Examination of the rate of peptide biosynthesis in neuroendocrine cell lines using a stable isotopic label and mass spectrometry
文献类型：期刊论文
| 作者 | Che, FY; Yuan, Q; Kalinina, E; Fricker, LD |
| 刊名 | JOURNAL OF NEUROCHEMISTRY
 |
| 出版日期 | 2004 |
| 卷号 | 90期号:3页码:585-594 |
| 关键词 | carboxypeptidase gamma-lipotropin insulin prohormone convertase proinsulin proopiomelanocortin |
| 通讯作者 | Fricker, LD (reprint author), Yeshiva Univ Albert Einstein Coll Med, Dept Mol Pharmacol, 1300 Morris Pk Ave, Bronx, NY 10461 USA.,fricker@aecom.yu.edu |
| 英文摘要 | 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 (d(10)-Leu) and used mass spectrometry to measure the biosynthetic rate of gamma-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 KCl or forskolin, the ratio of d- to H-labeled gamma-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 gamma-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 mum chloroquine for 3 or 6 h significantly reduced the rate of formation of gamma-lipotropin in both cells and media. Similarly, INS-1 cells treated with 10, 30, or 60 mum 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 d(10)-Leu provides a sensitive method to examine the rate of peptide formation in neuroendocrine cell lines. |
| 学科主题 | Biochemistry & Molecular Biology; Neurosciences & Neurology |
| 类目[WOS] | Biochemistry & Molecular Biology ; Neurosciences |
| 关键词[WOS] | NEWLY SYNTHESIZED INSULIN ; QUANTITATIVE PROTEOMICS ; SECRETORY VESICLES ; ATT-20 CELLS ; IN-VIVO ; PROSAAS ; QUANTIFICATION ; OCCURS ; IDENTIFICATION ; NEUROPEPTIDES |
| 收录类别 | SCI |
| 语种 | 英语 |
| WOS记录号 | WOS:000222720500008 |
| 版本 | 出版稿 |
| 源URL | [http://202.127.25.143/handle/331003/2052]  |
| 专题 | 上海生化细胞研究所_上海生科院生化细胞研究所 |
| 推荐引用方式 GB/T 7714 | Che, FY,Yuan, Q,Kalinina, E,et al. Examination of the rate of peptide biosynthesis in neuroendocrine cell lines using a stable isotopic label and mass spectrometry[J]. JOURNAL OF NEUROCHEMISTRY,2004,90(3):585-594. |
| APA | Che, FY,Yuan, Q,Kalinina, E,&Fricker, LD.(2004).Examination of the rate of peptide biosynthesis in neuroendocrine cell lines using a stable isotopic label and mass spectrometry.JOURNAL OF NEUROCHEMISTRY,90(3),585-594. |
| MLA | Che, FY,et al."Examination of the rate of peptide biosynthesis in neuroendocrine cell lines using a stable isotopic label and mass spectrometry".JOURNAL OF NEUROCHEMISTRY 90.3(2004):585-594. |
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