Release of highly active Fet3 from membranes of the yeast Pichia pastoris by limited proteolysis.
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.
Bonaccorsi di Patti
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CNR Center of Molecular Biology, University of Rome, "La Sapienza," P.le A. Moro 5, Rome, 00185, Italy. bonaccorsi@axrma.uniroma1.it
Bellenchi
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GC
Bielli
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Calabrese
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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
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