Radioactivity is incorporated essentially exclusively into ribosomal protein when [3H] tetracycline is irradiated in the presence of ribosomes. Such incorporation is shown to arise from three different processes: photoincorporation of native tetracycline, photoincorporation of tetracycline photoproduct, and, in the absence of P-mercaptoethanol, light-independent incorporation of tetracycline photoproduct. When both the rate of tetracycline to tetracycline photoproduct conversion and the protein labeling pattern produced by tetracycline photoproduct (utilizing both polyacrylamide gel electrophoresis and specific immunoprecipitation) are determined separately, it is possible to subtract out the contribution of tetracycline photoproduct to the overall labeling pattern obtained on irradiation of tetracycline and ribosomes and thus determine the labeling pattern due to native tetracycline incorporation. In this way we show that protein S7 is the major protein labeled by native tetracycline in both the presence and absence of @-mercaptoethanol. High labeling of proteins S18 and S4

4w-Diazomalonyldigitoxin and its isomer, 3#w-diazomalonyldigitoxin have been synthesized at high specific radioactivity and used as photolabels for the NaK-ATPase (ATP phosphohydrolase, EC 3.6.1.3) purified from Elkctrophos electricus. Photoaffinity labeling experiments using both type I and type II complexes of enze with both photolabels showed ouabain-protectable labeling of the a as well as the , subunit. These data suggest that, in the purified eel enzyme, the a and , subunits are in intimate contact, at least in the region of the third digitoxose of the "sugar-specific" binding site.Cardiac glycosides are specific inhibitors of NaK-ATPase (ATP phosphohydrolase, EC 3.6.1.3) (1). It has been suggested that this enzyme is the physiological receptor for the cardiotonic actions of these drugs (2-4).The purified NaK-ATPase from several sources (5, 6) consists of a large nonglycosylated (a) subunit (Mr = 95,000-100,000) and a small, glycosylated ((O subunit (Mr = 40,000-75,000) probably arranged in an a2j32 (7) or a214 (8) structure. The a subunit contains the phosphorylation site of the enzyme and at least a portion of the cardiac glycoside binding site as shown initially by Ruoho and Kyte (9) using a diazomalonylcymarin (DAM-cymarin) photolabel on the enzyme purified from dog kidney and by Hegyvary (10) using a periodate-oxidized ouabain affinity label. These data have been confirmed and extended more recently by Hall and Ruoho (11) using 2-nitro-4-azidophenyl (NAP)-glycyldigitoxigenin, Forbush et al. (12) using 2-nitro-5-azidobenzoyl (NAB)-ouabain, and Rogers and Lazdunski using NAP-ouabain (13). In addition to specific labeling of the a subunit, Forbush et al. (12) and Rogers and Lazdunski (13,14) reported ouabain-protectable photoincorporation of radioactivity into a low molecular weight (17,000) proteolipid component. Specific labeling of the (3 subunit has not been studied.In an attempt to probe the "sugar-specific" binding site region of the cardiotonic steroid site of Na,K-ATPase, we synthesized a tritiated DAM derivative of digitoxin (specific activity, 7.4 Ci/mmol; 1 Ci = 3.7 X 1010 becquerels) in which the photoactive group is on the 4T-hydroxyl of the third sugar (Fig.

The photoincorporation of p-azido[3H]puromycin [6-(dimethylamino)-9-[3'-deoxy-3'-[(p-azido-L-phenylalanyl)amino]-beta-D-ribofuranosyl]purine] into specific ribosomal proteins and ribosomal RNA [Nicholson, A. W., Hall, C. C., Strycharz, W. A., & Cooperman, B. S. (1982) Biochemistry (preceding paper in this issue)] is decreased in the presence of puromycin, thus demonstrating that labeling is site specific. The magnitudes of the decreases in incorporation into the major labeled 50S proteins found on addition of different potential ribosome ligands parallel the abilities of these same ligands to inhibit peptidyltransferase. This result provides evidence that p-azidopuromycin photoincorporation into these proteins occurs at the peptidyltransferase center of the 50S subunit, a conclusion supported by other studies of ribosome structure and function. A striking new finding of this work is that puromycin aminonucleoside is a competitive inhibitor of puromycin in peptidyltransferase. The photoincorporation of p-azidopuromycin is accompanied by loss of ribosomal function, but photoincorporated p-azidopuromycin is not a competent peptidyl acceptor. The significance of these results is discussed. Photolabeling of 30S proteins by p-azidopuromycin apparently takes place from sites of lower puromycin affinity than that of the 50S site. The possible relationship of the major proteins labeled, S18, S7, and S14, to tRNA binding is considered.

We have developed a method for the rapid localization of sites of ribosomal RNA labeling to limited regions (approximately 200 bases). The method is based on the formation and polyacrylamide gel electrophoretic separation of hybrids between restriction fragments of rrnB DNA and isotopically labeled rRNA and the subsequent determination of radioactivity across the gel. Using [3H]adenine-labeled rRNA as a control sample, we optimized experimental conditions with respect to a number of variables, including rRNA:DNA stoichiometric ratio, temperature of the annealing step, and levels of nucleases. An important result is that different rRNA X DNA hybrid fragments are obtained in different yields. The method was then applied to analyses of C3H3-labeled rRNA, giving results in good accord with known and proposed sites of rRNA methylation, and of rRNA that has been photoaffinity-labeled with 5-azido-2-nitrobenzoyl-[3H]Phe-tRNAPhe, a probe directed toward the peptidyltransferase center. The latter study showed a single major site of RNA labeling, falling within bases 2445-2668 of 23S rRNA. The extent of labeling was shown to be dependent on light-induced formation of a reactive intermediate and to be decreased in the absence of poly(uridylic acid) or in the presence of puromycin. The location of this major site of labeling is consistent with recent results obtained with an analogous tRNA photoaffinity label [Barta, A., Steiner, G., Brosius, J., Noller, H. F., & Kuechler, E. (1984) Proc. Natl. Acad. Sci. U.S.A. 81, 3607-3611] and with related genetic and biochemical studies of antibiotic interaction with ribosomes suggesting that the peptidyltransferase center falls within region V (bases 2043-2625) of 23S rRNA.

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