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Microvillus-specific Mr 75,000 plasma membrane protein of human choriocarcinoma cells
Abstract:
We have previously purified from cultured JEG-3 choriocarcinoma cells an Mr 75,000 protein, originally detected using antibodies to a retrovirus-related synthetic peptide. Using polyclonal antibodies, we have now localized this protein immunocytochemically in JEG-3 cells at both light and electron microscopic levels. In immunofluorescence microscopy of saponin-permeabilized cells, the antigen appeared as dots and short strands at the apical cell surface. In pre-embedding immunoperoxidase electron microscopy, the Mr 75,000 protein was specifically localized to microvilli on the apical cell surface. Immunoferritin electron microscopy was used to assess more quantitatively the antigen distribution in the plane of the plasma membrane, and to define the position of the antigenic site(s) with respect to the membrane. The immunoferritin results confirmed the microvillus specificity of the Mr 75,000 protein and showed that the antigenic portion of the protein is within a few nanometers from, and on the cytoplasmic side of, the lipid bilayer. In detergent extraction experiments, the Mr 75,000 antigen was highly enriched in the soluble fractions. These results demonstrate that the Mr 75,000 protein is a membrane protein highly specific for microvilli.
Insights
Researchers identified a Mr 75,000 protein in JEG-3 choriocarcinoma cells. This protein is specifically located on the microvilli of the apical cell surface, indicating its role as a microvillus-specific membrane protein.
Area of Science:
- Cell Biology
- Molecular Biology
- Biochemistry
Background:
- Previously purified a Mr 75,000 protein from JEG-3 choriocarcinoma cells.
- Initial detection was via antibodies to a retrovirus-related synthetic peptide.
Purpose of the Study:
- To immunocytochemically localize the Mr 75,000 protein in JEG-3 cells.
- To determine the precise location and orientation of the protein within the cell membrane.
Main Methods:
- Immunofluorescence microscopy of saponin-permeabilized cells.
- Pre-embedding immunoperoxidase electron microscopy.
- Immunoferritin electron microscopy for quantitative distribution analysis.
- Detergent extraction experiments.
Main Results:
- The Mr 75,000 protein was observed as dots and short strands at the apical cell surface.
- Specific localization to microvilli on the apical cell surface was confirmed.
- Antigenic sites are located on the cytoplasmic side of the lipid bilayer, within nanometers of the membrane.
- The protein was highly enriched in soluble fractions after detergent extraction.
Conclusions:
- The Mr 75,000 protein is a membrane protein.
- This protein exhibits high specificity for microvilli.
- The findings provide insights into the composition and organization of microvillar membranes.