Limits...
Molecular and Kinetic Characterization of Babesia microti Gray Strain Lactate Dehydrogenase as a Potential Drug Target.

Vudriko P, Masatani T, Cao S, Terkawi MA, Kamyingkird K, Mousa AA, Adjou Moumouni PF, Nishikawa Y, Xuan X - Drug Target Insights (2014)

Bottom Line: The result showed that the 0.99 kbp BmLDH gene codes for a barely soluble 36 kDa protein (332 amino acids) localized in both the cytoplasm and nucleus of the parasite.The K m values of NAD(+) and lactate were 8.7 ± 0.57 mM and 99.9 ± 22.33 mM, respectively.The IC50 value for gossypol was 0.345 μM, while at 2.5 μM, gossypol caused 100% inhibition of BmLDH catalytic activity.

View Article: PubMed Central - PubMed

Affiliation: National Research Center for Protozoan Diseases (NRCPD), Obihiro University of Agriculture and Veterinary Medicine, Inada-cho, Obihiro, Hokkaido, Japan. ; Department of Veterinary Pharmacy, Clinics and Comparative Medicine, College of Veterinary Medicine, Animal Resources and Biosecurity, Makerere University, Kampala, Uganda.

ABSTRACT
Babesia microti is an emerging zoonotic protozoan organism that causes "malaria-like" symptoms that can be fatal in immunocompromised people. Owing to lack of specific therapeutic regiment against the disease, we cloned and characterized B. microti lactate dehydrogenase (BmLDH) as a potential molecular drug receptor. The in vitro kinetic properties of BmLDH enzyme was evaluated using nicotinamide adenine dinucleotide (NAD(+)) as a co-factor and lactate as a substrate. Inhibitory assay was also done using gossypol as BmLDH inhibitor to determine the inhibitory concentration 50 (IC50). The result showed that the 0.99 kbp BmLDH gene codes for a barely soluble 36 kDa protein (332 amino acids) localized in both the cytoplasm and nucleus of the parasite. In vitro enzyme kinetic studies further revealed that BmLDH is an active enzyme with a high catalytic efficiency at optimal pH of 10.2. The K m values of NAD(+) and lactate were 8.7 ± 0.57 mM and 99.9 ± 22.33 mM, respectively. The IC50 value for gossypol was 0.345 μM, while at 2.5 μM, gossypol caused 100% inhibition of BmLDH catalytic activity. These findings, therefore, provide initial evidence that BmLDH could be a potential drug target, although further in vivo studies are needed to validate the practical application of lactate dehydrogenase inhibitors against B. microti infection.

No MeSH data available.


Related in: MedlinePlus

SDS-PAGE (A) showing non-induced control (lane 1), expressed protein in pellet (lane 2), and supernatant/soluble fraction (lanes 3); SDS-PAGE (B) for purified GST-BmLDH protein (lanes 4) and PreScission™ Protease cleaved BmLDH (lane 5).
© Copyright Policy - open-access
Related In: Results  -  Collection


getmorefigures.php?uid=PMC4125376&req=5

f2-dti-8-2014-031: SDS-PAGE (A) showing non-induced control (lane 1), expressed protein in pellet (lane 2), and supernatant/soluble fraction (lanes 3); SDS-PAGE (B) for purified GST-BmLDH protein (lanes 4) and PreScission™ Protease cleaved BmLDH (lane 5).

Mentions: The recombinant GST-fussed BmLDH (GST-BmLDH) had a molecular weight of approximately 62 kDa (Fig. 2B, lane 4). Since the molecular weight of GST is 26 kDa, it is estimated that the molecular weight of BmLDH protein is 36 kDa. Over 70% of the recombinant protein was insoluble in water upon over expression in E. coli (Fig. 2A, lane 2). The average yield of the GST-BmLDH and GST-free BmLDH from the soluble fraction was approximately 200 μg/mL (Fig. 2B, lanes 4 and 5).


Molecular and Kinetic Characterization of Babesia microti Gray Strain Lactate Dehydrogenase as a Potential Drug Target.

Vudriko P, Masatani T, Cao S, Terkawi MA, Kamyingkird K, Mousa AA, Adjou Moumouni PF, Nishikawa Y, Xuan X - Drug Target Insights (2014)

SDS-PAGE (A) showing non-induced control (lane 1), expressed protein in pellet (lane 2), and supernatant/soluble fraction (lanes 3); SDS-PAGE (B) for purified GST-BmLDH protein (lanes 4) and PreScission™ Protease cleaved BmLDH (lane 5).
© Copyright Policy - open-access
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC4125376&req=5

f2-dti-8-2014-031: SDS-PAGE (A) showing non-induced control (lane 1), expressed protein in pellet (lane 2), and supernatant/soluble fraction (lanes 3); SDS-PAGE (B) for purified GST-BmLDH protein (lanes 4) and PreScission™ Protease cleaved BmLDH (lane 5).
Mentions: The recombinant GST-fussed BmLDH (GST-BmLDH) had a molecular weight of approximately 62 kDa (Fig. 2B, lane 4). Since the molecular weight of GST is 26 kDa, it is estimated that the molecular weight of BmLDH protein is 36 kDa. Over 70% of the recombinant protein was insoluble in water upon over expression in E. coli (Fig. 2A, lane 2). The average yield of the GST-BmLDH and GST-free BmLDH from the soluble fraction was approximately 200 μg/mL (Fig. 2B, lanes 4 and 5).

Bottom Line: The result showed that the 0.99 kbp BmLDH gene codes for a barely soluble 36 kDa protein (332 amino acids) localized in both the cytoplasm and nucleus of the parasite.The K m values of NAD(+) and lactate were 8.7 ± 0.57 mM and 99.9 ± 22.33 mM, respectively.The IC50 value for gossypol was 0.345 μM, while at 2.5 μM, gossypol caused 100% inhibition of BmLDH catalytic activity.

View Article: PubMed Central - PubMed

Affiliation: National Research Center for Protozoan Diseases (NRCPD), Obihiro University of Agriculture and Veterinary Medicine, Inada-cho, Obihiro, Hokkaido, Japan. ; Department of Veterinary Pharmacy, Clinics and Comparative Medicine, College of Veterinary Medicine, Animal Resources and Biosecurity, Makerere University, Kampala, Uganda.

ABSTRACT
Babesia microti is an emerging zoonotic protozoan organism that causes "malaria-like" symptoms that can be fatal in immunocompromised people. Owing to lack of specific therapeutic regiment against the disease, we cloned and characterized B. microti lactate dehydrogenase (BmLDH) as a potential molecular drug receptor. The in vitro kinetic properties of BmLDH enzyme was evaluated using nicotinamide adenine dinucleotide (NAD(+)) as a co-factor and lactate as a substrate. Inhibitory assay was also done using gossypol as BmLDH inhibitor to determine the inhibitory concentration 50 (IC50). The result showed that the 0.99 kbp BmLDH gene codes for a barely soluble 36 kDa protein (332 amino acids) localized in both the cytoplasm and nucleus of the parasite. In vitro enzyme kinetic studies further revealed that BmLDH is an active enzyme with a high catalytic efficiency at optimal pH of 10.2. The K m values of NAD(+) and lactate were 8.7 ± 0.57 mM and 99.9 ± 22.33 mM, respectively. The IC50 value for gossypol was 0.345 μM, while at 2.5 μM, gossypol caused 100% inhibition of BmLDH catalytic activity. These findings, therefore, provide initial evidence that BmLDH could be a potential drug target, although further in vivo studies are needed to validate the practical application of lactate dehydrogenase inhibitors against B. microti infection.

No MeSH data available.


Related in: MedlinePlus