Dataset Information

Kinetic analysis of the amino terminal end of active site loop of lactate deyhdrogenase from Plasmodium vivax.

ABSTRACT: OBJECTIVE: In this study, kinetic analysis was performed to understand the functional importance of the amino terminal of the active site of previously mutated Plasmodium vivax Lactate Dehydrogenase enzyme by mimicking Toxoplasma gondii I, II, Eimeria acervulina and Eimeria tenella LDH's. MATERIAL AND METHODS: Mutant LDH genes were amplified by PCR and 6xHistag was added to the C-terminal of the enzymes. Then LDH enzymes are overproduced as recombinant in E. coli cells, purified by Ni-NTA agarose matrix and kinetic properties were analysed. RESULTS: Observing increase of Km values of mutant enzymes showed that mutations in this place caused decreasing affinity of enzyme for its substrate. However kcat values were about the same throughout all mutant proteins. CONCLUSION: Sensitivity of the studied region emphasizes the significance of this site for drug design studies for both Plasmodium and some other Apicomplexans.

SUBMITTER: Mutlu O

PROVIDER: S-EPMC4115892 | biostudies-literature | 2012 Dec

REPOSITORIES: biostudies-literature

ACCESS DATA

Publications

Kinetic analysis of the amino terminal end of active site loop of lactate deyhdrogenase from Plasmodium vivax.

Mutlu Ozal O Balık Dilek Turgut DT

Balkan medical journal 20121201 4

<h4>Objective</h4>In this study, kinetic analysis was performed to understand the functional importance of the amino terminal of the active site of previously mutated Plasmodium vivax Lactate Dehydrogenase enzyme by mimicking Toxoplasma gondii I, II, Eimeria acervulina and Eimeria tenella LDH's.<h4>Material and methods</h4>Mutant LDH genes were amplified by PCR and 6xHistag was added to the C-terminal of the enzymes. Then LDH enzymes are overproduced as recombinant in E. coli cells, purified by ...[more]

PMID: 25207035

Similar Datasets

Project description:Background:Plasmodium lactate dehydrogenase (pLDH) is a major target in diagnosing the erythrocytic stage of malaria parasites because it is highly expressed during blood-stage parasites and is distinguished from human LDH. Rapid diagnostic tests (RDTs) for malaria use pLDH as a target antigen; however, genetic variations in pLDH within the natural population threaten the efficacy of pLDH-based RDTs.Methods:Genetic polymorphisms of Plasmodium vivax LDH (PvLDH) and Plasmodium falciparum LDH (PfLDH) in Myanmar isolates were analysed by nucleotide sequencing analysis. Genetic polymorphisms and the natural selection of PvLDH and PfLDH were analysed using DNASTAR, MEGA6, and DnaSP ver. 5.10.00 programs. The genetic diversity and natural selection of global PvLDH and PfLDH were also analysed. The haplotype network of global PvLDH and PfLDH was constructed using NETWORK ver. 5.0.0.3. Three-dimensional structures of PvLDH and PfLDH were built with YASARA Structure ver. 18.4.24 and the impact of mutations on structural change and stability was evaluated with SDM ver. 2, CUPSAT and MAESTROweb.Results:Forty-nine PvLDH and 52 PfLDH sequences were obtained from Myanmar P. vivax and P. falciparum isolates. Non-synonymous nucleotide substitutions resulting in amino acid changes were identified in both Myanmar PvLDH and PfLDH. Amino acid changes were also identified in the global PvLDH and PfLDH populations, but they did not produce structural alterations in either protein. Low genetic diversity was observed in global PvLDH and PfLDH, which may be maintained by a strong purifying selection.Conclusion:This study extends knowledge for genetic diversity and natural selection of global PvLDH and PfLDH. Although amino acid changes were observed in global PvLDH and PfLDH, they did not alter the conformational structures of the proteins. These suggest that PvLDH and PfLDH are genetically well-conserved in global populations, which indicates that they are suitable antigens for diagnostic purpose and attractive targets for drug development.

Project description:BACKGROUND: Assaying for the parasitic lactate dehydrogenase (pLDH) is widely used as a rapid diagnostic test (RDT), but the efficacy of its serological effectiveness in diagnosis, that is antibody detection ability, is not known. The genetic variation of Korean isolates was analysed, and recombinant protein pLDH was evaluated as a serodiagnostic antigen for the detection of Plasmodium vivax malaria. METHODS: Genomic DNA was purified, and the pLDH gene of P. vivax was amplified from blood samples from 20 patients. The samples came from five epidemic areas: Bucheon-si, Gimpo-si, and Paju-si of Gyeonggi Province, Gangwha-gun of Incheon metropolitan city, and Cheorwon-gun of Gangwon Province, South Korea, from 2010 to 2011. The antigenicity of the recombinant protein pLDH was tested by western blot and enzyme-linked immunosorbent assay (ELISA). RESULTS: Sequence analysis of 20 Korean isolates of P. vivax showed that the open reading frame (ORF) of 951 nucleotides encoded a deduced protein of 316 amino acids (aa). This ORF showed 100% identity with the P. vivax Belem strain (DQ060151) and P. vivax Hainan strain (FJ527750), 89.6% homology with Plasmodium falciparum FCC1_HN (DQ825436), 90.2% homology with Plasmodium berghei (AY437808), 96.8% homology with Plasmodium knowlesi (JF958130), and 90.2% homology with Plasmodium reichenowi (AB122147). A single-nucleotide polymorphism (SNP) at nucleotide 456 (T to C) was also observed in the isolate from Bucheon, but it did not change in the amino acid sequence. The expressed recombinant protein had a molecular weight of approximately 32 kDa, as analysed by sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) analysis. Of the 40 P. vivax patients, 34 (85.0%) were positive by ELISA. CONCLUSIONS: The pLDH genes of 19 isolates of P. vivax were identical, except one for SNP at nucleotide 456. This observation indicates that this gene is relatively stable. Based on these results, the relationship between antibody production against pLDH and the pattern of disease onset should be investigated further before using pLDH for serodiagnosis.

Dataset Information

Kinetic analysis of the amino terminal end of active site loop of lactate deyhdrogenase from Plasmodium vivax.

Publications

Kinetic analysis of the amino terminal end of active site loop of lactate deyhdrogenase from Plasmodium vivax.

Similar Datasets

OmicsDI is part of the ELIXIR infrastructure

Tweets