Molecular-level Analysis for the Cyanobacterial Response of Heavy Metal Stress from Environment

(1)采用反相高效液相色谱（RP-HPLC）和电感耦合等离子质谱（ICPMS）相结合的方法，证实了在Zn^<2+>或Cd^<2+>胁迫下培养的蓝藻Anacystis nidulans R-2诱导两种主要的金属硫蛋白（MT）异构体。这两种异构体在氨基酸组成上只有轻微的差异。两种Zn-MT同工异构体在含有Cd^<2+>的培养基中孵育，导致金属交换，反映MT对Cd^<2+>的亲和力高于Zn^<2+>。在这一过程中，两种同工异构体的相对丰度几乎保持不变。Zn^<2+>剂量（1 ~ 20 muM）对两种异构体的诱导率为恒定，Cd^<2+>剂量（0.5 ~ 10 muM）对两种异构体的诱导率随剂量水平的变化而变化。这些结果证明了RP-HPLC/ICPMS系统在阐明MT同工异构体功能方面的潜力。(2)硒是一种有毒元素，会造成环境污染。采用HPLC/ICPMS研究了Zn^<2+>-诱导的金属硫蛋白（MT）在蓝藻中的吸收和结合。当我们在富含亚硒酸盐的培养基中培养细胞时，水溶性细胞组分中多达60%的硒被掺入到MT中，MT以两种主要的同工异构体存在，但在总蛋白质中所占的比例很小。首次证明了硒在蓝藻MT中掺入的两条途径；一种是S-Se键，可被2-巯基乙醇还原破坏；另一种是硒代半胱氨酸的形成，并与MT肽链结合。亚硒酸盐和硒酸盐在硒的吸收和结合方面没有本质差异。目前的HPLC/ICPMS系统可以在分子水平上阐明具有环境化学意义的生物过程。

(1) By combining reversed-phase high-performance liquid chromatography (RP-HPLC) with inductively coupled plasma mass spectrometry (ICPMS), the induction of two major metallothionein (MT) isoforms has been demonstrated for a cyanobacterium Anacystis nidulans R-2, cultured under a stress of Zn^<2+> or Cd^<2+>. The two isoforms showed only a slight difference in the amino acid composition. Incubation of two Zn-MT isoforms in a medium containing Cd^<2+> led to a metal exchange, reflecting the higher affinity of MT to Cd^<2+> than to Zn^<2+>. During this process the relative abundance of the two isoforms remained practically unchanged. The two isoforms were induced in a constant ratio by the Zn^<2+> dosage (1-20 muM), while by the Cd^<2+> dosage (0.5-10 muM) the ratio showed a change with the dose level. These results demonstrate the potential of the present RP-HPLC/ICPMS system to elucidate the function of MT isoforms.(2) Selenium is a toxic element which can cause environmental pollution. Its uptake and incorporation by Zn^<2+>-induced metallothionein (MT) biosynthesis in a cyanobacterium were studied by means of HPLC/ICPMS.When we cultured the cells in selenite-enriched media, as muchas 60% of the total selenium in the water-soluble cell components was incorporated into MT,which exists as two major isoforms but occupies a very minor fraction of the overall proteins. Existence of two pathways has been shown for the first time for selenium incorporation into the cyanobacterial MT ; one is the S-Se bonding which can be reductively broken by 2-mercaptoethanol, and the other is the formation of selenocysteine followed by its incorporation into the MT peptide chain. No essential difference was noted between selenite and selenate regarding the uptake or the incorporation of Se. The present HPLC/ICPMS system can elucidate biological processes of environmental chemical importance at the molecular level.