Rat metallothionein-2 contains N-alpha-acetylated and unacetylated isoforms

Mammalian metallothioneins (MT), are characteristically N-alpha-acetylated and the presence of an unblocked N-terminus has not previously been reporte d. On-line capillary electrophoresis-electrospray mass spectrometry of hepa tic MT-2 from rats injected with zinc revealed two isoforms differing by a mass equivalent to that of a single acetyl group. The lower mass component constituted > 20% of total MT-2 protein and both MT-2 isoforms were separat ed by reversed-phase high-performance liquid chromatography. The identity o f each fraction was confirmed by matrix-assisted laser desorption ionisatio n mass spectrometry, and amino acid analysis and N-terminal sequencing reve aled that the lower mass isoform was unblocked at the N-terminus and had an amino acid composition and sequence which is characteristic of rat MT-2. T hus the complementary techniques of mass spectrometry and K-terminal sequen cing demonstrated conclusively that purified MT-2 from zinc-treated rats co ntains an unacetylated isoform. We propose that the cotranslational acetyla tion of rat MT-2 may under some circumstances be inefficient compared to th at in other nonrodent species, where we have detected only trace levels of unacetylated MT isoforms.