A colorimetric assay for measuring peptidylglycine alpha-amidating monooxygenase using high-performance liquid chromatography.
In many peptide hormones and neuropeptides, the carboxy-terminal alpha-amide structure is essential in eliciting biological activity. In the present study, a rapid and sensitive assay method for the determination of peptidylglycine alpha-amidating monooxygenase (PAM) activity has been reported. This method is based on the monitoring of the absorption at 460 nm of 4-dimethylaminoazobenzene-4'-sulfonyl-Gly-L-Phe-NH2 (Dabsyl-Gly-Phe-NH2), enzymatically formed from the substrate 4-dimethylaminoazobenzene-4'-sulfonyl-Gly-L-Phe-Gly, after separation by high-performance liquid chromatography (HPLC) using a C-18 reversed-phase column by isocratic elution. This method is sensitive enough to measure Dabsyl-Gly-Phe-NH2 at concentrations as low as 1 pmol and yield highly reproducible results and requires less than 5 min per sample for separation and quantitation. The concentrations of copper and ascorbic acid required for maximal enzyme activity were 1 microM and 2 mM, respectively. The pH optimum for PAM activity was 5.0 to 5.5. The Km and Vmax values were respectively 3.5 microM and 100 pmol/micrograms/h with the use of enzyme extract obtained from bovine pituitary. By using this method, PAM activity could be readily detected in a single rat saliva. The sensitivity of this assay method will also aid in the effort to examine the regulation of in vivo PAM activity.
Chikuma
T
T
Department of Pharmaceutical Analytical Chemistry, Showa College of Pharmaceutical Sciences, Tokyo, Japan.
Hanaoka
K
K
Loh
Y P
YP
Kato
T
T
Ishii
Y
Y
D000595:Amino Acid Sequence; D000818:Animals; D002417:Cattle; D002851:Chromatography, High Pressure Liquid / Q000379:methods; D003124:Colorimetry / Q000379:methods; D007700:Kinetics; D008297:Male; D006899:Mixed Function Oxygenases / Q000737:chemistry*; D008969:Molecular Sequence Data; D009097:Multienzyme Complexes; D010902:Pituitary Gland / Q000201:enzymology; D051381:Rats; D011919:Rats, Inbred Strains; D012463:Saliva / Q000201:enzymology; D013379:Substrate Specificity
D009097:Multienzyme Complexes; D006899:Mixed Function Oxygenases; C035189:peptidylglycine monooxygenase