This thesis describes the results obtained from the phytochemical analysis of Matricaria chamomilla, and the microbial transformation of oxymatrine (85) and harmine (87), as summarized below. 1. Chemical investigation of the crude methanolic extract of Matricaria chamomilla resulted in the isolation of a new natural product, matriisobenzofuran (72), along with four known compounds: apigenin (73), apigenin-7-O-β-glucopyranoside (74), scopoletin (75), and fraxidin (76). The structures of compounds 72-76 were elucidated with the aid of extensive NMR and mass spectroscopic studies. All of the aforementioned compounds showed moderate to good inhibitory activities against glutathione S-transferase, an enzyme which has been implicated in the resistance of cancer cells to chemotherapeutic agents. These compounds were also evaluated for antioxidant activity and displayed moderate to good free radical scavenging activity. Additionally, compounds 72-76 were screened for anti-leishmanial activity. Compounds 75 and 76 were significantly active in this assay, while the remaining compounds were weakly active. In the antibacterial and antifungal assays, compounds 72-76 were not active. 2. The second part of this thesis deals with the biotransformation studies on oxymatrine (85) and harmine (87). Oxymatrine (85) was metabolized to the deoxy analogue, matrine (84) by Penicillum chrysogeneum (ATCC 9480), Cunninghamella bainieri (ATCC 9244), Cunninghamella blakesleena (ATCC 9245 and 8688A), Curvularia lunata (ATCC 12017), and Fusarium sp. In the time-based analysis of this transformation, the metabolism of oxymatrine (85) could be detected after 48 hours of incubation. Additionally, incubation of harmine (87) with Mucor plumbeus (ATCC 4740) resulted in the isolation of harmine-N-oxide (94). The biotransformed products (84 and 94) were identified using IR, UV, NMR, and mass spectroscopic techniques. Compound 94 was evaluated for its ability to inhibit the enzyme acetylcholinestrase, whose overexpression has been linked to Alzheimer’s disease, and was found to possess weaker activity than harmine (87).

Plants can not escape from biotic and abiotic stress factors such as, extreme temperatures, high light intensity, drought, UV radiation, heavy metals, and pathogen attack. Plants have versatile defens systems against such stress conditions. In this study, the role of glutathione S-transferases (GSTs) in cold stress conditions were examined. Glutathione S-transferases are the enzymes that detoxify natural and exogenous toxic compounds by conjugation with glutathione. Glutathione, an endogenous tripeptide, is important as reducing agent, nucleophilic scavenger, and alleviate the chemical toxicity in the plants by the reaction of GSTs. Glutathione conjugates can be transported to the vacuoles or apoplast and are generally much less toxic than the parent compounds. In plants there are four distinct families of the soluble GSTs, namely Phi (F), Type I / Zeta (Z), Type II / Tau (U), Type III / Theta (T), Type IV. By contrast with the mammalian families of GST, relatively little is known about the plant GST families. Up to date, there is not any study on GST isolation and characterization from Turkish red pine, in this respect, this study well play a frontier role the future research dealing with this topic. In this study, some properties of Turkish red pine GST activity towards CDNB (1-chloro-2,4 dinitrobenzene) were examined. The average specific activity of Turkish red pine GST towards CDNB was found as 200&plusmn / 50 (Mean&plusmn / SE, n= 18) nmole/min/mg cytosolic protein. GSTs in cytosol prepared from Turkish red pine needles retained its activity without loss for four weeks at -80&amp / #61616 / C. The rate of conjugation reactions were linear up to 0.8mg of Turkish red pine cytosolic protein and 0.4 mg cytosolic protein was routinely used. The Turkish red pine GST showed its maximum activity at pH 8.0 in 25 mM phosphate buffer and 42 &amp / #730 / C. The measurements were carried out at room temperature (RT) of 25 &amp / #61616 / C. Turkish red pine GST seemed to be saturated at 1 mM CDNB and 1 mM GSH concentrations. The Vmax and Km values of Turkish red pine GST for CDNB was 416nmole/min/mg protein and 0,8 mM, respectively, and for GSH 106.4 nmole/min/mg protein and 0.10 mM, respectively. Turkish red pine cytosol was applied on DEAE-Sepharose fast flow column but almost no purification was achieved with respect GST activity. In order to examine the effects of cold stress on Turkish red pine GST activity, the GST activity was determined in 240 seedlings at &ndash / 3&amp / #61616 / , 0&amp / #61616 / and 13 &amp / #61616 / C environmental temperatures. It was observed that GST activity was the highest at -3&amp / #730 / C and the lowest at 13&amp / #730 / C in both cold resistant and sensitive families with the exception of Yaylaalan and &Ccedil / ameli.

Helicoverpa armigera is a polyphagous pest. Due to excessive use of insecticides, the field populations of H. armigera have become resistant to synthetic pyrethroids by one or combination of three mechanisms / reduced penetration through the cuticle, decreased nerve sensitivity and enhanced metabolism by the detoxification enzymes especially glutathione S-transferases. In this study, gut sections of H. armigera were obtained from Adana and Antalya field populations and susceptible populations from Israel. Each gut section was homogenized separately in 1.0 ml, 40 mM and pH 7.5 phosphate buffers. GST activity was determined using CDNB as substrate. Product formation linearly increased up to 29.5&micro / g proteins in 20mM, pH 7.5 phosphate buffers. Maximum reaction rate was reached at 30&amp / #9702 / C. The Vmax and Km values for GST towards CDNB and GSH were calculated with Lineweaver-Burk and Eadie-Scatchard plots as CDNB Vmax / 6.54&micro / mol/min/mg, 6.35&micro / mol/min/mg , Km / 0.29mM, 0.28mM ,respectively and as GSH Vmax / 6.42&micro / mol/min/mg, 6.65&micro / mol/min/mg, Km / 0.22mM, 0.23mM, respectively. Cytosolic GST activity of each individual from Adana, Antalya and susceptible populations were determined under optimized conditions. The mean of GST activity in Adana population (n=50) and Antalya population (n=50) were found 7.824&micro / mol/min/mg and 9.518&micro / mol/min/mg, respectively. The mean of GST activity in susceptible population (n=50) was determined as 3.272&micro / mol/min/mg. According to these results, GST activities of Adana and Antalya field populations&rsquo / showed statistically significant increase (p&lt / 0.05) than susceptible H. armigera populations with ANOVA method. In addition, Antalya population showed statistically increase (p&lt / 0.05) GST activity than Adana.

In the recent years, environmental pollution becomes a health threatening issue for human beings. Technological developments introduce industrial wastes and heavy metals, and developments in agriculture introduce pesticides into the world that we live. All these toxic wastes accumulate in drinking water and food consumed by humans. Therefore, detection of toxic wastes in all kinds of environmental samples, and development of new detection techniques become an important issue. In this study, development of a protein-based biosensor for detection of heavy metals in environmental samples, by expressing genetically modified glutathione S-transferase (GST-(His)6) protein in E.Coli BL21 (DE3) expression system, was designed. Recombinant GST proteins was expressed in E.Coli BL21 (DE3) expression system and purified with Glutathione Sepharose 4B affinity column and Ni-NTA spin kit. GST activities were determined using the GST substrate 1-chloro-2,4-dinitrobenzene (CDNB). Protein expression was tested by SDS-PAGE and Western blot analysis. Product formation linearly increased up to 1 mM CDNB, 1 mM GSH, 1.7 &micro / g proteins in 0.05 M, pH 6.9 phosphate buffer in the final volume of 1.0 ml at 25&amp / #9702 / C. The Vmax and Km values for GST-(His)6 towards CDNB and GSH were calculated with Lineweaver-Burk as CDNB Vmax / 22.88 &micro / mol/min/mg, Km / 4.29 mM,and as GSH Vmax / 6.42 &micro / mol/min/mg, 24.45 &micro / mol/min/mg, Km / 3.69 mM, respectively. Biosensor working electrode was prepared by immobilizing the GST-(His)6 by 1-(3-dimethylaminopropyl)-3-ethyl-carbodiimide hydrochloride (EDC) coupling on the gold surface. Electrode preparation was confirmed by cyclic voltammetry measurements. The biosensor was inserted as the working electrode in the constructed three(four)-electrode flow cell. The conformational change resulting from the binding of the metal ions to the recombinant protein causing a capacitance change proportional to the concentration of the metal ions was determined. After the working electrode is standardized and calibrated, the heavy metal concentration in water samples was measured. The GST-(His)6 biosensor has a large operational range between 1 fM and 10 mM and a storage stability of approximately 2 weeks. The GST-(His)6 biosensor is very sensitive to, Cu+2&gt / Cd+2&gt / Zn+2&gt / Hg+2 metal ions, at low concentrations.

A screening method was developed for identification of catalytically active enzymes in combinatorial cDNA libraries of mutated glutathione transferase (GST) derivatives expressed in E. coli. The method is based on spraying monochlorobimane (MCB) directly over bacterial colonies growing on agar. The substrate MCB become fluorescent under UV light, when the bacterial colony contains active GSTs catalyzing the conjugation with endogenous glutathione. Eleven out of twelve GSTs investigated where active with MCB. This method can be used to screen libraries generated from most cytosolic GSTs in the search for proteins with altered functions and structures. Azathioprine (Aza), a thiopurine that has been used clinically for 40 years was investigated with 14 GSTs. Three enzymes showed prominent catalytic activities with Aza and all of them are highly expressed in the liver. We estimated the contribution of the three enzymes GSTs A1-1, A2-2 and M1-1 bioactivation of Aza in the liver and concluded that it was about 2 orders of magnitude more effective than the uncatalyzed reaction. GST bioactivation of Aza could clarify aspects of idiosyncratic reactions observed in some individuals. Two other thiopurine prodrugs, cis-acetylvinylthiopurine (cAVTP) and trans-acetylvinylthioguanine (tAVTG), were investigated with the same 14 GSTs. The results displayed diverse catalytic activities. A mechanism of consecutive reactions was proposed. The studies contribute to knowledge under what conditions the drug should optimally be administered. A study of the same prodrugs with several mutants from the Mu class characterized by a point mutation of a hypervarible residue. We conclude that the effects of the mutations were qualitatively parallel for cAVTP and tAVTG, but they vary significantly in magnitude; steric hindrance may interfere with transition-state stabilization. From the evolutionary perspective the data show that a point mutation can alternatively enhance or attenuate the activity with a particular substrate and illustrate the functional plasticity of GSTs.

Diss. (sammanfattning) Stockholm : Karolinska institutet, 2007. / Härtill 4 uppsatser.

Diss. (sammanfattning) Stockholm : Karolinska institutet, 2005. / Härtill 4 uppsatser.

Diss. (sammanfattning) Stockholm : Karol. inst., 2003. / Härtill 5 uppsatser.

Diss. (sammanfattning) Stockholm : Karol. inst., 2002. / Härtill 5 uppsatser.

Diss. (sammanfattning) Stockholm : Karol. inst., 2004. / Härtill 5 uppsatser.