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Anthracycline Photoaffinity Labeling of a Mitochondrial Polypeptide in P388 Murine Leukemic Cell Lines

Laboratory of Biological Chemistry, Developmental Therapeutics Program, Division of Cancer Treatment, National Cancer Institute, Bethesda, Maryland 20892

N-(p-Azido[3,5-³H]benzoyl)daunorubicin ([³H]NABD), a radioactive photoactive anthracycline analogue, was used to photoaffinity label anthracycline binding polypeptides in P388 murine leukemic cell lines. Whole cell homogenates were mixed with 6 x 10^-8 M [³H]NABD, exposed to ultraviolet light, and analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis for radiolabel incorporation. Autoradiofluorography showed incorporation of radioactivity into a M_r 18,000 component independent of polypeptides prominently stained with Coomassie blue. Photolabeling of subcellular fractions showed predominant mitochondrial localization of the M_r 18,000 radiolabel. The protein composition of the photolabeled constituents was confirmed by treatment with proteinase K, DNase and RNase, or by lipid extraction with organic solvent. [³H]NABD photolabeling of homogenates from anthracycline sensitive and resistant cells resulted in M_r 18,000 radiolabel incorporation of 3966 ± 355 and 6487 ± 533 dpm per 50 µg cellular protein for anthracycline sensitive and resistant cells, respectively (P < 0.005). These studies characterize the photoaffinity labeling of a low molecular weight mitochondrial polypeptide using a photoactive anthracycline analogue. The role for this polypeptide as a mediator of anthracycline activity remains to be determined.

¹ Present address: Division of Clinical Pharmacology, Department of Pharmacology, Uniformed Services University of the Health Sciences, Bethesda, MD 20814-4799. To whom requests for reprints should be addressed, at Clinical Pharmacology Branch, Division of Cancer Treatment, National Cancer Institute, NIH, Building 10, Room 6N119, Bethesda, MD 20892.

Received 2/ 4/86. Revised 6/27/86. Accepted 8/13/86.