Degradation of horseradish peroxidase after microinjection into mammalian cells.
Horseradish peroxidase (HRP) has been microinjected into mammalian cells in tissue culture by the erythrocyte ghost-mediated technique. This protein was selected because it can be localized and quantified after injection by cytochemical and spectrophotometric methods. HRP labeled by reductive methylation retained full catalytic activity, was efficiently loaded into erythrocyte ghosts, and did not associate to a significant degree with ghost membranes. A combination of cytochemical staining and autoradiography established that HRP injected into rat L6 myoblasts, HE(39)L human diploid fibroblasts, or HeLa cells was intracellular and uniformly distributed throughout the cell, while cell lysis techniques showed that the catalytically active HRP was not membrane bound. Inactivation of labeled HRP after injection paralleled the disappearance of the 40-kDa polypeptide, and was always more rapid than its overall degradation. This difference was associated with a pool of water-insoluble radioactivity in the injected cells. This material was of smaller molecular size than the native protein: many labeled peptides were detected in the range of 10 to 38 kDa. By the use of inhibitors of autophagic proteolysis or lysosomal function it was established that HRP degradation was not subjected quantitatively to the same regulatory processes as the average endogenous protein labeled in the same cultures.
Knowles
S E
SE
CSIRO (Australia) Division of Human Nutrition, Adelaide.
Hopgood
M F
MF
Ballard
F J
FJ
D000818:Animals; D002478:Cells, Cultured; D004910:Erythrocyte Membrane; D005347:Fibroblasts / Q000378:metabolism; D006367:HeLa Cells / Q000378:metabolism; D006735:Horseradish Peroxidase / Q000008:administration & dosage / Q000378:metabolism*; D006801:Humans; D007700:Kinetics; D008297:Male; D008845:Microinjections; D009132:Muscles / Q000166:cytology / Q000378:metabolism*; D010222:Parainfluenza Virus 1, Human / Q000235:genetics; D010544:Peroxidases / Q000378:metabolism*; D014316:Tritium
D014316:Tritium; D006735:Horseradish Peroxidase; D010544:Peroxidases