@article {2437,
	title = {Identification of Nitroxyl-induced Modifications in Human Platelet Proteins Using a Novel Mass Spectrometric Detection Method},
	journal = {Molecular \& Cellular Proteomics},
	volume = {8},
	number = {5},
	year = {2009},
	note = {ISI Document Delivery No.: 446JCTimes Cited: 5Cited Reference Count: 67Hoffman, Michael D. Walsh, Geraldine M. Rogalski, Jason C. Kast, Juergen},
	month = {May},
	pages = {887-903},
	type = {Article},
	abstract = {Nitroxyl (HNO) exhibits many important pharmacological effects, including inhibition of platelet aggregation, and the HNO donor Angeli{\textquoteright}s salt has been proposed as a potential therapeutic agent in the treatment of many diseases including heart failure and alcoholism. Despite this, little is known about the mechanism of action of HNO, and its effects are rarely linked to specific protein targets of HNO or to the actual chemical changes that proteins undergo when in contact with HNO. Here we study the presumed major molecular target of HNO within the body: protein thiols. Cysteine-containing tryptic peptides were reacted with HNO, generating the sulfinamide modification and, to a lesser extent, disulfide linkages with no other long lived intermediates or side products. The sulfinamide modification was subjected to a comprehensive tandem mass spectrometric analysis including MS/MS by CID and electron capture dissociation as well as an MS3 analysis. These studies revealed a characteristic neutral loss of HS(O)NH2 (65 Da) that is liberated from the modified cysteine upon CID and can be monitored by mass spectrometry. Upon storage, partial conversion of the sulfinamide to sulfinic acid was observed, leading to coinciding neutral losses of 65 and 66 Da (HS(O)OH). Validation of the method was conducted using a targeted study of nitroxylated glyceraldehyde-3-phosphate dehydrogenase extracted from Angeli{\textquoteright}s salt-treated human platelets. In these ex vivo experiments, the sample preparation process resulted in complete conversion of sulfinamide to sulfinic acid, making this the sole subject of further ex vivo studies. A global proteomics analysis to discover platelet proteins that carry nitroxyl-induced modifications and a mass spectrometric HNO dose-response analysis of the modified proteins were conducted to gain insight into the specificity and selectivity of this modification. These methods identified 10 proteins that are modified dose dependently in response to HNO, whose functions range from metabolism and cytoskeletal rearrangement to signal transduction, providing for the first time a possible mechanistic link between HNO-induced modification and the physiological effects of HNO donors in platelets. Molecular \& Cellular Proteomics 8: 887-903, 2009.},
	keywords = {ACTIVATION, ALDEHYDE, ALZHEIMERS-DISEASE, BIOLOGICAL-ACTIVITY, DEHYDROGENASE, DETERRENT AGENT CYANAMIDE, INTEGRIN, NITRIC-OXIDE, NITROGEN-OXIDE, NMDA RECEPTOR, S-NITROSYLATION, SUCCINIMIDE FORMATION},
	isbn = {1535-9476},
	url = {<Go to ISI>://000266116900002},
	author = {Hoffman, M. D. and Walsh, G. M. and Rogalski, J. C. and Kast, J.}
}
@article {859,
	title = {Active site residues and mechanism of UDP-glucose dehydrogenase},
	journal = {European Journal of Biochemistry},
	volume = {271},
	number = {1},
	year = {2004},
	note = {ISI Document Delivery No.: 756CXTimes Cited: 16Cited Reference Count: 33},
	month = {Jan},
	pages = {14-22},
	type = {Article},
	abstract = {UDP-glucose dehydrogenase catalyzes the NAD(+)-dependent twofold oxidation of UDP-glucose to give UDP-glucuronic acid. A sequestered aldehyde intermediate is produced in the first oxidation step and a covalently bound thioester is produced in the second oxidation step. This work demonstrates that the Streptococcus pyogenes enzyme incorporates a single solvent-derived oxygen atom during catalysis and probably does not generate an imine intermediate. The reaction of UDP-[6",6"-di-H-2]-D-glucose is not accompanied by a primary kinetic isotope effect, indicating that hydride transfer is not rate determining in this reaction. Studies with a mutant of the key active site nucleophile, Cys260Ala, show that it is capable of both reducing the aldehyde intermediate, and oxidizing the hydrated form of the aldehyde intermediate but is incapable of oxidizing UDP-glucose to UDP-glucuronic acid. In the latter case, a ternary Cys260Ala/aldehyde intermediate/NADH complex is presumably formed, but it does not proceed to product as both release and hydration of the bound aldehyde occur slowly. A washout experiment demonstrates that the NADH in this ternary complex is not exchangeable with external NADH, indicating that dissociation only occurs after the addition of a nucleophile to the aldehyde carbonyl. Studies on Thr118Ala show that the value of k(cat) is reduced 160-fold by this mutation, and that the reaction of UDP-D-[6",6"-di-H-2]-glucose is now accompanied by a primary kinetic isotope effect. This indicates that the barriers for the hydride transfer steps have been selectively increased and supports a mechanism in which an ordered water molecule (H-bonded to Thr118) serves as the catalytic base in these steps.},
	keywords = {ACID, ALDEHYDE DEHYDROGENASE, CONSERVED CYSTEINE RESIDUES, DEHYDROGENASE, ENZYME, GDP-MANNOSE DEHYDROGENASE, GROUP-A STREPTOCOCCI, HISTIDINOL, MOLECULAR CHARACTERIZATION, PSEUDOMONAS-AERUGINOSA, URIDINE DIPHOSPHOGLUCOSE DEHYDROGENASE},
	isbn = {0014-2956},
	url = {<Go to ISI>://000187449200002},
	author = {Ge, X. and Penney, L. C. and van de Rijn, I. and Tanner, M. E.}
}
