Hyperhomocyst(e)inemia is believed to injure endothelial cells in vivo through a number of mechanisms, including the generation of hydrogen peroxide (H 2 O 2 ).Earlier in vitro studies demonstrated that homocyst(e)ine (Hcy) decreases the biological activity of endothelium-derived relaxing factor and that this decrease can be reversed by preventing the generation of hydrogen peroxide. Here we show that Hcy treatment of bovine aortic endothelial cells leads to a dose-dependent decrease in NO x (p ‫؍‬ 0.001 by one-way analysis of variance) independent of endothelial nitric-oxide synthase activity or protein levels and nos3 transcription, suggesting that Hcy affects the bioavailability of NO, not its production. We hypothesized that, in addition to increasing the generation of H 2 O 2 , Hcy decreases the cell's ability to detoxify H 2 O 2 by impairing intracellular antioxidant enzymes, specifically the intracellular isoform of glutathione peroxidase (GPx). To test this hypothesis, confluent bovine aortic endothelial cells were treated with a range of concentrations of Hcy, and intracellular GPx activity was determined. Compared with control cells, cells treated with Hcy showed a significant reduction in GPx activity (up to 81% at 250 M Hcy). In parallel with the decrease in GPx activity, steady-state GPx mRNA levels were also significantly decreased compared with control levels after exposure to Hcy, which appeared not to be a consequence of message destabilization. These data suggest a novel mechanism by which Hcy, in addition to increasing the generation of hydrogen peroxide, may selectively impair the endothelial cell's ability to detoxify H 2 O 2 , thus rendering NO more susceptible to oxidative inactivation.Hyperhomocyst(e)inemia is a disease caused by an abnormality in either an enzyme (cystathionine ␤-synthetase or temperature-sensitive methylenetetrahydrofolate reductase) or a cofactor (folate, vitamin B 12 , or vitamin B 6 ) required for homocysteine metabolism. These abnormalities lead to elevations in plasma concentrations of homocyst(e)ine (Hcy) 1 and its precursor methionine as well as a reduction in plasma concentrations of cysteine (1-5). In its most severe form, hyperhomocyst(e)inemia confers a significant risk for thromboembolic complications that are often fatal (6). In contrast, the less severe form of the disease is commonplace and indolent, not presenting with clinical sequelae until the third or fourth decade of life. These individuals manifest atherosclerosis as well as recurrent episodes of acute arterial and venous thrombosis (6) with near normal levels of fasting plasma homocyst(e)ine; following a methionine challenge, however, levels rise significantly compared with normal levels. Many studies demonstrate that hyperhomocyst(e)inemia is an independent risk factor for atherosclerosis in the coronary, cerebral, and peripheral vasculature (7-11), and up to 40% of patients with coronary or cerebrovascular atherosclerosis have hyperhomocyst(e)inemia.The mechanism by which Hcy damages the vessel ...

Therapeutic strategies targeted at modulating Lrrk2 kinase activity may be important to treat patients with genetically defined familial or typical sporadic Parkinson's disease.

Acetylcholinesterase (AChE) contains a narrow and deep active site gorge with two sites of ligand binding, an acylation site (or A-site) at the base of the gorge, and a peripheral site (or P-site) near the gorge entrance. The P-site contributes to catalytic efficiency by transiently binding substrates on their way to the acylation site, where a short-lived acyl enzyme intermediate is produced. A conformational interaction between the A- and P-sites has recently been found to modulate ligand affinities. We now demonstrate that this interaction is of functional importance by showing that the acetylation rate constant of a substrate bound to the A-site is increased by a factor a when a second molecule of substrate binds to the P-site. This demonstration became feasible through the introduction of a new acetanilide substrate analogue of acetylcholine, 3-(acetamido)-N,N,N-trimethylanilinium (ATMA), for which a = 4. This substrate has a low acetylation rate constant and equilibrates with the catalytic site, allowing a tractable algebraic solution to the rate equation for substrate hydrolysis. ATMA affinities for the A- and P-sites deduced from the kinetic analysis were confirmed by fluorescence titration with thioflavin T as a reporter ligand. Values of a >1 give rise to a hydrolysis profile called substrate activation, and the AChE site-specific mutant W86F, and to a lesser extent wild-type human AChE itself, showed substrate activation with acetylthiocholine as the substrate. Substrate activation was incorporated into a previous catalytic scheme for AChE in which a bound P-site ligand can also block product dissociation from the A-site, and two additional features of the AChE catalytic pathway were revealed. First, the ability of a bound P-site ligand to increase the substrate acetylation rate constant varied with the structure of the ligand: thioflavin T accelerated ATMA acetylation by a factor a(2) of 1.3, while propidium failed to accelerate. Second, catalytic rate constants in the initial intermediate formed during acylation (EAP, where EA is the acyl enzyme and P is the alcohol leaving group cleaved from the ester substrate) may be constrained such that the leaving group P must dissociate before hydrolytic deacylation can occur.