Abstract
A neural cell adhesion molecule (N-CAM) was purified in milligram quantities from detergent extracts of embryonic chick brain membranes. N-CAM has an unusual carbohydrate content and structure, is polydisperse in solution, and is associated with proteolytic activity leading to its spontaneous cleavage. The carbohydrate composition of N-CAM includes 13 mol of sialic acid but only 1.4 mol of galactose/100 mol of amino acids, suggesting the presence of a sialic acid to protein linkage not previously observed in higher organisms. N-CAM appears to be an integral membrane protein in that its extraction from membranes required detergent. Although soluble, the purified molecule was aggregated (Mr = 0.5 to 1.2 X 10(6)) and polydisperse in detergent-free solutions. N-CAM from brain also migrated as a broad but continuously stained region from Mr = 200,000 to Mr = 250,000 on sodium dodecyl sulfate-polyacrylamide gel electrophoresis; the molecule from retina was similar but had a somewhat faster mobility. Desialation of N-CAM did not significantly change its behavior in solution, but converted both brain and retinal N-CAM to components migrating on sodium dodecyl sulfate-polyacrylamide gel electrophoresis as material of about Mr = 140,000. Despite the apparent heterogeneity, amino acid sequence analysis and comparison of proteolytic fragments suggest that all forms of the glycoprotein are derived from the same polypeptide chain. On prolonged incubation at neutral pH, N-CAM undergoes apparent proteolysis to yield a polypeptide that contains little sialic acid and has a Mr = 65,000 on sodium dodecyl sulfate-polyacrylamide gel electrophoresis, a separate sialic acid-rich component, and a variety of small peptides. The 65,000-dalton polypeptide appeared to contain all of the antigenic determinants of intact N-CAM that neutralize the adhesion-blocking ability of anti-retinal cell Fab' fragments.
Highlights
neural cell adhesion molecule (N-CAM) has an unusual carbohydrate content and structure, is polydisperse in solution, and is associated with proteolytic activity leading to its spontaneous cleavage. ”he carbohydrate composition of N-CAM includes 13 mol of sialic acid but only 1.4 mol of galactose/100 mol of amino acids, suggesting the presence of a sialic acid to protein linkage not previously observed in higher organisms
The amount of N-CAM in a sample was estimated by its ability to was incorporated into the protein to account for all of the neutralize the adhesion-blocking ability of Fab’ prepared from the sera of rabbits immunized with retinal cells.One unit of N-CAM
The abundance of NCAM in brain tissue combined with the efticiency of immunoaffiity purification (78%yield) allowed the routine isolation of greater than15 mg of highly purified N-CAM from the brains of 1200 embryonic chicks and make practical a detailed structural analysis of this molecule
Summary
The amount of N-CAM in a sample was quantitated by its ability subtypes IgGl and IgG2b. The pellets (160 ml) rotaryshaker for 1.5 h with either the partially purified N-CAM were resuspended with 320 ml of 2.25 M sucrose in salt/Pi buffer, obtained by the chromatography procedure described above, or the placed in 12 centrifuge tubes, each aliquot overlaid with 10 ml of 0.8 total Nonidet P-40 extract of membranes from 600 brains. Detergent was removed suspended in 600 ml of salt/Pi buffer plus 1 m~ EDTA and 0.5% from the N-CAM sample by incubation with Bio-Beads SM-2 and by Nonidet P-40. In both cases, the extracts were centrifuged for 40 min dialysis twice uersus 12 liters of H10 for 4 h at 4 "C.
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