Metal ions, such as Ca2+ and Mn2+, are necessary for the generation of cofactor activity following reconstitution of factor VIII from its isolated light chain (LC) and heavy chain (HC). Titration of EDTA-treated factor VIII with Mn2+ showed saturable binding with high affinity (K(d) = 5.7 +/- 2.1 microM) as detected using a factor Xa generation assay. No significant competition between Ca2+ and Mn2+ for factor VIII binding (K(i) = 4.6 mM) was observed as measured by equilibrium dialysis using 20 microM Ca2+ and 8 microM factor VIII in the presence of 0-1 mM Mn2+. The intersubunit affinity measured by fluorescence energy transfer of an acrylodan-labeled LC (fluorescence donor) and fluorescein-labeled HC (fluorescence acceptor) in the presence of 20 mM Mn2+ (K(d) = 53.0 +/- 17.1 nM) was not significantly different from the affinity value previously obtained in the absence of metal ion (K(d) = 53.8 +/- 14.2 nM). The sensitization of phosphorescence of Tb3+ bound to factor VIII subunits was utilized to detect Mn2+ binding to the subunits. Mn2+ inhibited the phosphorescence of Tb3+ bound to HC and LC, as well as the HC-derived A1 and A2 subunits with a relatively wide range of estimated inhibition constant values (K(i) values = 169-1147 microM), whereas Ca2+ showed no effect on Tb3+ phosphorescence. These results suggest that factor VIII cofactor activity can be generated by Mn2+ binding to site(s) on factor VIII that are different from the high-affinity Ca2+ binding site. However, like Ca2+, Mn2+ did not alter the affinity for HC and LC association. Thus, Mn2+appears to generate factor VIII cofactor activity by a similar mechanism as observed for Ca2+following its association at nonidentical sites on the protein.
Key words: Alfalfa, joint stress, cadmium, napropamide. INTRODUCTIONThe study on biochemical responses of plants to joint stress of metals and herbicides is an important area of ecotoxicology. Metals are environmental pollutants released from both industrial and agricultural sources affecting the biosphere in many places worldwide. Among them, cadmium (Cd), a nonessential element present in the atmosphere, soil, and water, is one of the most aggressive and persistent element in natural environments. Cd released into the environment may be concentrated in the soil, where it is available for the rooted plants. Due to its great solubility in water and high mobility in the soil-plant *Corresponding author. E-mail: hjyyzx@sina.com. Tel: +86-518-85895409.system, Cd is readily taken up by the roots (KreveŠan et al., 2003). Agricultural soil may be contaminated with Cd as a result of industrialization, land applications of sewage sludge, and use of different fertilizers, pesticides, and insecticides (Mench, 1998;Sanita Di Toppi and Gabbrielli, 1999). The high solubility of Cd makes this element an environmental concern especially because it is easily assimilated by plants and it disturbs their metabolism (Benavides et al., 2005). Herbicide such as paraquat would also cause a significant activation of all antioxidant enzymes (Ekmekci and Terzioglu, 2005). The inhibition on protein biosynthesis is one of the popular ways in preventing plant growth by exogenous toxic chemicals. Pesticides are an indispensable controller of plant diseases and weeds for modern agriculture. Such pesticides get accumulated in crops or other organisms and may find their way into food chain to cause a series of secondary contaminations (Eberle and Gerber, 1976;Pylypiw et al., 1993;Nagami et al., 2004).One of the visible toxic symptoms of the plants exposed to metals and herbicides is the change of chlorophyll content in leaves. In fact, most of the herbicides kill weeds by inhibiting their photosynthesis and then inhibiting their growth. Napropamide is a selective systemic herbicide used to limit the growth of grasses and weeds in much agricultural cultivation. When the napropamide concentration exceeds the maximum soil holding capacity, it may transfer to the surface or ground water and consequently bring contamination to aquatic or ecological systems. Commercial napropamide can easily pass into tissues of living organisms and is readily accumulated in crops and exposure of napropamide induced substantial production of O 2•− , H 2 O 2 and oxidative injury to Brassica napus Biswas et al., 2007).Alfalfa (Medicago sativa), a legume, is one of the most popular species used for perennial grazing and ubiquitously cultured on the global scale (Sengupta-Gopalan et al., 2007). At present, few reports are involved in the joint stress of cadmium and napropamide. However, the use of chemical fertilizers and other pesticides may introduce metals such as Cd to the terrestrial systems, which leads to combined pollution of cadmium and napropamide.In this work,...
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