PHOTOAFFINITY-LABELING OF CREATINE-KINASE WITH 2-AZIDOADENOSINE AND 8-AZIDOADENOSINE TRIPHOSPHATE - IDENTIFICATION OF 2 PEPTIDES FROM THE ATP-BINDING DOMAIN

Two different analogs of ATP, [gamma-P-32]2N(3)ATP and [gamma-P-32]8N( 3)ATP, were used to photoaffinity label the MM and BB isoforms of rabb it cytosolic creatine kinase. Evidence that photoinsertion was within the ATP-binding domain was as follows: (1) Assays for creatine phospha te production demonstrated that [gamma-(32)]2N(3)ATP and [gamma-(32)p] 8N(3)ATP are substrates for creatine kinase. (2) Enzymatic activity wa s inhibited by photolabeling with either analog. (3) Saturation of pho toinsertion was observed for both analogs. Half-maximal saturation was observed at 5 mu M [gamma-P-32]2N(3)ATP or 12 mu M [gamma-P-32]8N(3)A TP. (4) Photoinsertion of both probes could be decreased by micromolar levels of ATP. Immobilized Al3+ affinity chromatography and HPLC were used to isolate the peptides modified by these probes. Overlapping se quence analysis of the isolated peptides from the tryptic and chymotry ptic digests of the photolabeled MM isoform revealed that [gamma-P-32] 8N(3)ATP photoinserted into the peptide region corresponding to Val(27 9)-Arg(291), whereas [gamma-P-32]2N(3)ATP photoinserted into Val(236)- Lys(241). The corresponding peptides (Ile(279)-Arg(291) and Val(236)-L ys(241)) from the BB isoform were shown to be selectively modified. We conclude that amino acid residues within the peptide regions 236-241 and 279-291 of rabbit cytosolic creatine kinase are localized within t he binding domain for the adenine moiety of ATP. The results also demo nstrate the effectiveness and selectivity of Al3+ as the chelating age nt in immobilized metal affinity chromatography for the isolation of p hotolabeled peptides as well as its potential to enhance retention of radiolabel during HPLC.