Immunophotoaffinity labeling of binders of 1-methyladenine, the oocyte maturation-inducing hormone of starfish

• Published in: Biochemical and biophysical research communications 2017-03, Vol.485 (1), p.41-46
• Main Authors: Toraya, Tetsuo, Kida, Tetsuo, Kuyama, Atsushi, Matsuda, Shinjiro, Tanaka, Seiichi, Komatsu, Yo, Tsurukai, Taro
• Format: Article
• Language: English
• Subjects: 1-Methyladenine; 60 APPLIED LIFE SCIENCES; Adenine - analogs & derivatives; Adenine - metabolism; Animals; BINDERS; Blotting, Western - methods; Female; HORMONES; Immunophotoaffinity labeling; LABELLING; Oocyte maturation; OOCYTES; Oocytes - cytology; Oocytes - metabolism; Oogenesis; Photoaffinity Labels - chemical synthesis; Photoaffinity Labels - chemistry; Photoaffinity Labels - metabolism; Receptor; RECEPTORS; Starfish; Starfish - physiology
• ISSN: 0006-291X; 1090-2104
• DOI: 10.1016/j.bbrc.2017.02.009

Starfish oocytes are arrested at the prophase stage of the first meiotic division in the ovary and resume meiosis by the stimulus of 1-methyladenine (1-MeAde), the oocyte maturation-inducing hormone of starfish. Putative 1-MeAde receptors on the oocyte surface have been suggested, but not yet been biochemically characterized. Immunophotoaffinity labeling, i.e., photoaffinity labeling combined with immunochemical detection, was attempted to detect unknown 1-MeAde binders including putative maturation-inducing hormone receptors in starfish oocytes. When the oocyte crude membrane fraction or its Triton X-100/EDTA extract was incubated with N6-[6-(5-azido-2-nitrobenzoyl)aminohexyl]carboxamidomethyl-1-methyladenine and then photo-irradiated, followed by western blotting with antibody that was raised against a 1-MeAde hapten, a single band with Mr of 47.5 K was detected. The band was lost when extract was heated at 100 °C. A similar 47.5 K band was detected in the crude membrane fraction of testis as well. Upon labeling with whole cells, this band was detected in immature and maturing oocytes, but only faintly in mature oocytes. As judged from these results, this 1-MeAde binder might be a possible candidate of the starfish maturation-inducing hormone receptors. •Synthesis of photoaffinity labeling reagents for 1-methyladenine binders of starfish.•Immunochemical detection of photoaffinity-labeled 1-methyladenine binders.•Immunophotoaffinity labeling of a 47.5 K 1-methyladenine binder in oocytes and testis.•A possible candidate of oocyte maturation-inducing hormone receptors of starfish.