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In C4 plants carbonic anhydrase catalyzes the critical first step of C4 photosynthesis, the hydration of CO2 to bicarbonate. The maximum activity of this enzyme in C4 leaf extracts, measured by H production with saturating CO2 and extrapolated to 250C, was found to be 3,000 to 10,000 times the maximum photosynthesis rate for these leaves. Similar activities were found in C3 leaf extracts. However, the calculated effective activity of this enzyme at in vivo C02 concentrations was apparently just sufficient to prevent the rate of conversion of CO2 to HC03-from limiting C4 photosynthesis. This conclusion was supported by the mass spectrometric determination of leaf carbonic anhydrase activities.The initial carboxylation reaction of C4 photosynthesis, catalyzed by PEP2 carboxylase, utilizes bicarbonate rather than CO2 as the inorganic carbon substrate (9). To sustain this process, atmospheric CO2 entering mesophyll cells must be rapidly converted to HCO3-and this reaction should rightly be regarded as the first step in C4 photosynthesis. Consistent with this concept is the fact that the CA of C4 leaves is very largely or exclusively confined to the cytosol of mesophyll cells (4, 6, 8), where PEP carboxylase is also located. In spite of this apparently critical role of CA in C4 photosynthesis, the quantitative aspects ofthe operation ofthis enzyme have been largely neglected. The implicit assumption has been that the CA is present in a substantial excess in C4 and also C3 leaves. Hence, most studies have focused on the physical and kinetic properties of the enzyme (10).In this paper we report on the maximum activity of CA in leaf extracts of C4 and C3 plants expressed in units which allow comparison with photosynthetic activity. An error in the leaf activities reported in an earlier study (4) MATERIALS AND METHODS MaterialsPlants were grown in soil in a naturally illuminated glasshouse maintained between 20 and 30°C. Biochemicals were obtained from either Sigma Chemical Co. or BoehringerMannheim (Australia). Preparation of Leaf ExtractsAbout 0.5 g of leaf tissue was vigorously ground for about 20 s in a chilled mortar with 1 mL of either 40 mM barbitone-KOH or 40 mm Hepes-KOH (pH 8.0), each containing 10 mM dithiothreitol. After adding an additional 1 mL of the buffer mixture the homogenate was filtered through Miracloth. A sample of this filtrate was used to determine the Chl content of the extract (1), and the remainder was diluted in the blending buffer as required (usually 1 to 10 diluted) and stored at 0°C prior to being assayed.Assay of Carbonic Anhydrase CA was assayed by two different procedures. In the more conventional assay, the rate of CO2 hydration was measured at 0°C by following the change ofpH traced on chart recorder. Reactions contained 25 mm barbitone-KOH buffer (pH 8.2), in a final volume of 1 mL.