A radioimmunoassay for measuring alpha-amidating enzyme activity.
A sensitive alpha-amidating enzyme (alpha AE) assay using C-terminal glycine-extended substance P (SP-Gly) as a substrate was developed. The product, substance P (SP), was measured by a radioimmunoassay with specific polyclonal antibodies which recognize SP with an affinity 10,000-fold higher than that of SP-Gly. The sensitivity of the radioimmunoassay was 5 fmol. Enzyme activity could be readily detected with 25 ng alpha AE partially purified from the conditioned medium of rat medullary thyroid carcinoma CA-77 cells. The Km and Vmax values were 2.0 +/- 0.2 microM and 1.7 +/- 0.1 nmol/mg/min (mean +/- SE, n = 3), respectively. The assay enabled the kinetic characterization of alpha AE from a single rat pituitary homogenate. Optimal Cu2+ required was 30 microM and greater than 3 mM of ascorbate was needed for maximal enzyme activity. The sensitivity of this assay will aid efforts to examine the regulation of in vivo alpha AE activity.
Research Department, CIBA-GEIGY, Corporation, Summit, New Jersey 07901.
A Y
AY
Jeng
M
M
Wong
S J
SJ
Lovato
M D
MD
Erion
J P
JP
Gilligan
D000818:Animals; D002277:Carcinoma / Q000473:pathology; D003470:Culture Media; D006041:Goats; D007700:Kinetics; D008297:Male; D006899:Mixed Function Oxygenases; D009097:Multienzyme Complexes; D000587:Oxidoreductases Acting on CH-NH Group Donors / Q000378:metabolism*; D010902:Pituitary Gland / Q000201:enzymology; D011817:Rabbits; D011863:Radioimmunoassay; D051381:Rats; D011919:Rats, Inbred Strains; D013373:Substance P / Q000031:analogs & derivatives / Q000378:metabolism; D013964:Thyroid Neoplasms / Q000473:pathology; D014020:Tissue Extracts / Q000378:metabolism; D014407:Tumor Cells, Cultured
D003470:Culture Media; D009097:Multienzyme Complexes; D014020:Tissue Extracts; D013373:Substance P; C046072:substance P, Gly(12)-; D006899:Mixed Function Oxygenases; C035189:peptidylglycine monooxygenase; D000587:Oxidoreductases Acting on CH-NH Group Donors