EFFECT OF RATE OF INTRACELLULAR-TRANSPORT AND DIACYTOSIS ON CYTOTOXICITY OF HYBRID TOXINS - STUDY WITH HYBRIDS USING HEPATIC ASIALOGLYCOPROTEIN RECEPTOR-MEDIATED ENDOCYTOSIS

The effects of diacytosis and intracellular transport rate on cytotoxi city of hybrid toxins were studied with conjugates of diphtheria toxin fragment A (DTA) to asialoorosomucoid (ASOR) and its reduced and carb oxymethylated cyanogen bromide fragment I (RC-ASCNBr-I) in cultured ra t hepatocytes. In the hepatocytes the kinetics of uptake of the conjug ate of asialoorosomucoid (DTA-ASOR) and that of the conjugate of the c yanogen bromide fragment (DTA-RC-ASCNBr-I) were quite similar, but the rate of accumulation of DTA moiety into the lysosomes, as determined by Percoll density gradient centrifugation, was found to be greater fo r the latter than the former. However, after internalization, DTA-RC-A SCNBr-I was diacytosed to a lesser extent than that of DTA-ASOR, parti cularly when colchicine was present during internalization. Analysis o f the subunits of DTA-ASOR internalized by the hepatocytes indicated t hat they were accumulated disproportionately in a time-dependent manne r so that the glycoprotein moiety was accumulated progressively more t han the toxin moiety. Cytotoxicity of DTA-ASOR toward the hepatocytes was 2-times as much as that of DTA-RC-ASCNBr-I. Colchicine enhanced th e toxicity of DTA-RC-ASCNBr-I (33-fold) to a greater extent than that of DTA-ASOR (12-fold). The difference in enhancement by colchicine was also observed in the rate of cell intoxication by the conjugates. Bot h conjugates were more toxic to the hepatocytes after incubation with the cells at 18 degrees C than at 37 degrees C. In the presence of van adate (0.2 mM), which enhanced diacytosis, toxicity of DTA-ASOR decrea sed by 5-fold. After incubation with the hepatocytes, a partial dissoc iation of DTA-ASOR was found to occur independently of the receptor-me diated endocytosis. Taken together, these results indicate that diacyt osis, subunit dissociation and rapid transport of conjugate toward lys osomes affect kinetically the rate of accumulation of the conjugate in to a yet unidentified compartment of toxin translocation.