Rat spleen lymphocytes were iodinated (125 I) with lactoperoxidase. Quantitative autoradiographic studies on cells fixed immediately after iodination showed 19-24% of intracytoplasmic grains at 3HD and over from the plasma membrane. Normalization of grain density distribution and comparison of resulting curves with the universal curve of grain scatter of 125 I showed that a significant percentage of intracytoplasmic grains (36%) originates from intracytoplasmic labeled sources rather than from scattering from the heavily labeled plasma membrane. Damaged cells had a threefold grain density than intact cells. Radioactivity counts in sliced polyacrylamide gels of iodinated cells revealed 65-72% of total radioactivity in five peaks of apparent mol wt of 44, 50, 57, 90 and 195 thousand daltons. Segregation and internalization of anti-immunoglobulin-Ig-horseradish peroxidase (HRP) complexes from the iodinated plasma membrane proteins of lymphocytes was studied with quantitative autoradiography (125 I) and peroxidase cytochemistry; 64% of grains at 1.5HD (1,500 A) from the plasma membrane were within the cap zone, and 36% of grains remained outside the capped immunoglobulins; 45-57% of grains internalized together with Fab-anti-Ig-Ig-HRP, and 68% of grains internalized together with anti-Ig-Ig-HRP. These studies indicate that (a) iodination of rat spleen lymphocytes results in a significant internal labeling and that (b) immunoglobulins segregate into caps and internalize together with other iodinated plasma membrane proteins while a significant percentage of iodinated proteins (36%) are excluded from the immunoglobulin caps or internalization sites (32-55%).
Quantitative ultrastructural autoradiographic studies of iodinated plasma membranes of lymphocytes during segregation and internalization of surface immunoglobulins.
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N K Gonatas, J O Gonatas, A Stieber, J C Antoine, S Avrameas; Quantitative ultrastructural autoradiographic studies of iodinated plasma membranes of lymphocytes during segregation and internalization of surface immunoglobulins.. J Cell Biol 1 September 1976; 70 (3): 477–493. doi: https://doi.org/10.1083/jcb.70.3.477
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