Recycling of 5'-nucleotidase in a rat hepatoma cell line.
Intracellular movement of cell surface 5'-nucleotidase was studied in H4S cells, a rat hepatoma cell line. Surface labelled cells were incubated for various periods at 37 degrees C and treated with neuraminidase at 0 degrees C. Removal of sialic acid residues from glycoproteins results in a change of their isoelectric points. Analysis with isoelectric focusing was then used to distinguish between cell surface and intracellular 5'-nucleotidase. Incubation of 125I-surface-labelled cells at 37 degrees C resulted in a gradual decrease of labelled 5'-nucleotidase at the plasma membrane until, at 60 to 90 min, a steady state was reached with 52% of the label on the cell surface and 48% intracellular. Pretreatment of the cells with the weak base primaquine had no influence on this distribution while at the same time uptake of iron via the transferrin receptor was inhibited. Using immunoelectron microscopy 5'-nucleotidase was found on the cell surface, in multivesicular endosomes and the Golgi complex. Preincubation of the cells in the presence of cycloheximide caused a reduction of labelling in the Golgi complex, whereas the label in the other compartments was retained. These results lead to the conclusion that 5'-nucleotidase does not recycle through the Golgi complex and that in contrast to the transferrin receptor the recycling of 5'-nucleotidase is not inhibited by primaquine.
van den Bosch
R A
RA
Department of Cell Biology, University of Utrecht, Medical School, The Netherlands.
du Maine
A P
AP
Geuze
H J
HJ
van der Ende
A
A
Strous
G J
GJ
D015720:5'-Nucleotidase; D000818:Animals; D002462:Cell Membrane / Q000187:drug effects / Q000201:enzymology*; D004591:Electrophoresis, Polyacrylamide Gel; D006056:Golgi Apparatus / Q000201:enzymology; D007150:Immunohistochemistry; D007425:Intracellular Membranes / Q000187:drug effects / Q000201:enzymology*; D007525:Isoelectric Focusing; D007526:Isoelectric Point; D008113:Liver Neoplasms; D008114:Liver Neoplasms, Experimental; D008854:Microscopy, Electron; D009708:Nucleotidases / Q000032:analysis / Q000378:metabolism*; D011233:Precipitin Tests; D011319:Primaquine / Q000494:pharmacology; D051381:Rats; D011990:Receptors, Transferrin / Q000187:drug effects / Q000378:metabolism; D014407:Tumor Cells, Cultured
D011990:Receptors, Transferrin; D009708:Nucleotidases; D015720:5'-Nucleotidase; D011319:Primaquine