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

(1)采用反相高效液相色谱和电感耦合等离子体质谱(ICPMS)相结合的方法，研究了蓝藻Anacystis nidulans R-2在锌和镉胁迫下产生两种主要的金属硫蛋白(MT)亚型。两种异构体的氨基酸组成仅略有不同。两种锌-金属硫蛋白亚型在含Cd^&lt；2+&gt；的介质中孵育产生金属交换，反映了MT与Cd^&lt；2+&gt；的亲和力高于对锌的亲和力。在这一过程中，两种异构体的相对丰度几乎保持不变。锌离子剂量(1-20um)和镉离子剂量(0.5-10um)诱导的两种异构体的比例随剂量的增加而变化。这些结果表明，本体系具有阐明MT亚型功能的潜力。(2)硒是一种有毒元素，可对环境造成污染。用高效液相色谱/离子交换光谱分析技术研究了锌离子诱导蓝藻对金属硫蛋白(MT)的吸收和掺入。当我们在富硒培养基中培养蓝藻时，有60%的水溶性细胞组分中的总硒被结合到MT中，MT以两种主要亚型存在，但在总蛋白中所占的比例很小。首次证实了硒进入蓝藻MT的两条途径：一条是S-Se键，可被2-巯基乙醇还原破坏；另一条是形成硒半胱氨酸，然后进入MT多肽链。亚硒酸盐和亚硒酸盐在吸收或并入Se方面没有本质区别。本系统可以从分子水平上解释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.