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Characterization of sheep brain ryanodine receptor ATP binding site by photoaffinity labeling
Author(s) -
Hadad Nurit,
Martin Cecile,
Ashley Richard H.,
Shoshan-Barmatz Varda
Publication year - 1999
Publication title -
febs letters
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.593
H-Index - 257
eISSN - 1873-3468
pISSN - 0014-5793
DOI - 10.1016/s0014-5793(99)00738-3
Subject(s) - ryanodine receptor , photoaffinity labeling , biochemistry , gtp' , binding site , chemistry , nucleotide , biology , receptor , enzyme , gene
Two high M r protein bands (440 and 420 kDa) in sheep brain microsomal membranes were labeled with the photoaffinity ATP analog, O ‐(4‐benzoyl)benzoyl adenosine 5′‐triphosphate (Bz 2 ATP). The 420 kDa band is labeled by [α‐ 32 P]‐Bz 2 ATP with about 1000‐fold higher affinity than the 440 kDa band. The heavily labeled 420 kDa band is identified as dynein heavy chain based on its partial amino acid sequence, and cross‐reactivity with anti‐dynein antibodies. The 440 kDa protein is immunologically identified as the type‐2 RyR. Bz 2 ATP binding is obtained in the absence of divalent cations. Bz 2 ATP and ATP increased the binding of ryanodine to its receptor up to 3‐fold, and increased the binding affinity up to 6‐fold. Other nucleotides stimulate ryanodine binding with decreasing effectiveness: Bz 2 ATP>ATP>ADP>AMP>AMP‐PNP>GTP>cAMP. With respect to nucleotide specificity, this binding site is similar to the skeletal muscle RyR (type 1). However, the brain RyR may have additional one or more sites with lower affinity with inhibitory effect on ryanodine binding. These results suggest that the major RyR isoform in sheep brain corresponds to the type‐2 isoform, and that modulation of ryanodine binding by ATP involves its binding to the RyR protein. The association of dynein with brain microsomal membranes may reflect a linkage of RyR to the cytoskeleton.