No abstract

Cystathionine beta-synthase is an unusual enzyme that requires the cofactors heme and pyridoxal phosphate (PLP) to catalyze the condensation of homocysteine and serine to generate cystathionine. This transsulfuration reaction represents one of two major cellular routes for detoxification of homocysteine, which is a risk factor for atherosclerosis. While the beta-replacement reaction catalyzed by this enzyme suggests a role for the pyridoxal phosphate, the role of the heme is uncertain. In this study we have examined the effect of changing one of the ligands to the heme on the activity of the enzyme. Binding of carbon monooxide results in the displacement of a thiolate ligand to the ferrous heme, and is accompanied by complete loss of cystathionine beta-synthase activity. Furthermore, inhibition by CO is competitive with respect to homocysteine, providing the first indication that the homocysteine binding site is in the proximity of heme. Binding of both CO and cyanide to ferrous cystathionine beta-synthase occurs in two distinct isotherms and indicates that the hemes are nonequivalent. We have employed fluorescence spectroscopy to characterize the bound PLP and its interaction with serine. PLP bound to cystathionine beta-synthase is weakly fluorescent and exists as a mixture of the protonated and unprotonated tautomers. Reaction with hydroxylamine releases the oxime and greatly enhances the associated fluorescence. Binding of serine is accompanied by a shift to the unprotonated tautomer of the external aldimine as well as the appearance of a new fluorescent species at approximately 400 nm that could be due to the aminoacrylate or to a gemdiamine intermediate. These data provide the first characterization of the PLP bound to cystathionine beta-synthase. Treatment of cystathionine beta-synthase with hydroxylamine releases two PLPs after 1 day and results in complete loss of activity. Incubation for an additional 3-4 days results in the release of two more PLPs. These data lead us to revise the PLP stoichiometry to 4 per tetramer, and to the conclusion that the heme and PLP sites in cystathionine beta-synthase are nonequivalent.

The central nervous system (CNS) presents both challenges and opportunities to researchers of redox biochemistry. The CNS is sensitive to oxidative damage during aging or disease; excellent transgenic models of specific neurodegenerative diseases have been created that reproduce oxidative stress components of the corresponding human disorder. Mouse models of familial amyotrophic lateral sclerosis (ALS) based on overexpressed mutant human Cu, Zn-superoxide dismutase (SOD1) are cases in point. These animals experience predictably staged, age-dependent motor neuron degeneration with profound cellular and biochemical damage to nerve fibers and spinal cord tissue. Severe protein and lipid oxidation occurs in these animals, apparently as an indirect consequence of protein aggregation or cytopathic protein-protein interactions, as opposed to aberrant redox catalysis by the mutant enzyme. Recent studies of G93A-SOD1 mice and rats suggest that oxidative damage is part of an unmitigated neuroinflammatory reaction, possibly arising in combination from mitochondrial dysfunction plus pathophysiologic activation of both astrocytes and microglia. Lesions to redox signal-transduction pathways in mutant SOD1+ glial cells may stimulate broad-spectrum upregulation of proinflammatory genes, including arachidonic acid-metabolizing enzymes [e.g., cyclooxygenase-II (COX-II) and 5-lipoxygenase (5LOX)]; nitric oxide synthase (NOS) isoforms; cytokines (particularly tumor necrosis factor alpha, TNF-alpha); chemokines; and immunoglobulin Fc receptors (FcgammaRs). The integration of these processes creates a paracrine milieu inconsistent with healthy neural function. This review summarizes what has been learned to date from studies of mutant SOD1 transgenic animals and demonstrates that the G93A-SOD1 mouse in particular is a robust laboratory for the study of neuroinflammation and redox biochemistry.

This study was designed to examine whether NO regulates protein glutathiolation. Exposure to NO donors increased protein glutathiolation in COS-7 or rat aortic smooth muscle cells as detected by anti-protein glutathione (GSH) antibodies. This process was reversible and saturable. Stimulation with acetylcholine (ACh) increased protein glutathiolation in isolated rat aortic rings. This was prevented by inhibiting endothelial NO synthase (eNOS). In ACh-treated rings, proteins showing positive immunoreactivity with the anti-PSSG antibody (Ab) were identified by matrix assisted laser desorption-time-of-flight mass spectrometry to be actin, vimentin, and heat shock protein 70. Purified actin was more readily glutathiolated by S-nitrosoglutathione than by oxidized GSH as determined by electrospray-ionization mass spectrometry, and nitrosylated actin was glutathiolated by reduced GSH. Relative to wild-type (WT) mice, increased protein glutathiolation was observed in hearts of mice with cardiac-specific expression of inducible NO synthase (iNOS). Proteins immunoprecipitated from transgenic hearts revealed GSH-adducted peptides corresponding to adenine nucleotide translocator and the alpha-subunit of F1F0ATPase. These data suggest that exogenous NO or NO generated by eNOS or iNOS regulates protein adduction with GSH. This could be due to a direct reaction of proteins with S-nitrosoglutathione or denitrosylation of S-nitrosylated proteins by reduced GSH. Glutathiolation of cytoskeletal and mitochondrial proteins may be a significant feature of NO bioreactivity.

Activation of protein kinase C (PKC) has been linked to the development of secondary diabetes complications. However, the underlying molecular mechanisms remain unclear. We examined the contribution of aldose reductase, which catalyzes the first, and the rate-limiting, step of the polyol pathway of glucose metabolism, to PKC activation in vascular smooth muscle cells (VSMCs) isolated from rat aorta and exposed to high glucose in culture. Exposure of VSMCs to high glucose (25 mmol/ l), but not iso-osmotic mannitol, led to an increase in total membrane-associated PKC activity, which was prevented by the aldose reductase inhibitors tolrestat or sorbinil or by the ablation of aldose reductase by small interfering RNA (