The Journal of Cell Biology, Vol 70, 477-493, Copyright © 1976 by The Rockefeller University Press
Quantitative ultrastructural autoradiographic studies of iodinated plasma membranes of lymphocytes during segregation and internalization of surface immunoglobulins
NK Gonatas, JO Gonatas, A Stieber, JC Antoine and S Avrameas
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%).
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