Martin M. N., Saftner R. A.
Horticultural Crops Quality Laboratory, Beltsville Agricultural Research Center, Agricultural Research Service, United States Department of Agriculture, Beltsville, Maryland 20705.
Plant Physiol. 1995 Jul;108(3):1241-1249. doi: 10.1104/pp.108.3.1241.
1-Aminocyclopropane-1-carboxylic acid (ACC) can be oxidized to ethylene or diverted to the conjugate 1-(malonylamino)cyclopropane-1-carboxylic acid (MACC) by an ACC N-malonyltransferase. We developed a facile assay for the ACC N-malonyltransferase that resolved [14C]MACC from [14C]ACC by thin-layer chromatography and detected and quantified them using a radioisotope-imaging system. Using this assay, we showed that ACC N-malonyltransferase activity has developmental and tissue-specific patterns of expression in tomato (Lycopersicon esculentum) fruit. In the pericarp, activity was elevated for several days postanthesis, subsequently declined to a basal level, increased 3-fold at the onset of ripening, and again declined in overripe fruit. In the seed, activity increased throughout embryogenesis, maturation, and desiccation. Treatment of fruit with ethylene increased activity 50- to 100-fold in the pericarp. ACC N-malonyltransferase was purified 22,000-fold to a specific activity of 22,000 nmol min-1 mg-1 protein using ammonium sulfate precipitation, DyeMatrex Green A affinity, anion-exchange, Cibacron Blue 3GA affinity, hydrophobic interaction, and molecular filtration chromatography. Native and sodium dodecyl sulfate-denatured enzyme showed molecular masses of 38 kD, indicating that the enzyme exists as a monomer. The enzyme exhibited a Km for ACC of 500 [mu]M, was not inhibited by D- or L-amino acids, and did not conjugate [alpha]-aminoisobutyric acid or L-amino acids.
1-氨基环丙烷-1-羧酸（ACC）可被ACC N-丙二酰基转移酶氧化为乙烯，或转化为共轭物1-（丙二酰氨基）环丙烷-1-羧酸（MACC）。我们开发了一种简便的ACC N-丙二酰基转移酶检测方法，该方法通过薄层色谱法将[14C]MACC与[14C]ACC分离，并使用放射性同位素成像系统对它们进行检测和定量。利用该检测方法，我们发现ACC N-丙二酰基转移酶活性在番茄（Lycopersicon esculentum）果实中具有发育和组织特异性的表达模式。在果皮中，开花后几天活性升高，随后降至基础水平，在成熟开始时增加3倍，在过熟果实中再次下降。在种子中，活性在整个胚胎发生、成熟和干燥过程中增加。用乙烯处理果实可使果皮中的活性提高50至100倍。使用硫酸铵沉淀、DyeMatrex Green A亲和、阴离子交换、Cibacron Blue 3GA亲和、疏水相互作用和分子过滤色谱法，将ACC N-丙二酰基转移酶纯化了22000倍，比活性达到22000 nmol min-1 mg-1蛋白质。天然和十二烷基硫酸钠变性的酶的分子量均为38 kD，表明该酶以单体形式存在。该酶对ACC的Km为500 μM，不受D-或L-氨基酸抑制，也不与α-氨基异丁酸或L-氨基酸共轭。
