Posttranslational processing of carboxypeptidase E, a neuropeptide-processing enzyme, in AtT-20 cells and bovine pituitary secretory granules

Lloyd D. Fricker, Lakshmi Devi

Research output: Contribution to journalArticle

23 Citations (Scopus)

Abstract

Carboxypeptidase E (CPE) functions in the posttranslational processing of peptide hormones and neurotransmitters. Like other peptide processing enzymes, CPE is present in secretory granules in soluble and membrane-associated forms that arise from posttranslational processing of a single precursor, "proCPE." To identify the intracellular site of proCPE processing, the biosynthesis and posttranslational processing were investigated in the mouse anterior pituitary-derived cell line, AtT-20. Following a 15-min pulse with [35S]Met, both soluble and membrane-bound forms of CPE were identified, indicating that the posttranslational processing event that generates these forms of CPE occurs in the endoplasmic reticulum or early Golgi apparatus. The relative proportion of soluble and membrane-bound forms of CPE changed when cells were chased for 2 h at 37°C but was unaffected when cells were chased at either 20 or 15°C, suggesting that further processing of membrane forms to the soluble form occurs in a post-Golgi compartment. Treatment of the cells with chloroquine did not alter the relative distribution of soluble and membrane forms, suggesting that an acidic compartment is not required for this processing event. Overexpression of CPE did not influence the distribution of soluble and membrane forms of CPE, indicating that the CPE-processing enzymes are not rate-limiting. To examine directly CPE-processing enzymes, bovine anterior pituitary secretory vesicles were isolated. An enzyme activity that releases the membrane-bound form of CPE was detected in the purified secretory vesicle membranes. This enzyme, which removes the C-terminal region of CPE, is partially inhibited by EDTA and phenylmethylsulfonyl fluoride and is activated by CaCl2. Together, the data indicate that posttranslational processing of CPE occurs in secretory granules and that this activity may be mediated by a prohormone convertase-like enzyme.

Original languageEnglish (US)
Pages (from-to)1404-1415
Number of pages12
JournalJournal of Neurochemistry
Volume61
Issue number4
StatePublished - Oct 1993

Fingerprint

Carboxypeptidase H
Secretory Vesicles
Neuropeptides
Enzymes
Processing
Membranes
Cells
Proprotein Convertases
Phenylmethylsulfonyl Fluoride
Peptide Hormones
Biosynthesis
Chloroquine
Enzyme activity
Golgi Apparatus
Edetic Acid

Keywords

  • Carboxypeptidase B-like enzyme
  • Carboxypeptidase H
  • Enkephalin convertase
  • Neuropeptide biosynthesis
  • Prohormone convertase

ASJC Scopus subject areas

  • Biochemistry
  • Cellular and Molecular Neuroscience

Cite this

@article{86c618d821a54cec875c8db351ae4be1,
title = "Posttranslational processing of carboxypeptidase E, a neuropeptide-processing enzyme, in AtT-20 cells and bovine pituitary secretory granules",
abstract = "Carboxypeptidase E (CPE) functions in the posttranslational processing of peptide hormones and neurotransmitters. Like other peptide processing enzymes, CPE is present in secretory granules in soluble and membrane-associated forms that arise from posttranslational processing of a single precursor, {"}proCPE.{"} To identify the intracellular site of proCPE processing, the biosynthesis and posttranslational processing were investigated in the mouse anterior pituitary-derived cell line, AtT-20. Following a 15-min pulse with [35S]Met, both soluble and membrane-bound forms of CPE were identified, indicating that the posttranslational processing event that generates these forms of CPE occurs in the endoplasmic reticulum or early Golgi apparatus. The relative proportion of soluble and membrane-bound forms of CPE changed when cells were chased for 2 h at 37°C but was unaffected when cells were chased at either 20 or 15°C, suggesting that further processing of membrane forms to the soluble form occurs in a post-Golgi compartment. Treatment of the cells with chloroquine did not alter the relative distribution of soluble and membrane forms, suggesting that an acidic compartment is not required for this processing event. Overexpression of CPE did not influence the distribution of soluble and membrane forms of CPE, indicating that the CPE-processing enzymes are not rate-limiting. To examine directly CPE-processing enzymes, bovine anterior pituitary secretory vesicles were isolated. An enzyme activity that releases the membrane-bound form of CPE was detected in the purified secretory vesicle membranes. This enzyme, which removes the C-terminal region of CPE, is partially inhibited by EDTA and phenylmethylsulfonyl fluoride and is activated by CaCl2. Together, the data indicate that posttranslational processing of CPE occurs in secretory granules and that this activity may be mediated by a prohormone convertase-like enzyme.",
keywords = "Carboxypeptidase B-like enzyme, Carboxypeptidase H, Enkephalin convertase, Neuropeptide biosynthesis, Prohormone convertase",
author = "Fricker, {Lloyd D.} and Lakshmi Devi",
year = "1993",
month = "10",
language = "English (US)",
volume = "61",
pages = "1404--1415",
journal = "Journal of Neurochemistry",
issn = "0022-3042",
publisher = "Wiley-Blackwell",
number = "4",

}

TY - JOUR

T1 - Posttranslational processing of carboxypeptidase E, a neuropeptide-processing enzyme, in AtT-20 cells and bovine pituitary secretory granules

AU - Fricker, Lloyd D.

AU - Devi, Lakshmi

PY - 1993/10

Y1 - 1993/10

N2 - Carboxypeptidase E (CPE) functions in the posttranslational processing of peptide hormones and neurotransmitters. Like other peptide processing enzymes, CPE is present in secretory granules in soluble and membrane-associated forms that arise from posttranslational processing of a single precursor, "proCPE." To identify the intracellular site of proCPE processing, the biosynthesis and posttranslational processing were investigated in the mouse anterior pituitary-derived cell line, AtT-20. Following a 15-min pulse with [35S]Met, both soluble and membrane-bound forms of CPE were identified, indicating that the posttranslational processing event that generates these forms of CPE occurs in the endoplasmic reticulum or early Golgi apparatus. The relative proportion of soluble and membrane-bound forms of CPE changed when cells were chased for 2 h at 37°C but was unaffected when cells were chased at either 20 or 15°C, suggesting that further processing of membrane forms to the soluble form occurs in a post-Golgi compartment. Treatment of the cells with chloroquine did not alter the relative distribution of soluble and membrane forms, suggesting that an acidic compartment is not required for this processing event. Overexpression of CPE did not influence the distribution of soluble and membrane forms of CPE, indicating that the CPE-processing enzymes are not rate-limiting. To examine directly CPE-processing enzymes, bovine anterior pituitary secretory vesicles were isolated. An enzyme activity that releases the membrane-bound form of CPE was detected in the purified secretory vesicle membranes. This enzyme, which removes the C-terminal region of CPE, is partially inhibited by EDTA and phenylmethylsulfonyl fluoride and is activated by CaCl2. Together, the data indicate that posttranslational processing of CPE occurs in secretory granules and that this activity may be mediated by a prohormone convertase-like enzyme.

AB - Carboxypeptidase E (CPE) functions in the posttranslational processing of peptide hormones and neurotransmitters. Like other peptide processing enzymes, CPE is present in secretory granules in soluble and membrane-associated forms that arise from posttranslational processing of a single precursor, "proCPE." To identify the intracellular site of proCPE processing, the biosynthesis and posttranslational processing were investigated in the mouse anterior pituitary-derived cell line, AtT-20. Following a 15-min pulse with [35S]Met, both soluble and membrane-bound forms of CPE were identified, indicating that the posttranslational processing event that generates these forms of CPE occurs in the endoplasmic reticulum or early Golgi apparatus. The relative proportion of soluble and membrane-bound forms of CPE changed when cells were chased for 2 h at 37°C but was unaffected when cells were chased at either 20 or 15°C, suggesting that further processing of membrane forms to the soluble form occurs in a post-Golgi compartment. Treatment of the cells with chloroquine did not alter the relative distribution of soluble and membrane forms, suggesting that an acidic compartment is not required for this processing event. Overexpression of CPE did not influence the distribution of soluble and membrane forms of CPE, indicating that the CPE-processing enzymes are not rate-limiting. To examine directly CPE-processing enzymes, bovine anterior pituitary secretory vesicles were isolated. An enzyme activity that releases the membrane-bound form of CPE was detected in the purified secretory vesicle membranes. This enzyme, which removes the C-terminal region of CPE, is partially inhibited by EDTA and phenylmethylsulfonyl fluoride and is activated by CaCl2. Together, the data indicate that posttranslational processing of CPE occurs in secretory granules and that this activity may be mediated by a prohormone convertase-like enzyme.

KW - Carboxypeptidase B-like enzyme

KW - Carboxypeptidase H

KW - Enkephalin convertase

KW - Neuropeptide biosynthesis

KW - Prohormone convertase

UR - http://www.scopus.com/inward/record.url?scp=0027482427&partnerID=8YFLogxK

UR - http://www.scopus.com/inward/citedby.url?scp=0027482427&partnerID=8YFLogxK

M3 - Article

C2 - 8376994

AN - SCOPUS:0027482427

VL - 61

SP - 1404

EP - 1415

JO - Journal of Neurochemistry

JF - Journal of Neurochemistry

SN - 0022-3042

IS - 4

ER -