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Kinetics of Alloxan-Induced Inhibition on δ-Aminolevulinate Dehydratase Activity in Mouse Liver Homogenates

Abstract

This study evaluated the effects of alloxan on the kinetics properties of the δ-aminolevulinate dehydratase (δ-ALA-D) using mouse liver homogenates. δ-ALA-D is an important sulfhydryl enzyme that catalyses the second step in heme biosynthesis and is commonly diminished in experimental and human diabetes. Despite the known effects of alloxan in models of experimental diabetes, there are no data in the literature demonstrating the effects of alloxan on the kinetics properties of the δ-ALA-D. The results showed that alloxan (1.25–20 μM) caused a concentration-dependent inhibition of hepatic δ-ALA-D activity. The inhibition constant (K i ) for alloxan-induced inhibition on δ-ALA-D was 3.64 μM. The alloxan (5 μM) caused a decrease in V max (65.8%) and in K m (53.1%), which is suggestive of an uncompetitive inhibition of enzyme. In addition, dithiothreitol (700 and 1,000 μM) completely prevented the δ-ALA-D activity inhibition induced by 10 and 20 μM alloxan. Similar protection was obtained in the presence of 2,000 μM glutathione. Therefore, this work showed that the inhibition of hepatic δ-ALA-D activity can be obtained in vitro at low micromolar levels of alloxan, and can also be prevented by reducing agents. Moreover, these results may help to understand the abnormalities in heme pathway found in models of experimental diabetes in vivo.

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Acknowledgements

This study was supported by FAPERGS (Fundação de Amparo à Pesquisa do Estado do Rio Grande do Sul), CAPES (Coordenação de Aperfeiçoamento de Pessoal de Nível Superior), and CNPq (Conselho Nacional de Desenvolvimento Científico e Tecnológico).

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Correspondence to Verônica B. Brito.

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Brito, V.B., Folmer, V., Guerra, I.I.R. et al. Kinetics of Alloxan-Induced Inhibition on δ-Aminolevulinate Dehydratase Activity in Mouse Liver Homogenates. Appl Biochem Biotechnol 166, 1047–1056 (2012). https://doi.org/10.1007/s12010-011-9492-8

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  • DOI: https://doi.org/10.1007/s12010-011-9492-8

Keywords

  • δ-ALA-D
  • Alloxan
  • Diabetes
  • Kinetics
  • Mice