1,720,976 research outputs found
Expression and characterization of recombinant bovine cytosolic 5'-nucleotidase IMP-GMP specific
No full text
Nitric Oxide and Platelet Aggregation in Migraineurs Children
No full textNO is a key molecule in the pathogenesis of migraine and other vascular headaches. These data were underlined because of particular interest as they concern migraine in children. It is difficult to prove the correlation between NO in the plasma and platelet activity from this study. Differing values of plasma NO were observed in migrainous children with respect TH and controls. Even if there is not an increased NOS activity in lymphocytes and macrophages, an increased endogenous synthesis of NO in platelet cannot be completely rejected. In fact cytosolic calcium of platelet is greater in controls than the migraineurs. This parameter is a sensitive index in revealing platelet function. The differing values of platelet calcium in migrainous children support the hypothesis of platelet hypofunction
Active and regulatory sites of cytosolic 5 '-nucleotidase
No full textCytosolic 5'-nucleotidase (cN-II), which acts preferentially on 6-hydroxypurine nucleotides, is essential for the survival of several cell types. cN-II catalyses both the hydrolysis of nucleotides and transfer of their phosphate moiety to a nucleoside acceptor through formation of a covalent phospho-intermediate. Both activities are regulated by a number of phosphorylated compounds, such as diadenosine tetraphosphate (Ap(4)A), ADP, ATP, 2,3-bisphosphoglycerate (BPG) and phosphate. On the basis of a partial crystal structure of cN-II, we mutated two residues located in the active site, Y55 and T56. We ascertained that the ability to catalyse the transfer of phosphate depends on the presence of a bulky residue in the active site very close to the aspartate residue that forms the covalent phospho-intermediate. The molecular model indicates two possible sites at which adenylic compounds may interact. We mutated three residues that mediate interaction in the first activation site (R144, N154, I152) and three in the second (F127, M436 and H428), and found that Ap4A and ADP interact with the same site, but the sites for ATP and BPG remain uncertain. The structural model indicates that cN-II is a homotetrameric protein that results from interaction through a specific interface B of two identical dimers that have arisen from interaction of two identical subunits through interface A. Point mutations in the two interfaces and gel-filtration experiments indicated that the dimer is the smallest active oligomerization state. Finally, gel-filtration and light-scattering experiments demonstrated that the native enzyme exists as a tetramer, and no further oligomerization is required for enzyme activation
Characterization of the adenine nucleoside specific phosphorylase of Bacillus cereus
No full textAdenosine phosphorylase, a purine nucleoside phosphorylase endowed with high specificity for adenine nucleosides, was purified 117-fold
from vegetative forms of Bacillus cereus. The purification procedure included ammonium sulphate fractionation, pH 4 treatment, ion exchange
chromatography on DEAE-Sephacel, gel filtration on Sephacryl S-300 HR and affinity chromatography on N6-adenosyl agarose. The enzyme
shows a good stability to both temperature and pH. It appears to be a homohexamer of 164±5 kDa. Kinetic characterization confirmed the
specificity of this phosphorylase for 6-aminopurine nucleosides. Adenosine was the preferred substrate for nucleoside phosphorolysis (kcat/Km
2.1×106 s−1 M−1), followed by 2′-deoxyadenosine (kcat/Km 4.2×105 s−1 M−1). Apparently, the low specificity of adenosine phosphorylase
towards 6-oxopurine nucleosides is due to a slow catalytic rate rather than to poor substrate binding.
© 2007 Elsevier B.V. All rights reserved
- …
