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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-32P]ATP
Journal article   Open access   Peer reviewed

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-32P]ATP

Kevin P Campbell and David H MacLennan
The Journal of biological chemistry, Vol.258(3), pp.1391-1394
02/10/1983
DOI: 10.1016/S0021-9258(18)32993-4
PMID: 6687385
url
https://doi.org/10.1016/S0021-9258(18)32993-4View
Published (Version of record) Open Access

Abstract

8-Azido-[~x-~~P]ATP (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 sarcoplasmic reticulum vesicles were incubated with 0.14-1.6 PM 8-N3-ATP. The presence of 100-500 PM ATP during the incubation inhibited the binding of 8-N3- ATP, while CAMP 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, K. P., and MacLennan, D. H. (1981) J. Biol. Chem 256, 4626-4632) are the 53,000- and 160,000-dalton that bind 8-N3-[a-32P]ATP and, therefore, these glycoproteins contain ATP binding sites. Antiserum to the 53,000-dalton glycoprotein specifically immunoprecipitated the 53,000- and 160,000-dalton 8-N3-ATP-labeled proteins from extracts of sarcoplasmic reticulum. When cardiac sarcoplasmic reticulum was labeled with 8-N3-ATP, label was incorporated into proteins of 53,000 and 130,000 daltons. These proteins were precipitated with antiserum against the skeletal muscle, 53,000-dalton glycoprotein and the molecular weights of these proteins were reduced to 49,000 and 125,000, respectively, after incubation with Endo8-N-acetylglucosaminidase H. Thus the 8-Na-ATP binding proteins were identified as the 53,000- and 130,000- dalton, intrinsic glycoproteins of cardiac sarcoplasmic reticulum.
 Rabbits Molecular Weight Glycoproteins - metabolism Phosphorus Radioisotopes Affinity Labels - metabolism Adenosine Triphosphate - analogs & derivatives Azides Animals Adenosine Triphosphate - metabolism Protein Binding Sarcoplasmic Reticulum - metabolism Muscles - metabolism Binding Sites

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