Generation of Pre-Neocarzinostatin and Its Antagonistic Effect on Neocarzinostatin-Induced DNA Strand Scission

Abstract

Pretreatment of the antitumor protein neocarzinostatin (NCS) with heat, UV or room light, and sulfhydryl reagents inactivates the drug, as measured by the cessation of phage T2 DNA strand scission in vitro. The inactive forms obtained are identical with pre-neocarzinostatin (pre-NCS) on the basis of isoelectric focusing, molecular weight determination, and changes in circular dichroism (CD) spectra. Addition of pre-NCS to incubations of T2 DNA and NCS inhibits strand breaking activity in a ratiodependent NCS/pre-NCS manner, indicating a competition between the two proteins for a limited number of DNA binding sites. A quantitative analysis demonstrates that pre-NCS binds at least 3 to 4 times more strongly than NCS at 37° C, and the two bind roughlyequally at 54° C. These different ratios could be explained by proportionately greater temperature effects on pre-NCS fine structure which may occur as a result of its looser overall conformation.