Isolation of a novel rat testicular metalloprotein binding cadmium and zinc.
Various testicular metal-binding proteins having apparent mol wt in the range of 10-30 kD have been demonstrated by gel filtration of 109Cd- or 65Zn-labeled cytosol, but in no case has a purified metalloprotein been isolated that contains stoichiometric amounts of the metal. The purpose of this work was to purify from rat testes a testes-specific 30 kD Cd-binding protein (Cd-testin) following in vitro addition of 109Cd to testis cytosol. Conventional purification methods similar to those used for purification of metallothionein could not be used because Cd was not retained in stoichiometric amounts by the 30 kD species when these methods were employed. However, using ammonium sulfate fractionation, hydrophobic interaction and gel filtration chromatography, a 30 kD protein containing 2.6 mol of Cd/mol of protein was isolated. Two-dimensional sodium dodecyl sulfate polyacrylamide gel electrophoresis demonstrated that the isolated protein contained one major polypeptide with a mol mass of 22 kD and a pI of 4.6 (22 kD/pI 4.6) and two minor polypeptides (16 kD/pI 4.6 and 10 +/- 4 kD/pI 6.3). Two-dimensional gel electrophoresis demonstrated that the 22 kD species is a major low mol mass (less than 60 kD) protein in rat testis cytosol. The 22 kD protein was not detectable in cytosol of rooster testis, a tissue that is insensitive to Cd-induced damage and devoid of the 30 kD Cd-binding protein. Gel filtration and hydrophobic interaction chromatography of 109Cd- and 65Zn-labeled cytosol demonstrated that 109Cd and 65Zn cochromatograph with the 30 kD protein. The function of this novel 30 kD testicular metal-binding protein is not known, but our work and other studies suggest that its occurrence in testes is linked to the production of a unique 22 kD polypeptide.
Kinoshita
C M
CM
Department of Nutritional Sciences, University of Wisconsin, Madison 53706.
Ganther
H E
HE
D000596:Amino Acids / Q000032:analysis; D000818:Animals; D002104:Cadmium / Q000032:analysis*; D002850:Chromatography, Gel; D003600:Cytosol / Q000378:metabolism; D015180:Electrophoresis, Gel, Two-Dimensional; D005982:Glutathione Transferase / Q000378:metabolism; D008297:Male; D008668:Metallothionein / Q000032:analysis* / Q000378:metabolism*; D051381:Rats; D013737:Testis / Q000032:analysis* / Q000201:enzymology; D015032:Zinc / Q000378:metabolism*
D000596:Amino Acids; C004203:cadmium-binding protein; D002104:Cadmium; D008668:Metallothionein; D005982:Glutathione Transferase; D015032:Zinc