Characterization of a bifunctional peptidylglycine alpha-amidating enzyme expressed in Chinese hamster ovary cells.
Peptidylglycine alpha-amidating enzyme (alpha-AE) catalyzes the conversion of glycine-extended prohormones to their biologically active alpha-amidated forms. We have derived a clonal Chinese hamster ovary cell line that secretes significant quantities of active alpha-AE. Enzyme production was increased by selection for methotrexate-resistant cells expressing a dicistronic message. Amplification of the alpha-AE gene was monitored by Southern blot analysis, enzyme activity, and immunoreactive protein throughout the selection process. The soluble enzyme is bifunctional as determined by the ability to convert either the glycine-extended substrate, dansyl-Tyr--Val--Gly, or the intermediate, dansyl-Tyr--Val--alpha-hydroxyglycine, to the dansyl-Tyr--Val--NH2 product. The recombinant alpha-AE was purified by a simple two-step chromatographic process. The purified enzyme is partially glycosylated and the glycosylated and nonglycosylated forms of the enzyme were separated on a Con A-Sepharose column. The kinetic constants for dansyl-Tyr--Val--Gly, dansyl-Tyr--Val--alpha-hydroxyglycine, ascorbate, and catechol were the same for both forms of alpha-AE. In addition, mimosine is competitive vs ascorbate with K(is) = 3.5 microM for the nonglycosylated alpha-AE and K(is) = 4.2 microM for the glycosylated alpha-AE. Therefore, the presence or absence of asparagine-linked oligosaccharide does not affect the catalytic efficiency of the enzyme. Overexpression of the recombinant enzyme in CHO cells greatly enhances expression of the endogenous gene, implicating a feedback mechanism on the alpha-AE gene.
Miller
D A
DA
Unigene Laboratories, Fairfield, New Jersey 07004.
Sayad
K U
KU
Kulathila
R
R
Beaudry
G A
GA
Merkler
D J
DJ
Bertelsen
A H
AH
D000595:Amino Acid Sequence; D000818:Animals; D015152:Blotting, Northern; D015139:Blotting, Southern; D015153:Blotting, Western; D016466:CHO Cells; D002852:Chromatography, Ion Exchange; D002999:Clone Cells; D006224:Cricetinae; D004247:DNA / Q000235:genetics / Q000302:isolation & purification; D004591:Electrophoresis, Polyacrylamide Gel; D007700:Kinetics; D006899:Mixed Function Oxygenases / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D008969:Molecular Sequence Data; D009097:Multienzyme Complexes; D010957:Plasmids; D012313:RNA / Q000235:genetics / Q000302:isolation & purification; D051381:Rats; D011994:Recombinant Proteins / Q000302:isolation & purification / Q000378:metabolism; D013379:Substrate Specificity; D013762:Tetrahydrofolate Dehydrogenase / Q000235:genetics / Q000378:metabolism; D014162:Transfection
D009097:Multienzyme Complexes; D011994:Recombinant Proteins; D012313:RNA; D004247:DNA; D006899:Mixed Function Oxygenases; C035189:peptidylglycine monooxygenase; D013762:Tetrahydrofolate Dehydrogenase