Examination of the rate of peptide biosynthesis in neuroendocrine cell lines using a stable isotopic label and mass spectrometry

Fa Yun Che, Quan Yuan, Elena Kalinina, Lloyd D. Fricker

Research output: Contribution to journalArticle

11 Scopus citations

Abstract

The biosynthesis of neuroendocrine peptides is typically examined by following the rate of appearance of a radioactive amino acid into mature forms of peptides. In the present study, we labeled cell lines with L-leucine containing 10 deuterium residues (d10-Leu) and used mass spectrometry to measure the biosynthetic rate of γ-lipotropin in the AtT-20 cell line and insulin in the INS-1 cell line. After 3 h of labeling, both peptides show detectable levels of the d-labeled form in the cells and media. The relative levels of the d-labeled forms are greater in the media than in the cells, consistent with previous studies that found that newly synthesized peptides are secreted at a higher rate than older peptides under basal conditions. When AtT-20 cells were stimulated with KCI or forskolin, the ratio of d- to H-labeled γ-lipotropin in the medium decreased, suggesting that the older peptide was in a compartment that could be released upon the appropriate stimulation. Overexpression of proSAAS in AtT-20 cells reduced the ratio of d- to H-labeled γ-lipotropin, consistent with the proposed role of proSAAS as an endogenous inhibitor of prohormone convertase-1. Labeling with d 10-Leu was also used to test whether altering the pH of the secretory pathway with chloroquine affected the rate of peptide biosynthesis. In AtT-20 cells, 30 μM chloroquine for 3 or 6 h significantly reduced the rate of formation of γ-lipotropin in both cells and media. Similarly, INS-1 cells treated with 10, 30, or 60 μM chloroquine for 6 h showed a significant decrease in the rate of formation of insulin in both cells and media. These results are consistent with the acidic pH optima for peptide processing enzymes. Stable isotopic labeling with d10-Leu provides a sensitive method to examine the rate of peptide formation in neuroendocrine cell lines.

Original languageEnglish (US)
Pages (from-to)585-594
Number of pages10
JournalJournal of Neurochemistry
Volume90
Issue number3
DOIs
Publication statusPublished - Aug 1 2004

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Keywords

  • Carboxypeptidase
  • Gamma-lipotropin
  • Insulin
  • Prohormone convertase
  • Proinsulin
  • Proopiomelanocortin

ASJC Scopus subject areas

  • Biochemistry
  • Cellular and Molecular Neuroscience

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