Receptor-mediated endocytosis of enterokinase by rat liver. Preliminary characterisation of low-density endosomes.
The endocytosis of enterokinase by rat hepatocytes has been studied both in a perfused liver system and in the intact, anaesthetised animal. 10 min after administration of the enzyme, only 70% of the activity was cleared by the perfused liver, whereas clearance was total in the intact animal. In both cases, about 85% of the internalised enzyme co-purified with the smooth microsomes and virtually all (more than 90%) of the catalytic activity was latent and could only be detected in the presence of detergent. After 10 min, 22.5% of the activity remained with the sinusoidal plasma membrane in the case of the perfused liver, while for the intact animal this figure was only 10%, confirming the more efficient clearance of enterokinase in the intact animal. Further subcellular fractionation showed that in the anaesthetised animal 8% of the internalised enzyme was associated with a low-density Golgi-like endosomal compartment (prepared from the mitochondrial pellet), whereas the corresponding value for the perfused liver was only 2.5%. Enterokinase specific activity was also up to 50-times greater in the low-density endosomes prepared from the intact animal. A second low-density Golgi-like compartment (purified from the smooth microsomes) also contained latent enterokinase, which together with the endosomes derived from the mitochondria accounted for 20% of the total enterokinase internalised by the liver 10 min after its administration to the intact animal. The passage of enterokinase through these two low-density compartments was shown not to be synchronous with its passage through the peripheral (sinusoidal membrane) and internal endosomes (smooth microsomes). There were qualitative differences in marker enzymes and polypeptide composition between the mitochondria and microsome-derived low-density endosomes. The sub-fractionation of low-density fractions on shallow sucrose gradients revealed a complex enzyme and polypeptide heterogeneity both between and within fractions. There was an apparent density-dependent separation of enterokinase from galactosyltransferase and the asialoglycoprotein receptor which was coincident with marked changes in the polypeptide composition of the endosomal membranes, particularly in the 30-45 kDa range.
Grant
D
D
Department of Surgery, St. George's Hospital Medical School, London, U.K.
Siddiqui
S
S
Graham
J
J
D000818:Animals; D002458:Cell Fractionation; D004705:Endocytosis; D004765:Enteropeptidase / Q000378:metabolism*; D008099:Liver / Q000378:metabolism* / Q000648:ultrastructure; D008862:Microsomes, Liver / Q000378:metabolism / Q000648:ultrastructure; D009940:Organoids / Q000378:metabolism* / Q000648:ultrastructure; D051381:Rats; D011956:Receptors, Cell Surface / Q000378:metabolism*; D012697:Serine Endopeptidases / Q000378:metabolism*; D013347:Subcellular Fractions / Q000378:metabolism
D011956:Receptors, Cell Surface; D012697:Serine Endopeptidases; D004765:Enteropeptidase