Isolation of a novel rat testicular metalloprotein binding cadmium and zinc |
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Authors: | Carol M Kinoshita H E Ganther |
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Institution: | (1) Department of Nutritional Sciences, University of Wisconsin, 53706 Madison, WI |
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Abstract: | Various testicular metal-binding proteins having apparent mol wt in the range of 10–30 kD have been demonstrated by gel filtration
of109Cd- or65Zn-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 of109Cd 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 (<60 kD) protein in rat testic 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 of109Cd- and65Zn-labeled cytosol demonstrated that109Cd and65Zn cochromatography 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. |
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Keywords: | Cadmium-binding protein in rat testes isolation zinc-binding protein in rat testes testis rat metal-binding protein in testis rat novel 22 kD polypeptide in rat testes metal-binding proteins rooster testes cytosolic proteins glutathione transferase testes |
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