Isolation and identification of metallothionein isoforms (MT-1 and MT-2) in the rat testis

It has been a long-lasting controversial issue as to whether or not the male genital organs, such as the testis and prostate, contain metallothioneins (MTs), a group of cysteine-rich heavy-metal-binding proteins that play a role in detoxifying heavy metals such as cadmium (Cd). Earlier studies reported that the rodent testis lacks MTs and concluded that this is why the testis is very susceptible to Cd, although other indirect experimental evidence suggests that MTs are present in this organ. A deficiency of MTs in the testis was originally suspected on the basis of amino acid composition analysis, since MT-like proteins isolated as Cd-binding proteins did not have a characteristic MT structure. In the present study, we demonstrate that the rat testis indeed expresses Cd-binding proteins with sequences identical to those previously described for MT-1 and MT-2, the major isoforms. To confirm that MT-1 and MT-2 are present in the rat testis, we purified and isolated Cd-binding proteins by homogenization using Cd-containing buffer, followed by sequential purification using Sephadex G-75 gel filtration chromatography and anion HPLC column chromatography, which yielded Cd-binding protein-1 (Cd-BP-1) and -2 (Cd-BP-2). After pyridylethylation, Cd-BP-1 and Cd-BP-2 were subjected to specific protein fragmentation by acids and endopeptidases, which revealed that these Cd-binding proteins have the same primary structures as MT-1 and MT-2 respectively. Thus we believe that the present results clearly resolve the long-standing debate about the presence of MTs in the testis, at least in the rodent.