The major objective is to synthesize nucleotide photoaffinity and to validate their selective interaction at the active site of enzymes and regulatory proteins. All of the prominent mononucleotides have been modified to make effective photoprobes of cAMP, cGMP, ATP, GTP, UTP, TTP, NAD+, CoA, UDPG, etc. These probes have proven very effective for the identification of specific nucleotide binding proteins, even in crude homogenates. They have been relatively ineffective in detecting active side peptides due to the sometimes low efficiency of insertion and the lability of the photoinserted label to all forms of HPLC. We have resolved this latter problem by taking advantage of the properties of the photoinserted nucleotide to selectively isolate the photolabeled peptide without the use of HPLC except to verify the purity of the final material. Using these procedures we propose to identify the tryptic peptides of various nucleotide binding proteins. Proteins that we will study include the various """"""""biological response modifiers"""""""" such as tumor necrosis factor, interferon-alphaA, glucagon, IL-1 alpha & beta, prolactin, certain commercially available key enzymes such as glutamate dehydrogenase, phosphoglycerate & pyruvate kinase & unique proteins found in the cerebral spinal fluid (CSF) if Alzheimer's (AD) & ALS diseased individuals. All of these are known to have specific nucleotide binding sites. We also propose the synthesis of new nucleotide photoprobes designed to: (i) remove inherent weaknesses of previous probes (e.g., [alpha32P]8N3GTPdeltaS to prevent hydrolysis during long preincubations) (ii) make isolation of the photolabeled peptides easier (e.g. probes with a cis hydroxyl group attached through the alpha-phosphate increase the selective retention by boronate columns) and (iii) be new probes that will be selective for uncommon nucleotide sites (e.g. cyclic-ADPribose, cyclic-diGMP, 7-Me- Guanosine) or are small nucleic acid probes for proteins that selectively bind known small sequences (e.g., [32P]8N3ApUp which binds to diphtheria toxin). The most obvious health related application of this research is the determination of the nucleotide binding properties of various polypeptide hormones which may help explain their mechanism of action, the detection of certain oncogene protein products viral induced proteins, CSF proteins unique to AD, ALS, etc. and the study of several enzymes that are important in metabolism and certain enzymes that may be potential indicators of neoplasia.
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