Characterization of sheep brain ryanodine receptor ATP binding site by photoaffinity labeling

Two high M-r protein bands (440 and 420 kDa) in sheep brain microsomal memb ranes mere labeled with the photoaffinity ATP analog, O-(4-benzoyl)benzoyl adenosine 5'-triphosphate (Bz(2)ATP), The 420 kDa band is labeled by [alpha -P-32]Bz2ATP with about 1000-fold higher affinity than the 440 kDa band. Th e heavily labeled 420 kDa band is identified as dynein heavy chain based on its partial amino acid sequence, and crossreactivity with anti-dynein anti bodies, 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 ryanodirie to its receptor up to 3-fold, and increased the binding affinity up to 6-fold. Other nucleotides stimulat e ryanodine binding with decreasing effectiveness: Bz(2)ATP > ATP > ADP > A MP > AMP-PNP > GTP > cAMP, With respect to nucleotide specificity, this bin ding site is similar to the skeletal muscle RyR (type 1). However, the brai n RyR may have additional one or more sites with lower affinity with inhibi tory effect on ryanodine binding, These results suggest that the major RyR isoform in sheep brain corresponds to the type-2 isoform, and that modulati on of ryanodine binding by ATP involves its binding to the RyR protein, The association of dynein with brain microsomal membranes may reflect a linkag e of RyR to the cytoskeleton. (C) 1999 Federation of European Biochemical S ocieties.