Includes bibliography / ix, 89 leaves : ill. / Title page, contents and abstract only. The complete thesis in print form is available from the University Library. / Thesis (Ph.D.1971)--University of Adelaide, Dept. of Agricultural Biochemistry, 1970

Thesis (M.Ag.Sc. 1973) from the Dept. of Agricultural Biochemistry and Dept. of Statistics, University of Adelaide.

Thesis (Ph.D. 1971) -- University of Adelaide, Dept. of Agricultural Biochemistry, 1970. / Includes bibliography.

Ribonucleoside diphosphate reductase (RR) from vaccinia virus was recently cloned and overexpressed rn Escherichia coli. The amino acid sequence identities of the small and large subunits between the mouse and the vaccinia virus reductase are approximately 80 and 72 percent, respectively. In addition, vaccinia virus RR displays similar complex allosteric regulation to the mouse enzyme and other eukaryotic reductases. The overall activity of the enzyme, which has two subunits (Rl and R2), is regulated through binding to ATP, which activates the enzyme, and dATP which seryes as an inhibitor. Both nucleotides bind to the same allosteric site, called the activity site, on the large subunit of RR. The specificity of the enzyme towards the four ribonucleoside diphosphate substrates is regulated by the binding of ATP, dATP, dTTP and GTp. Each of these nucleotides affects the reduction of a specific nucleoside diphosphate. Although this enzyme's allosteric regulation is kinetically well understood it has not been possible so far to gain further structural information about the location of the activity site and specificity site. The use of deletion mutants and photoaffinity labeling of the large subunit to identify the location of the binding sites is the incentive for this thesis. With the introduction of 6xHis/Nickel Nitrilo-tri-acetic acid (Ni-NTA) chromatography, the purification of the large subunit was improved in the E. coli and vaccinia virus/T7 RNA polymerase hybrid system. The purification of several deletion mutant forms of the large subunit was also attempted, but it was not possible to purify any of them from either of the expression systems. The purified full-length large subunit obtained with the Ni-NTA-chromatography system was used for a photoaffinity labeling experiment with [³²P]dATP and [³²P]dTTP. The labeled proteins were proteolytically digested to find out about the specificity of the labeling experiment and also to map the binding site of the nucleotide. It was found that labeling of dATP yielded few discrete bands indicating specific binding, while a comparable experiment with dTTP indicated less specific binding, based on a larger number of labeled bands. In competition experiments with non-radioactive nucleotides, vaccinia virus R1 featured the same properties as the mouse and E. coli counterparts. This is consistent with data from kinetic experiments, which also establish the same kinetic properties between vaccinia virus RR with those of mouse and E. coli (RR). To identify the sequence of the fragments carrying the label the digests were subjected to mass spectrometric analysis. However, it was not possible to determine the sequence of the labeled fragment by mass spectrometry due to poor spectral resolution. / Graduation date: 1994

Diss. (sammanfattning) Stockholm : Karol. inst. / Härtill 6 uppsatser.

Steroid hydroxylases are members of a group of enzymes termed "mixed function oxidases". These enzymes catalyze the introduction of an atom of molecular oxygen into the substrate molecule concomitant with the oxidation of NADPH. This thesis describes studies carried out with a steroid 11β-hydroxylase prepared from an acetone powder of bovine adrenal mitochondria. The conversion of deoxycorticosterone to corticosterone has been employed to measure the 11β-hydroxylase activity. The modes of action of two inhibitors of the 11β-hydroxylation reaction, namely, dicumarol and Metopirone have been examined as a means of obtaining information concerning the mechanism of 11β hydroxylation. Kinetic examination of dicumarol inhibition of 11β-hydroxylation indicated that this compound had at least two inhibitory actions. At concentrations below 100 μmoles/1, dicumarol was a noncompetitive inhibitor of 11β-hydroxylation. At dicumarol concentrations above 100 μmoles/1 a second inhibitory action became apparent. This second inhibition could be greatly diminished by increasing the substrate concentration. Kinetic examination of Metopirone inhibition of 11β-hydroxylation indicated that this compound was a competitive inhibitor of the 11β-hydroxylase reaction. In addition Metopirone had a higher affinity for the 11β-hydroxylase system than did substrate deoxycorticosterone. The K𝒾 for Metopirone was 1.0 x 10ˉ⁷ moles/1 while the K𝘮 for deoxycorticosterone was 5.5 x 10ˉ⁶ moles/1. Cytochrome P-450 has been shown to be both the oxygen-activating and substrate binding component of steroid hydroxylases. The interactions of steroid substrate, dicumarol, and Metopirone with this hemoprotein were therefore examined. The ability of cytochrome P-450 to bind carbon monoxide forming a complex exhibiting an absorption maximum at 450 mμ was employed to measure this cytochrome in the 11β-hydroxylase preparation. Cytochrome P-450 present in the mitochondrial acetone powder preparation was found to be unstable, undergoing spontaneous decomposition at temperatures above 30° C to cytochrome P-420, a hemoprotein that does not function in 11β-hydroxylation. However, the rate and extent of cytochrome P-450 decomposition was diminished by the addition of steroid substrate, suggesting that substrate was binding to and stabilizing the hemoprotein. A similar stabilization of cytochrome P-450 was produced upon addition of Metopirone and of low concentrations of dicumarol. Hence these inhibitors could also bind to cytochrome P-450. Dicumarol at high concentrations enhanced the rate of breakdown of cytochrome P-450 to cytochrome P-420. Thus this compound had two opposing effects on cytochrome P-450. The binding of deoxycorticosterone to cytochrome P-450 resulted in spectral changes in the hemoprotein that could be measured by the technique of difference spectrophotometry. The substrate concentration required for half-maximal spectral change, and hence half-maximal binding to cytochrome P-450 was almost identical to its K𝘮 for 11β-hydroxylation. The deoxycorticosterone-induced spectral change in cytochrome P-450 was diminished by addition of Metopirone or by high concentrations of dicumarol but not by low concentrations of dicumarol. Metopirone inhibits 11β-hydroxylation by binding to cytochrome P-450 and preventing the concomitant binding of substrate deoxycorticosterone. The binding of Metopirone and deoxycorticosterone to cytochrome P-450 is competitive in nature, hence competitive kinetics are observed with Metopirone inhibition of the 11β-hydroxylation reaction. Dicumarol exerts two inhibitory actions on 11β-hydroxylation. At low concentrations this compound binds to cyto chrome P-450 but does not affect substrate binding to the hemo- protein, resulting in noncompetitive inhibition of 11β-hydroxylation. The binding of dicumarol at these concentrations therefore must inhibit the interaction of steroid substrate and oxygen to diminish the rate of 11β-hydroxylation Dicumarol, at high concentrations, inhibits the binding of deoxycorticosterone to cytochrome P-450 thus producing its second inhibitory action on 11β-hydroxylation. In addition, binding of dicumarol at high concentrations results in the breakdown of cytochrome P-450. / Medicine, Faculty of / Biochemistry and Molecular Biology, Department of / Graduate

Thesis (doctoral)--Lund University, 1994. / Added t.p. with thesis statement inserted.

Thesis (doctoral)--Lund University, 1994. / Added t.p. with thesis statement inserted.

No description available.

by Simon, Ka-wa Cheung. / Thesis (M.Ph.)--Chinese University of Hong Kong, 1986 / Bibliography: leaves 118-126