Tissue Cd-Binding Activity
The Cd-hemoglobin affinity assay, a standard method of analysis based on 109Cd binding to heat-resistant tissue pro teins, was used to estimate MT-like binding activity in staged testicular tissues. Liver, kidney, heart, and muscle were used as controls. In brief, ~200 mg of frozen tissue was homogenized in 4 vol of homogenization buffer (HB; Tris-HCl, 10 mM, pH 7.4) and centrifuged at 10 000 X g for 10 min; the supernatant was boiled for 2 min. After centrifuging at 10 000 Xg for 10 min, 100 ^l of supernatant was incubated with the same volume of 109Cd solution (2 ^g/ml CdCl2 and 1 ^Ci 109Cd in HB) for 10 min at room temperature. Addition of bovine hemoglobin (2% in 100 ^l HB) followed by boiling for 2 min, cooling on ice for 5 min, and centrifugation (10 000 Xg for 5 min) and then addition of hemoglobin, boiling, cooling and centrifuging again, separated free and bound fractions. Radioactivity in the supernatant (200 ^l) was measured in a gamma counter (Searle Analytic, Des Plaines, IL). buy ventolin inhalers
Blanks (no tissue extract) and total 109Cd (no tissue extract, no hemoglobin) were used as controls. To minimize isotope dilution by endogenous Cd (after Cd treatment) and to assure ligand availability, samples were diluted prior to assay such that bound 109Cd was no more than 20% of total added. Values were expressed as ^g Cd bound/g tissue. MT II from rabbit liver (Sigma Chemical Company, St. Louis, MO) was used to standardize the assay.