|Budget Amount *help
¥4,500,000 (Direct Cost : ¥4,500,000)
Fiscal Year 1995 : ¥1,500,000 (Direct Cost : ¥1,500,000)
Fiscal Year 1994 : ¥1,500,000 (Direct Cost : ¥1,500,000)
Fiscal Year 1993 : ¥1,500,000 (Direct Cost : ¥1,500,000)
To clarify the subunit structure of NADPH oxidase (O_2^- generating enzyme), we have tried to fix the enzyme complex activated in a semi-recombinant system by crosslinkers. After trying several linkers and conditions, we realized that the enzyme activated in the semirecombinant system is very labile and is not efficiently stabilized by crosslinkers, different from that in the cell-free system containing cytosol. The result lead us to search for the stabilizing factor in cytosol. As a result, we found that actin in cytosol may be involved in the stabilization (and possibly in the activation). Considering the idea, we are planning to modify the cross-linking experiments ot fit the system.
We realized that we need a large amount of recombinant proteins for this type of experiment. So we have started to produce these recombinant proteins in E.coli or Sf9 cells by ourselves. We have learned from Dr.Lambeth's group how to grow the cells and let them produce the proteins. Now we have some stoc
ks for doing experiments described above.
In the other part of this project, we have searched for the signaling molecules which control the activation of NADPH oxidase in the cell, and found that phosphatidic acid (PA) elicits the oxidase activation and spermine, a cellular polymine, suppresses it. When added to permeabilized neutrophils, PA at micromolar concentrations elicited the activity quickly. The activation was found independent of Ca^<2+>, diacylglycerol, or protein kinase c. And the rate of O_2^- generation was similar to that by physiological stimuli. These results show that PA may searve as a second messenger to activate NADPH oxidase in the cell.
On the other hand, spermine was found to suppress the cell-free activation of the oxidase (IC_<50>=18muM). The inhibition was specific for spermine over its precursor amines. The amine also inhibited semi-recombinant cell-free system. The kinetic sutdies showed that spermine may interfere with the assembly of the enzyme by binding to the cytosolic subunits, especially to p67phox.