title: Reversible modification of arginine residues in neocarzinostatin. Isolation of a biologically active 89-residue fragment from the tryptic hydrolysate.
abstract: Reaction of the antitumor protein neocarzinostatin with 1,2-cyclohexanedione in 0.25 M borate buffer, pH 9.0, resulted in complete modification of arginine residues in positions 66, 67, and 78. The arginine-modified protein lost its native structure and was biologically inactive in the inhibition of growth of HeLa cells, inhibition of DNA synthesis, and in vitro DNA strand scissions. Trypsin hydrolysis of 1,2-cyclohexanedione-modified neocarzinostatin resulted in selective cleavage of the Lys-Val (positions 20 and 21) bond of the primary structure yielding NH2-terminal 1-20 and the COOH-terminal 21-109 residue fragments. The latter contained modified arginine residues. Both peptide fragments were biologically inactive. Treatment of the arginine-modified neocarzinostatin and the arginine-protected 89-residue fragment with 0.25 M Tris-acetate buffer, pH 9.0, for 15 h resulted in the release of 1,2-cyclohexanedione, regenerating all three arginine residues. The regenerated protein and the 89-residue fragment were fully active biologically. Further, the regenerated 89-residue fragment possessed 70% of the reactivity of neocarzinostatin with antibody raised against the native protein. The conformation of the 89-residue fragment was almost identical with that of the native protein in CD spectral properties.
mesh_terms: D000596:Amino Acids / Q000032:analysis; D000903:Antibiotics, Antineoplastic; D001120:Arginine / Q000032:analysis*; D001665:Binding Sites; D002942:Circular Dichroism; D003510:Cyclohexanes; D003512:Cyclohexanones; D010446:Peptide Fragments / Q000032:analysis; D011485:Protein Binding; D011487:Protein Conformation; D014357:Trypsin; D009353:Zinostatin
chemical_list: D000596:Amino Acids; D000903:Antibiotics, Antineoplastic; D003510:Cyclohexanes; D003512:Cyclohexanones; D010446:Peptide Fragments; C018031:1,2-cyclohexanedione; D009353:Zinostatin; D001120:Arginine; D014357:Trypsin