Abstract
In cells of an antibody-synthesizing lymph node, newly synthesized soluble polypeptides were shown to represent 80% to 85% of the proteins in the post-mitochondrial supernatant. The presence of free light chains and free heavy chains was detected after a short pulse with radioactive amino acids and gel filtration in 1 M propionic acid. Free light chains were also observed by gel filtration in neutral buffers without detergents. Part of the free light and heavy chains detected in the cell at low pH or in the presence of detergents (SDS) could have been a result of the dissociation of IgG molecules or intermediates with an incomplete set of covalent inter-chain disulfide bonds.
Light chains could be dissociated even from secreted IgG molecules by using 1 M propionic acid or hydrochloric acid, but not PBS. This dissociation occurred without reducing agents, suggesting that the S-S bonds between light and heavy chains are formed later than the disulfide bond between the two heavy chains.
Nascent polypeptide chains were released from ribosomes within the cells by adding puromycin to the cell suspension after a short pulse with radioactive amino acids. The release of completed native peptides was reduced by the simultaneous addition of streptovitacin or cycloheximide with puromycin, but this yielded an increased fraction of nascent peptides in the total population of polypeptides released after a short pulse. Incomplete heavy chains were detected after this treatment by gel filtration of the released material in 1 M propionic acid and subsequent immunologic analysis on specific immunoadsorbents.
Footnotes
This investigation was supported by United States Public Health Service Research Grant HE-04598 from the National Heart Institute and United States Public Health Service Training Grant 5 T1 AI 154 from the National Institute of Allergy and Infectious Diseases.
