Changes in pulmonary adenosine triphosphate-binding proteins detected by nucleotide photoaffinity labeling following treatment of mice with the tumor-modulatory agent butylated hydroxytoluene.

Protein kinases play a central role in the regulation of normal and neoplastic growth. A novel approach to evaluating this role is to apply the photoaffinity analogue of adenosine 5'-triphosphate (ATP), 8-azido[gamma-32P]adenosine 5'-triphosphate (8-N3-[gamma-32P]ATP), to extracts obtained from nondividing and dividing tissues in order to label high affinity ATP receptors, including protein kinases. A pattern of ATP-binding proteins was observed on autoradiograms prepared from lung extracts which were reproducible upon repeated experimentation and was identical among extracts prepared from several inbred strains. Seven specifically photolabeled proteins were detected with molecular weights of 120,000, 84,000, 53,000, 51,000, 43,000, 41,000, and 33,000. The pneumotoxicity that results from treating mice with butylated hydroxytoluene (BHT) is followed by a stimulation of lung cell proliferation as part of the regenerative repair phase. Increased photoincorporation of 8-N3-[gamma-32P]ATP into proteins with molecular weights of 41,000, 37,000 and 33,000 resulted from BHT treatment. The following correlations between increased photoincorporation and lung repair, as defined by an increased lung weight/body weight ratio, were observed: (a) these changes shared a similar BHT dose dependency; (b) both followed the same time course and were reversible; (c) both lung damage and enhanced photolabeling were abolished if the mice were pretreated with cedrene, an inducer of drug-detoxifying enzymes; (d) the effect of BHT on ATP-binding proteins was agent specific, since urethan treatment did not affect photoincorporation. These results demonstrate that the specific photolabeling of ATP receptors in tissue extracts is a sensitive indicator of a particular growth-related repair process.