Isolation, purification and kinetic characterization of prolyl endopeptidase from Titicum aestivum
- Authors: Abrahams, Adriam Mark
- Date: 2013
- Subjects: Endopeptidases , Wheat , Chemical kinetics , Pharmacokinetics
- Language: English
- Type: Thesis , Masters , MSc (Biochemistry)
- Identifier: vital:11269 , http://hdl.handle.net/10353/d1004356 , Endopeptidases , Wheat , Chemical kinetics , Pharmacokinetics
- Description: PEP activity has been described in several locations and has mostly been linked to a variety of neurological disorders such as schizophrenia, amnaesia, depression as well as other disease states such as anorexia nervosa, bulimia nervosa and blood pressure regulation. The enzyme has also been previously isolated from a variety of archae, microorganisms and several eukaryotic species but no prolyl endopeptidases have been isolated from plants. Plants have high levels of proline and glutamine rich peptides in seeds. We therefore hypothesize plants must express PEPs during germination. Bioinformatics tools were used to identify known PEPs and putative plant PEPs. A global sequence alignment of putative plant PEPs and other known PEPs indicated that the active site amino acids Ser, His and Asp are conserved in putative plant PEP sequences. Furthermore, putative plant PEPs showed similar secondary structures to known PEPs and when a rice PEP was modelled onto porcine brain PEP structure, a high degree of similarity was found. Germination studies of wheat seed showed an increase of PEP activity over time with maximum PEP activity reached after 4 days of germination, which remained at this level until 9 days of germination, implying a function for PEP in plant seed germination. Wheat PEP was purified using ion exchange and gel filtration chromatography with a final yield of less than 1 percent and a relative purity (only 2 bands detected by SDS-PAGE). The purified wheat PEP had a molecular weight of approximately 55kDa, substrate specificity for chymotrypsin-like substrates (N-Suc-Ala-Ala-Pro-Phe-pNa, Km value of 0.58 mM, Kcat of 29.37 s–1; Kcat /Km 50813.14s–1 M–1); a pH optimum of 7.9; temperature optima of 37oC and a high sensitivity to temperature as indicated by loss of activity at temperatures above 40oC. Inhibition studies using E64, Leupeptin and PMSF confirmed that the wheat PEP is from the Serine protease family and is most likely a trypsin-like protease.
- Full Text:
- Date Issued: 2013
- Authors: Abrahams, Adriam Mark
- Date: 2013
- Subjects: Endopeptidases , Wheat , Chemical kinetics , Pharmacokinetics
- Language: English
- Type: Thesis , Masters , MSc (Biochemistry)
- Identifier: vital:11269 , http://hdl.handle.net/10353/d1004356 , Endopeptidases , Wheat , Chemical kinetics , Pharmacokinetics
- Description: PEP activity has been described in several locations and has mostly been linked to a variety of neurological disorders such as schizophrenia, amnaesia, depression as well as other disease states such as anorexia nervosa, bulimia nervosa and blood pressure regulation. The enzyme has also been previously isolated from a variety of archae, microorganisms and several eukaryotic species but no prolyl endopeptidases have been isolated from plants. Plants have high levels of proline and glutamine rich peptides in seeds. We therefore hypothesize plants must express PEPs during germination. Bioinformatics tools were used to identify known PEPs and putative plant PEPs. A global sequence alignment of putative plant PEPs and other known PEPs indicated that the active site amino acids Ser, His and Asp are conserved in putative plant PEP sequences. Furthermore, putative plant PEPs showed similar secondary structures to known PEPs and when a rice PEP was modelled onto porcine brain PEP structure, a high degree of similarity was found. Germination studies of wheat seed showed an increase of PEP activity over time with maximum PEP activity reached after 4 days of germination, which remained at this level until 9 days of germination, implying a function for PEP in plant seed germination. Wheat PEP was purified using ion exchange and gel filtration chromatography with a final yield of less than 1 percent and a relative purity (only 2 bands detected by SDS-PAGE). The purified wheat PEP had a molecular weight of approximately 55kDa, substrate specificity for chymotrypsin-like substrates (N-Suc-Ala-Ala-Pro-Phe-pNa, Km value of 0.58 mM, Kcat of 29.37 s–1; Kcat /Km 50813.14s–1 M–1); a pH optimum of 7.9; temperature optima of 37oC and a high sensitivity to temperature as indicated by loss of activity at temperatures above 40oC. Inhibition studies using E64, Leupeptin and PMSF confirmed that the wheat PEP is from the Serine protease family and is most likely a trypsin-like protease.
- Full Text:
- Date Issued: 2013
Interaction of terpenes and oxygenated terpenes with some drugs
- Authors: Ajayi, Emmanuel Olusegun
- Date: 2012
- Subjects: Terpenes -- Essences and essential oils , Lavenders -- Monoterpenes -- Drug interactions , Drugs -- Camphor -- Medicinal plants
- Language: English
- Type: Thesis , Masters , MSc (Chemistry)
- Identifier: vital:11332 , http://hdl.handle.net/10353/418 , Terpenes -- Essences and essential oils , Lavenders -- Monoterpenes -- Drug interactions , Drugs -- Camphor -- Medicinal plants
- Description: SFME and HD for the extraction of essential oil in Lavandula officinalis in Alice have been reported. A total of 59 compounds were identified with the major compound being 1,8-cineole, an oxygenated monoterpene, with 46.89% and 44.84% yield obtained for HD and SFME respectively. Charge transfer (CT) complexes formed between α-pinene, 1,8-cineole and camphor as electron donors with iodine as the electron acceptor have been studied spectrophotometrically in methylene chloride solution. The Benesi- Hildebrand equation has been applied to estimate the formation constant (Kf) and molecular extinction coefficient (εCT). The value of Kf is the highest in camphor-I2 complex compared to the other two complexes. Antibacterial assessment was carried out on the various reagents, determining the MIC of individual reagents and in combination. The results show an improvement, on combination of the various reagents than when tested alone.
- Full Text:
- Date Issued: 2012
- Authors: Ajayi, Emmanuel Olusegun
- Date: 2012
- Subjects: Terpenes -- Essences and essential oils , Lavenders -- Monoterpenes -- Drug interactions , Drugs -- Camphor -- Medicinal plants
- Language: English
- Type: Thesis , Masters , MSc (Chemistry)
- Identifier: vital:11332 , http://hdl.handle.net/10353/418 , Terpenes -- Essences and essential oils , Lavenders -- Monoterpenes -- Drug interactions , Drugs -- Camphor -- Medicinal plants
- Description: SFME and HD for the extraction of essential oil in Lavandula officinalis in Alice have been reported. A total of 59 compounds were identified with the major compound being 1,8-cineole, an oxygenated monoterpene, with 46.89% and 44.84% yield obtained for HD and SFME respectively. Charge transfer (CT) complexes formed between α-pinene, 1,8-cineole and camphor as electron donors with iodine as the electron acceptor have been studied spectrophotometrically in methylene chloride solution. The Benesi- Hildebrand equation has been applied to estimate the formation constant (Kf) and molecular extinction coefficient (εCT). The value of Kf is the highest in camphor-I2 complex compared to the other two complexes. Antibacterial assessment was carried out on the various reagents, determining the MIC of individual reagents and in combination. The results show an improvement, on combination of the various reagents than when tested alone.
- Full Text:
- Date Issued: 2012
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