Effects of inhibitors on aldolase breakdown after its microinjection into HeLa cells.

1. The regulation of protein breakdown as well as the generation of intermediates in the pathway from intact protein to amino acids was investigated by using 3H-labelled N-ethylmaleimide-modified aldolase (NEM-aldolase) as an indicator protein after its microinjection into HeLa cells. 2. NEM-aldolase degradation to acid-soluble products proceeded at a slower rate than that of endogenously labelled total cell protein, and was inhibited to a greater extent by 3-methyladenine, leupeptin and NH4Cl. The combination of leupeptin plus NH4Cl was particularly effective, decreasing the NEM-aldolase breakdown rate by 90%. 3. Measurements of the loss of radioactivity from the aldolase band located from fluorograms after SDS/polyacrylamide-gel electrophoresis showed that NEM-aldolase breakdown was much more rapid when measured by this method. The effects of insulin, 3-methyladenine, leupeptin and NH4Cl on this breakdown were also substantial. 4. Substantial amounts of peptide intermediates in the breakdown pathway of NEM-aldolase accumulated in cells. The production of small intermediates (less than 30 kDa) accounted for approx. 40% of the NEM-aldolase degraded in control cultures. Addition of NH4Cl increased the proportion of these intermediates. Large intermediates, between 31 and 38 kDa, were particularly evident in the presence of the cysteine proteinase inhibitor leupeptin, but almost no small intermediates were detected. 5. The results are best explained by the degradation of NEM-aldolase being predominantly a lysosomal process, with cysteine proteinases involved in early proteolytic steps and other proteinases that have acid pH optima required for the complete catabolism of small intermediates.