost at both 0 and 25°C when the pH was about 7.8 or higher. Addition of bovine serum albumin to extracts incubated at 25°C and pH 8.2 not only prevented inactivation with several species, but resulted in a substantial increase in activity. The addition of dithiothreitol plus Mg2+ to extracts from some of these species reduced or prevented inactivation. With extracts maintained at 0°C, addition of either bovine serum albumin or dithiothreitol was effective only in reducing the rate of inactivation in extracts. Phosphoenolpyruvate carboxylase activity remained stable, or increased substantially, when extracts buffered between pH 7.4 and 6.9 were incubated at either 0 or 25°C. Activation was usually complete within an hour and was often significantly greater at 25°C or when bovine serum albumin was added. The activity of partially purified phosphoenolpyruvate carboxylase from Zea mays was similarly affected by pH, temperature, and bovine serum albumin. The present studies raise doubts about the accuracy of phosphoenolpyruvate carboxylase determinations made during the course of some previous studies on C4 species. Reliable procedures for the determination of phosphoenolpyruvate carboxylase activity in C4 plant extracts are described. Possible physiological implications of the results are considered.