Degradation of native and modified forms of fructose-bisphosphate aldolase microinjected into HeLa cells.
The uptake and degradation of radiolabelled rabbit muscle fructose-bisphosphate aldolase (EC 4.1.2.13) was studied in HeLa cells microinjected by the erythrocyte ghost fusion system. Labelled aldolase was progressively modified by treatment with GSSG or N-ethylmaleimide (NEM) before microinjection to determine whether these agents, which inactivate and destabilize the enzyme in vitro, affect the half-life of the enzyme in vivo. Increasing exposure of aldolase to GSSG or NEM before microinjection increased the extent of aldolase transfer into the HeLa cells and decreased the proportion of the protein that could be extracted from the cells after water lysis. Some degradation of the GSSG- and NEM-inactivated aldolases was observed in the ghosts before microinjection; thus a family of radiolabelled proteins was microinjected in these experiments. In spite of the above differences, the 40 kDa subunit of each aldolase form was degraded with a half-life of 30 h in the HeLa cells. In contrast, the progressively modified forms of aldolase were increasingly susceptible to proteolytic action in vitro by chymotrypsin or by cathepsin B and in ghosts. These studies indicate that the rate of aldolase degradation in cells is not determined by attack by cellular proteinases that recognize vulnerable protein substrates; the results are most easily explained by a random autophagic process involving the lysosomal system.
Hopgood
M F
MF
CSIRO, Division of Human Nutrition, Adelaide, Australia.
Knowles
S E
SE
Bond
J S
JS
Ballard
F J
FJ
D002401:Cathepsin B / Q000494:pharmacology; D002918:Chymotrypsin / Q000494:pharmacology; D046508:Culture Techniques; D004591:Electrophoresis, Polyacrylamide Gel; D004910:Erythrocyte Membrane / Q000201:enzymology; D005033:Ethylmaleimide / Q000494:pharmacology; D005634:Fructose-Bisphosphate Aldolase / Q000378:metabolism*; D005978:Glutathione / Q000031:analogs & derivatives / Q000494:pharmacology; D019803:Glutathione Disulfide; D006207:Half-Life; D006367:HeLa Cells / Q000201:enzymology*; D006801:Humans; D008845:Microinjections; D010446:Peptide Fragments / Q000032:analysis; D011506:Proteins / Q000378:metabolism
D010446:Peptide Fragments; D011506:Proteins; D002918:Chymotrypsin; D002401:Cathepsin B; D005634:Fructose-Bisphosphate Aldolase; D005978:Glutathione; D005033:Ethylmaleimide; D019803:Glutathione Disulfide