Shinga J, Kashiwagi K, Tashiro K, Igarashi K, Shiokawa K
Department of Biological Sciences, Graduate School of Science, University of Tokyo, Japan.
Biochim Biophys Acta. 1996 Jul 31;1308(1):31-40. doi: 10.1016/0167-4781(96)00020-6.
From Xenopus tailbud cDNA library, we isolated the cDNA for S-adenosylmethionine decarboxylase (SAMDC), an enzyme which provides putrescine and spermidine with the aminopropyl group to form spermidine and spermine, respectively. The cDNA coded for 335 amino acids whose sequence had high homology (ca. 83%) to other vertebrate SAMDCs, preserving the sequences reportedly essential for enzyme activity, proenzyme processing, and putrescine stimulation of the enzyme activity. Northern blot analysis showed one major mRNA signal of ca. 3.5 kb, with a minor signal of ca 2.0 kb which may probably be due to cross-hybridization. In oocytes the SAMDC mRNA occurred from stage I, and its amount peaked at stage II, then gradually decreased from stage III to VI. The decreased level of the mRNA was maintained during oocyte maturation, further decreased from the cleavage to early neurula stage, and then increased greatly due to the zygotic expression during late neurula stages (stage 21-25), reaching a plateau level at the late tailbud stage (stage 28). Enzyme assays showed that the changing level of the SAMDC mRNA was reflected in the level of the functional enzyme, suggesting strongly that the zygotic expression of the mRNA leads to a large increase in the amount of SAMDC, albeit in the pre-neurula embryo the amount of the enzyme is very small. We found that the relative composition of polyamines is the eukaryote-type (high-level spermine) at the beginning of oogenesis, but it changes to the prokaryote-type, or more appropriately Escherichia coli-type (high-level putrescine but background level spermine) during oocyte maturation, and remains E. coli-type throughout embryogenesis. We assume that the E. coli-type polyamine composition is a necessary factor for the normal embryogenic development in Xenopus and its maintenance, especially that in pre-neurula stages, can be explained by the low level of both SAMDC mRNA and SAMDC.
我们从非洲爪蟾尾芽cDNA文库中分离出S-腺苷甲硫氨酸脱羧酶(SAMDC)的cDNA,该酶分别为腐胺和亚精胺提供氨丙基以形成亚精胺和精胺。该cDNA编码335个氨基酸,其序列与其他脊椎动物的SAMDC具有高度同源性(约83%),保留了据报道对酶活性、酶原加工以及腐胺对酶活性的刺激至关重要的序列。Northern印迹分析显示一个约3.5 kb的主要mRNA信号,以及一个约2.0 kb的次要信号,后者可能是由于交叉杂交所致。在卵母细胞中,SAMDC mRNA从I期开始出现,其数量在II期达到峰值,然后从III期到VI期逐渐减少。mRNA水平的降低在卵母细胞成熟过程中保持,从卵裂到早期神经胚阶段进一步降低,然后由于在神经胚后期阶段(21 - 25期)的合子表达而大幅增加,在尾芽后期阶段(28期)达到平台期水平。酶活性测定表明,SAMDC mRNA水平的变化反映在功能性酶的水平上,这强烈表明mRNA的合子表达导致SAMDC数量大幅增加,尽管在神经胚前期胚胎中该酶的数量非常少。我们发现,多胺的相对组成在卵子发生开始时是真核生物类型(高水平精胺),但在卵母细胞成熟过程中变为原核生物类型,或者更确切地说是大肠杆菌类型(高水平腐胺但精胺为背景水平),并且在整个胚胎发育过程中都保持为大肠杆菌类型。我们假设大肠杆菌类型的多胺组成是非洲爪蟾正常胚胎发育及其维持的必要因素,特别是在神经胚前期阶段,这可以通过SAMDC mRNA和SAMDC的低水平来解释。