The Digestive Function of Lysosomes as Studied by the Turnover of Ingested Foreign Macromolecules

The persistence of small foreign molecules in lysosomes described by Dr Dingle stands quite in contrast with the fast turnover of foreign macromolecules captured by these organelles. Since Dr de Duve chose to talk today about peroxysomes rather than lysosomes, it might perhaps add to the Discussion to present some data related to this important function, i. e. the ability of lysosomes to digest foreign macromolecular material. As part of our study of the penetration of macro­molecules into mammalian cells (Ryser 1968), we have been studying for some time the fate of 131I albumin ingested by Sarcoma S180 cells in culture (Ryser 1968; Gabathuler & Ryser 1967). Cells are first labelled with albumin under conditions enhancing albumin penetration (Ryser & Hancock 1965). They are then washed and re-incubated in label-free medium. The fate of the ingested albumin can be followed in two ways; first, by measuring as a function of time the decrease in specific activity of the cellular proteins; secondly, by measuring the radioactivity appearing with time in the re-incubation medium. Figure 60 shows a result of the first type. The value given on the logarithmic ordinate at time zero corresponds to the level of albumin uptake obtained during the labelling period. The loss of acid-insoluble label from the cells is represented by the descending straight lines. This loss is biphasic. Although the second phase has a rather flat slope, it cannot be accounted for by an isotope dilution due to cell growth in vitro . Half-lives of ingested albumin can easily be read from such diagrams, and turn out to be of the order of 50 to 90 min. The rate of breakdown can be influenced to some extent by changing conditions of re-incubation as shown by the difference between the two curves of figure 60, due to a different serum concentration in the medium. Simultaneously with the loss of acid-insoluble activity from the cells, there is a pro­gressive appearance of radioactivity in the re-incubation medium. It was of interest to determine the molecular size of the label appearing in the medium by means of gel filtration on Sephadex G 75. Figure 61 shows the elution diagrams of samples taken from the re-incubation medium at three different times. In all three diagrams there is a strong initial peak of activity excluded by the gel and corres­ponding, therefore, either to unchanged or only slightly modified albumin. It is not yet clear whether this peak results from a desorption of surface-bound albumin or to a regurgitation of ingested material or both. In the upper diagram (sample taken at time zero), radioactivity is found in this initial peak only. As time goes on, however, activity of a different form appears in the medium, as indicated by the occurrence of a second peak, which coincides with the elution of the small-sized marker phenol red (PhR). This peak corresponds to free tyrosin or a dipeptide containing tyrosin. It is evident, therefore, that ingested albumin is broken down not only rapidly, but also thoroughly to the level of free amino acids or dipeptides. Similar evidence has been obtained with phagocytic cells (Mego, Bertini & McQueen 1967; Ehrenreich & Cohn 1968; Unanue & Askonas 1968). Electron microscopy of normal and tumour cells indicates that ingested foreign protein such as ferritin, haemoglobin, peroxydase and 125I-albumin are essentially localized in secondary lysosomes (multivesicular bodies, phagosomes) (Maunsbach 1966; Neustein & Maunsbach 1966; Friend & Farquhar 1967; J. P. Revel & H. J. -P. Ryser 1967, unpublished data; Ryser, Caulfield & Aub 1962). It can be assumed, therefore, that the intracellular degradation of these proteins takes place in these structures.