Purification and Molecular Characterization of a Novelb5-Type Cytochrome of the Parasitic Nematode,Ascaris suum

Ab5-type cytochrome was extracted fromAscaris suummuscle at pH 4.5 with 0.3% aluminum sulfate and purified by ammonium sulfate fractionation, ion-exchange chromatography on CM-500 Cellulofine, and gel filtration on Sephadex G-75. The hemoprotein displayed a typical absorption spectrum of cytochromebwith a midpoint redox potential of 78 mV. The N-terminal amino acid sequence was determined and revealed the N-terminus to be highly homologous to the heme-binding domain of vertebrate cytochromeb5.Using an oligonucleotide probe synthesized based on the amino acid sequence of the purified protein, the cDNA clone encodingA. suumcytochromeb5was isolated from a λZAP II cDNA library. The entire nucleotide sequence of 563 bases comprised an open reading frame of 339 bases encoding a precursor protein of 112 amino acid residues. The purified cytochromeb5was predicted to contain 82 amino acids with a molecular mass of 9141 Da, matching the 9140 Da obtained from electrospray ionization mass spectrometry, and to lack a membrane–anchor domain at the C-terminus. In contrast, an N-terminal extension of 30 amino acids, characteristic of signal peptides, was apparent. Immunoblots revealed the presence of anA. suumcytochromeb5of 82 amino acids, but no protein with an N-terminal extension. These results demonstrate a novel cytochromeb5possessing a presequence.