ISOLATION OF 3 COPPER METALLOTHIONEIN ISOFORMS FROM THE BLUE-CRAB (CALLINECTES-SAPIDUS)

In the blue crab Callinectes sapidus, a direct correlation exists between levels of copper(I)-metallothionein (CuMT) and Cu(I)-hemocyanin during the molt cycle (Engel and Brouwer, 1987). This phenomenon makes these organisms unique model systems to study the involvement of MTs in metalloprotein activation and metal detoxification. To further clarify the role of MT in metal-regulatory processes we have determined how many different MT isoforms are present in blue crabs and how these different forms respond to elevated dietary levels of Cu. Anion-exchange HPLC showed two Zn- and Cu-containing MT isoforms, eluting at 40 and 87 mM Tris, in hepatopancreas from control crabs. Three forms (CuMT-I, CuMT-II, and CuMT-III), eluting at 42, 55 and 93 mM Tris, were present in hepatopancreas from Cu-treated animals. CuMT-III was induced to a much greater extent than CuMT-I or CuMT-II. Purification of the metal-free MTs by reverse-phase HPLC and amino acid analysis showed that each isoform from Cu-treated animals had a unique amino acid composition indicating the presence of three different Class I MTs. Functional implications of these findings are discussed.