sodium-dodecyl-sulfate and sapropterin

Nitric oxide (NO) is produced by NO synthase (NOS) in many cells and plays important roles in the neuronal, muscular, cardiovascular, and immune systems. In various disease conditions, all three types of NOS (neuronal, inducible, and endothelial) are reported to generate oxidants through unknown mechanisms. We present here the first evidence that peroxynitrite (ONOO(-)) releases zinc from the zinc-thiolate cluster of endothelial NOS (eNOS) and presumably forms disulfide bonds between the monomers. As a result, disruption of the otherwise SDS-resistant eNOS dimers occurs under reducing conditions. eNOS catalytic activity is exquisitely sensitive to ONOO(-), which decreases NO synthesis and increases superoxide anion (O(2)(.-)) production by the enzyme. The reducing cofactor tetrahydrobiopterin is not oxidized, nor does it prevent oxidation of eNOS by the same low concentrations of OONO(-). Furthermore, eNOS derived from endothelial cells exposed to elevated glucose produces more O(2)(.-), and, like eNOS purified from diabetic LDL receptor-deficient mice, contains less zinc and fewer SDS-resistant dimers. Hence, eNOS exposure to oxidants including ONOO(-) causes increased enzymatic uncoupling and generation of O(2)(.-) in diabetes, contributing further to endothelial cell oxidant stress. Regulation of the zinc-thiolate center of NOS by ONOO(-) provides a novel mechanism for modulation of the enzyme function in disease.

Topics: Animals; Antioxidants; Biopterins; Cattle; Cell Line; Detergents; Diabetes Mellitus, Experimental; Dimerization; Endothelium, Vascular; Glucose; Male; Mice; Mice, Inbred NOD; Mice, Knockout; Nitric Oxide Synthase; Nitric Oxide Synthase Type II; Nitric Oxide Synthase Type III; Oxidants; Oxidation-Reduction; Peroxynitrous Acid; Receptors, LDL; Recombinant Proteins; Sodium Dodecyl Sulfate; Zinc

The major enzyme isoform that synthesizes nitric oxide (NO) in first trimester human placentae is endothelial or type III NO-synthase (NOS III) which exhibits high specific activity in the microsomal fraction. In the present study, we investigated the possible protective and enzyme-stabilizing role of tetrahydropterin (BH4). The anionic detergent, sodium dodecyl sulphate (SDS) and thermal stress (freeze-thaw) were used as non-specific 'subunit-dissociating' agents, and alterations in enzyme activity and subunit structure were investigated. SDS (> or =0.05% w/v) resulted in significant inhibition both of basal and BH4-stimulated activities of NOS III, but the latter responded more sensitively. Preincubation of microsomes with SDS (> or =0.1%, w/v), followed by incubation in an SDS-depleted reaction mixture led to an inhibition of BH4-stimulated enzyme activity, while no change in the basal activity was noted. This indicated that the SDS effect is only fully reversible in the case of basal activity. Considering that basal activity is due to the presence of endogenous BH4 tightly bound to the enzyme, this differential sensitivity of basal and BH4-stimulated enzyme activities to SDS may be related to a putative differential protective effect of BH4 on the two subunits of the NOS III dimer. Western blot analysis revealed that the SDS-induced inhibition of enzyme activity could not be ascribed to disruption of the dimeric structure. This finding confirms the view that SDS may affect NOS III activity without necessarily deteriorating quaternary protein structure. Nevertheless, BH4 is essential in maintaining dimeric structure under denaturing conditions, e.g. SDS treatment and freezing/thawing; it is even able to reverse the dissociation caused by SDS. A model describing the interaction between BH4 and NOS III, and its implications on the physiology and pathology of the human placenta, is discussed.

Topics: Antioxidants; Biopterins; Dimerization; Dose-Response Relationship, Drug; Female; Freezing; Humans; Immunoblotting; Microsomes; Nitric Oxide Synthase; Placenta; Pregnancy; Sodium Dodecyl Sulfate

Nitric oxide synthases (NOSs), which catalyze the formation of the ubiquitous biological messenger molecule nitric oxide, represent unique cytochrome P-450s, containing reductase and mono-oxygenase domains within one polypeptide and requiring tetrahydrobiopterin as cofactor. To investigate whether tetrahydrobiopterin functions as an allosteric effector of NOS, we have analyzed the effect of the pteridine on the conformation of neuronal NOS purified from porcine brain by means of circular dichroism, velocity sedimentation, dynamic light scattering and SDS-polyacrylamide gel electrophoresis. We report for the first time the secondary structure of NOS, showing that the neuronal isozyme contains 30% alpha-helix, 14% antiparallel beta-sheet, 7% parallel beta-sheet, 19% turns and 31% other structures. The secondary structure of neuronal NOS was neither modulated nor stabilized by tetrahydrobiopterin, and the pteridine did not affect the quaternary structure of the protein, which appears to be an elongated homodimer with an axial ratio of approximately 20/1 under native conditions. Low temperature SDS-polyacrylamide gel electrophoresis revealed that tetrahydrobiopterin and L-arginine synergistically convert neuronal NOS into an exceptionally stable, non-covalently linked homodimer surviving in 2% SDS and 5% 2-mercaptoethanol. Ligand-induced formation of an SDS-resistant dimer is unprecedented and suggests a novel role for tetrahydrobiopterin and L-arginine in the allosteric regulation of protein subunit interactions.

Topics: Allosteric Regulation; Amino Acid Oxidoreductases; Animals; Arginine; Biopterins; Brain Chemistry; Hot Temperature; Neurons; Nitric Oxide Synthase; Protein Conformation; Protein Folding; Protein Structure, Secondary; Sodium Dodecyl Sulfate; Swine