Phosphoenolpyruvate carboxylase in Crassulacean acid metabolism plants during the day exists in dimeric form the activity of which is strongly inhibited by malate. Enzyme purified from Crassula leaves collected during the day and stored at -70°C for 49 days shows a steady progression of change from dimer to tetramer, and this change in oligomeric state is accompanied by a decrease in the sensitivity of the enzyme to inhibition by malate. At 10 minutes preincubation of enzyme after 11 days storage-which is composed of an equilibrium mixture of dimer and tetramer-with malate causes most of the enzyme to be converted to dimer and increases the sensitivity of the enzyme to malate inhibition during assay. Preincubation with phosphoenolpyruvate shifts the equilibrium toward the tetrameric form and reduces the maximal inhibition produced by 5 millimolar malate to less than 20%. However, none of the treatments used resulted in shifting the oligomerization equilibrium completely in either direction. (Crassula argentea Thunb.) were grown in a greenhouse as described previously (17).Chemicals. The chemicals used in this study were identical with those described earlier (17, 18) and were of the highest purity commercially available.Enzyme Assay. The activity of PEP carboxylase was measured at 25°C in 1.0 ml of 50 mm Mops (pH 7.2) containing 0.15 mm NADH, 5 mM MgSO4, 10 mM NaHCO3, 5 mM PEP, and 1 IU of malate dehydrogenase (from Sigma). The reaction was started by the addition of enzyme and the oxidation of NADH was followed at 340 nm in the cell compartment of a Beckman DU-50 spectrophotometer controlled at 25°C. Kinetic parameters were determined by fitting assays at varying concentrations of PEP to the Michaelis-Menten equation modified to provide estimates of the Hill number as well as Vm and Km (17).Enzyme Purification. The enzyme was purified from day leaves collected after 4 to 5 h of light. The purification procedure was a modification ofthe method previously described (18). All steps were performed at 0 to 4°C. Leaves were homogenized in 2 volumes of 0.05 M Hepes buffer (pH 7.2), with 1 mM EDTA, 1 mM DTT and 20% (v/v) glycerol. A Polytron homogenizer was run in an ice bath at full speed for two 15 s bursts with a 15 s cooling interval. After filtering through two layers of fine nylon mesh, the homogenate was centrifuged at 5OOg for 10 min and the supernatant liquid discarded. The pellet was extracted with the extraction buffer containing 2% Triton X-1 14 but omitting glycerol, following the method of Pryde (10) for solubilizing membrane-associated proteins. After centrifugation, the material soluble in Triton X-1 14 was fractionated by ammonium sulfate precipitation, with the fraction precipitating between 35 and 60% ammonium sulfate saturation collected by centrifugation at 16,000g for 15 min and suspended in the extraction buffer without either Triton or glycerol. The preparation was then desalted using a 2 x 20 cm Sephadex G-25 column and applied to either a DEAE-cellulose