| Abstract: | A double-label protocol was used to estimate the extent of leakage and relocation artifacts that affect exocrine pancreatic proteins in cell fractionation experiments. Guinea pig pancreatic lobules were pulsed in vitro with a mixture of 14C-amino acids to enable the lobules to produce and process endogenously labeled exocrine proteins. At the end of the pulse (10 min) or after an appropriate chase interval, the lobules were homogenized in 0.3 M sucrose to which a complete mixture of 3H-labeled exocrine pancreatic proteins was added as an exogenous tracer. The distribution of both labels was studied in each cell fraction of interest at the level of TCA-insoluble proteins and individual exocrine proteins resolved by using a two-dimensional gel system. Based on the premises that the exogenous and endogenous label behave identically during homogenization-fractionation and that all endogenously labeled exocrine proteins found in the postmicrosomal supernate come from intracellular compartments ruptured during tissue homogenization, a series of equations was derived to quantitate leakage and adsorption and to define the ratio of endogenous label still in its primary location to total label (primary location index or PLI) for each cell fraction. Leakage was found to be uniform for all exocrine proteins, but unequal in extent from different cell compartments (condensing vacuoles is greater than zymogen granules is greater than rough endoplasmic reticulum) ; it increased with exposure to shearing forces especially in the case of zymogen granules and condensing vacuoles, and was substantially reduced from rough microsomes by adding 10 mM KCl to the homogenization media. Relocation of exogenous label by adsorption to other subcellular components was extensive (approximately 55%), uneven (free polysomes is greater than rough microsomes is greater than smooth microsomes and zymogen granules), preferential (cationic proteins are massively adsorbed to ribosomes and membranes, resulting in a complementary enrichment of the post-microsomal supernate with anionic exocrine proteins), and reversible (with successive 50-100 mM KCl washes). After correction for adsorption and leakage, the kinetics of intracellular transport derived from cell fractionation data were found to be nearly identical to those obtained from quantitative autoradiographic studies. |
