The biochemistry and molecular biology of amidases from M. methylotrophus was investigated. Acetamidase purification from whole cells exhibiting low specific activities yielded pure low-activity acetamidase (specific activity 6-15 umol min-1 [mg protein]-1), the activity of which could be reactivated to a level approaching that of the high-activity acetamidase by heating (1-6 h, 60 °C) with an activator component. Identical purifications from whole cells with high specific activities produced pure 'high-activity' acetamidases exhibiting a wide range of generally diminished activities (19-108 umol min-1 [mg protein]-1) and an unexpected propensity for heat-reactivation similar to that of low-activity acetamidase. It was concluded that high-activity acetamidases underwent varying degrees of 'switch-off of activity both pre- and post-purification. The physico-chemical properties of purified acetamidases were investigated in vitro to elucidate the nature of the putative acetamidase post-transcriptional modification and its role in the reversible regulation of acetamidase activity. High- and low-activity acetamidases exhibited significantly different properties, although their respective MW values differed only by 52Da. Low-activity acetamidase was significantly more stable than high-activity acetamidases which were labile. Results suggested that high- and low-activity acetamidases existed in different conformational states and that the regulation of acetamidase activity probably involved an allosteric mechanism. Cloning of the M. methylotrophus acetamidase structural gene (amiE) was unsuccessful, but the formamidase structural gene (fmd) was successfully cloned and heterologously- expressed in E. coli. The DNA sequences of fmd and three putative ORFs showed that (i) the formamidase primary sequence exhibited 57% strict identity with that of the Mycobacterium smegmatis 'acetamidase' (Mahenthiralingam et al., 1993), and (ii) ORF2 and ORF3 apparently respectively encoded a zinc finger DNA-binding protein and an AmiC-type regulatory protein. The evolutionary and regulatory implications of these findings were discussed.

No description available.

Proinsulin connecting peptide (C-peptide) joins the A and B-chain of proinsulin and plays an important role in coordinating the folding of insulin. For many years this peptide was simply considered an inert by-product of insulin biosynthesis and was used mainly as an alternative marker for insulin secretion. Recent evidence, however, demonstrates convincingly that C-peptide has biological function and establishes C-peptide as an attractive therapeutic agent to provide protection against chronic diabetic complications. Little is known about C-peptide signalling in pancreatic β cells with studies focussing on antioxidant effects. C-peptide could have protective roles in these cells. For example, pancreatic β cells exposed to immune complexes in type 2 diabetes require protection possibly via C-peptide. Data presented here demonstrate that C-peptide induced a concentration-dependent phosphorylation (activation) of rpS6, at S235/S236 and S240/244, as well as phosphorylation of components of the upstream signalling pathways of rpS6 (ERK1/2, Akt and S6K) in the pancreatic β cell line, INS-1E. C-peptide also caused concentration-dependent increases in phospo-ERK1/2 and phospho-rpS6 (S240/244) in HEK293 and SH-SY5Y, but not in HEK293A. Stimulatory effects of C-peptide on two important intracellular signalling pathways, ERK1/2 and rpS6, can deliver cytoprotective effects and may, therefore, be of potential importance in the treatment of diabetes. A major limitation of current work on C-peptide is that the receptor(s) have not been identified convincingly. Some recent evidence suggests that the orphan GPCR, GPR146, may be the C-peptide receptor. Here, stable overexpression of C-terminally EGFP-tagged GPR146 in HEK293 and HEK293A cells showed predominant membrane localisation of the receptor. However, C-peptide responses in these were unaffected and C-peptide-evoked internalisation/co-localisation of GPR146-EGFP was not observed. An expression cloning approach in Xenopus oocytes was used to screen pools of cDNA library clones for Gαi-evoked responses of C-peptide given the pertussis toxin sensitivity of responses. This approach did not, however, reveal any C-peptide-evoked responses in any of the approximately 130,000 clones screened. Furthermore, the published C-peptide receptor candidates, GPR146 and α-enolase did not respond to C-peptide when expressed in oocytes. Taken together, signalling events in a pancreatic β cell line suggest relevance of C-peptide to events such as cell survival and proliferation. However, the present work provides no evidence that GPR146 is the C-peptide receptor, which still remains elusive.

Histone deacetylase 3 (HDAC3) acts as the catalytic core of the SMRT/NCoR co-repressor complex which regulates chromatin structure and gene expression. It was recently shown that HDAC3 binds, and is regulated in vitro, by the binding of inositol phosphates (IP). We used transcriptional reporter assays to interrogate whether HDAC3-mediated repression in vivo is dependent of IP. Manipulation of intracellular IP levels through chemical inhibition of enzymes involved in IP metabolism or RNAi-mediated protein knockdown were inconclusive. However, mutation of key IP binding residues in both SMRT and HDAC3 directly impacts the repressive ability of the co-repressor complex, presumably through an impaired ability to bind IP and failure to fully activate the enzyme. Germline deletion of HDAC3 in the mouse results in early embryonic lethality (around e9.5) suggesting it plays an essential role in embryogenesis. To further investigate the role of HDAC3 in embryonic development, I have generated a conditional knockout embryonic stem cell line in which HDAC3 can be specifically inactivated. Loss of the protein occurs within 3 days suggesting a half-life of approximately 24 hours and correlates with concomitant decrease in co-repressor complex components, indicating HDAC3 contributes to co-repressor integrity. Unlike deletion of HDAC1 and -2, loss of HDAC3 does not cause a significant reduction in total deacetylase activity with only minor changes in the acetylation levels of histones. However, the proliferative capacity of knockout cells is inhibited with a delay in cell doubling time. Upon differentiation, we find that embryoid bodies (EBs) lacking HDAC3 are significantly smaller and morphologically different compared to controls. Microarray analysis over a 7-day time course of EB differentiation reveals that endodermal cell markers are over-expressed at both early and late stages of development, suggesting that HDAC3 plays an important role in regulating gene expression during embryonic development.

Sister chromatid cohesion (SCC) is mediated by the cohesin complex whose core components are Smc1, Smc3, Scc1, and Scc3 (SA1 and SA2 in vertebrates) and is regulated by associated factors that include Pds5 (Pds5A and Pds5B in vertebrates) and the acetyltransferases Esco1 and Esco2. The correct establishment and maintenance of sister chromatid cohesion is important in safeguarding genome integrity. Previous studies have shown that Pds5 is important in the establishment and maintenance of SCC (in yeast and fungi) as well as sister chromatid resolution (in humans and Xenopus). In humans, both Esco1 and Esco2 acetylate Smc3 during DNA replication to establish SCC. Cohesin participates in DNA replication by modulating higher-order organisation of replication factories. Although disputed in Xenopus egg extracts, cohesin acetylation is reported to speed replication forks in human somatic cells. However, the mechanistic consequences of cohesin acetylation are still poorly understood and while Pds5’s role in SCC maintenance and resolution has been explored, no study to date has reported its role in DNA replication. Pilot studies (unpublished) in our lab have implicated Pds5 in DNA replication. In light of the foregoing, the aim of this project was to analyse the role of Pds5, Esco1, and Esco2 in DNA replication. Here, using an siRNA approach and DNA combing techniques in mammalian cells, I show that Pds5, Esco1, and Esco2 are functionally important in DNA replication. Depletion of either Esco1 or Esco2 results in precocious separation of sister chromatids, delay in DNA replication, apoptosis, or senescence. Depletion of Pds5 proteins is characterised by DNA damage which eventually activates the intra-S-phase DNA damage checkpoint that delays replication fork progression. Depletion of the anti-establishment complex, Pds5A and Wapl, rescues the defect in DNA replication observed when Pds5A is depleted alone. These results provide a novel insight into the role of Esco1, Esco2, and Pds5 in the regulation of DNA replication and suppression of aneuploidy in mammalian cells.

No description available.

In this thesis a study has been made of the interaction of the components of the mitochondrial respiratory chain with particular reference to ubiquinone (Coenzyme Q). The results may be summarized as follows:- i) A potentiometric method of measuring the succinate ferricyanide reductase activity of a heart muscle preparation has been described. It has been used to define two sites of reaction of the electron acceptor, potassium ferricyanide with the succinate oxidase respiratory chain. Above 0.6 mM, the reaction of ferricyanide is mainly at the succinate dehydrogenase-non-haem iron level, below this concentration of ferricyanide the main activity is at the cytochrome c level. ii) Non-haem iron function as an electron carrier has been investigated using metal chelating agents as inhibitors of the enzymic reactions of the succinate oxidase system of heart muscle preparations. Three sites of involvement have been tentatively identified. These agree with those suggested by other workers approaching the problem from different angles. The formation of a metal chelate in heart muscle preparation treated with the chelating agent., 2-thenoyltrifluoroacetone has been demonstrated. iii) The function of the lipid cofactor, ubiquinone, in electron transfer processes has been investigated in tightly coupled rat liver mitochondria. Examination of the oxidation-reduction state of the quinone in different metabolic conditions, and in the presence of different uncoupling agents and electron transfer inhibitors by rapid chemical extraction of the intramito- chondrial ubiquinone has reinforced the idea that ubiquinone is an electron carrier. The possibility that ubiquinone is also involved in oxidative phosphorylation has not been eliminated. iv) A newly discovered stimulation of succinate oxidase by ubiquinone homologues in cytochrome c deficient heart muscle preparations is described. The results suggested two possible explanations (a) that the added quinone might by-pass the cytochrome c deficient site or (b) that the electron flux through residual cytochrome c might be increased by the boosting of an inter-chain conductor pathway by the added quinone.

Preliminary results had indicated that the isocitric dehydrogenase (IDH) of the bacterium Acinetobacter lwoffi 4B was activated by low levels of glyoxylate. Furthermore, in the presence of glyoxylate the enzyme had been found to be more stable to heat or high concentrations of urea. Subsequent studies have shown that two isoenzymes of NADP-linked IDH exist in A. lwoffii and that only one of these is stimulated and protected by glyoxylate. It has also been shown that pyruvate has a similar activating and protecting effect as that of glyoxylate. On addition of either of these effectors the activity of the sensitive enzyme is markedly changed in its dependence on substrate concentration and pH (the former resulting from changes in both the apparent Km and Vm for the substrates). The isoenzymes have been separated by DEAE-cellulose chromatography, cellulose acetate electrophoresis, gel-filtration and preparative rate zonal ultracentrifugation. Investigations using the latter two techniques have shown the sensitive enzyme to be a much larger molecule than the insensitive one. The use of preparative zonal ultracentrifugation for the separation of isoenzymes was novel and these studies have shown it to be a potentially powerful technique for both the separation and purification of isoenzymes. Studies on the separated isoenzymes have shown them to differ in their sensitivities to heat and urea and to have different dependences on pH, temperature and substrate concentration. Experiments undertaken with the aim of elucidating the physiological role of both isoenzymes are also described and discussed.

A survey of the occurrence of bacterial cytochromes demonstrated that the presence of cytochrome oxidase isozymes was a common feature, and confirmed that the complements aa3+o and a1+a2+o, were present mainly in Gram-positives and Gram-negatives respectively. Cytochrome a2, which never occurred as the sole oxidase, was confined to highly adaptable organisms. There was a close correlation between cytochrome complement and general taxonomy. Bacteria were selected for experimental study: Kurthia zopfii, Xanthomonas hyacinthi and Acinetobacter Iwoffi (o); Acetobacter [T71] (a1+o); Chromobacterium violaceum (a2+o); Escherichia coli (a1+a2+o); Microbacterium thermosphactum and Bacillus subtilis (aa3), and a Keilin-Hartree preparation (aa3) from pig heart for comparison. Membrane preparations containing cytochrome oxidases a1,aa3 o were similarly inhibited both quantitatively and qualitatively by CO, indicating similarities in their active site. Each oxidase-CO complex was photodissociable, the Keilin-Hartree preparation being particularly sensitive. A CO-insensitive oxidase was present in K.zopfii. Inhibition, by cyanide with respect to electrons was uncompetitive in preparations containing a1 or aa3 and non-competitive in those containing o. Oxidases aa3 and o were inhibited non-competitively with respect to oxygen. Relative sensitivities were variable. Cytochromes a1 and o were only partially inhibited and multiple, independent sites were affected. Copper was present in the membranes equivalent to approximately 1 Cu : 1 a1, 1 o or 0.5 a3. Cytochrome a1 displayed a much higher second order velocity constant (∼ 23 x 107 M-1. S-1) than did aa3 and o (∼ 3 x 107 M-1. s-1) at 30°. No oxidase class bestowed a particularly high capacity for reaction. No preparation mediated the reduction of natural, alternative inorganic electron acceptors, but aa3 and a2 bound to nitrite. Estimates of P/O ratios in intact cells suggested only one site of oxidative phosphorylation in Ac. [T71] (containing a1 as major whereas three sites were present in cells containing aa3 or o. These findings were confirmed in E. coli utilising either o or a1+a2+o. The results allowed broad discussion and speculation on the reasons for cytochrome oxidase multiplicity.

The pathway and regulation of the gluconeogenic conversion of pyruvate to glyceraldehyde 3-phosphate (G3P) and dihydroxyacetone phosphate (DHAP) in Escherichia coli K12 and Salmonella typhimurium LT2 has been investigated. It has been shown that the conversion of pyruvate to phosphoenol-pyruvate (PEP) in S.typhimurium LT2 is catalysed by the enzyme PEP synthetase. Three genes involved in the synthesis of this enzyme have been identified. One of these genes (PPSA), situated at approximately minute 46 of the S. typhimurium chromosome linkage map appears to be a structural gene for PEP synthetase, whilst the ppsB and ppsC genes, situated at approximately minute 46 and minute 3 respectively of the S. typhimurium chromosome linkage map appear to have a regulatory function. Possible models to explain the control of PEP synthetase formation in S. typhimurium LT2 are discussed and it appears likely that the synthesis of this enzyme is subject to a positive control type of regulation, analogous to the regulation of the synthesis of the maltose catabolising enzymes of E. coli. E. coli K12 3-phosphoglycerate kinase has been studied and shown to be subject to control by the adenylate energy charge, when assayed in the gluconeogenic direction. Little or no effect of energy charge on the activity of this enzyme was seen when the enzyme was assayed in the glycolytic direction. Some kinetic properties of this enzyme have been investigated and the physiological significance of adenylate energy charge control is discussed. Evidence is also presented for a new reaction involved in bacterial gluconeogenesis. This enzyme appears to catalyse a gluconeogenic reaction between PEP and the triose phosphates DHAP and G3P, possibly at a step after 3-phosphoglycerate. Mutants have been isolated which, unlike the parental strain, are able to synthesise this novel enzyme after growth on gluconate or a mixture of 25mM pyruvate plus 10mM glucose.