Purification, characterization, and localization of 70 kDa calcium-sensitive protein (calelectrin) from mammary glands.
Mammary glands contain a group of calcium-sensitive proteins that bind to membranes in a calcium-dependent manner. Using the calcium-dependent binding to hydrophobic surfaces in combination with conventional techniques, we have purified the 70 kDa mammary calcium-binding protein (70 kDa M-CBP) to homogeneity. Antisera prepared to the 70 kDa M-CBP or to bovine liver 67 kDa calelectrin reacted in immunoblot analysis with the 70 kDa M-CBP antigen and with several additional mammary CBP species in crude tissue homogenates. Limited proteolysis of the 70 kDa M-CBP produced smaller immunoreactive species; extensive proteolysis resulted in more complete degradation of the protein. Identical data were obtained with digestion of 67 kDa calelectrin. The pl for the 70 kDa M-CBP was determined to be approximately 5.8; the same value reported for 67 kDa calelectrin. Phosphorylation of 70 kDa M-CBP was not detected in epithelial cell culture metabolic labeling. Immunohistochemical localization showed the protein to be located in ductal epithelia of virgin mouse mammary glands with a pattern of increased staining of the basal portions of the cells. Some stromal cells were also reactive. Apparently, the 70 kDa M-CBP and 67 kDa calelectrin are the same protein. Furthermore, like the 32.5 calelectrin (endonexin) and calpactin I/p36/lipocortin II, the 70 kDa protein appears to be a ductal epithelial cell associated protein in the mammary gland.
Lozano
J J
JJ
Biology Board of Studies, University of California, Santa Cruz 95064.
Haindl
A H
AH
Rocha
V
V
D000818:Animals; D017302:Annexins; D002135:Calcium-Binding Proteins / Q000276:immunology / Q000302:isolation & purification* / Q000378:metabolism; D002417:Cattle; D002478:Cells, Cultured; D002918:Chymotrypsin / Q000494:pharmacology; D004591:Electrophoresis, Polyacrylamide Gel; D004847:Epithelial Cells; D004848:Epithelium / Q000032:analysis / Q000378:metabolism; D005260:Female; D007150:Immunohistochemistry; D007525:Isoelectric Focusing; D008099:Liver / Q000032:analysis / Q000166:cytology / Q000378:metabolism; D008321:Mammary Glands, Animal / Q000032:analysis* / Q000166:cytology / Q000378:metabolism; D051379:Mice; D010766:Phosphorylation; D011247:Pregnancy
D017302:Annexins; D002135:Calcium-Binding Proteins; D002918:Chymotrypsin