Purification, characterization, and amino acid sequence of the mating pheromone Er-10 of the ciliate Euplotes raikovi.
The mating pheromone Er-10 from mat-10 homozygous Euplotes raikovi was purified by a three-step purification procedure with an overall yield of 62%. It was identified as a protein of molecular weight 8000 having an isoelectic point of 3.9. Its complete primary structure was determined by automated Edman degradation of the whole protein after performic acid oxidation and of peptides generated by cyanogen bromide and Staphylococcus aureus V8 protease. The proposed sequence is Asp1-Leu-Cys-Glu-Gln-Ser-Ala-Leu-Gln-Cys10-Asn-Glu-Gln-Gly-Cys-His -Asn-Phe-Cys- Ser20-Pro-Glu-Asp-Lys-Pro-Gly-Cys-Leu-Gly-Met30-Val-Trp-Asn- Pro-Glu-Leu-Cys- Pro38. The calculated molecular weight of 4191.7, which is in good agreement with the value of m/z 4190.7 obtained by fission fragment ionization mass spectrometry, suggests that the native structure is a dimer with three intrachain disulfide bonds in each subunit. The amino acid sequence is 43% identical with that of the E. raikovi mating pheromone Er-1, with the identities concentrated in the amino-terminal half. The half-cystine locations are conserved, but Er-10 is two residues shorter than Er-1. Prediction of the secondary structure suggests that Er-10 may also contain a helical structure at the amino terminus. These results indicate that the mating pheromones of E. raikovi form a homologous family.
Raffioni
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Department of Biological Chemistry, California College of Medicine, University of California, Irvine 92717.
Luporini
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Bradshaw
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D000595:Amino Acid Sequence; D000596:Amino Acids / Q000032:analysis; D000818:Animals; D002851:Chromatography, High Pressure Liquid; D016798:Ciliophora / Q000032:analysis*; D003488:Cyanogen Bromide; D004591:Electrophoresis, Polyacrylamide Gel; D007525:Isoelectric Focusing; D013058:Mass Spectrometry; D008565:Membrane Proteins; D008969:Molecular Sequence Data; D010446:Peptide Fragments / Q000302:isolation & purification; D010447:Peptide Hydrolases; D010675:Pheromones / Q000302:isolation & purification*; D015800:Protozoan Proteins / Q000302:isolation & purification*; D012689:Sequence Homology, Nucleic Acid; D012724:Sex Attractants / Q000302:isolation & purification*
D000596:Amino Acids; D008565:Membrane Proteins; D010446:Peptide Fragments; D010675:Pheromones; D015800:Protozoan Proteins; D012724:Sex Attractants; C057783:mating pheromone Er-1, Euplotes raikovi; C060870:mating pheromone Er-10, Euplotes raikovi; D010447:Peptide Hydrolases; D003488:Cyanogen Bromide