title: Release of highly active Fet3 from membranes of the yeast Pichia pastoris by limited proteolysis.
abstract: A soluble derivative of Fet3 has been obtained from the methylotrophic yeast Pichia pastoris by limited proteolysis of membrane suspensions with trypsin. The soluble protein and the membrane-bound parent Fet3 have been purified to apparent homogeneity. Soluble Fet3 had molecular mass 100 kDa, while the full-length protein had molecular mass 110 kDa, in line with the expected decrease for cleavage and loss of a single transmembrane helix and a small cytoplasmic domain. The optical and EPR spectra of Fet3 were typical of the multicopper oxidases, indicating the presence of one type 1 blue copper site and a type 2/type 3 copper trinuclear cluster. V(max) values for iron oxidation by P. pastoris Fet3 were obtained similar to human ceruloplasmin and much higher than those reported for Saccharomyces cerevisiae Fet3.
authors.affiliation: CNR Center of Molecular Biology, University of Rome, "La Sapienza," P.le A. Moro 5, Rome, 00185, Italy. bonaccorsi@axrma.uniroma1.it
authors.forename: M C
authors.initials: MC
authors.lastname: Bonaccorsi di Patti
authors.forename: G C
authors.initials: GC
authors.lastname: Bellenchi
authors.forename: P
authors.initials: P
authors.lastname: Bielli
authors.forename: L
authors.initials: L
authors.lastname: Calabrese
mesh_terms: D000595:Amino Acid Sequence; D001665:Binding Sites; D002384:Catalysis; D002462:Cell Membrane / Q000378:metabolism*; D002570:Ceruloplasmin / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism*; D003300:Copper / Q000378:metabolism / Q000494:pharmacology; D004578:Electron Spin Resonance Spectroscopy; D010450:Endopeptidases / Q000378:metabolism*; D005656:Fungal Proteins / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism; D006031:Glycosylation; D006801:Humans; D007501:Iron / Q000378:metabolism / Q000494:pharmacology; D007502:Iron Chelating Agents / Q000494:pharmacology; D007700:Kinetics; D008565:Membrane Proteins / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism; D008969:Molecular Sequence Data; D008970:Molecular Weight; D010843:Pichia / Q000166:cytology / Q000187:drug effects / Q000201:enzymology / Q000378:metabolism*; D012441:Saccharomyces cerevisiae / Q000201:enzymology; D029701:Saccharomyces cerevisiae Proteins; D012995:Solubility
chemical_list: D005656:Fungal Proteins; D007502:Iron Chelating Agents; D008565:Membrane Proteins; D029701:Saccharomyces cerevisiae Proteins; D003300:Copper; D007501:Iron; D002570:Ceruloplasmin; C085513:FET3 protein, S cerevisiae; D010450:Endopeptidases