title: Isolation and functional expression of human pancreatic peptidylglycine alpha-amidating monooxygenase.
abstract: Pancreastatin (PST) is processed from chromogranin A and the C-terminal amide of the peptide is an absolute requirement for biological activities. Human pancreatic carcinoma cells QGP-1 which produce both chromogranin A and PST were used to isolate cDNAs encoding two forms of peptidylglycine alpha-amidating monooxygenase (PAM). The two forms are a full length bifunctional enzyme and a variant lacking the transmembrane domain-coding region. When the cDNAs of these two forms were expressed in COS-7 cells, cells transfected with the predicted soluble form released into the culture medium a very much higher amidating activity which converts human chromogranin A-(273-302) to PST-29. The optimal pH for amidating activity was 5.4 and Cu2+, ascorbate and catalase were required as cofactors for the both forms of PAM. Km values for the membrane-bound and the soluble forms of PAM were 15.7 +/- 3.1 microM and 12.4 +/- 1.6 microM, respectively. These results demonstrate that both forms of PAM can function in the posttranslational processing of chromogranin A to PST in the environment of a secretory vesicle.
authors.affiliation: First Department of Biochemistry, School of Medicine, Fukuoka University, Japan.
authors.forename: K
authors.initials: K
authors.lastname: Tateishi
authors.forename: F
authors.initials: F
authors.lastname: Arakawa
authors.forename: Y
authors.initials: Y
authors.lastname: Misumi
authors.forename: A M
authors.initials: AM
authors.lastname: Treston
authors.forename: M
authors.initials: M
authors.lastname: Vos
authors.forename: Y
authors.initials: Y
authors.lastname: Matsuoka
mesh_terms: D000595:Amino Acid Sequence; D001483:Base Sequence; D002460:Cell Line; D002462:Cell Membrane / Q000201:enzymology; D018076:DNA, Complementary; D006801:Humans; D006899:Mixed Function Oxygenases / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism; D008969:Molecular Sequence Data; D009097:Multienzyme Complexes; D010179:Pancreas / Q000201:enzymology*; D012333:RNA, Messenger / Q000378:metabolism; D011994:Recombinant Proteins; D014407:Tumor Cells, Cultured
chemical_list: D018076:DNA, Complementary; D009097:Multienzyme Complexes; D012333:RNA, Messenger; D011994:Recombinant Proteins; D006899:Mixed Function Oxygenases; C035189:peptidylglycine monooxygenase