Relationships between paraoxon and 2-coumaranone hydrolytic activities in sera genotyped for PON1 Q192R polymorphism
ABSTRACT To set-up a method for a direct evaluation in human serum of paraoxonase enzymatic activities, establishing a possible correlation with Q192R genotype polymorphism.
101 different human serum samples were genotyped for paraoxonase Q192R polymorphism by PCR restriction analysis, and evaluated spectrophotometrically with regard to paraoxon and 2-coumaranone hydrolytic activities. Both activities of paraoxonase were assayed, quantified through normalization by arylesterase activity, and compared with the data concerning Q/R genetic polymorphism.
The mean normalized paraoxonase activity was found to be significantly higher in RR than in QQ human sera (3.99+/-0.6 versus 1.32+/-0.44; P<0.0001); instead, the 2-coumaranone hydrolysis showed an opposite trend (0.10+/-0.02 versus 0.23+/-0.04, in RR and QQ sera respectively; P<0.0001).
These methods were successfully applied to the whole serum, suggesting a possible use of this approach for a clinically relevant phenotypic characterization.
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ABSTRACT: Cholesterol is a 27-carbon steroid that is an essential component of the cell membrane, the immediate precursor of steroid hormones, the substrate for the formation of bile acids, and is required for the assembly of very low density lipoprotein in the liver. Because as much as two-thirds of total body cholesterol in patients is of endogenous origin, an effective means to control cholesterogenesis may occur by inhibition of its biosynthesis. Cholesterol is biosynthesized in a series of more than 25 separate enzymatic reactions that initially involve the formation of 3-hydroxy-3-methylglutaryl coenzyme A (HMG CoA). Early attempts to pharmacologically block cholesterol synthesis focused only on steps later in the biosynthetic pathway and resulted in compounds with unacceptable toxicity. Recent research had identified that HMG CoA reductase is a key rate-limiting enzyme in this pathway and is responsible for the conversion of HMG CoA to mevalonate. Additional research with fungal metabolites identified a series of compounds with potent inhibiting properties for this target enzyme, from which lovastatin was selected for clinical development. A reduction in cholesterol synthesis by lovastatin has been subsequently confirmed in cell culture, animal studies and in humans. A resultant decrease in circulating total and low-density lipoprotein (LDL) cholesterol has also been demonstrated in animals and humans. Because hepatic LDL receptors are the major mechanism of LDL clearance from the circulation, further animal research has confirmed that these declines in cholesterol are accompanied by an increase in hepatic LDL receptor activity. Lovastatin effectively diminishes endogenous cholesterol synthesis providing useful therapeutic properties for patients with hypercholesterolemia.The American Journal of Cardiology 12/1988; 62(15):10J-15J. DOI:10.1016/0002-9149(88)90002-1 · 3.43 Impact Factor
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ABSTRACT: Serum paraoxonases (PONs) are a group of enzymes that play a key role in organophosphate (OP) detoxification and in prevention of atherosclerosis. However, their structure and mechanism of action are poorly understood. PONs seem like jacks-of-all-trades, acting on a very wide range of substrates, most of which are of no physiological relevance. Family shuffling and screening lead to the first PON variants that express in a soluble and active form in Escherichia coli. We describe variants with kinetic parameters similar to those reported for PONs purified from sera and others that show dramatically increased activities. In particular, we have evolved PON1 variants with OP-hydrolyzing activities 40-fold higher than wild type and a specificity switch of >2,000-fold, producing PONs specialized for OP rather than ester hydrolysis. Analysis of the newly evolved variants provides insights into the evolutionary relationships between different family members.Proceedings of the National Academy of Sciences 01/2004; 101(2):482-7. DOI:10.1073/pnas.2536901100 · 9.81 Impact Factor