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Abstract
This study was undertaken to further establish the nature of the low molecular weight metal-binding proteins in rat testes. In all cases, control testes were compared to livers of zinc-treated rats, which are known to contain high concentrations of metallothionein. Gel filtration of testicular and hepatic cytosol revealed a major zinc- and/or cadmium-binding protein in the low molecular weight range in both tissues. This protein could be partially purified from either source by a combination of heat treatment and sequential acetone precipitation. When such partially purified preparations were further fractionated by high performance liquid chromatography using a linear gradient of 25%-40% acetonitrile in 0.1% trifluoroacetic acid, two major forms with similar retention times were seen in each tissue. The utility of this high performance liquid chromatography system for separating isoforms of metallothionein was verified by separation of commercially available purified rabbit hepatic metallothionein into a total of five separate forms. Amino acid analysis of the two proteins derived from rat liver was consistent with the known amino acid composition of metallothionein. However, the two testicular forms separated by high performance liquid chromatography were notably different in amino acid composition from metallothionein, with a distinctly lower content of cysteine. These results indicate that the major low molecular weight cadmium/zinc-binding proteins in rat testes are not metallothioneins.
Highlights
This study was undertaken to further establish the Several reports have presented evidence for the presence of nature of the low molecular weight metal-binding pro- metallothionein in rodenttestes [17,18,19,20,21,22]
Ing isoforms of metallothionein was verified by sepa- Following cadmium-induced necrosis, testicular interstitial ration of commercially available purified rabbit he- cell tumors develop in high incidence inboth the rat and patic metallothionein into a total of fiveseparate mouse [29, 30]
These results indicate that the major low molecular weight cadmium/zinc-binding proteins in rat testes are not metallothioneins
Summary
Marked “advertisement” in accordance with 18 U.S.C. Section 1734 Trifluoroacetic acid and acetonitrile for high performance liquid solely to indicate this fact. Gel Filtration-Gel filtration samples of testicular cytosol were either used as prepared or initially saturated with cadmium by the addition of1.25 pmol ofCd"/g wet weight equivalent Acetone precipitate from heat-treated cytosol reconstituted in 2.0 ml of buffer/g wet tissue equivalent were saturated with cadmium in a similar fashion. Partial Purification-The recovery of the hepatic and testicular low molecular weight metal-binding protein following partial purification by heat treatment ansdequential acetone precipitation was estimated by cadmium-binding capacity. HPLC-Reverse-phase HPLC was used to characterize hepatic and testicular preparations of partially purified metal-binding proteins, using the method of Klauser et al [39], which previously has been used for isolation of hepatic metallothionein isoforms from various species. Amino Acid Analysis-The subfractions of hepatic and testicular metal-binding proteins eluted from the HPLC column were divided into two equal parts and lyophilized. The resulting data were entered into the National Institutes of Health DEClO computer and analyzed for best fit composition
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