Monoclonal antibodies to a proenkephalin A fusion peptide synthesized in Escherichia coli recognize novel proenkephalin A precursor forms.
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.
Spruce
B A
BA
Department of Biochemistry, Imperial College, South Kensington, London, United Kingdom.
Jackson
S
S
Lowry
P J
PJ
Lane
D P
DP
Glover
D M
DM
D000313:Adrenal Medulla / Q000032:analysis; D000818:Animals; D000911:Antibodies, Monoclonal / Q000276:immunology*; D000936:Antigen-Antibody Complex; D002417:Cattle; D002837:Chromaffin Granules / Q000032:analysis; D004745:Enkephalins / Q000032:analysis / Q000235:genetics / Q000276:immunology*; D000939:Epitopes / Q000032:analysis*; D004926:Escherichia coli / Q000235:genetics; D015151:Immunoblotting; D011498:Protein Precursors / Q000032:analysis / Q000235:genetics / Q000276:immunology*; D011993:Recombinant Fusion Proteins / Q000276:immunology*; D011994:Recombinant Proteins / Q000276:immunology*; D001616:beta-Galactosidase / Q000235:genetics
D000911:Antibodies, Monoclonal; D000936:Antigen-Antibody Complex; D004745:Enkephalins; D000939:Epitopes; D011498:Protein Precursors; D011993:Recombinant Fusion Proteins; D011994:Recombinant Proteins; C033660:preproenkephalin; D001616:beta-Galactosidase