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）和电感耦合等离子体质谱（ICPMS）相结合的方法，研究了在Zn^2+或Cd^2+胁迫下，蓝细菌Anacystis nidulans R-2中两种主要的金属硫蛋白（MT）亚型的诱导表达。两种亚型的氨基酸组成仅略有差异。在含Cd^<2+>的培养基中孵育两种Zn-MT异构体导致金属交换，反映出MT对Cd^<2+>的亲和力高于对Zn^<2+>的亲和力。在此过程中，两种异构体的相对丰度几乎保持不变。Zn^2+剂量（1-20 μ M）以恒定的比例诱导两种亚型，而Cd^2+剂量（0.5-10 μ M）的比例随剂量水平而变化。这些结果证明了本RP-HPLC/ICPMS系统阐明MT亚型功能的潜力。(2)硒是一种有毒元素，可造成环境污染。用HPLC/ICPMS方法研究了Zn^<2+>诱导的蓝细菌金属硫蛋白（MT）生物合成对硒的吸收和掺入.当我们在富硒培养基中培养蓝细菌时，细胞水溶性组分中高达60%的硒被掺入到MT中，MT以两种主要的异构体存在，但在总蛋白中所占比例很小.存在两种途径已被证明为第一次硒掺入到蓝藻MT ;一个是S-Se键合，它可以被2-巯基乙醇还原性地打破，另一个是硒代半胱氨酸的形成，然后将其纳入MT肽链。硒的吸收或掺入硒之间没有本质的区别。目前的HPLC/ICPMS系统可以在分子水平上阐明环境化学重要的生物过程。

(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.