CHARACTERIZATION OF A BIFUNCTIONAL PEPTIDYLGLYCINE ALPHA-AMIDATING ENZYME EXPRESSED IN CHINESE-HAMSTER OVARY CELLS

Peptidylglycine α-amidating enzyme (α-AE) catalyzes the conversion of glycine-extended prohormones to their biologically active α-amidated forms. We have derived a clonal Chinese hamster ovary cell line that secretes significant quantities of active α-AE. Enzyme production was increased by selection for methotrexate-resistant cells expressing a dicistronic message. Amplification of the α-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-TyrValGly, or the intermediate, dansyl-TyrValα-hydroxyglycine, to the dansyl-TyrValNH2 product. The recombinant α-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-TyrValGly, dansyl-TyrValα-hydroxyglycine, ascorbate, and catechol were the same for both forms of α-AE. In addition, mimosine is competitive vs ascorbate with Kis =3.5 μM for the nongly-cosylated α-AE and Kis = 4.2 μM for the glycosylated α-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 α-AE gene. © 1992.