Purification and characterization of cytochrome b5-like hemoprotein associated with outer mitochondrial membrane of rat liver.

Abstract

The cytochrome b5-like hemoprotein associated with the outer membrane of rat liver mitochondria (OM-cytochrome) was found to be more susceptible to solubilization with trypsin [EC 3.4.4.4] than microsomal bound cytochrome b5. Based on this finding, a condition for tryptic digestion was set up under which OM-cytochrome could be preferentially released from crude outer mitochondrial membrane preparations leaving most, if not all, cytochrome b5 of contaminating microsomes still attached to the membrane. The OM-cytochrome thus solubilized was highly purified by DEAE-cellulose column chromatography and Sephadex G-75 gel filtration. The purified cytochrome obtained in one experiment (Prep I) was separated into two hemoprotein bands upon polyacrylamide gel electrophoresis, whereas the sample purified in a second experiment (Prep II) gave three hemoprotein bands. Both preparations were, however, free of any other protein contaminants. Evidence was obtained to indicate that the extra band in Prep II was mainly due to cytochrome b5 derived from contaminating microsomes. The other two bands of Prep II as well as those of Prep I seemed to correspond to OM-cytochrome. The separation of OM-cytochrome into two bands suggested that the solubilized hemoprotein had suffered certain proteolytic modifications during tryptic digestion. The molecular weight (about 12,000) and behavior upon DEAE-cellulose column chromatography of purified OM-cytochrome were closely similar to those of trypsin-solubilized microsomal cytochrome b5. However, the two cytochromes differed from each other in immunological properties. Small differences were also detected in their absorption spectra. Furthermore, the tryptic peptide map of apo-OM-cytochrome was clearly different from that of apo-cytochrome b5