Studies on nucleotide interactive proteins in control versus ischemic rat brains. I.~Increased glutamine synthetase synthesis and photolabeling. II.~Decreased phosphorylation of calcium-ATPase. III.~Identification of peptides in the NADP(+) binding site

Brain damage from global ischemia is caused by a reduction in the total cerebral blood flow which results in regionally selective neuronal cell death. Striatal neurons are damaged irreversibly and die within 24 hrs after ischemia, hippocampal neurons show a delay of 48-72 hrs before neuronal death whereas neurons of the paramedian cortex are the least permanently damaged. The exact reason for this regionally selective vulnerability is not known.; Ischemia/reperfusion injury results in a series of metabolic and biochemical changes that eventually lead to neuronal death. These changes include; depletion of ATP, disturbance in calcium homeostasis, increase in extracellular glutamate levels, and production of free radicals. Photoaffinity analogs were used to study the changes in nucleotide interactive proteins in control versus ischemic rat brain. Changes in both nucleotide binding and phosphorylation were observed that may explain the selective vulnerability displayed by different regions of the brain.; A 43 kDa protein with pI 6.8 showed increased photoinsertion of ({dollar}alphasp{lcub}32{rcub}{dollar}P) 8N{dollar}sb3{dollar}ATP in striatal and hippocampal ischemic samples reperfused for 1 hr. This protein was identified as glutamine synthetase by Western blotting and by exploiting certain metal binding properties of glutamine synthetase. A 130 kDa protein showed decreased phosphorylation in all ischemic samples. This protein was identified as CaATPase by Western blotting. There are other proteins that also show changes in ischemic samples relative to the control samples.; Isocitrate dehydrogenase (IDH) is an important enzyme both in citric acid cycle and in glutamate metabolism. This enzyme was shown inactivated by free radicals produced during ischemia. The structure of prokaryotic E. Coli NADP{dollar}sp+{dollar}-IDH is known, but the structure of mammalian NADP{dollar}sp+{dollar}-IDH is not defined. There is very little sequence homology between the pig heart NADP{dollar}sp+{dollar}-IDH and E. Coli NADP{dollar}sp+{dollar}-IDH. Pig heart NADP{dollar}sp+{dollar}-IDH is readily available, and if it is assumed that the brain isozyme would be homologous to the heart isozyme, the peptides in the NADP{dollar}sp+{dollar} binding site of pig heart NADP{dollar}sp+{dollar}-IDH isolated using (2{dollar}sp{lcub}prime32{rcub}{dollar}P) 2N{dollar}sb3{dollar}NADP{dollar}sp+{dollar} and ({dollar}alphasp{lcub}32{rcub}{dollar}P) 2N{dollar}sb3{dollar}NAD{dollar}sp+{dollar} are presumably similar to the brain enzyme. Two peptides, Ile{dollar}sp{lcub}244{rcub}{dollar}-Arg{dollar}sp{lcub}249{rcub}{dollar} and Leu{dollar}sp{lcub}121{rcub}{dollar}-Arg{dollar}sp{lcub}133{rcub}{dollar} were identified. It was also found that the ATP photoaffinity analogs ({dollar}alphasp{lcub}32{rcub}{dollar}P) 8N{dollar}sb3{dollar}ATP and ({dollar}gammasp{lcub}32{rcub}{dollar}P) 2N{dollar}sb3{dollar}ATP bind at the NADP{dollar}sp+{dollar} site of the enzyme.