| Budget Amount *help |
¥3,400,000 (Direct Cost: ¥3,400,000)
Fiscal Year 2004: ¥1,300,000 (Direct Cost: ¥1,300,000)
Fiscal Year 2003: ¥2,100,000 (Direct Cost: ¥2,100,000)
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| Research Abstract |
Three kinds of subtypes have been identified as PAF-acetylhydrolase (PAF-AH) which degrades PAF ; mediator of inflammation and allergy. Those consist of plasma-type, distributed in body fluids such as plasma, and intracellular type I and type II. Type I PAF-AH forms oligomer consisting of catalytic subunits termed α_1 or α_2. In the human blood, 70 % of enzyme activity is detected in plasma, while 30 % of enzyme activity is detected in red blood cells. Total enzyme activity of white blood cells such as polymorphonuclear leukocytes and platelets was less than 1 %. Despite high occupancy of enzyme activity, subtype of this enzyme and physiological role remain unknown. In this study, enzyme purification was undertaken from easily available pig blood. As a result, it was clarified that pig red blood cells contained α_1/ α_2 heterodimer and α2/α2 homodimer. Although ai subunit is highly expressed in fetal brain, expression of this subunit is very low in the adult except spermatogonium. By contrast, white blood cells such as polymorphonuclear leukocytes and platelets contained α_2/α_2 homodimer alone. This fact suggests that this subunit plays an important role in regulation of red blood cells function. In addition, Western blotting analysis using specific antibodies to these subunits showed that human red blood cells contained α_1/α_2 heterodimer and α_2/α_2 homodimer, as well as human red blood cells.
Since red blood cells do not produce PAF and lack PAF receptor, physiological substrate towards this enzyme is not thought to be PAR Thus, we added recombinant PAF-AH to the lipids extracted from red blood cells membranes and analyzed the lipid constituents by mass-spectrometry. As a result, it was shown that recombinant PAF-AH degraded oxidatively modified lipids of the red blood cells membranes exposed by Cu^<2+> as the substrates.
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