Processing-independent radioimmunoanalysis: a general analytical principle applied to progastrin and its products.
Most peptide hormone assays measure only fully processed bioactive peptides. Such assays are unsuited to detect hormone gene expression by alternative or attenuated prohormone processing (tissue- or cell-specific processing). The gastrin system is expressed in several different tissues and is therefore useful for studies of tissue-specific processing. Consequently we have developed a simple processing-independent radioimmunoanalysis for progastrin. Using antisera against the NH2-terminus of a sequence, devoid of processing sites (preprogastrin76-86) after trypsination of neighboring cleavage sites, the assay quantitates the mRNA product irrespective of degree of processing. Used together with a conventional assay for the mature carboxyamidated gastrins, the processing-independent analysis shows that in different tissues only 1 to 55% of the total translation product is processed to bioactive gastrins. Thus processing-independent analysis greatly improves the detection of gastrin gene expression at the peptide level. The principle of the assay should be applicable to all protein and peptide systems.
Bardram
L
L
University Department of Gastroenterology C, Rigshospitalet, Copenhagen, Denmark.
Rehfeld
J F
JF
D000595:Amino Acid Sequence; D002850:Chromatography, Gel; D005753:Gastric Mucosa / Q000032:analysis; D015408:Gastrinoma / Q000032:analysis; D005755:Gastrins / Q000032:analysis* / Q000235:genetics; D006801:Humans; D008175:Lung Neoplasms / Q000032:analysis; D008969:Molecular Sequence Data; D010446:Peptide Fragments / Q000032:analysis; D011498:Protein Precursors / Q000032:analysis* / Q000235:genetics; D011499:Protein Processing, Post-Translational; D011863:Radioimmunoassay / Q000379:methods*; D014357:Trypsin
D005755:Gastrins; D010446:Peptide Fragments; D011498:Protein Precursors; C013706:big gastrin; D014357:Trypsin