Fathi M, Schnarrenberger C
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.
Plant Physiol. 1990 Mar;92(3):710-7. doi: 10.1104/pp.92.3.710.
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 C(3) 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 C(4) 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 C(4) plants, 0.5% in the CAM plant, and less than 0.01% in C(3) 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 C(3) 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 C(3) and CAM plants than to those of C(4) plants.
采用常规技术对玉米（Zea mays L.）叶片中的NADP - 苹果酸酶进行纯化，经十二烷基硫酸钠 - 聚丙烯酰胺凝胶电泳判断，该酶达到了表观均一性。用此蛋白免疫家兔制备的抗体经纯化后，与蛋白A - 琼脂糖CL - 4B共价偶联，用作免疫亲和树脂，用于纯化C₃植物菠菜（Spinacia oleracea L.）和小麦（Triticum aestivum L.）、景天酸代谢（CAM）植物落地生根（Bryophyllum daigremontianum R. Hamed et Perr. de la Bathie）以及C₄植物玉米、甘蔗（Saccharum officinarum L.）和大花马齿苋（Portulaca grandiflora L.）中的NADP - 苹果酸酶。除大花马齿苋有两条带外，经十二烷基硫酸钠 - 聚丙烯酰胺凝胶电泳判断，这些方法均得到了单一蛋白条带的均一蛋白制剂。纯化蛋白的比活性在56至91单位（每毫克蛋白）之间。NADP - 苹果酸酶在C₄植物中占总可溶性蛋白的比例高达1%，在CAM植物中占0.5%，在C₃植物中则小于0.01%。在涉及抗玉米叶片酶抗血清对活性进行免疫沉淀和免疫抑制的免疫滴定试验中，玉米和甘蔗的NADP -苹果酸酶表位几乎完全相同，而C₃植物和CAM植物的NADP - 苹果酸酶在这些试验中的交叉反应分别为二十分之一和四分之一。大花马齿苋的NADP - 苹果酸酶表现出的特性与C₃植物和CAM植物的酶更为接近，而与C₄植物的酶关系较远。
