PARTIAL-PURIFICATION AND CHARACTERIZATION OF LYSINE-KETOGLUTARATE REDUCTASE IN NORMAL AND OPAQUE-2 MAIZE ENDOSPERMS
PARTIAL-PURIFICATION AND CHARACTERIZATION OF LYSINE-KETOGLUTARATE REDUCTASE IN NORMAL AND OPAQUE-2 MAIZE ENDOSPERMS
复制标题
DOI：
10.1104/pp.98.3.1139
复制
发表时间：
1992-03-01
期刊：
影响因子：
7.4
通讯作者：
ARRUDA, P
中科院分区：
文献类型：
--
作者：
BROCHETTOBRAGA, MR;LEITE, A;ARRUDA, P
赖氨酸酮戊二酸还原酶催化玉米（Zea mays L.）胚乳该酶使用NADPH作为辅因子将L-赖氨酸和α-酮戊二酸缩合成糖精。它是胚乳特异性的，并且具有活动的时间模式，随着籽粒发育的开始而增加，在授粉后20至25天达到峰值，此后随着籽粒接近成熟而减少。从发育中的玉米胚乳中提取该酶，并通过硫酸铵沉淀、DEAE-纤维素阴离子交换层析和Blue-SepharoseCL-6 B亲和层析进行部分纯化。从亲和层析获得的制剂富集275倍，并且具有每毫克蛋白质每分钟411纳摩尔的比活性。通过聚丙烯酰胺凝胶电泳测定，天然和变性的酶是140千道尔顿蛋白质。该酶表现出其底物的特异性，并没有被抑制，无论是氨乙基半胱氨酸或谷氨酸。稳态产物抑制研究表明，糖精是一种非竞争性抑制剂α-酮戊二酸和竞争性抑制剂赖氨酸。这表明快速平衡有序结合机制，结合顺序为赖氨酸、α-酮戊二酸、NADPH。以正常胚乳和opaque-2胚乳的两个玉米自交系为材料，研究了胚乳酶活性的变化。赖氨酸-酮戊二酸还原酶的活性模式与胚乳发育过程中玉米醇溶蛋白的积累速率相协调。在不透明-2胚乳中观察到酶活性和玉米醇溶蛋白积累的协调调节，因为活性和玉米醇溶蛋白水平比正常胚乳低两到三倍。从授粉后20天的L1038正常和不透明-2中提取的酶通过DEAE-纤维素层析部分纯化。两种基因型显示出相似的洗脱模式，在约0.2摩尔KCl处洗脱出单一活性峰。正常和opaque-2酶的分子量和物理性质基本相同。我们认为，Opaque-2基因，这是一个反式激活蛋白的22千道尔顿玉米醇溶蛋白基因，可能参与调节赖氨酸-酮戊二酸还原酶基因在玉米胚乳。此外，由opaque-2突变引起的还原酶活性降低可以至少部分地解释opaque-2胚乳中赖氨酸浓度升高的原因。
Lysine-ketoglutarate reductase catalyzes the first step of lysine catabolism in maize (Zea mays L.) endosperm. The enzyme condenses L-lysine and alpha-ketoglutarate into saccharopine using NADPH as cofactor. It is endosperm-specific and has a temporal pattern of activity, increasing with the onset of kernel development, reaching a peak 20 to 25 days after pollination, and thereafter decreasing as the kernel approaches maturity. The enzyme was extracted from the developing maize endosperm and partially purified by ammonium-sulfate precipitation, anion-exchange chromatography on DEAE-cellulose, and affinity chromatography on Blue-Sepharose CL-6B. The preparation obtained from affinity chromatography was enriched 275-fold and had a specific activity of 411 nanomoles per minute per milligram protein. The native and denaturated enzyme is a 140 kilodalton protein as determined by polyacrylamide gel electrophoresis. The enzyme showed specificity for its substrates and was not inhibited by either aminoethyl-cysteine or glutamate. Steady-state product-inhibition studies revealed that saccharopine was a noncompetitive inhibitor with respect to alpha-ketoglutarate and a competitive inhibitor with respect to lysine. This is suggestive of a rapid equilibrium-ordered binding mechanism with a binding order of lysine, alpha-ketoglutarate, NADPH. The enzyme activity was investigated in two maize inbred lines with homozygous normal and opaque-2 endosperms. The pattern of lysine-ketoglutarate reductase activity is coordinated with the rate of zein accumulation during endosperm development. A coordinated regulation of enzyme activity and zein accumulation was observed in the opaque-2 endosperm as the activity and zein levels were two to three times lower than in the normal endosperm. Enzyme extracted from L1038 normal and opaque-2 20 days after pollination was partially purified by DEAE-cellulose chromatography. Both genotypes showed a similar elution pattern with a single activity peak eluted at approximately 0.2 molar KCL. The molecular weight and physical properties of the normal and opaque-2 enzymes were essentially the same. We suggest that the Opaque-2 gene, which is a transactivator of the 22 kilodalton zein genes, may be involved in the regulation of the lysine-ketoglutarate reductase gene in maize endosperm. In addition, the decreased reductase activity caused by the opaque-2 mutation may explain, at least in part, the elevated concentration of lysine found in the opaque-2 endosperm.