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Differential hydrolysis of homocysteine thiolactone by purified human serum (192)Q and (192)R PON1 isoenzymes.

Abstract
Human serum paraoxonase 1 (PON1) is a HDL-associated enzyme that catalyzes the hydrolysis of a variety of aromatic carboxylic acid esters and several organophosphates. Recently it has been suggested that a physiological substrate of serum PON1 is homocysteine thiolactone which is a putative risk factor in atherosclerosis. In this study, human (192)Q and (192)R PON1 isoenzymes were purified from the respective phenotype human serum, using a protocol consisting of ammonium sulfate precipitation and four chromatography steps: gel filtration, ion-exchange, non-specific affinity, and a second ion-exchange. Using paraoxon as substrate, overall purification fold was found as 742 for (192)R PON1 and 590 for (192)Q PON1. The final purified enzymes were shown as single protein bands close to 45kDa on SDS-PAGE and confirmed by Western blot. Substrate kinetics were studied with phenyl acetate, paraoxon and homocysteine thiolactone. Both PON1 isoenzymes showed mixed type inhibition with phenyl acetate. K(m) values of (192)Q and (192)R PON1 for homocysteine thiolactone were 23.5mM and 22.6mM respectively. For (192)R PON1, the V(max) was 2.5-fold and k(cat)/K(m) was 2.6-fold higher than those for (192)Q PON1 when homocysteine thiolactone is used as substrate. The present data suggest that defining (192)Q and (192)R PON1 isoforms could be a good predictor and prognostic marker in the cardiovascular risk assessment.
AuthorsAhmet Bayrak, Tülin Bayrak, Ediz Demirpençe, Kamer Kılınç
JournalJournal of chromatography. B, Analytical technologies in the biomedical and life sciences (J Chromatogr B Analyt Technol Biomed Life Sci) Vol. 879 Issue 1 Pg. 49-55 (Jan 01 2011) ISSN: 1873-376X [Electronic] Netherlands
PMID21123122 (Publication Type: Journal Article, Research Support, Non-U.S. Gov't)
CopyrightCopyright © 2010 Elsevier B.V. All rights reserved.
Chemical References
  • Isoenzymes
  • Phenylacetates
  • Homocysteine
  • homocysteine thiolactone
  • Aryldialkylphosphatase
  • PON1 protein, human
  • Paraoxon
  • Ammonium Sulfate
Topics
  • Ammonium Sulfate (chemistry)
  • Aryldialkylphosphatase (blood, chemistry)
  • Atherosclerosis (metabolism)
  • Blotting, Western
  • Chemical Precipitation
  • Chromatography, Liquid
  • Homocysteine (analogs & derivatives, chemistry, metabolism)
  • Humans
  • Isoenzymes (blood, chemistry)
  • Kinetics
  • Paraoxon (chemistry)
  • Phenylacetates (chemistry)

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