Degradation and regurgitation of extracellular proteins by cultured mouse peritoneal macrophages and baby hamster kidney fibroblasts. Kinetic evidence that the transfer of proteins to lysosomes is not irreversible.

S Buktenica,S J Olenick,R Salgia,A Frankfater

doi:10.1016/s0021-9258(18)47957-4

Abstract

The uptake and degradation of bovine serum albumin (BSA), bovine liver catalase, and rabbit muscle enolase have been studied in cultured mouse peritoneal macrophages (MPM) and baby hamster kidney fibroblasts (BHK cells). Rates constant for the uptake of the three proteins by MPM were similar. In addition, BSA accumulation was independent of BSA concentration in the uptake medium and was not inhibited by a large excess of serum, suggesting that protein accumulation was by fluid phase pinocytosis. Following an overnight uptake, 20-30% of the accumulated protein was subsequently regurgitated into the medium in a trichloroacetic acid/phosphotungstic acid-precipitable form. This material co-migrated with the authentic protein during molecular sieve chromatography on Sephadex G-50. The rates of appearance of trichloroacetic acid/phosphotungstic acid-insoluble products were greater than expected for cell death and leakage. The observed first order rate constants, kobs, for the appearance of trichloroacetic acid/phosphotungstic acid-soluble and trichloroacetic acid/phosphotungstic acid-insoluble products in the culture medium were identical, indicating that both products were released in parallel from MPM and BHK cells. The kobs for intracellular BSA degradation and regurgitation were independent of the initial BSA concentration in the uptake medium, but were decreased about 35% when degradation was allowed to proceed in the presence of high concentrations of serum. Degradation was also inhibited by chloroquine and pepstatin. Inhibition of degradation was accompanied by an increase in the total amount of regurgitated protein appearing in the medium. Remarkably, however, these inhibitors also decreased kobs for regurgitation, thereby preserving the similarity in the observed rate constants for the appearance of trichloroacetic acid/phosphotungstic acid-soluble and trichloroacetic acid/phosphotungstic acid-insoluble products. These and other results were inconsistent with desorption of proteins from the surface of the culture dish or the surface of cells as the source of the trichloroacetic acid/phosphotungstic acid-insoluble label appearing in the medium.(ABSTRACT TRUNCATED AT 400 WORDS)

Highlights

The uptake and degradaotifobnovine serum albumin to a degradative compartment, we suggest that the (BSA),bovine liver catalase, and rabbmituscle enolase transfer of extracellular proteins tloysosomes maynot have been studied in cultured mouse peritoneal mac- be unidirectional and that protein substrates within rophages (MPM) and baby hamster kidney fibroblasts the lysosome could reappear in the medium during a (BHK cells)
The radioactivity remaining in the cellular monolayer at the end of the experiment was multiplied by R, the fraction of the total label released by the cells during the preceding period which appeared in a trichloroacetic acid/ phosphotungstic acid-soluble form
This product was added to the totaltrichloroacetic acid/phosphotungstic acidsoluble radioactivity released during the period of observation

Summary

EXPERIMENTAL PROCEDURES

Preparation ofLabeledSubstrates-Bovine liver catalase and rabbit muscle enolase (Sigma) were analyzed by electrophoresis prior to labeling. Accumulation and Degradation of Labeled Substrates-At the beginning of an experiment, the old medium was removedby aspiration and either 1.0 ml (multiwell plates) or 3.0 ml(35 X 10-mm dishes) of fresh medium containing 50% fetal calf serum and a specific radiolabeled protein was added. This concentration of serum was found to reduce the nonspecific binding of labeled proteins to the surface of the plastic culture dishes and has been shown to stimulate pinocytosis by cultured macrophages (34). Spectrophotometric measurements were performed with either a GCA/McPherson Model 707 double-beam recording spectrophotometer or a Perkin-Elmer Model 320 double-beam spectrophotometer thermostated at 25.0 k 0.2 "C

RESULTS

Part 2. Effect of Droteinase inhibitors

DISCUSSION