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NaCl, 1% Triton X-100, 1% sodium deoxycholate, 0.1% SDS and 1mM EDTA) as described previously.5) Cell extracts were immunoprecipitated with an excess amount of anti-p60 serum raised against p60v~src which is produced in Escherichia coli;e:> 4 fA of anti-p60 serum was added to 80 fA of lysate (prepared from 1/20 of one 90 mm-dish culture) and kept on ice for 1 hour. After protein A-Sepharose 4B-CL were added, the mixture were rotated at 4°Cfor 90 minutes. The immunoprecipitates were washed five times with RIPA buffer and further washed three times with 40mMTris-HCl (pH 7.2). Washedimmunoprecipitates were resuspended in 30 [A of kinase assay buffer contain- obtained by excising the p60T~src bands from the gel and counting as described.6) By this reaction, p60v'src is phosphorylated on tyrosine-4166) and this residue is the site of phosphorylation of

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A new inhibitor of protein kinase C (PKC), UCN-01, was isolated from the culture broth of Streptomyces sp. N-126. Wehave found that this strain also produces UCN-02 which is a stereoisomer of UCN-01.The inhibitors have the molecular formula C28H26N4O4 and have an indolo[2,3-#]carbazole chromophore. Their structures have been elucidated by mass and NMR spectra. UCN-01has been shown to inhibit PKCand protein kinase A (PKA) with IC5o values of 0.0041 and 0.042^m, respectively, and UCN-02has been shown to inhibit PKCand PKAwith IC50 values of 0.062 and 0.25^m, respectively. UCN-01and UCN-02also showedthe cytotoxic effect on the growth of HeLaS3 cells.In the course of our screening program for new selective inhibitors of protein kinase C (PKC)1~3), a Streptomyces strain N-1264) was found to produce new selective inhibitors of PKC, UCN-01and UCN-02. UCN-01is a novel compound that has a hydroxyl group at C-7 of staurosporine5>6), and UCN-02is a stereoisomer at C-7 of UCN-01. They have been shown to inhibit PKCat extremely low concentration and showed the cytotoxic effect on the growth of HeLa S3 cells. In this paper, their purification, physico-chemical properties, structures and biological activities are described.Isolation and Purification TLC and HPLCanalysis were used to monitor UCN-01 and UCN-02 during isolation from the culture broth. The whole broth (30 liters) was filtered and the solid cake was extracted with acetone. Acetone extract was diluted with equivalent volume of water and then the extract was mixed with the filtrate of the broth. The mixed solution was applied on a column (2 liters) of non-ionic porous resin, Diaion HP-20 (Mitsubishi Chemical Industries Limited). After washing with water and then with aqueous methanol (2 : 1) to remove impurities, the column was eluted with acetone. The acetone eluate was evaporated to remove acetone. The aqueous solution was adjusted to pH10 and extracted with ethyl acetate. The resulting organic phase was extracted with 0.1 n hydrochloric acid and after the separation the aqueous phase was immediately adjusted to pH 10 by addition of NaOHand extracted with ethyl acetate. The ethyl acetate extract was applied to silica gel (Wakogel C-200) column chromatography with chloroform -methanol as a elution solvent. The 97 %-chloroform eluate was concentrated to give a pale yellow syrup, which was recrystallized from methanol to obtain 200 mg of