PURIFICATION AND CHARACTERIZATION OF THE LIGHT AND DARK FORMS OF PHOSPHOENOLPYRUVATE CARBOXYLASE FROM THE DICOT PLANT AMARANTHUS VIRIDIS L - AN EXAMINATION OF ITS KINETIC AND REGULATORY PROPERTIES IN THE PRESENCE OF WATER-ALCOHOL BINARY SOLVENTS

The light and dark forms of phosphoenolpyruvate (PEP) carboxylase (PEP C) from the dicot plant Amaranthus viridis L. were purified and their kinetic properties were studied in water-based or binary alcohol-water solvents. At pH 7.3, the specific activity of the purified light form was about 2.7-fold higher than that presented by the dark form of PEP C under optimal conditions, while K-m remained virtually unchanged in both forms. The enzyme's light form was better activated by glucose 6- phosphate and less inhibited by L-malate than the dark PEPC. From the organic solvents studied, methanol showed the most important effect, e nhancing PEPC activity by two-fold at 20% (v/v). Ethanol, ethylene gly col, tert-butanol and 2-propanol were also activators to a lesser degr ee, but at high concentrations (typically greater than 20%, v/v) the e ffect was reduced or turned to inhibition. K-m (PEP) was reduced by an order of magnitude in the presence of 20% (v/v) methanol (i.e. from 0 .32 to 0.022 mM for the light form of the enzyme). The inhibitory effe ct of malate at low PEP was lessened by methanol for both forms (i.e. I-50 0.25 mM in aqueous medium to 0.48 mM in binary mixture for the da rk form), while glucose-6-P activation of PEPC was not affected by met hanol. The results suggest that the kinetics of PEPC in a medium that mimics more closely in vivo conditions are different from those observ ed by standard procedures consisting of aqueous media, and provide a n ew insight on the properties of PEPC as related to its regulation in v ivo.