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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.! _( E: W- q3 @
I( b5 R0 y5 L% m) pEnzymes (black deposits of reaction product) colocalize with substrates (ingested ferritin; small black particles) in lysosomes.1 c+ u4 [/ s2 f7 Q0 M K- B
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) G2 ?9 S# O* d! k u8 kFritz Miller and George Palade injected rats and mice with two proteins, hemoglobin and ferritin, that were readily recognizable by their characteristic density or shape; they were also known to end up in two lysosomal structures. Sections of the rodent kidney cells were also treated with the enzyme reactions in one of the first examples of combined cytochemistry and EM. The results were plainly obvious when the lead reaction products showed up alongside the foreign proteins "within the same membrane-bounded structures," they wrote.
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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 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.
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/ U8 b& h( E: d- _de Duve, C., et al. 1955. Biochem. J. 60:604–617.
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+ x' ~, I6 V7 d% F& Ide Duve, C. 1963. in Ciba Foundation Symposium on Lysosomes. A.V.S. de Reuck and M.P. Cameron, editors. London, J., and A. Churchill, Ltd.
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( x4 q- H7 t7 @0 O+ f+ H& ]" Y7 q% P4 uEssner, E., and A.B. Novikoff. 1961. J. Biophys. Biochem. Cytol. 9:773–784.
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Miller, F., and G.E. Palade. 1964. J. Cell Biol. 23:519–552.
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Novikoff, A.B., and S.J. Holt. 1957. J. Cell Biol. 3:127–129.
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Smith, R.F., and M.G. Farquhar. 1966. J. Cell Biol. 31:319–347.(By 1963, lysosomes were well established) |
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