Labeling of high affinity ATP binding sites on the 53,000- and 160,000-dalton glycoproteins of the sarcoplasmic reticulum with the photoaffinity probe 8-N3-alpha-32PATP.

lL6 8-Azido-~x-~~PATP (8-N3-ATP) was used as a photoaffinity label for ATP binding sites in the sarcoplasmic reticulum membrane.The radioactive 8-N3-ATP was specifically incorporated into proteins of 53,000, 105,000, and 160,000 daltons when intact sar- coplasmic reticulum vesicles were incubated with 0.14-1.6PM 8-N3-ATP.The presence of 100-500 PM ATP during the incubation inhibited the binding of 8-N3- ATP, while C A M P and AMP did not affect binding.Analysis of various membrane fractions during purification of the sarcoplasmic reticulum from muscle homogenates showed concomitant purification of the 53,000-, 105,000-, and 160,000-dalton proteins that bound 8-N3-ATP.The 8-N3-ATP-labeled proteins had identical mobilities to the 53,000-dalton glycoprotein, the 105,000-dalton (Ca2+ + M&+)-ATPase and the 160,-000-dalton glycoprotein, respectively.8-N3-ATP labeling of deoxycholate extracts of sarcoplasmic reticulum resulted in the specific labeling of two proteins of 40,000 and 53,000 daltons, while calsequestrin (63,000 daltons) was not labeled.The 53,000-and 160,000-dalton labeled proteins bound to Con A Sepharose columns and were eluted by a-methyl-D-mannoside. Endo-/3-N-acetylglucosaminidase H digestion of 8-Ns-ATP-labeled proteins reduced the 53,000-and 160,000-dalton 8-N3-ATP-labeled proteins to 49,000 and 155,000 daltons, respectively.These observations show that the major intrinsic glycoproteins of the sarcoplasmic reticulum (Campbell,