Identification of a spectrin-like protein in Sertoli cells.
Sertoli cells prepared from rats ages 15 and 25 days were shown to contain a spectrin-like protein. Indirect immunofluorescence with monospecific antimouse erythrocyte immunoglobulin G (IgG) and with monospecific antimouse brain spectrin IgG revealed specific staining in Sertoli cells. Both antibodies precipitated two spectrin-like peptides of 240,000 and 235,000 daltons from cells solubilized with octyl glucoside. Proteins from Sertoli cell membranes were separated by electrophoresis on polyacrylamide gels containing sodium dodecyl sulfate and electrophoretically transferred to nitrocellulose membrane. Incubation of nitrocellulose membrane with either of the two antibodies, followed by horseradish peroxidase conjugated to second antibody, revealed only the larger, or alpha, spectrin subunit (Western blots). Both antibodies were used to provide immunoautoradiographic identification of the spectrin-like protein. In this procedure, spectrin and Sertoli cell membranes were shown to compete with [125I]-labeled spectrin from mouse erythrocytes for binding to antimouse erythrocyte spectrin IgG. Finally, two-dimensional proteolytic mapping of the 240,000- and 235,000-dalton peptides demonstrated limited spot homology with rat erythrocyte spectrin. However, subcellular fractions from Sertoli cells all contained a spectrin-like protein showing high homology from fraction to fraction. It is concluded that Sertoli cells contain a spectrin-like protein that is seen in cell fractions prepared by centrifugation, i.e., mitochondria, microsomes, nuclei, cytoplasm, and plasma membranes. Although homology with spectrin from erythrocytes or brain is not seen in peptide maps, the alpha subunit shares antigenic determinants with spectrin from erythrocytes. The beta subunit is believed to be precipitated by antispectrin as the result of binding to the alpha subunit, since the beta subunit shows no detectable antigenic homology with that of spectrin.
K
K
Borland
S
S
Osawa
D
D
Kew
D B
DB
Coleman
S R
SR
Goodman
P F
PF
Hall
D000818:Animals; D001345:Autoradiography; D001923:Brain Chemistry; D002478:Cells, Cultured; D002918:Chymotrypsin; D000939:Epitopes / Q000276:immunology; D004912:Erythrocytes / Q000032:analysis; D005455:Fluorescent Antibody Technique; D006651:Histocytochemistry; D007124:Immunoenzyme Techniques; D007074:Immunoglobulin G; D007163:Immunosorbent Techniques; D008297:Male; D008854:Microscopy, Electron; D010446:Peptide Fragments / Q000032:analysis; D051381:Rats; D012708:Sertoli Cells / Q000032:analysis*; D013049:Spectrin / Q000032:analysis* / Q000276:immunology
D000939:Epitopes; D007074:Immunoglobulin G; D010446:Peptide Fragments; D013049:Spectrin; D002918:Chymotrypsin