Ischemia and cardiac hypertrophy can lead to alterations in the function of cardiac sarcoplasmic reticulum. In the case of cardiac ischemia this can occur as early as 7.5 minutes after normothermic global ischemia. The natural history of the development of damage in hypertrophy has not been described. The proposed careful characterization of the damage to the sarcoplasmic reticulum avoids, where possible, the use of oxalate because the kinetics of the precipitation of calcium oxalate confound the interpretation of results. The parameters of activity which will be measured are: 1) nucleotide binding capacity, 2) steady state calcium loading, 3) phosphoenzyme maximum, 4) enzyme turnover rate, 5) calcium influx rate, 6) calcium efflux rate, 7) ATP greater than ADP flux rate and 8) ADP greater than ATP flux rate. With these measurements it will be possible to determine modifications of partial reactions, damage to the (Ca, Mg) ATPase and the effect of these pathological conditions on the coupling of calcium transport to ATPase activity. Characterization of the active site of the Ca, Mg-ATPase of normal and damaged sarcoplasmic reticulum will also be pursued. The active site will be photolabeled with 3 feet-0-(4-benzoyl)benzoic [Alpha-32P]ATP. Specificity of site labeling will be determined from the stoichiometry between incorporation and inhibition of ATPase activity. To identify the active site the labeled protein will be subjected to tryptic digestion and the amino acid sequences of the 32P labeled peptides will be determined. By determining the sequences of overlapping peptides and the aminoacids containing photoincorporated 3 feet-0-(4-benzoyl)benzoic [Alpha-32P]ATP the active site will be identified. In damaged sarcoplasmic reticulum the presence and position of altered amino acids in the region of the active site will be identified.
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