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

Multiple amino acid sequence alignment and identity score between BmLDH and LDH for P. falciparum (PfLDH), B. bovis (BbLDH), T. gondii (TgLDH), T. annulata (TaLDH), Mus musculus (mouse LDH), Cricetulus griseus (hamster LDH), and human (H. sapiens LDH).
© Copyright Policy - open-access
Related In: Results  -  Collection


getmorefigures.php?uid=PMC4125376&req=5

f1-dti-8-2014-031: Multiple amino acid sequence alignment and identity score between BmLDH and LDH for P. falciparum (PfLDH), B. bovis (BbLDH), T. gondii (TgLDH), T. annulata (TaLDH), Mus musculus (mouse LDH), Cricetulus griseus (hamster LDH), and human (H. sapiens LDH).

Mentions: The amplified BmLDH gene was approximately 0.99 kbp. Nucleotide sequence analysis revealed that BmLDH gene has an open reading frame of 996 nucleotides that codes for 332 amino acids. There were no introns in the BmLDH DNA. Alignment of the translated BmLDH amino acid sequence with that of related apicomplexan parasites and selected mammalian LDH gave contrasting result. The amino acid sequence for BmLDH was less than 28% identical to that of Plasmodium falciparum (Accession No. AAK12097), T. gondii (Accession No. XP_002368488), B. bovis (Accession No. EDO07479), and T. annulata (Accession No. ADG45564). However, there was high frequency of identical amino acid sequence between BmLDH and LDH mammals like hamster (70.3%, Accession No. NP_001230979), mouse (69.1%, Accession No. CAA26360), and humans (61.9%, Accession No. CAE11711). Approximately 43 amino acid residues were highly conserved in all the eight LDH sequences aligned (Fig. 1).


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)

Multiple amino acid sequence alignment and identity score between BmLDH and LDH for P. falciparum (PfLDH), B. bovis (BbLDH), T. gondii (TgLDH), T. annulata (TaLDH), Mus musculus (mouse LDH), Cricetulus griseus (hamster LDH), and human (H. sapiens LDH).
© Copyright Policy - open-access
Related In: Results  -  Collection

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

f1-dti-8-2014-031: Multiple amino acid sequence alignment and identity score between BmLDH and LDH for P. falciparum (PfLDH), B. bovis (BbLDH), T. gondii (TgLDH), T. annulata (TaLDH), Mus musculus (mouse LDH), Cricetulus griseus (hamster LDH), and human (H. sapiens LDH).
Mentions: The amplified BmLDH gene was approximately 0.99 kbp. Nucleotide sequence analysis revealed that BmLDH gene has an open reading frame of 996 nucleotides that codes for 332 amino acids. There were no introns in the BmLDH DNA. Alignment of the translated BmLDH amino acid sequence with that of related apicomplexan parasites and selected mammalian LDH gave contrasting result. The amino acid sequence for BmLDH was less than 28% identical to that of Plasmodium falciparum (Accession No. AAK12097), T. gondii (Accession No. XP_002368488), B. bovis (Accession No. EDO07479), and T. annulata (Accession No. ADG45564). However, there was high frequency of identical amino acid sequence between BmLDH and LDH mammals like hamster (70.3%, Accession No. NP_001230979), mouse (69.1%, Accession No. CAA26360), and humans (61.9%, Accession No. CAE11711). Approximately 43 amino acid residues were highly conserved in all the eight LDH sequences aligned (Fig. 1).

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