Roles of chromophore and apo-protein in neocarzinostatin action.

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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 the growth of HeLa [human cervical carcinoma] 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 was localized to fractions eluting at > 90% methanol and having fluorescence emission at 420 nm (excitation at 340 nm). The apo-protein of NCS is inactive by itself but comlexes with the chromophore so as to regulate its availability during the in vitro reaction. In DNA strand scission the chromophore acts rapidly at 0 and 37.degree. C; native and reconstituted NCS are inactive at 0.degree. C and slowly active at 37.degree. C. Complex formation with apo-NCS stabilizes the chromophore. Reconstitution of NCS (pI [isoelectric point] 3.3) from chromophore and apo-protein (pI 3.2) was shown by 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 polyacrylamide gels and ability to complex with isolated chromophore to form material with all the properties of native NCS.