Controlled proteolysis of flavocytochrome b2. Characterization of a 15000-dalton heme-binding core and comparison with detergent solubilized cytochrome b5.
It is known that each subunit of the tetrameric flavocytochrome b2 can be cleaved by yeast proteases to fragments of molecular weight 33-36000 and 21 000, with some modification of catalytic properties, but without destruction of the oligomeric state of the protein. We report here experimental conditions which enabled us to simulate this specific cleavage in a controlled fashion with chymotrypsin and subtilisin. With trypsin and papain, on the other hand, it was not found possible to stop the digestion in such a way as to obtain a homogeneous still active product. A characterization of the enzymatic forms obtained by digestion with chymotrypsin and subtilisin at 0 degrees C shows that modification of enzymatic and solubility properties occurs in a stepwise fashion. It is also ccluded that cleavage by yeast proteases is accompanied by loss of 10 to 25 residues. At 37 degrees C, chymotrypsin digestion yields a heme-binding core of molecular weight 15 000, larger than the already characterized tryptic heme-binding core by about 40 residues. Although the latter is known to be very similar to trypsin-solubilized cytochrome b5, the lack of aggregation of the former in aqueous solution, its amino acid composition and circular dichroism spectra do not point to a similarity of its additional peptide segment with the hydrophobic tail of detergent-solubilized cytochrome b5.
D000596:Amino Acids / Q000032:analysis; D000818:Animals; D002417:Cattle; D002918:Chymotrypsin / Q000378:metabolism; D002942:Circular Dichroism; D003580:Cytochromes; D006801:Humans; D007700:Kinetics; D008970:Molecular Weight; D010206:Papain; D010446:Peptide Fragments / Q000032:analysis; D010447:Peptide Hydrolases; D011487:Protein Conformation; D011817:Rabbits; D012441:Saccharomyces cerevisiae / Q000201:enzymology; D013045:Species Specificity; D013056:Spectrophotometry, Ultraviolet; D013381:Subtilisins; D013552:Swine; D013696:Temperature
D000596:Amino Acids; D003580:Cytochromes; D010446:Peptide Fragments; D010447:Peptide Hydrolases; D013381:Subtilisins; D002918:Chymotrypsin; D010206:Papain