The following thesis examines the effects of ionic strength and solution composition on the thermal stability and. activity of bovine erythrocyte superoxide dismutase (BESOD). An indirect assay procedure was employed. The oxidation of xanthine, catalyzed by xanthine oxidase, served as a generator of superoxide radicals (02) and ferricytochrome c functioned as a scavenger of these radicals. The assays were monitored and recorded spectrophotometrically. Percent superoxide dismutase activity remaining after a 30 minute heat treatment and 241 hours after the heat treatment were determined. As the concentration and computed ionic strength values of the ionic solutions increased, the percent BESOD activity remaining after 30 minutes at 80 C decreased. Recovery of BESOD activity after 24 hours was minimal in all ionic solutions. BESOD denaturation was apparently similar in the chloride and sulfate solutions, but much greater in the phosphate solutions. Mathematical interpretation confirmed the similar relationships between chloride and sulfate solutions, and the much greater effects of phosphate solutions on thermal denaturation of SOD. At concentrations of 10-1 M or less, thermal denaturation of SOD was greatest, implying the appearance of a denaturation threshold. A specific phosphate effect may be apparent and was described by two models: 1- complexation of Cut + ions with phosphate anions, and 2- conformational alteration due to charge repulsion between molecular strands of the SOD molecule. Both of these models could be possible explanations as to the much greater thermal denaturation observed in the phosphate solutions.

No description available.

sn-Glycerol-3-phosphate acyltransferase [G3PAT, PlsB (E.coli), EC 2.3.1.15] is an enzyme involved in glycerolipid biosynthesis, catalysing the acylation of glycerol-3-phosphate (G3P) to produce lysophosphatidic acid (LPA). Chilling tolerance in plants is linked to the acyl-group composition of membranes, which is linked to acyltransferases with a higher selectivity for unsaturated acyl-substrates. Plant soluble G3PAT is located in the chloroplast and uses acyl-acyl carrier protein (acyl-ACP) as substrate. Soluble G3PAT exhibits strong substrate selectivity for acyl-ACP, the plastidial substrate in vivo, over acyl-CoA. cDNAs encoding soluble G3PATs have previously been cloned from several plant species and both oleate-selective and non-selective forms identified. The purpose of this thesis is to study the mechanism of plastidial G3PAT and attempt to identify factors important in determining substrate selectivity. An in vitro assay has been optimised to distinguish selective and non-selective enzyme forms under physiologically relevant conditions. The assay has been adapted to determine enzyme activity with a range of acyl-ACP and acyl-CoA substrates and to measure the kinetic constants Km and Vmax. Kinetic measurements have been made on a G3PAT protein from the chilling sensitive plant squash (Cucurbita moschata) and the L261F mutant protein containing a single amino acid substitution that significantly alters substrate selectivity. The mutation was found to increase selectivity by raising Km for unsaturated acyl-substrate. Mutant squash G3PAT proteins have been investigated to determine the importance of particular regions or amino acid residues. The mutations E142A, K193S, R235S and R237S resulted in enzymes that were completely inactive. The mutations H194S and L261F altered catalytic or substrate binding characteristics without enzyme inactivation. The catalytic mechanism and order of substrate binding for squash G3PAT have been determined, the reaction was found to proceed via a compulsory-ordered ternary complex with acyl-ACP binding before glycerol-3-phosphate.

No description available.

Poly(ADP-ribose) polymerase (PADPRP) is a nuclear enzyme with a well documented role in DNA repair. Inhibitors of PADPRP, (e.g. 3' substituted benzamides) potentiate the cytotoxicity of a wide range of antitumour drugs. The results presented in this thesis represent, to the best of my knowledge, the first comprehensive and quantitative assessment of the ability of a range of P ADPRP inhibitors to modulate the cellular responses to damaging agents. Two novel PADPRP inhibitors, 8-hydroxy-2-methyl quinazolin-4(3H)-one (NU1025) and 3,4 dihydro-5-methoxyisoquinolin-1-(2H)-one (PD 128763) were compared with two "classical" PADPRP inhibitors, 3-aminobenzamide (3AB) and benzamide (BZ). The relative potencies for 3AB, BZ, NU1025 and PD 128763 as PADPRP inhibitors in vitro were 1.0, ~1.0, ~43 and ~53 respectively. All compounds potentiated the growth inhibition and cytotoxicity of the monofunctional alkylating agent temozolomide (TM) in L1210 cells. For example, 10/-lM NUI025 and PD 128763 gave dose enhancement factors (DEF) of ~2 at 100/0 survival, whereas ImM 3AB and 0.5mM BZ where required to give similar DEF values. Cellular NADl- levels were depleted up to 50% by 1-2mM TM and this depletion was completely prevented by coincubation with 50-100µM PD 128763 and 1-3mM 3AB. TM induced DNA single strand break levels were increased in a concentration dependent manner by the P ADPRP inhibitors. Overall, the relative potencies for ability of the compounds to potentiate TM induced growth inhibition, cytotoxicity and DNA single strand breaks showed good correlation with those determined in an in vitro inhibition study, with both NU1025 and PD 128763 exhibiting ~60 fold increased inhibitory activity as compared to 3AB. The PADPRP inhibitors per se did not effect the growth or survival of the L 121 0 cells, nor increase DNA strand breakage. NAD+ is the substrate for PADPRP. A L1210 cell line made resistant to tiazofurin (TZ) utilising a step wise selection protocol was shown to be deficient in nicotinamide mononucleotide adenyl transferase (NMNAT) , the final enzyme required for NAD+ biosynthesis. The consequences of a reduced NMNAT activity (<3% of the parental line ) and an ~40% reduction in intracellular NAD+ levels were determined. The resistant cells showed an ~3 fold increased sensitivity to TM as compared to the parental cells. Upon coincubation with increasing concentrations of NU1025 in the presence of a fixed concentration of TM, growth inhibition was potentiated ~70 fold in the resistant cells but only ~10 fold in the parental cell line, demonstrating the reduced level of competition between NAD+ and NUI025 for PADPRP. However, DNA single strand breaks were increased in the resistant compared to the parental cell line only when NU1025 was coincubated with TM. In contrast, in the presence of the PADPRP inhibitors alone, equivalent growth inhibitory effects were observed in each of the cell lines, suggesting inhibition of PADPRP was not the cytotoxic effector. The ~40% NAD+ depletion observed could therefore suggest, that NAD+ levels in the resistant cells were reduced to, or near to the KmNAD+ for PADPRP.

Human angiotensin-converting enzyme (ACE) is a key enzyme in the regulation of blood pressure via the renin-angiotensin and kallikrein-kinin systems. A number of orally active drugs have been developed over the years that target somatic ACE, for the treatment of hypertension, myocardial infarction and congestive heart failure. Protein structural information about ACE is an important key for the understanding of the mechanism and substrate-specificity of the enzyme. However, this information has only begun to be elucidated in the past year, with the solution of crystal structures of human testis ACE (tACE), and homologues Drosophila AnCE and human ACE2. tACE is identical to the C-terminal domain of somatic ACE, which consists of two homologous domains, each having a slightly different substrate-specificity. This thesis describes the purification, crystallisation and X-ray crystal structure-determination of a glycosylation-deficient mutant of tACE, tACEG1,3, to 2.9 &Aring / .

MBR suppliers are largely recommending NaOCl as the cleaning agent. Despite the popularity of this chemical for membrane cleaning, there is a lack of publications regarding NaOCl optimum cleaning conditions. To properly conduct this optimization study (i.e. obtain the required level of reproducibility and confidence), a rigorous methodology is still required. The potential effect of NaOCl on polymeric materials ageing has drawn attention and recent studies have been dedicated to assess the impact of its exposure on numerous membrane materials (except polyvinylidene fluoride (PVDF)). PVDF and polypropylene (PP) hollow fibers were investigated using unwashed yeast as model fouling solution, while mixture of sodium alginate and bovine serum albumin (BSA) acted as feed solution for PVDF flat sheet. The cleaning efficiency and optimum NaOCl concentration were found to vary between the different membrane materials and between single and cyclical cleanings. During cyclical cleaning, foulant was more difficult to remove. When 2% NaOCl was used, Fourier Transform Infra Red (FTIR) Spectroscopy showed a change in membrane function groups on PVDF flat sheet, indicating ageing occurrence. NaOCl agemg caused changes in membrane properties. PP hollow fibers became more brittle with 60 % elongation decrease after 13 weeks. PVDF flat sheet membrane exhibited two-steps-degradation mechanism; firstly, the removal of its surface modification substance, and secondly, the increase of its hydrophilicity. These results were confirmed by X-ray Photoelectron Spectroscopy (XPS), FTIR Spectroscopy, contact angle and hydraulic measurement. Enzyme is an alternative option for membrane cleaning. However, the enzymatic cleaning study did not present encouraging results. Optimum cleaning efficiency for protease (68%) and amylase (73%) were found to be lower compared to NaOCl cleaning (95%). Lowry and Dubois methods found that residual foulants were present on the membrane after the cleaning process, which caused fouling to occur faster when membrane was re-used.

viii, 120 leaves : ill. ; 28 cm. / Title page, contents and abstract only. The complete thesis in print form is available from the University Library. / Thesis (Ph.D.)--University of Adelaide, Dept. of Biochemistry, 1974

vi, 100 leaves : graphs ; 30 cm. / Title page, contents and abstract only. The complete thesis in print form is available from the University Library. / Thesis (Ph.D.)--University of Adelaide, Dept. of Biochemistry, 1979

xix, 122 leaves : ill., fold., diags. ; 28 cm. / Title page, contents and abstract only. The complete thesis in print form is available from the University Library. / Thesis (Ph.D.)--University of Adelaide, Dept. of Biochemistry, 1971