Differential interaction of nogalamycin with DNA of varying base composition.

Nogalamycin, a new antibiotic produced by Streptomyces nogalater var. nogalater, has a varied spectrum of biological effects. The isolation, antibacterial activity, and whole-animal toxicity of the antibiotic will be separately described.' This paper summarizes studies on the cytotoxicity of nogalamycin, and proposes a biochemical basis of its action on mammalian cells. Experimental Methods and Materials.-Cytotoxicity of nogalamycin against KB cells was determined by the method of Smith et al.2 The effects of nucleic acids and their derivatives, amino acids, and vitamins on cytotoxicity of a given substance were determined as described previously.3 To determine the inhibition of incorporation of radioactive precursors into DNA, RNA, and protein of intact cells, the precursors were added to KB cell monolayers at concentrations which permitted maximal incorporation rates and the cells were harvested after 2 hr. Cells were washed thrice with 10 per cent cold trichloroacetic acid and then fractionated to separate RNA and DNA by the Schmidt-Thannhauser method.4 RNA was determined by the orcinol method,5 and DNA by Burton's modification of the diphenylamine method.6 Cell protein was determined by the method of Oyama and Eagle.7 Radioactive samples were counted in a Packard Tri-carb scintillation counter. Bacterial DNA was extracted from cells in the log phase by the method of Marmur.8 Calf thymus DNA, yeast RNA, and E. coli sRNA were obtained from Worthington Biochemical Corp., Calbiochem, and General Biochemicals, respectively. Apurinic DNA was prepared from calf thymus DNA by the method of Tamm et al.9 The melting temperature (Tm) of DNA was determined with a recording thermospectrophotometer (Gilford Instruments Co.).10 The accepted GC content of DNA was that reported by Sueoka."I The difference spectra were measured in the Cary spectrophotometer, in specially designed cuvettes described by Trowne,'2 by the method of Goldberg et al."3 For the direct demonstration of binding of nogalamycin to DNA, mixtures of the drug and DNA in 0.1 M potassium phosphate buffer (pH 7) were extracted with ethyl acetate as described under Figure 2. The effect of nogalamycin on vaccinia and PR8 influenza virus was determined by the method of Siminoff,14 on herpes simplex by a modification of the method of Crowther and Melnick.15