DISCONTINUED This is a recently-commenced project being carried out collaboratively with Dr Kevin Ridge of the CARB facility (NIST/UMD) in Rockville MD. """"""""The NDP kinases are involved with GDP/GTP cycling in signal transduction, function as protein kinases, and are possibly associated with myc transcriptional regulation and with propensity to metastasize in a variety of human tumors. Little is known about the post-translational processing and oligomerization of the NDP kinases, and the effect of these processes on the biochemical functions."""""""" """"""""In the initial studies carried out so far, I have used both recombinant bovine NDPK (principally NBR2) and protein purified from bovine retina. These studies have been carried out by LC-ESI-MS on intact proteins, and by direct-infusion ESI-MS. Two components of the recombinant material have masses differing by 37 Da, in accordance with the two amino acid substitutions identified by cloning and sequencing approaches. However, the material purified from retina shows at least four major components, and the 37Da mass difference is lost. This loss indicates the presence of different post-translational modifications which are protein isoform specific. The mass difference between the quantitatively most significant proteins from retina is 80Da, indicating a post-translational phosphorylation. Considering the LC conditions, this must be an acid-stable phosphate, unlike the histidine- phosphate known to be an intermediate in the high-energy transfer of PO4 from ATP to GDP. Ongoing analyses are principally being carried out on digested proteins to identify the site of this phosphorylation, and the identity and localization of the other post-translational modifications. These studies involve chemical or endoproteolytic digestion and LCMS peptide analysis. Also, SDS- PAGE identified a major protein not represented by the cloned variants, and digestion/LCMS is being used to identify this protein.""""""""
