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Monoclonal antibodies to a proenkephalin A fusion peptide synthesized in Escherichia coli recognize novel proenkephalin A precursor forms

Spruce, Barbara A. and Jackson, Susan and Lowry, Philip J. and Lane, David P. and Glover, David M. (1988) Monoclonal antibodies to a proenkephalin A fusion peptide synthesized in Escherichia coli recognize novel proenkephalin A precursor forms. Journal of Biological Chemistry, 263 (36). pp. 19788-19795. ISSN 0021-9258. https://resolver.caltech.edu/CaltechAUTHORS:20200819-121045051

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Abstract

Monoclonal antibodies have been generated to a chimeric peptide comprised of Escherichia coli beta-galactosidase fused to the amino acid sequence 69-207 of human preproenkephalin A. Two monoclonal antibodies, PE-1 and PE-2, were identified by their ability to recognize the same segment of proenkephalin A fused to the cII gene product of the E. coli bacteriophage lambda. The binding domains of PE-1 and PE-2 have been broadly located, with respect to the primary translation product, within the amino acid sequences 152-207 and 84-131, respectively. Immunoblot analysis of total bovine adrenomedullary chromaffin granule lysate reveals PE-1 and PE-2 immunoreactive forms of observed molecular mass 35, 33, 29, 24, 22, and 15 kDa, and an 18-kDa PE-1 immunoreactive form. Separation of granule membranes from their contents reveals differential membrane association of these high molecular weight polypeptides. There is preliminary evidence that PE-1 may be detecting a subset of polypeptides where shortening from the NH2 terminus has occurred. We postulate that the 35-kDa form represents the intact bovine enkephalin precursor of predicted molecular mass 27.3 kDa. This experimental approach should be generally applicable to the generation of antibodies which will recognize intact peptide precursors together with their post-translational cleavage products.


Item Type:Article
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https://www.jbc.org/content/263/36/19788.abstractPublisherArticle
ORCID:
AuthorORCID
Glover, David M.0000-0003-0956-0103
Additional Information:© 1988 American Society for Biochemistry and Molecular Biology. (Received for publication, August 3, 1988) We are grateful to the following people for supplying invaluable reagents: the late Dr. E. Herbert for one of the human preproenkephalin cDNA clones; Dr. Kiyoshi Nagai for the vector pLcII; Drs. Rüther and Muller-Hill for the PUR vectors; and Dr. Dan Cutler for the p38 antiserum. In receipt of a Medical Research Council training fellowship and now supported by Medical Research Council Project Grant G8618653-CB. Supported by the Cancer Research Campaign. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
Funders:
Funding AgencyGrant Number
Medical Research Council (UK)G8618653-CB
Cancer Research CampaignUNSPECIFIED
Issue or Number:36
Record Number:CaltechAUTHORS:20200819-121045051
Persistent URL:https://resolver.caltech.edu/CaltechAUTHORS:20200819-121045051
Official Citation:B A Spruce, S Jackson, P J Lowry, D P Lane, and D M Glover Monoclonal antibodies to a proenkephalin A fusion peptide synthesized in Escherichia coli recognize novel proenkephalin A precursor forms. J. Biol. Chem. 1988 263: 19788-19795.
Usage Policy:No commercial reproduction, distribution, display or performance rights in this work are provided.
ID Code:105027
Collection:CaltechAUTHORS
Deposited By: George Porter
Deposited On:19 Aug 2020 22:18
Last Modified:19 Aug 2020 22:19

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