S-Adenosylmethionine-Dependent Inactivation and Radiolabeling of 1-Aminocyclopropane-1-Carboxylate Synthase Isolated from Tomato Fruits

Abstract

l-Aminocyclopropane-l-carboxylic acid (ACC) synthase was partially purified from the homogenate of wounded tomato (Lycoperiscon escukntum Mill.) Ethylene is a plant hormone regulating many aspects of plant growth and development, such as seed germination, seedling elongation, fruit ripening, and tissue senescence (1,12). It has been established that ethylene is biosynthesized from methionine via SAM3 and ACC and that the rate-limiting step in ethylene biosynthesis is the conversion of SAM to ACC, catalyzed by ACC synthase (21). Since ACC synthase is rapidly turned over in plant tissues, the concentration of the enzyme in plant tissue is determined not only by the synthesis of the enzyme but also by its inactivation (8,20).Recently, Satoh and Esashi (18) reported that ACC synthase obtained from mung bean hypocotyls is inactivated by its substrate, SAM, during its catalytic action. Similar finding was independently reported in a meeting abstract by Boller (3) with ACC synthase isolated from tomato pericarp tissue. Since the half-life of ACC synthase in vivo was similar to that in vitro,

Turnover of 1-Aminocyclopropane-1-Carboxylic Acid Synthase Protein in Wounded Tomato Fruit Tissue

“…These results indicate that 50 ML of immunoaffinity gel is enough to bind most, if not all, ACC synthase protein in the extract prepared from 2.5 g of tomato tissue. Because only one radioactive band was observed, our results confirmed that the present immunoaffinity purification is highly specific for ACC synthase (4,5,17). Hence, this one-step purification method was used for our subsequent experiments.…”

“…In another experiment, a half-life of 48 min was observed (data not shown). In wounded tomato fruit discs treated with CHI, Kende and Boller (10) (17) showed that the rate of ACC synthase inactivation in vitro depends upon the concentration of AdoMet and that in the presence of AVG, a competitive inhibitor of ACC synthase with respect to AdoMet (7), the half-life of the enzyme activity in vitro increases from 54 to 108 mmn. Later work showed that this AdoMet-dependent inactivation results from an irreversible covalent linkage of the 2-aminobutyrate moiety of AdoMet to the enzyme (18,25).…”

“…Because the half-life of ACC synthase acti the presence of AdoMet is similar to the half-li because the competitive inhibitor AVG increas of the enzyme activity in vitro as well as in v been suggested that the AdoMet-induced ina( first step leading to the rapid inactivation c found in the tissue (16,17). We have, therefo: effect of AOA, a well-known competitive inh synthase with respect to AdoMet (27), on ti ACC synthase protein in vivo.…”

“…1A, lane 1). This band was judged to be ACC synthase on the basis that its apparent molecular size matched that previously reported for tomato ACC synthase (4,5,17,18,23) and it was specifically bound to an anti-ACC synthase monoclonal antibody (4,5,17). To ascertain that under the present experimental conditions our immunoaffinity gel was effective in removing all the ACC synthase protein, a repetitive purification procedure was carried out.…”

“…demonstrated that incubation of a partially purified tomato ACC synthase preparation with AdoMet results in inactivation of the enzyme activity and that this inactivation is due to a covalent linkage of the aminobutyrate portion of AdoMet to the enzyme. Because the half-life of ACC synthase inactivation by AdoMet in vitro (16,17) is similar to that in vivo (10,26), and because AVG, a competitive inhibitor of ACC synthase activity with respect to AdoMet, increases the enzyme half-life both in vitro (17) and in vivo (26), AdoMet-induced inactivation of the enzyme is suggested to be the cause of the rapid decrease in enzyme activity observed in vivo (16,17 40C. An immunoaffinity matrix was prepared by coupling 3 mg of the dialyzed antibody to 1 mL of activated Affi-Gel 10 (Bio-Rad).…”