| Budget Amount *help |
¥2,100,000 (Direct Cost: ¥2,100,000)
Fiscal Year 1992: ¥700,000 (Direct Cost: ¥700,000)
Fiscal Year 1991: ¥1,400,000 (Direct Cost: ¥1,400,000)
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| Research Abstract |
Soluble form of guanylate cyclase is a dimer of 70k and 82k subunits containing heme and is activated by NO. In this project, first, we tried to reconstruct the enzyme from the dissociated subunits to clarify the subunit function in the activation process by NO. Second, we studied kinetics of cyclic GMP levels in platelets in response to NO(SNP).
For reconstruction experiment, large amounts of the highly purified enzyme was required. We developed a new elution method of immunoaffinity column using a single hollow fiber concentrator and succeeded in obtaining enough amounts of the enzyme. The enzyme showed two bands of the subunits on SDS-PAGE, contained 0.8 mole of heme and activated 40-fold by SNP. After complete denaturation in 8M urea or 6M Gua-HCl, the enzyme solution was diluted 100-fold with 20 mM Tris-HCl (pH=7)/20 % glycerol/1 mM DTT and incubated for up to 24 hr to recover the activity. The enzyme activity, however, could not be obtained. One of the problems must relate to the extremely unstable nature of the purified enzyme in dilute conditions, although glycerol stabilized it substantially.
The dimeric guanylate cyclase similar to that in rat lung was demonstrated in human platelet and shown to be activated 200-fold by SNP. Cyclic GMP in platelet quickly rose to maximal levels in 10 sec after SNP addition and declined to the basal levels after 30 sec. IBMX, however, blocked the decline. These implied importance of degradation as well as synthesis to regulate intracellular cyclic GMP levels.
Through this project, microsequencing technique was developed for the co-operational work on cDNA cloning of guanylate cyclase and applied successfully on other several proteins.
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