| Budget Amount *help |
¥2,200,000 (Direct Cost: ¥2,200,000)
Fiscal Year 1991: ¥600,000 (Direct Cost: ¥600,000)
Fiscal Year 1990: ¥1,600,000 (Direct Cost: ¥1,600,000)
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| Research Abstract |
Using 500 MHz ^1H-NMR we studied the interaction between carbonic anhydrase II and acetazolamide which is currently used for the therapy of glaucoma. The following results were obtained.
(1) As the material, we employed carbonic anhydrase II prepared from bovine red blood cells. Using CPMG method, we succeeded in the detection of 9 signals derived from C_2 proton of histidine residues. Bovine carbonic anhydrase has 11 histidines but two of them are located inside the molecule.
(2) We attempted to assign the 9 signals to the primary structure by means of comparison of the deuterization rate of each signal determined by NMR with the deuterization ratio of each residue determined by FAB mass spectrometry. However, we failed because we could not solve the problem of the noise due to FAB.
(3) In the same way as described in (1), we studied the complex of bovine carbonic anhydrase with acetazolamide, and found that acetamide moiety of acetazolamide interacted with one of histidines of the enzyme in addition to the co-ordination of sulfamoyl moiety of acetazolamide to the essential zinc ion of the enzyme.
(4) Other than acetazolamide, we also studied 2-methylamino-1,3,4-thiadiazole-5-sulfonamide, 2-formylamino-1,3,4-thiadiazole-5-sulfonamide and sulfanilamide. The former two compound formed a similar complex with carbonic anhydrase to acetazolamide, but sulfanilamide did not.
(5) In order to assign the signal of the residue which interact with acetazolamide, we attempted to carboxymethylate His-64 but this carboxymethylation altered also other important part of the spectrum. Therefore we attempted molecular mechanic study based on the co-ordinate obtained by X-ray diffraction. By this calculation, we thought His-64 may be a candidate for the residue involved in the interaction.
(6) In the case of isozyme I, we did not observe such an interaction as that we detected in the case of isozyme II.
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