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.
Release of highly active Fet3 from membranes of the yeast Pichia pastoris by limited proteolysis / BONACCORSI DI PATTI, Maria Carmela; Bellenchi, G. C.; Bielli, P.; Calabrese, Lilia. - In: ARCHIVES OF BIOCHEMISTRY AND BIOPHYSICS. - ISSN 0003-9861. - 372:(1999), pp. 295-299.
Release of highly active Fet3 from membranes of the yeast Pichia pastoris by limited proteolysis.
BONACCORSI DI PATTI, Maria Carmela;CALABRESE, Lilia
1999
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.I documenti in IRIS sono protetti da copyright e tutti i diritti sono riservati, salvo diversa indicazione.