Purification and characterization of cytosolic liver protein facilitating heme transport into apocytochrome b5 from mitochondria. Evidence for identifying the heme transfer protein as belonging to a group of glutathione S-transferases.
The results of the present experiment are shown in terms of the transport of protoheme from mitochondria to apocytochrome b5 when fresh rat liver mitochondria, apocytochrome b5, and cytosol were incubated. The heme transfer protein was purified from rat liver cytosol up to approximately 133-140-fold with a 43% yield by the procedure discussed herein, including Sephadex G-75 and CM-cellulose column chromatography. The final preparation showed apparent homogeneity upon polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate. Its native form was found to be a dimeric protein with a Mr = 45,000 which consists of a subunit with a Mr = 23,000. In the transporting system, the heme transfer depended on the concentration of mitochondria (donor), apocytochrome b5 (acceptor), and purified transfer protein, respectively. Omission of one of these components led to an almost complete loss of the transfer activity. The transport of mitochondrial protoheme was a rapid reaction which showed approximate linearity until 1.5 min and after that it became saturated. When the functional capacity was tested by the NADH-cytochrome c reductase system, the reconstituted cytochrome b5 expressed its complete original catalytic properties, as well as its characteristic absorption spectra for the hemoprotein. Furthermore, the detailed physicochemical and immunological characterization of the transfer protein provided evidence that the protein is identical with soluble glutathione S-transferase, which conjugates glutathione with a variety of electrophilic compounds. At least one of the glutathione S-transferase isozymes observed was identified as GST-C2, which comprises the subunit of Yb'Yb' by the immunoprecipitation reaction using various anti-glutathione S-transferase isozyme antibodies.
D000818:Animals; D003573:Cytochrome b Group / Q000378:metabolism*; D045303:Cytochromes b; D003600:Cytosol / Q000378:metabolism; D004591:Electrophoresis, Polyacrylamide Gel; D005982:Glutathione Transferase / Q000302:isolation & purification* / Q000378:metabolism; D006418:Heme / Q000378:metabolism*; D005779:Immunodiffusion; D007700:Kinetics; D008099:Liver / Q000378:metabolism*; D008930:Mitochondria, Liver / Q000378:metabolism*; D008970:Molecular Weight; D051381:Rats; D011919:Rats, Inbred Strains
D003573:Cytochrome b Group; D006418:Heme; D045303:Cytochromes b; D005982:Glutathione Transferase