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

J Biol Chem. 1990 Oct 15;265(29):17694-9.

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.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Amino Acid Sequence
  • Animals
  • Base Sequence
  • Cell Line
  • Genetic Vectors
  • Kinetics
  • Mice
  • Mixed Function Oxygenases / genetics
  • Mixed Function Oxygenases / isolation & purification*
  • Mixed Function Oxygenases / metabolism
  • Molecular Sequence Data
  • Multienzyme Complexes*
  • Mutagenesis, Site-Directed
  • Oligonucleotide Probes
  • Rats
  • Recombinant Proteins / biosynthesis
  • Recombinant Proteins / isolation & purification
  • Recombinant Proteins / metabolism
  • Transfection

Substances

  • Multienzyme Complexes
  • Oligonucleotide Probes
  • Recombinant Proteins
  • Mixed Function Oxygenases
  • peptidylglycine monooxygenase