Degradation of horseradish peroxidase after microinjection into mammalian cells

Journal Article · Fri Jan 01 04:00:00 EST 1988 · Exp. Cell Res.; (United States)

DOI:https://doi.org/10.1016/0014-4827(88)90160-7· OSTI ID:5513840

Knowles, S E; Hopgood, M F; Ballard, F J

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.

• Research Organization: CSIRO (Australia) Division of Human Nutrition, Adelaide
• OSTI ID: 5513840
• Journal Information: Exp. Cell Res.; (United States), Journal Name: Exp. Cell Res.; (United States) Vol. 174:1; ISSN ECREA
• Country of Publication: United States
• Language: English

Related Subjects

550501* -- Metabolism-- Tracer Techniques
59 BASIC BIOLOGICAL SCIENCES
ANIMAL CELLS
AUTORADIOGRAPHY
CELL CONSTITUENTS
CELL MEMBRANES
CONNECTIVE TISSUE CELLS
ENZYME ACTIVITY
ENZYMES
FIBROBLASTS
HELA CELLS
LABELLED COMPOUNDS
MEMBRANES
METABOLISM
MOLECULAR WEIGHT
MUSCLES
ORGANIC COMPOUNDS
OXIDOREDUCTASES
PEPTIDES
PEROXIDASES
PROTEINS
SOMATIC CELLS
TISSUE CULTURES
TRITIUM COMPOUNDS