| Budget Amount *help |
¥1,900,000 (Direct Cost: ¥1,900,000)
Fiscal Year 1988: ¥400,000 (Direct Cost: ¥400,000)
Fiscal Year 1987: ¥1,500,000 (Direct Cost: ¥1,500,000)
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| Research Abstract |
Peptidylarginine deiminase (protein L-arginine iminohydrolase, EC 3.5.3.15), A Ca^<2+>-dependent protein-modulating enzyme, catalyzes the deimination of arginyl residues in proteins. Chemical modification studies using iodoacetamide, and cysteine-specific reagent, have been carried out on the enzyme from mouse skeletal muscle. From kenetics studies with [1-^<14>C]iodoacetamide, we find that incorportion of two iodoacetamide molecule to two cysteinly residues of the enzyme resulted in complete loss of the enzymic activity. The co-existence of the substrate Bz-L__=-Arg-O__--Et and Ca^<2+>, cofactors for the enzyme offered complete protection against inodoacetamide modification, and either of the above ligands gave partial protection. These results indicate the involvement of the cysteinyl residues in catalysis. Fractionation of [1-^<14>C] iodoacetamide-modified enzyme oxidized with cyanogen bromide on Sephadex G-50, revealed two major radioactive peaks of approximately equal area.
Digestion of each peak with lysylendopeptidase and chromatography on C18 reverse-phase HPLC resulted in pure peptides, FI-P9 and FII-P8. Peptide Fi-p9 contained one modified cysteiny 1 residue, while peptide fii-p8 contained four modified cysteinyl residues. manual edman degradation revealed the sequences as follows: Fi-P9, Ala-Ser-Trp-Thr-Trp-Gly-Pro-Asn-Gly-Cmcys-(Asx3,Glx2,Arg,Ala,Pro,Val,Ile,Leu3,Phe)lys and FII-P8, Thr-Pro-Asn-Ile-Leu-Pro-Pro-Val-Ser-Val-Cal-(Asx2,Glx,CmCys4,Ser,Gly,Thr,Ala,Pro2,Val,Ile,Phe2)-Met.
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