The inhibition of M-MLV and HIV-1 reverse transcriptase by polyphenols extracted from the resurrection plant Myrothamnus flabellifolia (Welw.)

 

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dc.contributor.advisor Brandt, Wolf F en_ZA
dc.contributor.advisor Lindsey, George G en_ZA
dc.contributor.author Kamng'ona, Arox Wadson en_ZA
dc.date.accessioned 2014-07-30T17:38:35Z
dc.date.available 2014-07-30T17:38:35Z
dc.date.issued 2008 en_ZA
dc.identifier.citation Kamng'ona, A. 2008. The inhibition of M-MLV and HIV-1 reverse transcriptase by polyphenols extracted from the resurrection plant Myrothamnus flabellifolia (Welw.). University of Cape Town. en_ZA
dc.identifier.uri http://hdl.handle.net/11427/4277
dc.description Includes abstract.
dc.description Includes bibliographical references (leaves 80-90).
dc.description.abstract Polyphenols have been shown to exhibit anti-viral activity in vitro, making them a promising starting point for the development of HIV treatment drugs. The main objective of this thesis was to assess the inhibitory effect of polyphenols extracted from Myrothamnus flabellifolia (Welw.) on M-MLV and HIV-I reverse transcriptases. The first part of the thesis was an attempt to isolate 3,4,5 tri-O-galloylquinic acid, the major polyphenol found in Namibian Myrothamnus flabellifolia plants. This polyphenol was successfully purified by column chromatography (Sephadex LH-20) and its purity was confirmed by HPLC and MALDI-TOF mass spectrometry. The second part of this thesis involved the development of a polymerase enzyme activity assay based on ethidium bromide fluorescence. A calibration curve for quantification of DNA was therefore prepared from the ethidium bromide fluorescence of Calf Thymus DNA. Results demonstrated that Calf Thymus DNA was a good standard for estimating the amount of cDNA synthesised during reverse transcription, thus enabling the monitoring of both M-ML V and HIV -1 reverse transcriptase activity. The reverse transcriptase activity assay was optimised using a poly (rA) template, an oligo (dTb primer and dTTP as a substrate. It was observed that the rate of catalysis for M-ML V and HIV -1 RTs decreased with increase in the concentration of dTTP, which suggested substrate inhibition. A decrease in M-MLV RT activity with increased substrate concentration was found to be due to depletion of Mg2+ ions by dTTP. True substrate inhibition was however observed for HIV-I RT, and analysis of the observed kinetics suggested the formation of an ineffective enzyme substrate complex with two substrate molecules binding to HIV -1 reverse transcriptase. A Hill coefficient of one was obtained at low dTTP concentration and less than one at high dTTP concentration, suggesting zero and negative cooperativity respectively. The final part of this thesis tested the inhibitory effect of pure and crude polyphenol fractions on the activity of M-MLV and HIV-1 RTs. Results showed that all polyphenol fractions inhibited M-ML V and HIV -I reverse transcriptase activity, with the highest inhibitory activity demonstrated by the fraction that contained pure 3,4,5 tri-O-galloylquinic acid. The 50 % inhibitory concentrations of 3,4,5 tri-O-galloylquinic acid was 0.5 μM for M-MLV RT and 34 μM for HIV-I RT. Lineweaver-Burk plots showed that 3,4,5 tri-O-galloylquinic acid inhibited both enzymes non-competitively. Pure non-competitive inhibition was observed for M-MLV RT and mixed non-competitive inhibition for HIV-I RT. Results showed that the binding of 3,4,5 tri-O-galloylquinic acid to M-MLV RT was irreversible, suggesting strong binding under the conditions tested. 3,4,5 Tri-O-galloylquinic acid, however, bound to HIV-I RT reversibly. A comparison of catalytic efficiencies showed that M-MLV RT was more efficient than HIV -1 RT under saturating substrate concentrations with Kcat (min-¹) values of II ± 3 and 1.31 ± 0.02 respectively. M-MLV RT and HIV-¹ RT were, however, equally efficient under limiting substrate concentrations with Kcat/Km (min-¹M-¹) values of 1.1 ± 0.3 x 10⁴ and 1.2 ± 0.2 x 10⁴ respectively. en_ZA
dc.language.iso eng en_ZA
dc.subject.other Cell Biology en_ZA
dc.title The inhibition of M-MLV and HIV-1 reverse transcriptase by polyphenols extracted from the resurrection plant Myrothamnus flabellifolia (Welw.) en_ZA
dc.type Master Thesis
uct.type.publication Research en_ZA
uct.type.resource Thesis en_ZA
dc.publisher.institution University of Cape Town
dc.publisher.faculty Faculty of Science en_ZA
dc.publisher.department Department of Molecular and Cell Biology en_ZA
dc.type.qualificationlevel Masters
dc.type.qualificationname MSc en_ZA
uct.type.filetype Text
uct.type.filetype Image
dc.identifier.apacitation Kamng'ona, A. W. (2008). <i>The inhibition of M-MLV and HIV-1 reverse transcriptase by polyphenols extracted from the resurrection plant Myrothamnus flabellifolia (Welw.)</i>. (Thesis). University of Cape Town ,Faculty of Science ,Department of Molecular and Cell Biology. Retrieved from http://hdl.handle.net/11427/4277 en_ZA
dc.identifier.chicagocitation Kamng'ona, Arox Wadson. <i>"The inhibition of M-MLV and HIV-1 reverse transcriptase by polyphenols extracted from the resurrection plant Myrothamnus flabellifolia (Welw.)."</i> Thesis., University of Cape Town ,Faculty of Science ,Department of Molecular and Cell Biology, 2008. http://hdl.handle.net/11427/4277 en_ZA
dc.identifier.vancouvercitation Kamng'ona AW. The inhibition of M-MLV and HIV-1 reverse transcriptase by polyphenols extracted from the resurrection plant Myrothamnus flabellifolia (Welw.). [Thesis]. University of Cape Town ,Faculty of Science ,Department of Molecular and Cell Biology, 2008 [cited yyyy month dd]. Available from: http://hdl.handle.net/11427/4277 en_ZA
dc.identifier.ris TY - Thesis / Dissertation AU - Kamng'ona, Arox Wadson AB - Polyphenols have been shown to exhibit anti-viral activity in vitro, making them a promising starting point for the development of HIV treatment drugs. The main objective of this thesis was to assess the inhibitory effect of polyphenols extracted from Myrothamnus flabellifolia (Welw.) on M-MLV and HIV-I reverse transcriptases. The first part of the thesis was an attempt to isolate 3,4,5 tri-O-galloylquinic acid, the major polyphenol found in Namibian Myrothamnus flabellifolia plants. This polyphenol was successfully purified by column chromatography (Sephadex LH-20) and its purity was confirmed by HPLC and MALDI-TOF mass spectrometry. The second part of this thesis involved the development of a polymerase enzyme activity assay based on ethidium bromide fluorescence. A calibration curve for quantification of DNA was therefore prepared from the ethidium bromide fluorescence of Calf Thymus DNA. Results demonstrated that Calf Thymus DNA was a good standard for estimating the amount of cDNA synthesised during reverse transcription, thus enabling the monitoring of both M-ML V and HIV -1 reverse transcriptase activity. The reverse transcriptase activity assay was optimised using a poly (rA) template, an oligo (dTb primer and dTTP as a substrate. It was observed that the rate of catalysis for M-ML V and HIV -1 RTs decreased with increase in the concentration of dTTP, which suggested substrate inhibition. A decrease in M-MLV RT activity with increased substrate concentration was found to be due to depletion of Mg2+ ions by dTTP. True substrate inhibition was however observed for HIV-I RT, and analysis of the observed kinetics suggested the formation of an ineffective enzyme substrate complex with two substrate molecules binding to HIV -1 reverse transcriptase. A Hill coefficient of one was obtained at low dTTP concentration and less than one at high dTTP concentration, suggesting zero and negative cooperativity respectively. The final part of this thesis tested the inhibitory effect of pure and crude polyphenol fractions on the activity of M-MLV and HIV-1 RTs. Results showed that all polyphenol fractions inhibited M-ML V and HIV -I reverse transcriptase activity, with the highest inhibitory activity demonstrated by the fraction that contained pure 3,4,5 tri-O-galloylquinic acid. The 50 % inhibitory concentrations of 3,4,5 tri-O-galloylquinic acid was 0.5 μM for M-MLV RT and 34 μM for HIV-I RT. Lineweaver-Burk plots showed that 3,4,5 tri-O-galloylquinic acid inhibited both enzymes non-competitively. Pure non-competitive inhibition was observed for M-MLV RT and mixed non-competitive inhibition for HIV-I RT. Results showed that the binding of 3,4,5 tri-O-galloylquinic acid to M-MLV RT was irreversible, suggesting strong binding under the conditions tested. 3,4,5 Tri-O-galloylquinic acid, however, bound to HIV-I RT reversibly. A comparison of catalytic efficiencies showed that M-MLV RT was more efficient than HIV -1 RT under saturating substrate concentrations with Kcat (min-¹) values of II ± 3 and 1.31 ± 0.02 respectively. M-MLV RT and HIV-¹ RT were, however, equally efficient under limiting substrate concentrations with Kcat/Km (min-¹M-¹) values of 1.1 ± 0.3 x 10⁴ and 1.2 ± 0.2 x 10⁴ respectively. DA - 2008 DB - OpenUCT DP - University of Cape Town LK - https://open.uct.ac.za PB - University of Cape Town PY - 2008 T1 - The inhibition of M-MLV and HIV-1 reverse transcriptase by polyphenols extracted from the resurrection plant Myrothamnus flabellifolia (Welw.) TI - The inhibition of M-MLV and HIV-1 reverse transcriptase by polyphenols extracted from the resurrection plant Myrothamnus flabellifolia (Welw.) UR - http://hdl.handle.net/11427/4277 ER - en_ZA


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