Expression and characterization of frog peptidylglycine alpha-hydroxylating monooxygenase.
We report here a recombinant Chinese hamster ovary cell system,which is able to stably express frog peptidylglycine alpha-hydroxylating monooxygenase (PHM, EC 1.14.17.3), the first enzyme responsible for the formation of peptide C-terminal amide. This system excreted PHM mostly into the medium and almost no PHM activity was detected in the cell lysate. Three differentiation inducers were examined to determine whether or not they would enhance the PHM expression. Addition of 4mM sodium butyrate into the medium increased the expression of PHM activity about 4-fold at 48 h after addition. Increases of about 2-fold were observed in the cases of sodium propionate or N,N(')-hexamethylene-bis-acetamide. Through a three-step purification procedure, we obtained 5mg purified PHM, which showed a single band at 40 kDa on SDS-PAGE, from 2-L of conventional monolayer culture medium. The reactions with three synthetic substrates, D-Tyr-Val-Gly, N-trinitrophenyl-D-Tyr-Val-Gly (TNPYVG), and hippuric acid (HA), were characterized. Of these, TNPYVG was the most active substrate. The pH optima for TNPYVG and HA were pH 5-6, while that for D-Tyr-Val-Gly was pH 7.5. There is a possibility that the substrate N-terminal structure may affect the interaction between the substrate and the enzyme catalytic site.
Takahashi
Kenichi
K
Department of Medical Biochemistry, Kurume University School of Medicine, 67 Asahi-machi, Kurume 830-0011, Japan.
Satani
Manabu
M
Harada
Saori
S
Noguchi
Masato
M
D000818:Animals; D001001:Anura / Q000235:genetics; D016466:CHO Cells; D006224:Cricetinae; D005260:Female; D015870:Gene Expression; D007700:Kinetics; D006899:Mixed Function Oxygenases / Q000737:chemistry / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism*; D009097:Multienzyme Complexes / Q000737:chemistry / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism*; D011994:Recombinant Proteins / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D013379:Substrate Specificity
D009097:Multienzyme Complexes; D011994:Recombinant Proteins; D006899:Mixed Function Oxygenases; C035189:peptidylglycine monooxygenase