14 research outputs found
Effect of caffeine on metabolic activity of ejaculated and epididymal spermatozoa of buffalo (Bubalus bubalis)
Genetic mapping of the gene encoding guanylate cyclase-A/atrial natriuretic factor receptor (Npra) to mouse Chromosome 3
A Constitutively âPhosphorylatedâ Guanylyl Cyclase-linked Atrial Natriuretic Peptide Receptor Mutant Is Resistant to Desensitization
Action of different components of the kallikrein kinin-system on sperm motility in vitro
PCR-based dENATURING GRADIENT GEL ELECTROPHORETICtypification and identification of the microbial consortium present in Kefir grains
Molecular mechanisms of action of natural amino acids and serotonin on infusorian adenylyl cyclase and guanylyl cyclase
Methylthioadenosine phosphorylase from the archaeon Pyrococcus furiosus. Mechanism of the reaction and assignment of disulfide bonds
The extremely heat-stable 5'-methylthioadenosine phosphorylase from the hyperthermophilic archaeon Pyrococcus furiosus was cloned, expressed to high levels in Escherichia coli, and purified to homogeneity by heat precipitation and affinity chromatography. The recombinant enzyme was subjected to a kinetic analysis including initial velocity and product inhibition studies. The reaction follows an ordered Bi-Bi mechanism and phosphate binding precedes nucleoside binding in the phosphorolytic direction. 5'-Methylthioadenosine phosphorylase from Pyrococcus furiosus is a hexameric protein with five cysteine residues per subunit. Analysis of the fragments obtained after digestion of the protein alkylated without previous reduction identified two intrasubunit disulfide bridges. The enzyme is very resistant to chemical denaturation and the transition midpoint for guanidinium chloride-induced unfolding was determined to be 3.0 M after 22 h incubation. This value decreases to 2.0 M in the presence of 30 mM dithiothreitol, furnishing evidence that disulfide bonds are needed for protein stability. The guanidinium chloride-induced unfolding is completely reversible as demonstrated by the analysis of the refolding process by activity assays, fluorescence measurements and SDS/PAGE. The finding of multiple disulfide bridges in 5'-methylthioadenosine phosphorylase from Pyrococcus furiosus argues strongly that disulfide bond formation may be a significant molecular strategy for stabilizing intracellular hyperthermophilic proteins