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Journal of Biochemistry 2005 138(4):509-517; doi:10.1093/jb/mvi144
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© 2005 The Japanese Biochemical Society

Regular Paper

Red Blood Cells Highly Express Type I Platelet-Activating Factor-Acetylhydrolase (PAF-AH) Which Consists of the {alpha}1/{alpha}2 Complex

Ken Karasawa*, Mariko Shirakura, Ayako Harada, Noriko Satoh, Kazuaki Yokoyama, Morio Setaka and Keizo Inoue

Faculty of Pharmaceutical Sciences, Teikyo University, Sagamiko, Kanagawa 199-0195

* To whom correspondence should be addressed. Tel: +81-426-85-3733, Fax: +81-426-85-3733, E-mail: karasawa{at}pharm.teikyo-u.ac.jp

Although red blood cells account for about 30% of total PAF-AH activity found in the blood, the physiological function of this enzyme is unknown. To understand the role and regulatory mechanism of this enzyme, we purified it from easily obtainable pig red blood cells. PAF-AH activity was mainly found in the soluble fraction of the red blood cells. Two peaks of enzyme activity appeared with increasing concentration of imidazole on column chromatography on nickel-nitroacetic acid (Ni-NTA) resin. We called these peaks of small and large enzyme activities fractions X and Y, respectively, and then further purified the enzymes by sequential chromatofocusing on Mono P and gel filtration on TSK G-3000. In the final preparation from fraction Y, two proteins bands corresponding to 26 kDa and 28 kDa were related to enzyme activity. Determination of the partial amino acid sequences of the proteins of 26 kDa and 28 kDa revealed that these proteins were identical to {alpha}1 and {alpha}2, respectively, both of which are catalytic subunits of Type I intracellular PAF-AH. On Western analysis, the 26 kDa and 28 kDa protein bands cross-reacted with specific monoclonal antibodies to {alpha}1 and {alpha}2, respectively. Since the apparent molecular weight of the natural enzyme was estimated to be about 60 kDa, the enzyme activity in fraction Y was thought to be that of a heterodimer consisting of {alpha}1 and {alpha}2. On the other hand, the enzyme activity in fraction X was thought to be that of a homodimer consisting of {alpha}2. Other blood cells such as polymorphonuclear leukocytes and platelets only contained the {alpha}2/{alpha}2 homodimer. It has been reported that the {alpha}1/{alpha}2 heterodimer is poorly expressed in adult animals except for in the spermatogonium. Taken altogether, these results suggest that high expression of the {alpha}1/{alpha}2 heterodimer is important for the physiological function of mature red blood cells.


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