Characterization of peptidylglycine alpha-amidating monooxygenase in bovine hypothalamus.
In many peptide hormones and neuropeptides, the carboxyl-terminal alpha-amide structure is essential in eliciting their biological activity. In the present study, an enzymatic activity capable of converting 4-dimethylaminoazobenzene-4'-sulfonyl-Gly-L-Phe-Gly(Dabsyl-Gly-Phe -Gly) to 4-dimethylaminoazo-benzene-4'-sulfonyl-Gly-L-Phe-NH2(Dabsyl- Gly-Phe-NH2) was investigated in bovine hypothalamus. The concentrations of copper ion and ascorbic acid required for maximal enzyme activity were 16 microM and 2 mM, respectively. Amidating activity showed a pH profile with two pH optima at acidic pH (around 6.0) and neutral pH (around 7.5). Kinetic studies with the enzyme obtained from bovine hypothalamus demonstrated two distinct Km and Vmax values. The first Km and Vmax values were 142.9 microM and 22.2 pmol/microgram/h and the second Km and Vmax values were 22.7 microM and 4.44 pmol/microgram/h, respectively. Two molecular forms of amidating activity were identified by size-exclusion chromatography and the molecular weight of the two enzymes were estimated to be 49 kDa and 69 kDa.
Chikuma
T
T
Department of Pharmaceutical Analytical Chemistry, Showa College of Pharmaceutical Sciences, Tokyo, Japan.
Kocha
T
T
Hanaoka
K
K
Kato
T
T
Ishii
Y
Y
Tanaka
A
A
D000595:Amino Acid Sequence; D000818:Animals; D001205:Ascorbic Acid / Q000494:pharmacology; D002417:Cattle; D002850:Chromatography, Gel; D003300:Copper / Q000494:pharmacology; D019327:Copper Sulfate; D004492:Edetic Acid / Q000494:pharmacology; D006863:Hydrogen-Ion Concentration; D007031:Hypothalamus / Q000201:enzymology*; D007700:Kinetics; D006899:Mixed Function Oxygenases / Q000037:antagonists & inhibitors / Q000737:chemistry / Q000378:metabolism*; D008969:Molecular Sequence Data; D008970:Molecular Weight; D009097:Multienzyme Complexes; D010618:Phenanthrolines / Q000494:pharmacology
D009097:Multienzyme Complexes; D010618:Phenanthrolines; D003300:Copper; D004492:Edetic Acid; D006899:Mixed Function Oxygenases; C035189:peptidylglycine monooxygenase; D019327:Copper Sulfate; D001205:Ascorbic Acid; C025205:1,10-phenanthroline