J. Biol. Chem., Vol. 255, Issue 8, 3420-3426, 04, 1980
Reversible modification of arginine residues in neocarzinostatin. Isolation of a biologically active 89-residue fragment from the tryptic hydrolysate
TS Samy, LS Kappen and IH Goldberg
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.
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