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 reported. On-line capillary electrophoresis-electrospray mass spectrometry of hepatic 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 separated by reversed-phase high-performance liquid chromatography. The identity of each fraction was confirmed by matrix-assisted laser desorption ionisation mass spectrometry, and amino acid analysis and N-terminal sequencing revealed 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. Thus the complementary techniques of mass spectrometry and N-terminal sequencing demonstrated conclusively that purified MT-2 from zinc-treated rats contains an unacetylated isoform. We propose that the cotranslational acetylation of rat MT-2 may under some circumstances be inefficient compared to that in other nonrodent species, where we have detected only trace levels of unacetylated MT isoforms.
Beattie
J H
JH
Trace Element and Gene Expression Group, Rowett Research Institute, Bucksburn, Aberdeen, UK. j.beattie@rri.sari.ac.uk
Wood
A M
AM
Duncan
G J
GJ
D000107:Acetylation; D000596:Amino Acids / Q000737:chemistry; D000818:Animals; D002851:Chromatography, High Pressure Liquid; D019075:Electrophoresis, Capillary / Q000379:methods*; D008099:Liver / Q000737:chemistry; D013058:Mass Spectrometry / Q000379:methods*; D008668:Metallothionein / Q000737:chemistry*; D020033:Protein Isoforms; D051381:Rats; D013997:Time Factors
D000596:Amino Acids; D020033:Protein Isoforms; D008668:Metallothionein