Roles of chromophore and apo-protein in neocarzinostatin action

Abstract

The methanol-extractable, nonprotein chromophore of the antitumor, protein antibiotic neocarzinostatin (NCS) has at least the full activity of the parent compound in inhibiting DNA synthesis and growth of HeLa cells and in causing DNA strand breaks in vivo and in vitro. In vitro DNA strand scission by the chromophore is markedly stimulated by 2-mercaptoethanol and is inhibited by guanidine hydrochloride and alpha-tocopherol. By high-pressure liquid chromatography, this activity has been localized to fractions eluting at greater than 90% methanol and having fluorescence emission at 420 nm (excitation at 340 nm). The apo-protein of NCS is inactive by itself but complexes with the chromophore so as to regulate its availability during the in vitro reaction. In DNA strand scission the chromophore acts rapidly at both 0 and 37 degrees C, whereas native and reconstituted NCS are inactive at 0 degrees C and slowly active at 37 degrees C. Complex formation with apo-NCS stabilizes the chromophore. Reconstitution of NCS (pI 3.3) from chromophore and apo-protein (pI 3.2) was shown by both activity studies and isoelectric focusing on polyacrylamide gels. "Pre-NCS," the biosynthetic precursor of NCS, is identical to apo-NCS in amino acid composition, spectral properties, isoelectric focusing on polyacryl-amide gels, and ability to complex with isolated chromophore to form material with all the properties of native NCS.