Identification of mouse brain proteins associated with isoform 3 of metallothionein.
Using immunological approaches and mass spectrometry, five proteins associated with metallothionein-3 in mouse brains have been identified. Metallothionein-3 and associated proteins were isolated using immunoaffinity chromatography over immobilized anti-mouse brain MT3 antibody. Proteins in the recovered pool were separated by SDS-polyacrylamide gel electrophoresis, and distinct bands were excised and the proteins digested using trypsin. Peptides were extracted and analyzed using electrospray ionization mass spectrometry. Initial identification was done comparing the identified peptide mass:charge ratios to the MASCOT database. Confirmation of proteins was accomplished by sequencing of selected peptides using tandem mass spectrometry and comparison to the MASCOT database. The proteins were heat-shock protein 84 (mouse variant of heat-shock protein 90), heat-shock protein 70, dihydropyrimidinase-like protein 2, creatine kinase, and beta actin. Independently using antibodies against metallothionein-3, creatine kinase, and heat-shock protein 84 showed that all three proteins were coimmunoprecipitated from whole mouse brain homogenates with each of the three antibodies. Mixing purified samples of metallothionein and human brain creatine kinase also generated a complex that could be immunoprecipitated either by anti-metallothionein-3 or anticreatine kinase antibody. These data are consistent with metallothionein-3 being present in the mouse brain as part of a multiprotein complex providing new functional information for understanding the role of metallothionein-3 in neuronal physiology.
Lahti
David W
DW
Department of Biochemistry, Molecular Biology, and Biophysics, University of Minnesota, 6-155 Jackson Hall, 321 Church Street, SE, Minneapolis, MN 55455, USA.
Hoekman
John D
JD
Tokheim
Abigail M
AM
Martin
Bruce L
BL
Armitage
Ian M
IM
D000595:Amino Acid Sequence; D000818:Animals; D001921:Brain / Q000378:metabolism*; D002850:Chromatography, Gel / Q000379:methods; D004591:Electrophoresis, Polyacrylamide Gel; D047468:Immunoprecipitation; D008668:Metallothionein / Q000378:metabolism*; D000090023:Metallothionein 3; D051379:Mice; D008969:Molecular Sequence Data; D020033:Protein Isoforms / Q000378:metabolism*; D021241:Spectrometry, Mass, Electrospray Ionization
D000090023:Metallothionein 3; C000715430:Mt3 protein, mouse; D020033:Protein Isoforms; D008668:Metallothionein