Purification and characterization of functional recombinant alpha-amidating enzyme secreted from mammalian cells

Beaudry, G.A.; Mehta, N.M.; Ray, M.L.; Bertelsen, A.H.

Journal of Biological Chemistry 265(29): 17694-17699


ISSN/ISBN: 0021-9258
PMID: 2211657
Accession: 041135053

Download citation:  

Article/Abstract emailed within 1 workday
Payments are secure & encrypted
Powered by Stripe
Powered by PayPal

A rat alpha-amidating enzyme (alpha-AE) cDNA has been expressed in mouse C127 cells using a bovine papilloma virus vector in which transcription was regulated by the mouse metallothionein 1 promoter. The cDNA encoding the full length alpha-AE protein was modified to terminate translation at a site preceding the transmembrane and cytoplasmic domains, thereby enabling functional enzyme to be secreted into the medium. Purification of recombinant alpha-AE to homogeneity indicated that the enzyme was synthesized and secreted as two proteins of 75-77 kDA. The observed heterogeneity was due to inefficient glycosylation at Asn660, as demonstrated by glycopeptidase F digestion. Using the synthetic peptide, dansyl-Tyr-Val-Gly, the specific activity of the recombinant enzyme at pH 7.0 was found to be 1.4 mumol/min/mg and the Km of the enzyme was determined to be 3 microM. The purified recombinant enzyme has maximal activity at pH 4.5-5.5; however, a rapid inactivation of the enzyme occurs in acidic solutions in vitro. This inactivation is diminished when activity is measured at pH 7.0-10.0. The availability of large amounts of readily purified, active recombinant alpha-AE should allow detailed probing of reaction mechanism, copper coordination chemistry, and turn-over-based inactivation events.