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