Purification and molecular characterization of a novel b5-type cytochrome of the parasitic nematode, Ascaris suum.
A b5-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 heme-binding domain of vertebrate cytochrome b5. Using an oligonucleotide probe synthesized based on the amino acid sequence of the purified protein, the cDNA clone encoding A. suum cytochrome b5 was isolated from the 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 B5 was predicted to contain 82 amino acids with a molecular mass of 9141 Da, matching the 9140 Da obtained from electrospray ionization mass spectometry, and to lack of 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 b5 of 82 amino acids, but no protein with an N-terminal extension. These results demonstrate a novel cytochrome b5 possessing a presequence.
Yu
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Y
Department of Parasitology, Juntendo University, School of Medicine, Tokyo, Japan.
Yamasaki
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H
Kita
K
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Takamiya
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S
D000595:Amino Acid Sequence; D000818:Animals; D017165:Ascaris suum / Q000737:chemistry*; D001483:Base Sequence; D015786:Cytochromes b5 / Q000737:chemistry* / Q000235:genetics; D018076:DNA, Complementary / Q000235:genetics; D005260:Female; D015723:Gene Library; D014644:Genetic Variation; D013058:Mass Spectrometry; D008969:Molecular Sequence Data; D009132:Muscles / Q000737:chemistry*; D011199:Potentiometry; D017421:Sequence Analysis; D017386:Sequence Homology, Amino Acid; D013045:Species Specificity; D013053:Spectrophotometry
D018076:DNA, Complementary; D015786:Cytochromes b5