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In JoVE (1)
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Articles by Matthew L. Fowler in JoVE
JoVE General
Detecção direta da Atividade Acetato de formação do Acetato Quinase Enzima
Department of Genetics and Biochemistry, Clemson University
Um método para a determinação da atividade da quinase de acetato é descrito. Este ensaio utiliza uma reação direta para determinar a atividade enzimática e cinética de acetato quinase na direção de formação de acetato com diferentes aceptores fosforil. Além disso, este método pode ser utilizado para a dosagem de fosfato de acetila ou outras enzimas acetil-CoA utilizando.
Other articles by Matthew L. Fowler on PubMed
The Anti-trypanosomal Agent Lonidamine Inhibits Trypanosoma Brucei Hexokinase 1
Glycolysis is essential to the parasitic protozoan Trypanosoma brucei. The first step in this metabolic pathway is mediated by hexokinase, an enzyme that transfers the gamma-phosphate of ATP to a hexose. The T. brucei genome (TREU927/4 GUTat10.1) encodes two hexokinases (TbHK1 and TbHK2) that are 98% identical at the amino acid level. Our previous efforts have revealed that TbHK2 is an important regulator of TbHK1 in procyclic form parasites. Here, we have found through RNAi that TbHK1 is essential to the bloodstream form parasite. Silencing the gene for 4 days reduces cellular hexokinase approximately 60% and leads to parasite death. Additionally, we have found that the recombinant enzyme is inhibited by lonidamine (IC(50)=850 microM), an anti-cancer drug that targets tumor hexokinases. This agent also inhibits HK activity from whole parasite lysate (IC(50)=965 microM). Last, lonidamine is toxic to cultured bloodstream form parasites (LD(50)=50 microM) and procyclic form parasites (LD(50)=180 microM). Interestingly, overexpression of TbHK1 protects PF parasites from lonidamine. These studies provide genetic evidence that TbHK1 is a valid therapeutic target while identifying a potential molecular target of the anti-trypanosomal agent lonidamine.
