A processing-independent assay for human procholecystokinin and its products.
In order to develop a processing-independent analysis for procholecystokinin (proCCK) and its products, antibodies were raised against the synthetic fragment 62-71 of human proCCK. All rabbits (n = 8) responded to the immunization. One (No. 89,009) produced antibodies of particularly high titer (1:350,000), homogeneity (Sips' index approximately 1.0) and binding affinity (K0 eff approximately 0.88 x 10(12) l/mol). A radioimmunoassay using this antiserum and [125I]tyrosine-extended fragment 62-71 measured the total CCK mRNA product after tryptic cleavage at Lys61 in normal and neoplastic tissue independent of the degree of precursor processing. In addition to previously known CCK producing tumors, CCK was found also in a thoracic round-cell tumor (Askin tumor) and in brain tumors (gliomas and astrocytomas). These tumors processed proCCK poorly. Thus, they contained 11 and 23 (mean n = 5) pmol/g of proCCK and its products before, versus 71 and 99 (mean) pmol/g after tryptic cleavage, respectively. Accordingly, gel chromatography revealed significant amounts of unprocessed proCCK, large molecular forms of glycine-extended CCKs and the well-known carboxyamidated and tyrosine O-sulfated bioactive CCK-83, -58, -33, -22 and -8. We conclude that monospecific antibodies directed against the N-terminus of sequence 62-71 of human proCCK are suitable for processing-independent analysis (PIA) for proCCK and its products. Moreover, we suggest that such PIA should be used for quantitation of CCK gene expression at peptide level in normal tissue and tumors.
Paloheimo
L I
LI
Department of Clinical Biochemistry, Rigshospitalet, University of Copenhagen.
Rehfeld
J F
JF
D000595:Amino Acid Sequence; D000818:Animals; D000906:Antibodies; D000918:Antibody Specificity; D002766:Cholecystokinin / Q000032:analysis* / Q000737:chemistry / Q000235:genetics* / Q000276:immunology / Q000378:metabolism; D004285:Dogs; D015870:Gene Expression; D006801:Humans; D007457:Iodine Radioisotopes; D008969:Molecular Sequence Data; D009369:Neoplasms / Q000378:metabolism*; D010446:Peptide Fragments / Q000276:immunology; D011498:Protein Precursors / Q000032:analysis* / Q000737:chemistry / Q000276:immunology; D012333:RNA, Messenger / Q000032:analysis*; D011863:Radioimmunoassay; D017385:Sequence Homology
D000906:Antibodies; D007457:Iodine Radioisotopes; D010446:Peptide Fragments; D011498:Protein Precursors; D012333:RNA, Messenger; C060249:procholecystokinin; D002766:Cholecystokinin