Aust. J. Plant Physiol., 1988, 15, 621-31 Organic Cosolutes Increase the Catalytic Efficiency of Phosphoenolpyruvate Carboxylase, from Cynodon dactylon (L.) Pers., Apparently Through Self-association of the Enzymic Protein K. Stamatakis, N. A. Gavalas and Y. Manetas Laboratory of Plant Physiology, Department of Biology, University of Patras, Patras, Greece. Abstract Several organic cosolutes (glycerol, sorbitol, betaine, proline, polyethylene glycol, polyvinylpyrrolidone) increase, to a large extent and in a concentration-dependent manner, the apparent affinity of phosphoenolpyruvate carboxylase for phosphoenolpyruvate, whereas the maximum activity remains unaffected. In absence of cosolutes, a similar response is obtained as the concentration of the enzymic protein in the assay medium is increased. The effect of the organic additives does not depend on the osmotic potential or the viscosity of the medium and it could be best interpreted with the exclusion volume theory. It is inferred that the inclusion of an appropriate cosolute in the assay medium promotes the self-association of the enzymic protein and, therefore, mimics the intracellular situation, where the enzyme is much concentrated. In light of these results, it is suggested that the physiological relevance of past data concerning the non-saturating activity and the regulation of the enzyme in vivo should be reevaluated. Introduction PEP* carboxylase (EC 4.1.1.3 l), the enzyme responsible for C02 fixation into oxaloacetate, plays a multitude of metabolic roles in plants (Latzko and Kelly 1983); it is of pivotal importance in C4-photosynthesis, crassulacean acid metabolism (Kluge 1983) and guard cell function (Willmer 1983). Knowledge on its properties and modulation in vitro has accumulated during the last two decades (Andreo et al. 1987), but our understanding of its regulation in vivo is still far from complete. It has been shown that light and darkness affect PEPCase in a way commensurate with metabolic requirements (Manetas 1982; Winter 1982; Karabourniotis et al. 1983, 1985; Huber and Sugiyama 1986), but the biochemical mechanism remains largely unknown. In earlier studies we found that glycerol stabilises the levels of PEPCase activity during and after extraction from plant tissues (Manetas 1982; Karabourniotis et al. 1983), as it is often the case with other enzymes. In addition, the presence of glycerol in the assay medium increased the measured enzymic activity when the assay was run at low PEP levels (Gavalas et al. 1982) or at a suboptimum pH (Uedan and Sugiyama 1976). The effective concentrations of glycerol, however, were so high (6-20% v/v) that a specific action, apart from effects on the physical properties of the assay medium, seemed extremely doubtful (Gavalas et al. 1982). It is also known that glycerol and other organic cosolutes (polyhydric and polyoxy compounds, some amino acids, etc.) promote self-association of proteins and stabilise *Abbreviations used: CAM, crassulacean acid metabolism; DEAE-, diethylaminoethyl-; PEG, poly- ethylene glycol; PEP, phosphoenolpyruvate; PEPCase, phosphoenolpyruvate carboxylase; PVP, polyvinylpyrrolidone; Tes, N-tris(hydroxymethyl)methyl-2-amino-ethanesulfonic acid; tris, tris(hydroxymethy1)aminomethane. 0310-7841 /88/050621$03.00