Characterization of L-CAM, a major cell adhesion molecule from embryonic liver cells.
We have developed a method for purifying L-CAM, the cell adhesion molecule from embryonic chicken liver cells, and have compared its properties with those of N-CAM, the neural cell adhesion molecule. L-CAM was released from membranes with trypsin, purified by a series of chemical techniques, and used to generate monoclonal antibodies which allowed the identification of the intact L-CAM molecule from membranes. The monoclonal antibodies were used to isolate trypsin-released L-CAM in a single step by affinity chromatography. Material purified by either technique was predominantly a component of M(r) 81,000 on NaDodSO(4)/polyacrylamide gel electrophoresis with a pI of 4.0-4.5. Rabbit antibodies to this component and to the M(r) 81,000 species that had been further purified on NaDodSO(4)/polyacrylamide gel electrophoresis displayed all of the activities of anti-L-CAM. Some of the trypsin-released L-CAM bound specifically to lentil lectin, suggesting that L-CAM is a glycoprotein. The apparent molecular weight of material having L-CAM antigenic determinants depended upon the procedures used to extract membranes; this appears to account for the various values reported previously in the literature. Both the rabbit serum antibodies and the monoclonal antibodies detected the M(r) 81,000 species on immunoblots of unfractionated trypsin-released material. Immunoblots of whole liver cell membranes with the same antibodies revealed a major M(r) 124,000 component, with minor components of M(r) 94,000 and 81,000. Active L-CAM derivatives released by trypsin in the presence of EGTA were detected as a species of M(r) 40,000. L-CAM derivatives obtained by extraction of membranes with EDTA alone appeared as species of M(r) 53,000, 62,000, and 81,000. The combined results suggest that L-CAM on the cell surface is an acidic glycoprotein of M(r) 124,000. In the presence of calcium, the molecule can be released from membranes by trypsin as a soluble M(r) 81,000 fragment; in the absence of calcium, it is released by either endogenous proteases or by trypsin as a variety of smaller fragments.
D000911:Antibodies, Monoclonal; D002118:Calcium / Q000502:physiology; D002448:Cell Adhesion; D002462:Cell Membrane / Q000032:analysis / Q000276:immunology; D008099:Liver / Q000032:analysis / Q000166:cytology*; D014357:Trypsin
D000911:Antibodies, Monoclonal; D014357:Trypsin; D002118:Calcium