Purification and characterization of a copper-binding protein from Asian periwinkle Littorina brevicula.
The Asian periwinkle, Littorina brevicula, is highly resistant to a wide range of heavy metal concentrations and its metal-binding protein(s) are induced in the presence of cadmium (Cd) and zinc (Zn). In this study, we isolated and characterized a novel copper-binding protein (Cu-BP). Following purification by Sephacryl S-100 chromatography, Cu-BP contained an equal amount of Zn in non-exposed physiological conditions. However, Zn is replaced by Cu at the binding site upon addition of excess Cu (100 microM CuCl(2)) to the cytosol or after a long period (60 days) of exposure of the periwinkles to the metal ion (150 microg/l CuCl(2)). The ligand was further purified by DEAE-Sepharose anion-exchange chromatography and C(18) reverse-phase HPLC. The molecular weight of the purified protein was determined as 11.38 kDa by MALDI-TOF MS analyses. This Cu-BP is distinct from common mollusk metallothionein (MT) in that it contains significantly lower number of Cys (8 residues) and high levels of aromatic amino acids, Tyr and Phe. The protein additionally contains His and Met, which are absent in the MT-like Cd-BP of L. brevicula. The finding that Cu-BP in the Asian periwinkle is distinct from MT-like Cd-BP suggests that the timely expression of specific metal-binding proteins allows added protection against each heavy metal in severely polluted conditions.
Ryu
Soo-Kyung
SK
Department of Life Sciences, Ewha Womans University, Seoul 120-750, Republic of Korea.
Park
Jin-Sung
JS
Lee
In-Sook
IS
D000596:Amino Acids / Q000032:analysis; D000818:Animals; D002352:Carrier Proteins / Q000032:analysis* / Q000737:chemistry; D002848:Chromatography, DEAE-Cellulose; D002850:Chromatography, Gel; D002851:Chromatography, High Pressure Liquid; D004591:Electrophoresis, Polyacrylamide Gel; D004784:Environmental Monitoring / Q000379:methods; D008668:Metallothionein / Q000032:analysis / Q000737:chemistry; D008970:Molecular Weight; D008974:Mollusca / Q000737:chemistry*
D000596:Amino Acids; D002352:Carrier Proteins; C023548:copper-binding protein; D008668:Metallothionein