RESUMEN
Inorganic pyrophosphatases are divided in two families, which differ both in structure and mechanism. All of them incorporate in its structure divalent metal cations. In 2003, it was reported for the first time that Rhodobacter capsulatus cytoplasmic pyrophosphatase belongs to family II. It is expected then, that this enzyme contains metal elements in its structure; however, this characterization has not been carried out yet. A fine application of accelerators is the use of proton beams to induce X-ray emission (PIXE) for analyzing the composition of biological macromolecules. The purpose of this work is to complement R. capsulatus cytoplasmic pyrophosphatase characterization by determining the presence of metal elements in its structure. Three different strategies were used: PAGE-PIXE, PAGE-Digestion-PIXE, and Dialysis-PIXE and when metals were found the metal/enzyme ratio was calculated. Only cobalt was found to be associated to the enzyme chemical structure in a ratio 3 Co/enzyme.
Asunto(s)
Metales/análisis , Pirofosfatasas/química , Rhodobacter capsulatus/enzimología , Coenzimas/análisis , Espectrometría por Rayos X/métodosRESUMEN
A cytoplasmic pyrophosphatase [E.C. 3.6.1.1.] was partially purified from Helicobacter pylori. The molecular mass was estimated to be 103 kDa by gel filtration. Results of SDS-PAGE suggested that the enzyme consists of six identical subunits of 19.1 kDa each. The enzyme specifically catalyzed the hydrolysis of pyrophosphate and was very sensitive to NaF, but not to sodium molybdate. The optimal pH for activity was 8.5. Mg2+ was required for maximal activity; Mn2+, Co2+, and Zn2+ poorly supported hydrolytic activity. The pyrophosphatase had an apparent K(m) for Mg-PP(i)2 hydrolysis of 90 microM, and a Vmax estimated at 24.0 micromol P(i) min(-1) mg(-1).