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Metabolism and Enzymology

Purification by Immunoadsorption and Immunochemical Properties of NADP-Dependent Malic Enzymes from Leaves of C₃, C₄, and Crassulacean Acid Metabolism Plants

Institute of Plant Physiology, Cell Biology and Microbiology, Free University of Berlin, Königin-Luise-Strasse 12-16a, D-1000 Berlin 33 (West), Federal Republic of Germany

NADP:malic enzyme from corn (Zea mays L.) leaves was purified by conventional techniques to apparent homogeneity as judged by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Antibodies raised against this protein in rabbits were purified, coupled covalently to protein A-Sepharose CL-4B, and used as an immunoaffinity resin to purify the NADP:malic enzymes of the C₃ plants spinach (Spinacia oleracea L.) and wheat (Triticum aestivum L.), of the Crassulacean acid metabolism (CAM) plant Bryophyllum daigremontianum R. Hamed et Perr. de la Bathie and the C₄ plants corn, sugarcane (Saccharum officinarum L.), and Portulaca grandiflora L. Such procedures yielded homogeneous protein preparations with a single protein band, as judged by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, except for P. grandiflora L. with two bands. The specific activities of the purified proteins ranged between 56 and 91 units (milligrams per protein). NADP:malic enzyme represented up to 1% of the total soluble protein in C₄ plants, 0.5% in the CAM plant, and less than 0.01% in C₃ plants. In immunotitration tests involving immunoprecipitation and immunoinhibition of activity by an antiserum against the corn leaf enzyme, the NADP:malic enzymes of corn and sugarcane showed virtually full identity of epitopes, while the NADP:malic enzymes of the C₃ and CAM plants exhibited a cross-reaction of one-twentieth and one-fourth by these tests, respectively. The NADP:malic enzyme of P. grandiflora exhibited characteristics more closely related to the enzymes of C₃ and CAM plants than to those of C₄ plants.