The activity of the Caenorhabditis elegans scavenger decapping enzyme (DcpS) on its natural substrates and dinucleotide cap analogs, modified with regard to the nucleoside base or ribose moiety, has been examined. All tested dinucleotides were specifically cleaved between beta- and gamma-phosphate groups in the triphosphate chain. The kinetic parameters of enzymatic hydrolysis (K(m), V(max)) were determined using fluorescence and HPLC methods, as complementary approaches for the kinetic studies of C. elegans DcpS. From the kinetic data, we determined which parts of the cap structure are crucial for DcpS binding and hydrolysis. We showed that m(3) (2,2,7)GpppG and m(3) (2,2,7)GpppA are cleaved with higher rates than their monomethylated counterparts. However, the higher specificity of C. elegans DcpS for monomethylguanosine caps is illustrated by the lower K(m) values. Modifications of the first transcribed nucleotide did not affect the activity, regardless of the type of purine base. Our findings suggest C. elegans DcpS flexibility in the first transcribed nucleoside-binding pocket. Moreover, although C. elegans DcpS accommodates bulkier groups in the N7 position (ethyl or benzyl) of the cap, both 2'-O- and 3'-O-methylations of 7-methylguanosine result in a reduction in hydrolysis by two orders of magnitude.

OBJECTIVES: We sought to determine concentrations of total and labile iron in substantia nigra from patients with Parkinson disease and from controls to assess if oxidative stress is triggered by an increased concentration of iron. METHODS: Total iron concentration in the whole substantia nigra was evaluated in 17 parkinsonian and 29 control samples. Concentrations of labile iron and copper were assessed in 6 parkinsonian and 8 control samples. The total iron concentration, the Fe(2+)/Fe(3+) ratio, and iron-binding compounds were determined by Mössbauer spectroscopy. Labile iron and copper were measured by electrothermal atomic absorption spectrometry. Activity of reactive oxygen species was evaluated by visible light fluorescence. RESULTS: The labile iron concentration was significantly higher and corresponded to significantly higher reactive oxygen species activity in parkinsonian vs control samples. No significant difference was found in the total concentrations of copper or iron in the whole substantia nigra between parkinsonian and control samples. Mössbauer spectroscopy detected no Fe(2+) in any samples. CONCLUSIONS: The substantia nigra of parkinsonian patients contained more labile iron compared with that of controls. This labile iron generated higher reactive oxygen species activity. The oxidative stress damage in parkinsonian substantia nigra may be related to an excess of labile iron and not of the total iron in the diseased tissue.

Histidine triad (HIT)-family proteins interact with different mono- and dinucleotides and catalyze their hydrolysis. During a study of the substrate specificity of seven HIT-family proteins, we have shown that each can act as a sulfohydrolase, catalyzing the liberation of AMP from adenosine 5'-phosphosulfate (APS or SO(4)-pA). However, in the presence of orthophosphate, Arabidopsis thaliana Hint4 and Caenorhabditis elegans DcpS also behaved as APS phosphorylases, forming ADP. Low pH promoted the phosphorolytic and high pH the hydrolytic activities. These proteins, and in particular Hint4, also catalyzed hydrolysis or phosphorolysis of some other adenylyl-derivatives but at lower rates than those for APS cleavage. A mechanism for these activities is proposed and the possible role of some HIT-proteins in APS metabolism is discussed.