Demonstration that bovine erythrocyte cytochrome b5 is the hydrophilic segment of liver microsomal cytochrome b5

Abstract

A structural comparison has been made between bovine erythrocyte cytochrome b5 and solubilized forms of bovine hepatic microsomal cytochrome b5. Two soluble forms of microsomal cytochrome b5 (designated Forms A and B) were generated by digestion of microsomes with a crude hepatic lysosomal cathepsin preparation and purified by successive chromatography on DEAE-cellulose,Bio-Gel P-60 and DEAE-Sephadex A-50.Amino acid analyses and terminal residue analyses identified Form A as the segment corresponding to residues 1*95 of the native microsomal protein and Form B as the segment corresponding to residues 1*107. Erythrocyte cytochrome b5 I was shown to be a protein which corresponds to a segment of the hepatic microsomal molecule containing residues 1*97, whereas erythrocyte cytochrome b5 II is a protein corresponding to residues 1*95. Like the native microsomal cytochrome and the cathepsin-solubilized forms of the cytochrome, no amino terminal residue could be detected in the erythrocyte cytochrome.Carboxypeptidases A and B released from erythrocyte Form I a residue eluting at the position of serine, but released no residue from Form II. The results are consistent with serine being the residue at position 97 of the native microsomal protein, and proline and serine being the residues in positions 94 and 95, respectively. The maps of the tryptic peptides derived from the apoprotein forms of erythrocyte cytochrome b5 I and II and cathepsin-solubilized microsomal Forms A and B were very similar, with eight of the expected twelve peptides displaying the same mobility on every map. Amino acid analyses of the isolated tryptic peptides from erythrocyte Form I and hepatic Form B confirmed the structural assignments of these proteins. These data demonstrate that the soluble forms of erythrocyte cytochrome b5 correspond to hydrophilic segments of the native membrane-bound microsomal cytochrome b5 and suggest that the hepatic lysosomal proteases serve as a good model for the putative erythroid proteases which solubilize microsomal cytochrome b5 during erythroid maturation.