Purification and characterization of functional recombinant alpha-amidating enzyme secreted from mammalian cells.
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.
Department of Molecular Biology, Unigene Laboratories, Inc., Fairfield, New Jersey 07004.
G A
GA
Beaudry
N M
NM
Mehta
M L
ML
Ray
A H
AH
Bertelsen
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D002460:Cell Line; D005822:Genetic Vectors; D007700:Kinetics; D051379:Mice; D006899:Mixed Function Oxygenases / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism; D008969:Molecular Sequence Data; D009097:Multienzyme Complexes; D016297:Mutagenesis, Site-Directed; D015345:Oligonucleotide Probes; D051381:Rats; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000302:isolation & purification / Q000378:metabolism; D014162:Transfection
D009097:Multienzyme Complexes; D015345:Oligonucleotide Probes; D011994:Recombinant Proteins; D006899:Mixed Function Oxygenases; C035189:peptidylglycine monooxygenase