The signal peptide of pro-opiomelancortin: validation of a specific radioimmunoassay.
The N-terminus portion of the POMC leader sequence (signal peptide) was synthesized, and an antiserum was raised against it. A radioimmunoassay was developed which is effective at a dilution of 1:500,000, and sensitive at less than 1 fmole/tube. Since leader sequences often exhibit structural homologies, and since synthetic peptides are not readily available, we resorted to an unusual procedure to establish specificity. This involved extraction of pituitary RNA, cell-free translation to produce the pre-prohormones, and purification by B-END and signal antibody affinity columns. The eluates were then tested by SDS gel electrophoresis and by multiple immunoprecipitations. All results showed that the signal antibody captured a single molecular species, approximately 30,000 in MW, which was also captured by the B-END column, and was immunoprecipitable by B-END and ACTH antisera. It therefore appears that this antibody selectively measures the POMC leader sequence and should be valuable in measuring the newly synthesized pre-prohormone.
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D010902:Pituitary Gland / Q000378:metabolism; D010908:Pituitary Hormones, Anterior / Q000138:chemical synthesis / Q000235:genetics*; D011333:Pro-Opiomelanocortin; D014176:Protein Biosynthesis; D011498:Protein Precursors / Q000138:chemical synthesis / Q000235:genetics*; D012313:RNA / Q000235:genetics; D011817:Rabbits; D011863:Radioimmunoassay; D012156:Reticulocytes / Q000378:metabolism
D010908:Pituitary Hormones, Anterior; D011498:Protein Precursors; D012313:RNA; D011333:Pro-Opiomelanocortin