Estrogen binding protein activity in Morris hepatoma 7777 compared with normal rat liver

Abstract

Estrogen binding protein activities were determined in the cytosol from adult male Buffalo rat liver and Morris hepatoma 7777. Estrogen receptors were prepared using the protamine sulfate precipitation technique of Chamness. The ability of various unlabeled steroids competing with [3H]estradiol was examined to establish the binding specificity. Estradiol binding in Morris hepatoma 7777 cytosol was greatly decreased compared with that present in hepatic cytosol prepared from normal rat liver. The receptor concentration expressed as femtomoles per milligram of cytoplasmic protein was 31.1 +/- 2.9 SD for normal rat liver and 0.41 +/- 0.88 SD for the hepatoma. Gel filtration chromatography revealed the presence of an estrogen binder in hepatoma cytosol which was not present in either normal liver or in the protamine sulfate precipitates of hepatoma cytosol. The molecular weight, binding specificity, and precipitation of this protein by specific antiserum suggests that it is alpha-fetoprotein.

Figure 1

Specific binding of the [³H]E₂ by cytosol of the normal male rat liver. Aliquots (200 μm) of whole cytosol (5 mg/ml) precipitated with protamine sulfate were incubated with six different concentrations of [³H]E₂ (0.15–3 nM) for 18 h at 0°C in the presence and absence of 100-fold unlabeled E_2. Specific binding was calculated by subtracting nonspecific binding from total binding. Each point is the average of triplicate determinations. Closed circles represent total binding, open circles nonspecific binding, triangles specific binding.

Figure 2

Scatchard plot analysis of specific [³H]E₂ binding in protamine sulfate precipitates of whole rat male liver cytosol. Aliquots (200 μl) of whole cytosol were precipitated with protamine sulfate and were incubated with six different concentrations of [³H]E₂ (0.15–3 nM) for 18 h at 0°C in the presence and absence of 100-fold unlabeled E₂.

Figure 3

Specific binding of the [³H]E₂ by cytosol from adult male rats and Morris hepatoma 7777. Aliquots (200 μl) of whole cytosol (5 mg/ml) were precipitated with protamine sulfate and were incubated with 1.5 mM of [³H]E₂ in the presence or absence of 100-fold unlabeled E₂. Specific binding was calculated by subtracting nonspecific binding from total binding and was expressed as femtomoles per milligram of cytosol protein. p < 0.001.

Figure 4

Specificity of binding to cytosolic proteins in male rat liver cytosol. Aliquots (200 μl) of whole male rat liver cytosol were precipitated with protamine sulfate and were incubated with 1.5 nM [³H]E₂ in the presence and absence of 100-fold excess of competing substance for 18 h at 0°C. Each bar represents triplicate determinations.

Figure 5

Gel filtration chromatography of estrogen binding proteins from male rat liver and Morris hepatoma 7777 cytosol. Cytosol (4 ml) from normal liver (open circles) was chromatographed on Sephadex G-100 and estrogen binders were detected as indicated in Methods. Hepatoma (open circles) cytosol was incubated for 2 h with 2 nM [³H]E₂ before chromatography on Sephadex G-100. A 400-μl aliquot of each fraction was assayed for [³H]E₂ content. Markers used to calibrate the column included blue dextran (V_o); bovine serum albumin (B), 68.000; ovalbumin (O), 43,000; trypsinogen (T), 24,000; myoglobin (M), 17,000.

Figure 6

Specificity of binding to hepatoma estrogen binder. Fractions 33–35 were pooled from an experiment identical to that illustrated in Figure 5 except that the hepatoma cytosol was not labeled with [³H]E₂ before chromatography; instead, each fraction was tested for [³H]E₂ binding activity as indicated in Methods. The pooled fractions were tested for specificity by incubating with 5 nM [³H]E₂ in the absence or presence of a 100-fold excess of the potentially competing substance for 16 h at 0°C. In one experiment, the pooled fractions were labeled with [³H]E₂ as above, except that after incubation and dextran-coated charcoal treatment, the sample was treated with protamine sulfate and the supernatant estrogen binding activity was determined.