By 1963, lysosomes were well established as an in vitro degradative entity localized to a few fractions (de Duve et al., 1955; de Duve, 1963). But a corresponding in vivo classification was trickier due to the heterogeneity of structures seen in different cell types and within cells. Christian De Duve had grouped lysosome-like entities into a system of four types of compartments: enzyme-storing granules, digestive vacuoles for reabsorbing proteins, autolytic vacuoles, and residual bodies containing the remnants of digestion.
These compartments had enzymes such as acid phosphatase. But did the same compartments have both enzymes and meaningful protein substrates? The advent of lysosomal enzyme tests, which gave a lead precipitate reaction product visible by EM (Novikoff and Holt, 1957; Essner and Novikoff, 1961), gave Miller and Palade (1964) a method to test for colocalization.
The experiments also led to the observation that the cells did not store lytic enzymes, but rather “the enzyme might be produced when needed…and transported by small vesicles.” That assumption, the authors write, “implies the enzyme may well pass through some elements of the Golgi complex,” but the evidence so far for this theory “cannot be considered sufficient proof in [this] case.” This hypothesis would be raised more forcefully by Smith and Farquhar (1966) as they traced excess secretory proteins to the lysosome using the acid phosphatase test.