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Numerous genetic variants displaying stereotypic circling behaviour have been described in rodents. The majority are recessive mutants expressing dopaminergic alterations in the striatum- often with associated vestibular defects. We describe a novel circling mouse (Cr) with intact vestibular function frequently obtained from crosses of non-transgenic wild type mice (WT) and transgenic growth hormone mice (TGM). We have characterized Cr with stereotypic circling, head bobbing, hyperkinesia, aggressiveness, and elevated dopamine when compared to the transgenic growth hormone mouse. The Cr also demonstrates self-mutilating behaviour found in mice with dysregulated striatal dopamine levels. Cr oppositely mirrors most traits of the TGM including alterations in sleep, activity, eating, and drinking. TGM displayed superior performance than WT in novel object recognition, but this decreased with aging. Comparatively, Cr performed poorly in this test. The memory of young TGM exceeded controls, whereas young Cr displayed poorer memory with an age related improvement. In a stepdown test of emotionality, TGM step down more readily, while Cr are more hesitant than WT. TGM and Cr also demonstrate opposite alterations in striatal dopamine. Further analysis demonstrated differential responsiveness of TGM and Cr under dopaminergic drugs, and potentially sexually dimorphic differences in behaviour associated with elevated GH in TGM. TGM are characterized by increased levels of circulating growth hormone and alterations in sleep and activity. We hypothesize that frequent generation of Cr may reflect unintended selection of modifier genes that counterbalance negative consequences of elevated GH in TGM. / Thesis / Master of Science (MSc)

In the present study, adenosine kinase (AK) was purified from a number of different sources (e.g. bovine liver, Syrian hamster liver, kidney and heart; Chinese hamster ovary cells and human placenta). The enzyme from bovine liver and Syrian hamster liver was purified to apparent homogeneity by a combination of ion-exchange chromatography, affinity chromatography and gel filtration chromatography. The purified enzyme showed a single band of 40 kDa in SDS-polyacrylamide gel electrophoresis, which was similar to that reported from other sources in the literature. A number of biochemical and enzymatic characteristics of AK were investigated. The reliability and reproducibility of the AK assay as established in previous studies was determined. The apparent Km of partially purified AK from Syrian hamster liver for adenosine was determined to be 0.16 uM, which is consistent with earlier reports. A novel result obtained in these studies is that AK activity was found to be completely dependent upon the presence of phosphate or other pentavalent ions. AK from different sources did not show any activity in the Tris-HCl buffer, pH 7.0, but the activity increased dramatically upon the addition of phosphate and it reached a maximum at 2 mM phosphate. There was no inhibition of AK activity when the phosphate concentration was increased up to 100 mM. AK could also be activated by substituting phosphate with either arsenate or vanadate, which have similar chemical structures to phosphate. The temperature inactivation kinetics of AK showed that AK from human fibroblast cells had higher thermal resistance than AK from Chinese hamster ovary cells at 50°C. The presence of phosphate had no effect on the thermal stability of AK. Antibodies to purified AK from Syrian hamster liver were raised in both rabbits and guinea pigs. Antiserum from the rabbit which gave the strongest response was used for further studies. AK was recognized by and reacted specifically with the antiserum at a dilution of up to 1: 16,000. The AK antibody which was covalently bound to Protein A Sepharose beads immunoprecipitated a 40 kDa protein from radio-labelled Chinese hamster ovary or baby hamster kidney cell extracts. In addition, this antibody preparation immunoprecipitated AK activity from Syrian hamster liver extracts. However, immunoblotting showed that all the antisera could not react with AK from Chinese hamster ovary cells and human cells, suggesting a strong species specificity. A partial protein sequence of AK was obtained by microsequencing of a cyanogen bromide fragment of purified AK from Syrian hamster liver. The sequence was Tyr-Val-Asp-Ser-Leu-Phe-Gly-Ala-Glu-Thr-Glu-Ala-Ala-Leu. Degenerate DNA probes for this sequence can now be made and used for either screening of eDNA libraries or carrying out PCR experiments to facilitate the cloning of the AK gene(s). Finally, conditions for selection of revertants from AK⁻ mutants of Chinese hamster ovary cells have been developed. The AK⁺ revertants from AK⁻ mutants can be obtained using adenosine, alanosine, uridine and erythro-9-(2-hydroxyl-3-nonyl) adenine in the growth medium. Three revertants have been isolated. These revertants regained their AK activities and lost their drug-resistance at same time. This method can also be used in the future to clone the AK gene by transfecting AK⁻ mutants with foreign DNA. / Thesis / Master of Science (MSc)

The homotetrameric enzyme dihydrodipicolinate synthase (DHDPS, E.C. 4.2.1.52) from Escherichia coli was used as a model for probing oligomeric structure in enzymes. Dimeric mutants of this enzyme have been found in previous work to be largely inactive, due to the trapping of a covalent adduct. Partial restoration of catalytic activity has been achieved by incubation in the presence of the substrate pyruvate to displace the adduct. It was hypothesized that the buttressing of dimeric units against one another in the wildtype tetrameric form of DHDPS provides stability in the dimer interface, necessary to maintain optimum catalytic performance and substrate specificity. We hypothesized that buttressing a dimeric DHDPS mutant against a surface would result in restoration of catalytic activity by mimicking the buttressing proposed to occur in the tetrameric structure. To test this hypothesis, dimeric DHDPS mutants were immobilised against an agarose support and the immobilised enzymes characterised. Three DHDPS mutants were prepared, the double mutant DHDPS-C20S/L167C was produced by mutagenesis and a crystal structure obtained in collaboration with Dr Renwick Dobson. Two other mutants, DHDPS-Ll67C and DHDPS-Ll97Y were also over expressed and purified. The quaternary structures of the three mutants were characterised in solution, DHDPS-Ll67C was determined to be tetrameric, DHDPS-C20S-Ll67C was found to equilibrate between tetramer and dimer and DHDPS-Ll97Y was confirmed as a dimer, consistent with previous findings. Modification experiments indicated that the sulfhydryl groups of DHDPS-C20S/L167C were available for immobilisation. Activation experiments indicated that both DHDPS-Ll67C and DHDPS-Ll97Y activated. These results were in accord with those of others in indicating that the displacement of an a-ketoglutarate adduct from the active site was responsible for the activation of mutant DHDPS enzymes. Wild-type DHDPS and the mutants were immobilised through amine and sulfhydryl groups. The free and immobilised enzymes were rigorously characterised, with thermal stability, pH optima, kinetic and lysine inhibition properties determined and compared to wild-type DHDPS. Following immobilisation, substrate affinity was found to decrease for wild-type and mutant enzymes, wild-type KmPyr = 0.26 mM free, 0.8-1.2 mM immobilised, Km(S)-ASA = 0.10 mM free, 1.5-2.5 mM immobilised. Lysine inhibition was determined to be largely unaffected by immobilisation. The largest change in K, was an increase to double that of the free enzyme. Restoration of some catalytic activity was found following the immobilisation of dimeric DHDPS-Ll97Y, the immobilised enzyme was 31 ± 12% more active than free DHDPS-Ll97Y. DHDPS-C20S/L167C was also found to immobilise as a dimer. Comparison ofthe immobilised DHDPS-C20S/L167C dimer with a derivatised free dimeric form ofthis enzyme indicated that an increase from 3% to 9% of wild-type activity had resulted from immobilisation. These results supported the hypothesis that buttressing of a dimeric mutant of DHDPS against a support surface would increase catalytic activity and that buttressing across the dimerdimer interface is essential for optimal catalytic activity in DHDPS enzymes.

No description available.

No description available.

No description available.

No description available.

The homotetrameric enzyme dihydrodipicolinate synthase (DHDPS, E.C. 4.2.1.52) from Escherichia coli was used as a model for probing oligomeric structure in enzymes. Dimeric mutants of this enzyme have been found in previous work to be largely inactive, due to the trapping of a covalent adduct. Partial restoration of catalytic activity has been achieved by incubation in the presence of the substrate pyruvate to displace the adduct. It was hypothesized that the buttressing of dimeric units against one another in the wildtype tetrameric form of DHDPS provides stability in the dimer interface, necessary to maintain optimum catalytic performance and substrate specificity. We hypothesized that buttressing a dimeric DHDPS mutant against a surface would result in restoration of catalytic activity by mimicking the buttressing proposed to occur in the tetrameric structure. To test this hypothesis, dimeric DHDPS mutants were immobilised against an agarose support and the immobilised enzymes characterised. Three DHDPS mutants were prepared, the double mutant DHDPS-C20S/L167C was produced by mutagenesis and a crystal structure obtained in collaboration with Dr Renwick Dobson. Two other mutants, DHDPS-Ll67C and DHDPS-Ll97Y were also over expressed and purified. The quaternary structures of the three mutants were characterised in solution, DHDPS-Ll67C was determined to be tetrameric, DHDPS-C20S-Ll67C was found to equilibrate between tetramer and dimer and DHDPS-Ll97Y was confirmed as a dimer, consistent with previous findings. Modification experiments indicated that the sulfhydryl groups of DHDPS-C20S/L167C were available for immobilisation. Activation experiments indicated that both DHDPS-Ll67C and DHDPS-Ll97Y activated. These results were in accord with those of others in indicating that the displacement of an a-ketoglutarate adduct from the active site was responsible for the activation of mutant DHDPS enzymes. Wild-type DHDPS and the mutants were immobilised through amine and sulfhydryl groups. The free and immobilised enzymes were rigorously characterised, with thermal stability, pH optima, kinetic and lysine inhibition properties determined and compared to wild-type DHDPS. Following immobilisation, substrate affinity was found to decrease for wild-type and mutant enzymes, wild-type KmPyr = 0.26 mM free, 0.8-1.2 mM immobilised, Km(S)-ASA = 0.10 mM free, 1.5-2.5 mM immobilised. Lysine inhibition was determined to be largely unaffected by immobilisation. The largest change in K, was an increase to double that of the free enzyme. Restoration of some catalytic activity was found following the immobilisation of dimeric DHDPS-Ll97Y, the immobilised enzyme was 31 ± 12% more active than free DHDPS-Ll97Y. DHDPS-C20S/L167C was also found to immobilise as a dimer. Comparison ofthe immobilised DHDPS-C20S/L167C dimer with a derivatised free dimeric form ofthis enzyme indicated that an increase from 3% to 9% of wild-type activity had resulted from immobilisation. These results supported the hypothesis that buttressing of a dimeric mutant of DHDPS against a support surface would increase catalytic activity and that buttressing across the dimerdimer interface is essential for optimal catalytic activity in DHDPS enzymes.