Lysosomal sequestration of intracellular protein as a regulatory step in hepatic proteolysis

W. F. Ward, J. R. Cox, G. E. Mortimore

Research output: Contribution to journalArticle

30 Citations (Scopus)

Abstract

Livers from fed rats previously injected with a complex of iron, sorbitol, and citrate were perfused for 60 min with and without additions of insulin and amino acids in amounts known to suppress total proteolysis maximally (50 to 60% inhibition). The iron treatment caused a generalized increase in lysosomal density, and at a total dose of 50 mg/100-g rat, more than 40% of the lysosomal marker enzyme, N-acetyl-β-D-glucosaminidase, which was applied to sucrose density gradients was recovered in gradient fractions free from other organelles. The purified lysosomal fractions had an enzyme enrichment of about 33 and the overall recovery was nearly 15%. In control perfused livers, the distribution of the marker enzyme activity was distinctly bimodal, a pattern previously observed in control perfused livers from untreated rats. The denser of the two peaks comprised 22% of the total enzyme activity on the gradient and was lowered significantly by additions of insulin and amino acids during perfusion. A similar effect of treatment was observed on the protein content of the dense peak. Insulin and amino acids reduced the quantity of protein by nearly 40%, an effect equivalent to 0.52 mg of protein/g of liver. No net protein or enzyme alterations were observed in the lighter lysosomal fractions. In control perfused livers previously labeled with L-[1-14C]valine in vivo, the specific radioactivities of protein in the dense peak were about 2-fold higher than the average values of supernatant and mitochondrial proteins, but were comparable to those in microsomal fractions. As in the above experiments, additions of insulin and amino acids during perfusion significantly lowered the specific radioactivity of the dense lysosomal peak without affecting lighter fractions. These and the foregoing observations are consistent with the notion that proteins associated with the endoplasmic reticulum are sequestered by lysosome during control perfusion and that the process is suppressible by insulin and amino acids. The subsequent degradation of sequestered proteins could in part explain the increase in proteolysis that occurs under the same conditions.

Original languageEnglish (US)
Pages (from-to)6955-6961
Number of pages7
JournalJournal of Biological Chemistry
Volume252
Issue number19
StatePublished - 1977
Externally publishedYes

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Proteolysis
Liver
Insulin
Amino Acids
Enzymes
Proteins
Perfusion
Rats
Radioactivity
Enzyme activity
Hexosaminidases
Enzyme inhibition
Mitochondrial Proteins
Valine
Lysosomes
Citric Acid
Endoplasmic Reticulum
Organelles
Sucrose
Iron

ASJC Scopus subject areas

  • Biochemistry

Cite this

Lysosomal sequestration of intracellular protein as a regulatory step in hepatic proteolysis. / Ward, W. F.; Cox, J. R.; Mortimore, G. E.

In: Journal of Biological Chemistry, Vol. 252, No. 19, 1977, p. 6955-6961.

Research output: Contribution to journalArticle

Ward, W. F. ; Cox, J. R. ; Mortimore, G. E. / Lysosomal sequestration of intracellular protein as a regulatory step in hepatic proteolysis. In: Journal of Biological Chemistry. 1977 ; Vol. 252, No. 19. pp. 6955-6961.
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abstract = "Livers from fed rats previously injected with a complex of iron, sorbitol, and citrate were perfused for 60 min with and without additions of insulin and amino acids in amounts known to suppress total proteolysis maximally (50 to 60{\%} inhibition). The iron treatment caused a generalized increase in lysosomal density, and at a total dose of 50 mg/100-g rat, more than 40{\%} of the lysosomal marker enzyme, N-acetyl-β-D-glucosaminidase, which was applied to sucrose density gradients was recovered in gradient fractions free from other organelles. The purified lysosomal fractions had an enzyme enrichment of about 33 and the overall recovery was nearly 15{\%}. In control perfused livers, the distribution of the marker enzyme activity was distinctly bimodal, a pattern previously observed in control perfused livers from untreated rats. The denser of the two peaks comprised 22{\%} of the total enzyme activity on the gradient and was lowered significantly by additions of insulin and amino acids during perfusion. A similar effect of treatment was observed on the protein content of the dense peak. Insulin and amino acids reduced the quantity of protein by nearly 40{\%}, an effect equivalent to 0.52 mg of protein/g of liver. No net protein or enzyme alterations were observed in the lighter lysosomal fractions. In control perfused livers previously labeled with L-[1-14C]valine in vivo, the specific radioactivities of protein in the dense peak were about 2-fold higher than the average values of supernatant and mitochondrial proteins, but were comparable to those in microsomal fractions. As in the above experiments, additions of insulin and amino acids during perfusion significantly lowered the specific radioactivity of the dense lysosomal peak without affecting lighter fractions. These and the foregoing observations are consistent with the notion that proteins associated with the endoplasmic reticulum are sequestered by lysosome during control perfusion and that the process is suppressible by insulin and amino acids. The subsequent degradation of sequestered proteins could in part explain the increase in proteolysis that occurs under the same conditions.",
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