AJP - Cell Physiology, Vol 255, Issue 1 C70-C75, Copyright © 1988 by American Physiological Society
Uptake and intracellular fate of [14C]sucrose-insulin in perfused rat livers
C. A. Surmacz, J. J. Wert Jr, W. F. Ward and G. E. Mortimore
Department of Physiology, Milton S. Hershey Medical Center, Pennsylvania State University, Hershey 17033.
Insulin was covalently linked to [14C]sucrose by means of cyanuric chloride
to provide a label that would remain entrapped within the vacuolar system.
The uptake of the conjugate by the perfused rat liver was rapid (half-life
= 2.9 min), competitively inhibited by native insulin, and abolished by
alkali denaturation. As assessed by its distribution on self-generating
gradients of colloidal silica-povidone, label in lysosome-enriched samples
of liver taken at different times after the addition of the conjugate moved
progressively during 15 min from the plasma membrane into an intermediate
peak and then to dense lysosomal fractions. After 30-60 min, the label had
equilibrated throughout the lysosomal-vacuolar system. The initial movement
from the plasma membrane to the intermediate peak occurred between 2 and 5
min. Because label in the peak could be physically separated from the
lysosomal marker, beta-acetylglucosaminidase, by dispersing the sample
through the gradient mixture before centrifugation rather than layering it,
we concluded that the intermediate particles in question were not lysosomal
in nature. On gel-filtration chromatography, label extracted from the
intermediate peak did not move with insulin but rather as a broad band of
lower molecular weight products, suggesting that insulin is subject to
early proteolytic attack within a nonlysosomal compartment.
