Document Detail


Proteolytic processing of chromogranin A in purified insulin granules. Formation of a 20 kDa N-terminal fragment (betagranin) by the concerted action of a Ca2+-dependent endopeptidase and carboxypeptidase H (EC 3.4.17.10).
MedLine Citation:
PMID:  2822006     Owner:  NLM     Status:  MEDLINE    
Abstract/OtherAbstract:
The nature and subcellular localization of the enzymic activities responsible for the production of the 20 kDa protein betagranin from its 100 kDa chromogranin-A-like precursor was investigated in transplantable insulinoma tissue. [35S]Methionine-labelled precursor was converted by lysed insulin-secretory granules into betagranin and one or more proteins of 47 kDa, via intermediates in the 60-65 kDa range. Lysosome-enriched fractions also processed the precursor, but not into the peptides found in vivo; other fractions, including those enriched in Golgi, were inactive. Conversion of the precursor by granules was quantitative and the products were stable. Inhibitor studies showed that processing occurred by initial endoproteolytic cleavage at sites marked by pairs of basic amino acids, followed by removal of these by carboxypeptidase H. The endopeptidase activity appeared to be a novel metalloenzyme, with a markedly acidic pH optimum (4.8-5). It was inhibited by alanyl-L-lysyl-L-arginyl chloromethane (K0.5 = 1.3 microM), but to a much lesser extent by inhibitor analogues of processing sites defined by single or unpaired basic amino acid residues, e.g. alanyl-L-norleucyl-L-arginylchloromethane (K0.5 greater than 100 microM), leupeptin (K0.5 = 150 microM) and antipain (K0.5 = 40 microM). p-Chloromercuribenzoate (K0.5 = 13 microM), Hg2+ (K0.5 = 16 microM), Zn2+ (K0.5 = 0.8 mM) and vanadate (K0.5 = 7 microM) also abolished activity, as did various anions (SCN- greater than I- greater than Cl- greater than SO4(2-). Group-specific inhibitors of serine, thiol and acidic endopeptidases were without effect. EDTA and CDTA (1,2-cyclohexanediaminetetra-acetic acid), but not 1,10-phenanthroline, abolished endoproteolytic activity. Several bivalent cations could restore activity after EDTA or CDTA inhibition, including Ca2+, Zn2+, Mn2+ and Sr2+; however, the ion of physiological importance appeared to be Ca2+ (K0.5 = 8 microM). The properties of the granule endopeptidase and its subcellular localization suggested that it is of importance in processing chromogranin A in the pancreatic beta-cell.
Authors:
J C Hutton; H W Davidson; M Peshavaria
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Publication Detail:
Type:  Journal Article; Research Support, Non-U.S. Gov't    
Journal Detail:
Title:  The Biochemical journal     Volume:  244     ISSN:  0264-6021     ISO Abbreviation:  Biochem. J.     Publication Date:  1987 Jun 
Date Detail:
Created Date:  1987-11-05     Completed Date:  1987-11-05     Revised Date:  2009-11-19    
Medline Journal Info:
Nlm Unique ID:  2984726R     Medline TA:  Biochem J     Country:  ENGLAND    
Other Details:
Languages:  eng     Pagination:  457-64     Citation Subset:  IM    
Affiliation:
Department of Clinical Biochemistry, University of Cambridge, Addenbrooke's Hospital, U.K.
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MeSH Terms
Descriptor/Qualifier:
Adenoma, Islet Cell / metabolism*
Animals
Calcium / metabolism
Carboxypeptidase H
Carboxypeptidases / pharmacology
Cations, Divalent / pharmacology
Chemical Precipitation
Chromogranin A
Chromogranins / metabolism*
Cytoplasmic Granules / metabolism
Endopeptidases / pharmacology
Insulin / secretion*
Insulinoma / metabolism*
Nerve Tissue Proteins / metabolism*
Pancreatic Neoplasms / metabolism*
Peptide Fragments / metabolism
Protease Inhibitors / pharmacology
Rats
Chemical
Reg. No./Substance:
0/Cations, Divalent; 0/Chromogranin A; 0/Chromogranins; 0/Nerve Tissue Proteins; 0/Peptide Fragments; 0/Protease Inhibitors; 0/chromogranin A, rat; 11061-68-0/Insulin; 7440-70-2/Calcium; EC 3.4.-/Carboxypeptidases; EC 3.4.-/Endopeptidases; EC 3.4.17.10/Carboxypeptidase H
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