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 Citations (Scopus)

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
StatePublished - Aug 2004

Fingerprint

Peptide Biosynthesis
Neuroendocrine Cells
Biosynthesis
Mass spectrometry
Labels
Mass Spectrometry
Cells
beta-Lipotropin
Cell Line
Peptides
Chloroquine
Labeling
Proprotein Convertase 1
Insulin
Deuterium
Secretory Pathway
Colforsin
Leucine

Keywords

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

ASJC Scopus subject areas

  • Biochemistry
  • Cellular and Molecular Neuroscience

Cite this

Examination of the rate of peptide biosynthesis in neuroendocrine cell lines using a stable isotopic label and mass spectrometry. / Che, Fa Yun; Yuan, Quan; Kalinina, Elena; Fricker, Lloyd D.

In: Journal of Neurochemistry, Vol. 90, No. 3, 08.2004, p. 585-594.

Research output: Contribution to journalArticle

@article{6d158178769b40f3a36097863954b234,
title = "Examination of the rate of peptide biosynthesis in neuroendocrine cell lines using a stable isotopic label and mass spectrometry",
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.",
keywords = "Carboxypeptidase, Gamma-lipotropin, Insulin, Prohormone convertase, Proinsulin, Proopiomelanocortin",
author = "Che, {Fa Yun} and Quan Yuan and Elena Kalinina and Fricker, {Lloyd D.}",
year = "2004",
month = "8",
doi = "10.1111/j.1471-4159.2004.02522.x",
language = "English (US)",
volume = "90",
pages = "585--594",
journal = "Journal of Neurochemistry",
issn = "0022-3042",
publisher = "Wiley-Blackwell",
number = "3",

}

TY - JOUR

T1 - Examination of the rate of peptide biosynthesis in neuroendocrine cell lines using a stable isotopic label and mass spectrometry

AU - Che, Fa Yun

AU - Yuan, Quan

AU - Kalinina, Elena

AU - Fricker, Lloyd D.

PY - 2004/8

Y1 - 2004/8

N2 - 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.

AB - 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.

KW - Carboxypeptidase

KW - Gamma-lipotropin

KW - Insulin

KW - Prohormone convertase

KW - Proinsulin

KW - Proopiomelanocortin

UR - http://www.scopus.com/inward/record.url?scp=3343013767&partnerID=8YFLogxK

UR - http://www.scopus.com/inward/citedby.url?scp=3343013767&partnerID=8YFLogxK

U2 - 10.1111/j.1471-4159.2004.02522.x

DO - 10.1111/j.1471-4159.2004.02522.x

M3 - Article

C2 - 15255936

AN - SCOPUS:3343013767

VL - 90

SP - 585

EP - 594

JO - Journal of Neurochemistry

JF - Journal of Neurochemistry

SN - 0022-3042

IS - 3

ER -