Detection of a zinc-containing protein as the primary cadmium-binding protein before induction of metallothionein in rat liver.

Abstract

A high-molecular-weight protein that binds cadmium primarily before the induction of metallothionein (MT) in rat liver was identified chromatographically. Female Wistar rats were injected intravenously with a single dose of 0.4 or 1.0 mg Cd/kg body weight and sacrificed 0.5 and 3 or 0.5 h, respectively, after the injection. The liver supernatants were subjected to gel filtration analyses on Sephadex G-75 and Asahipak GS-520 columns, the former being used as a conventional column and the latter as an HPLC column. Cadmium (Cd) in the eluate was determined simultaneously with copper, iron, phosphorus, sulfur and zinc on an inductively coupled argon plasma-atomic emission spectrometer (ICP). Cadmium was eluted as a sharp peak at a retention time of 13.2 min accompanied with minor peaks at 11.5 min (broad peak), 14.5 min (MT) and others on a GS column before induction of MT at the low Cd dose. The major peak at 13.2 min from a GS column corresponded to the Cd peak eluted at Ve/Vo=1.20 on a Sephadex column. This major Cd-binding protein was separated on a cation exchange column and identified as a constitutive zinc-and sulfurcontaining protein without other metals. The Cd-binding zinc protein was identical with the Cd-binding protein of 40000 daltons that had been detected by competitive binding assay using a western blotting technique.