γ-Glutamyltransferases in higher plants and catabolism of glutathiones

γ-谷氨酰基转移酶(GGT)催化谷胱甘肽等γ-谷氨酰肽的γ-谷氨酰基部分的水解和转位。本研究发现：(1)萝卜子叶亚细胞分离和液泡分离表明，可溶性GGT是一种液泡酶。谷胱甘肽转移酶抑制剂Acivicin可介导内源性谷胱甘肽与外源性一氯丁烷生成的谷胱甘肽S结合物的体内分解代谢抑制。因此，可溶性GGT可能参与谷胱甘肽和谷胱甘肽S结合物的分解代谢。(2)编码异源二聚体GGT亚型的萝卜全长cDNAs(RsGGT1、RsGGT2和RsGGT3)转化烟草植株。表达分析表明，RsGGT表现出不同的器官表达模式。RsGGT1和RsGGT2cDNAs的过表达而不是RsGGT3cDNAs的过表达导致了转基因烟草中盐可提取结合的GGT活性的增加。这些结果表明，RsGGT1和RsGGT2cDNAs编码异二聚体结合的GGT亚型。(3)在可能的GGT基因AtGGT1、AtGGT2和AtGGT3中插入T-DNA或转座子的拟南芥突变体被鉴定。基因表达分析表明，所鉴定的atggt1-1、atggt2-1、atggt2-2和atggt3-1植株均为GGT缺失突变体。GGT活性分析表明，atggt1突变等位基因在细胞壁结合的GGT中缺失，而atggt2突变等位基因和atggt3突变等位基因在可溶性GGT中缺失。野生型和atggt1植株的GSH含量和GSH分解速率表明，AtGGT1参与了GSH的分解代谢。根据它们的序列，AtGGT1、2、3似乎与RsGGT-1、2、3相对应。然而，这些基因之间仍然存在差异。

γ-Glutamyltransferase (GGT) catalyzes the hydrolysis and transpeptidation of the γ-glutamyl moiety of γ-glutamyl peptides such as glutathione. We found the followings in this study.(1) Subcellular fractionation of radish (Raphanus sativus) cotyledon and isolation of vacuole revealed that soluble GGT is a vacuolar enzyme. Acivicin, a GGT inhibitor, mediated the in vivo catabolism inhibition of the glutathione S-conjugate generated from endogenous glutathione and exogenously supplied monochlorobimane. Thus, soluble GGT is possibly involved in the catabolism of glutathione and glutathione S-conjugates.(2) Tobacco plants transformed with□□ radish full-length cDNAs (RsGGT1, RsGGT2 and RsGGT3) encoding putative heterodimeric GGT isoforms. Expression analysis indicated that RsGGTs showed different organ expression patterns. The overexpression of RsGGT1 and RsGGT2cDNAs, but not that of RsGGT3cDNA, resulted in an increase of NaCl-extractable bound GGT activity in transgenic tobacco plants. These results suggest that RsGGT1 and RsGGT2cDNAs encode heterodimeric bound GGT isoforms.(3) Arabidopsis mutants harboring T-DNA or transposon insertions in putative GGT genes AtGGT1, AtGGT2 and AtGGT3 were characterized. Gene expression analysis indicated that the identified atggt1-1, atggt2-1, atggt2-2 and atggt3-1 plants were GGT-null mutants. GGT activity analysis indicated that the atggt1 mutant allele was deficient in cell wall bound GGT, whereas the two atggt2 mutant alleles and the atggt3 mutant allele were deficient in soluble GGT. GSH contents and GSH catabolic rates in wild type and atggt1 plants suggest that AtGGT1 is involved in GSH catabolism. AtGGT1, 2, 3 seem to be corresponding to RsGGT-1, 2, 3. based on their sequences. However there still be discrepancy between these genes.