Carbonic Anhydrase of Spinach

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Abstract
Carbonic anhydrase activity was determined in spinach (Spinacia oleracea) leaf organelles isolated on sucrose density gradients and was found to be predominantly in the intact chloroplast fraction. The small amount of activity associated with the mitochondrial fractions was probably due to intact chloroplast contamination. No activity could be associated with the broken chloroplast or microbody fractions. Based upon inhibitor studies, carbonic anhydrase was found to be around 2 mm in the chloroplast. Ethoxzolamide, an inhibitor of carbonic anhydrase, reduced CO2 fixation in intact chloroplasts. The concentration required to inhibit CO2 fixation 20 to 40% was in excess of that required to inhibit the purified enzyme. The inhibition was partially reversed by CO2. Ethoxzolamide had no effect on photosynthetic NADP reduction or photophosphorylation measured by methyl viologen reduction. The physiological role of carbonic anhydrase was shown not to be associated with CO2 diffusion or CO2 concentration. It is proposed that other functions of carbonic anhydrase could be the protection against denaturation by transient localized changes in pH or the hydration of compounds other than CO2.