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PMID: 1520272 Published · ppublish English Journal Article Research Support, Non-U.S. Gov't

Kinetic analysis of the type-1 proinsulin endopeptidase by a monoclonal antibody-based immunoadsorbent assay.

The Biochemical journal ·Vol. 286 ( Pt 1) ·1992-08-15 ·Pages 223-9

Bailyes EM, Hutton JC

Abstract

A simple, rapid and sensitive assay for the type-1 endopeptidase (Arg-Arg cleaving) was developed by using an antiproinsulin monoclonal immunoadsorbent to separate reaction products from the substrate. The values obtained by this assay were identical with those obtained by an h.p.l.c.-based procedure and yielded similar values for the pH optimum (5.6) and Ca2+ activation (K0.5 = 2 mM). It was shown that the type-1 endopeptidase was readily solubilized by Triton X-114 (87 +/- 3%, n = 12) and partitioned principally into the aqueous phase at 30 degrees C (90.1 +/- 2.6%, n = 12). Activity was lost on gel filtration, but could be restored by adenosine 5'-[gamma-thio]triphosphate (K0.5 = 6 microM), 50 microM-dithiothreitol or 50 microM-Ca(2+)-trans-1,2-diaminocyclohexane-NNN'N'-tetra-acetic acid (CDTA), indicating that the enzyme was particularly sensitive to heavy metal ions. The Km obtained with proinsulin as substrate (13 +/- 1.7 microM) indicated that the enzyme works at close to its Vmax. in the nascent secretory granule. The Vmax. of the enzyme prepared from insulin granules (0.6% proinsulin converted/min) corresponded closely to the rate measured in vivo in rat islets. The type-1 endopeptidase also appears to be capable of binding to proinsulin in the region of the C-peptide/A-chain junction, since a peptide spanning this region was found to inhibit the 125I-proinsulin processing measured by this assay.

MeSH Terms
Amino Acid Sequence Animals Antibodies, Monoclonal Endopeptidases/analysis,metabolism Humans Immunosorbent Techniques Insulinoma/enzymology Kinetics Macromolecular Substances Molecular Sequence Data Pancreatic Neoplasms/enzymology Peptides/chemical synthesis,pharmacology Proinsulin/metabolism Protein Processing, Post-Translational Rats Substrate Specificity Thermodynamics
Chemicals
Antibodies, Monoclonal Macromolecular Substances Peptides Proinsulin Endopeptidases proinsulin endopeptidase I
Authors & Affiliations
2 authors, click to expand affiliations / ORCID
Bailyes E M
Department of Clinical Biochemistry, University of Cambridge, Addenbrookes Hospital, U.K.
Hutton J C
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Article Info
Journal
The Biochemical journal
Abbr.
Biochem J
ISSN
0264-6021
DOI
10.1042/bj2860223
Published
1992-08-15
Pages
223-9
Language
English
Region
England
NLM ID
2984726R
PMCID
PMC1133043
Subset
IM
Grants
Wellcome Trust · United Kingdom
External Links
PubMed source DOI: 10.1042/bj2860223 PMC Full Text View journal details
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