Document Detail


Expression and characterization of human bifunctional peptidylglycine alpha-amidating monooxygenase.
MedLine Citation:
PMID:  12699694     Owner:  NLM     Status:  MEDLINE    
Abstract/OtherAbstract:
We report the purification and characterization of human bifunctional peptidylglycine alpha-amidating monooxygenase (the bifunctional PAM) expressed in Chinese hamster ovary cells. PAM is in charge of the formation of the C-terminal amides of biologically active peptides. The bifunctional PAM possesses two catalytic domains in a single polypeptide, peptidylglycine alpha-hydroxylating monooxygenase (PHM, EC 1.14.17.3) and peptidylamidoglycolate lyase (PAL, EC 4.3.2.5). By introducing a stop codon at 835 Glu, we were able to eliminate the membrane-spanning domain in the C-terminal region and succeeded in purifying a soluble form of bifunctional PAM that was secreted into the medium. Through a three-step purification procedure, we obtained 0.3mg of the purified PAM, which showed a single band at 91 kDa on SDS-PAGE, from 1L of monolayer culture medium. Metals contained in the purified PAM were analyzed and chemical modifications were performed to gain insight into the mechanism of the PAL reaction. Inductively coupled plasma detected 0.62 mol of Zn(2+) and 1.25 mol of Cu(2+) per mol of bifunctional PAM. Further, the addition of 1mM EDTA reduced the PAL activity by about 50%, but the decreased activity was recovered by the addition of an excess amount of Zn(2+). In a series of chemical modifications, phenylglyoxal almost completely eliminated the PAL activity and diethyl pyrocarbonate suppressed activity by more than 70%. These findings implied that Arg and His residues might play crucial roles during catalysis.
Authors:
Manabu Satani; Kenichi Takahashi; Hiroshi Sakamoto; Saori Harada; Yasuhiko Kaida; Masato Noguchi
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Publication Detail:
Type:  Journal Article; Research Support, Non-U.S. Gov't    
Journal Detail:
Title:  Protein expression and purification     Volume:  28     ISSN:  1046-5928     ISO Abbreviation:  Protein Expr. Purif.     Publication Date:  2003 Apr 
Date Detail:
Created Date:  2003-04-17     Completed Date:  2003-12-16     Revised Date:  2006-11-15    
Medline Journal Info:
Nlm Unique ID:  9101496     Medline TA:  Protein Expr Purif     Country:  United States    
Other Details:
Languages:  eng     Pagination:  293-302     Citation Subset:  IM    
Affiliation:
Department of Medical Biochemistry, Kurume University School of Medicine, 67 Asahi-machi, Kurume 830-0011, Japan.
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MeSH Terms
Descriptor/Qualifier:
Amidine-Lyases / genetics,  metabolism
Animals
CHO Cells
Catalysis / drug effects
Copper / pharmacology
Cricetinae
Electrophoresis, Polyacrylamide Gel
Female
Gene Expression Regulation, Enzymologic
Humans
Mixed Function Oxygenases / genetics*,  isolation & purification,  metabolism*
Multienzyme Complexes / genetics*,  isolation & purification,  metabolism*
Recombinant Proteins / genetics,  isolation & purification,  metabolism
Zinc / pharmacology
Chemical
Reg. No./Substance:
0/Multienzyme Complexes; 0/Recombinant Proteins; 7440-50-8/Copper; 7440-66-6/Zinc; EC 1.-/Mixed Function Oxygenases; EC 1.14.17.3/peptidylglycine monooxygenase; EC 4.3.2.-/Amidine-Lyases; EC 4.3.2.5/peptidylamidoglycolate lyase

From MEDLINE®/PubMed®, a database of the U.S. National Library of Medicine


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