Selective degradation of insulin within rat liver endosomes

Journal Article · Mon Jan 01 00:00:00 EST 1990 · Journal of Cell Biology; (USA)

DOI:https://doi.org/10.1083/jcb.110.1.35· OSTI ID:7105589

Doherty, II, J J; Kay, D G; Lai, W H; Posner, B I; Bergeron, J J ^[1]

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1. McGill Univ., Montreal (Canada)

To characterize the role of the endosome in the degradation of insulin in liver, we employed a cell-free system in which the degradation of internalized 125I-insulin within isolated intact endosomes was evaluated. Incubation of endosomes containing internalized 125I-insulin in the cell-free system resulted in a rapid generation of TCA soluble radiolabeled products (t1/2, 6 min). Sephadex G-50 chromatography of radioactivity extracted from endosomes during the incubation showed a time dependent increase in material eluting as radioiodotyrosine. The apparent Vmax of the insulin degrading activity was 4 ng insulin degraded.min-1.mg cell fraction protein-1 and the apparent Km was 60 ng insulin.mg cell fraction protein-1. The endosomal protease(s) was insulin-specific since neither internalized 125I-epidermal growth factor (EGF) nor 125I-prolactin was degraded within isolated endosomes as assessed by TCA precipitation and Sephadex G-50 chromatography. Significant inhibition of degradation was observed after inclusion of p-chloromercuribenzoic acid (PCMB), 1,10-phenanthroline, bacitracin, or 0.1% Triton X-100 into the system. Maximal insulin degradation required the addition of ATP to the cell-free system that resulted in acidification as measured by acridine orange accumulation. Endosomal insulin degradation was inhibited markedly in the presence of pH dissipating agents such as nigericin, monensin, and chloroquine or the proton translocase inhibitors N-ethylmaleimide (NEM) and dicyclohexylcarbodiimide (DCCD). Polyethylene glycol (PEG) precipitation of insulin-receptor complexes revealed that endosomal degradation augmented the dissociation of insulin from its receptor and that dissociated insulin was serving as substrate to the endosomal protease(s). The results suggest that as insulin is internalized it rapidly but incompletely dissociates from its receptor.

• OSTI ID: 7105589
• Journal Information: Journal of Cell Biology; (USA), Vol. 110:1; ISSN 0021-9525
• Country of Publication: United States
• Language: English

Related Subjects

59 BASIC BIOLOGICAL SCIENCES
INSULIN
METABOLISM
PEPTIDE HYDROLASES
ENZYME ACTIVITY
ATP
CHROMATOGRAPHY
ELECTRON MICROSCOPY
ENZYME INHIBITORS
EPIDERMIS
FRACTIONATION
GROWTH FACTORS
INHIBITION
IODINE 125
ISOTOPE DILUTION
LIVER
LTH
ORGANOIDS
RATS
RECEPTORS
TIME DEPENDENCE
TYROSINE
ULTRACENTRIFUGATION
AMINO ACIDS
ANIMAL TISSUES
ANIMALS
BETA DECAY RADIOISOTOPES
BODY
CARBOXYLIC ACIDS
CELL CONSTITUENTS
CENTRIFUGATION
DAYS LIVING RADIOISOTOPES
DIGESTIVE SYSTEM
ELECTRON CAPTURE RADIOISOTOPES
ENZYMES
EPITHELIUM
GLANDS
GONADOTROPINS
HORMONES
HYDROLASES
HYDROXY ACIDS
INTERMEDIATE MASS NUCLEI
IODINE ISOTOPES
ISOTOPE APPLICATIONS
ISOTOPES
MAMMALS
MEMBRANE PROTEINS
MICROSCOPY
MITOGENS
NUCLEI
NUCLEOTIDES
ODD-EVEN NUCLEI
ORGANIC ACIDS
ORGANIC COMPOUNDS
ORGANS
PEPTIDE HORMONES
PITUITARY HORMONES
PROTEINS
RADIOISOTOPES
RODENTS
SEPARATION PROCESSES
SKIN
TISSUES
TRACER TECHNIQUES
VERTEBRATES
550501* - Metabolism- Tracer Techniques