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A new technique has been developed to identify active proteinases in endosomes that does not require prior isolation of organelles and extraction of the active enzymes. [125I]Iodotyrosylalanyldiazomethane was reversibly conjugated to transferrin to selectively deliver it to endosomes. The protein was conjugated to the inhibitor via a disulphide bond using N-succinimidyl 3-(2-pyridyldithio)propionate. The inhibitor portion of the conjugate bound irreversibly to active cathepsins B and L, and subsequently the reacted enzymes were separated from the transferrin after SDS/PAGE under reducing conditions. Uptake of the protein-inhibitor conjugate and incorporation of inhibitor into cathepsins was blocked at 4 degreesC, demonstrating that the conjugate enters cells by receptor-mediated endocytosis. Furthermore, endocytosed transferrin-inhibitor conjugate could be recycled back to the extracellular medium and binding to the transferrin receptor could be blocked by native transferrin. Labelling of the enzymes was not blocked by incubating cells at 16 degreesC, consistent with the majority of the reagent being targeted to endosomes. The inhibited enzymes remained conjugated to transferrin, showing that the disulphide bond between the transferrin and inhibitor was not reduced in the endosome. Results from these studies show that endosomes contain both intermediate and late biosynthetic forms of active cathepsin B, which are indistinguishable from those found in mature lysosomes. These results indicate that the active enzymes in endosomes are not early biosynthetic forms in transit to lysosomes but most probably enter the endosome via retrograde traffic from the lysosome.

Design of a transferrin–proteinase inhibitor conjugate to probe for active cysteine proteinases in endosomes