HOMEPRODUCTSCOMPANYCONTACTFAQResearchDictionaryPharmaSign Up FREE or Login

Mass spectrometric kinetic analysis of human tyrosylprotein sulfotransferase-1 and -2.

Abstract
Protein tyrosine O-sulfation, a widespread post-translational modification, is mediated by two Golgi enzymes, tyrosylprotein sulfotransferase-1 and -2. These enzymes catalyze the transfer of sulfate from the universal sulfate donor 3'-phosphoadenosine-5'-phosphosulfate (PAPS) to the hydroxyl group of tyrosine residues to form tyrosine O-sulfate ester and PAP. More than 60 proteins have been identified to be tyrosine sulfated including several G protein-coupled receptors, such as CC-chemokine receptor 8 (CCR8) that is implicated in allergic inflammation, asthma, and atherogenesis. However, the kinetic properties of purified tyrosylprotein sulfotransferase (TPST)-1 and -2 have not been previously reported. Moreover, currently there is no available quantitative TPST assay that can directly monitor individual sulfation of a series of tyrosine residues, which is present in most known substrates. We chose an MS-approach to address this limitation. In this study, a liquid chromatography electrospray ionisation mass spectrometry (LC/ESI-MS)-based TPST assay was developed to determine the kinetic parameters of individual TPSTs and a mixture of both isozymes using CCR8 peptides as substrates that have three tyrosine residues in series. Our method can differentiate between mono- and disulfated products, and our results show that the K(m,app) for the monosulfated substrate was 5-fold less than the nonsulfated substrate. The development of this method is the initial step in the investigation of kinetic parameters of the sequential tyrosine sulfation of chemokine receptors by TPSTs and in determining its catalytic mechanism.
AuthorsLieza M Danan, Zhihao Yu, Adam J Hoffhines, Kevin L Moore, Julie A Leary
JournalJournal of the American Society for Mass Spectrometry (J Am Soc Mass Spectrom) Vol. 19 Issue 10 Pg. 1459-66 (Oct 2008) ISSN: 1044-0305 [Print] United States
PMID18672380 (Publication Type: Journal Article, Research Support, N.I.H., Extramural)
Chemical References
  • Membrane Proteins
  • Recombinant Proteins
  • Phosphoadenosine Phosphosulfate
  • Sulfotransferases
  • TPST2 protein, human
  • protein-tyrosine sulfotransferase
Topics
  • Catalysis
  • Chromatography, Liquid (methods)
  • Humans
  • Kinetics
  • Membrane Proteins (chemistry, genetics, metabolism)
  • Phosphoadenosine Phosphosulfate (chemistry, metabolism)
  • Recombinant Proteins (chemistry, metabolism)
  • Spectrometry, Mass, Electrospray Ionization (methods)
  • Sulfotransferases (chemistry, genetics, metabolism)

Join CureHunter, for free Research Interface BASIC access!

Take advantage of free CureHunter research engine access to explore the best drug and treatment options for any disease. Find out why thousands of doctors, pharma researchers and patient activists around the world use CureHunter every day.
Realize the full power of the drug-disease research graph!


Choose Username:
Email:
Password:
Verify Password:
Enter Code Shown: