A b(5)-type cytochrome was extracted from Ascaris suum muscle 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 cytochrome b with 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 hemebinding domain of vertebrate cytochrome bg. Using an oligonucleotide probe synthesized based on the amino acid sequence of the purified protein, the cDNA clone encoding A. suum cytochrome b(5) was isolated from a lambda ZAP 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 cytochrome b(5) was 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 an A. suum cytochrome b(5) of 82 amino acids, but no protein with an N-terminal extension. These results demonstrate a novel cytochrome b(5) possessing a presequence. (C) 1996 Academic Press, Inc.