Inulin hydrolysis can occur as a result of the action of exoinulinases and endoinulinases acting alone or synergistically. Exoinulinases cleave the non-reducing β-(2, I) end of inulin releasing fructose while endoinulinases act on the internal linkages randomly to release inulotrioses (F₃), inulotetraoses (F₄) and inulopentaoses (F₅) as major products. Fructosyltransferases act by cleaving a sucrose molecule and then transferring the liberated fructose molecule to an acceptor molecule such as sucrose or another oligosaccharide to elongate the short chain fructooligosaccharide. The production of high yields of oligosaccharides of specific chain length from simple raw materials such as inulin and sucrose is a challenge. Oligosaccharides of chain length up to degree of polymerisation (DP) 5 and fructose were produced using preparations of three commercial microbial enzymes. Production of these novel oligosaccharides was achieved by employing response surface methodology (RSM) with central composite experimental design (CCD) for optimising product yield. Using a crude Novozyme 960 endoinulinase preparation isolated from Aspergillus niger, the following conditions gave a high inulooligosaccharide (lOS) yield, temperature (60 ºC), 150 g/L inulin concentration, 48 h incubation; pH 6.0 and enzyme dosage of 60 U/ml. Under these conditions, inulotrioses (70.3 mM), inulotetraoses (38.8 mM), and inulopentaoses, (3.5 mM) were produced. Response surface regression predicted similar product levels under similar conditions. The crude endoinulinase was purified through a three step purification procedure with a yield of 1.11 % and 3.5 fold purification. The molecular weight of this endoinulinase was estimated to be 68 .1 kDa by SDS-PAGE and its endoinulinase nature was confirmed by native PAGE. The purified endoinulinase was more efficient in production of lOS than the crude endoinulinase preparation. The purified endoinulinase demonstrated a high affinity for the inulin substrate (Km[subscript] 3.53 mM, Vmax[subscript] 666.67 μmol/min/ml). Pectinex Ultra SP-L, a commercial crude enzyme preparation isolated from Aspergillus aculeatus is a cocktail of several enzymes including a fructosyltransferase. The crude enzyme showed both transfructosylation and hydrolytic activity in 200 to 600 g/L sucrose. The main fructooligosaccharides produced from sucrose were l-kestose (GF₂), nystose (GF₃) and fructofuranosyl nystose (GF₄). After the first RSM, with the coded independent variables of temperature, incubation time, pH and sucrose concentration, the highest levels of GF₂, was 68.61 mM, under sucrose concentration 600 g/L, temperature 60°C, enzyme dosage 20 U/ml , pH 5.6, after 4 h incubation. A sucrose concentration of 400 g/L favoured the synthesis of high levels of GF₃ and GF₄. In the second RSM the maximal yields of GF₂, GF₃ and GF₄ were 152.07 mM, 131.38 mM and 43.99 mM respectively. A purified fructosyltransferase did not synthesise GF₄. Ammonium ions were demonstrated to enhance the yield of FOS. A mixture of glucose and fructose was used as substrate for FOS synthesis and no FOS were formed. Glucose was shown to be an end product inhibitor of the fructosyltransferase and therefore hinders the formation of high FOS yield. Fructozyme, isolated from Aspergillus ficuum is a mixture of exo and endoinulinases with the former being predominant was used for fructose production from inulin hydrolysis. The exoinulinase was purified to electrophoretic homogeneity by a three step purification procedure. The molecular weight of the enzyme was estimated to be 53 kDa with a 2 I % yield and 4.2-fold. Response surface regression was used to predict the maximum fructose levels achievable under the combinations of temperature, enzyme dosage and incubation time. A reaction time (48 h), enzyme dosage (100 U/ml) and inulin concentration (150 g/l) at pH 5.0 at 50°C gave higher fructose levels (106.6 mg/ml) using crude exoinulinase as compared to 98.43 mg/ml using the purified exoinulinase. These findings indicate that higher levels of fructose require longer incubation periods and higher inulin substrate concentrations with higher enzyme dosage. The crude exoinulinase preparation gave fairly higher levels of fructose than the purified exoinulinase and this is due to the presence of other hydrolytic enzymes in the crude preparation. The conditions established by RSM and CCO were adequate in producing high yield of oligosaccharides and fructose and can therefore be applied for their industrial production since they are in high demand due to their health benefits as prebiotics.

DNA polymerase activity associated with purified nuclei of rat intestinal mucosa was studied. Two DNA polymerase activities have been isolated, partially purified and characterized. One of the enzymes was extracted from purified nuclei with 10 mM Tris-HCl, pH 8.0, containing 5 mM dithiothreitol while the second enzyme, which was associated with the nuclear deoxyribonucleoprotein complex, was extracted only in a high ionic strength medium containing 1 M NaCl in 0.1 M Tris-HCl, pH 8.0 and 5 mM dithiothreitol. The molecular weights of these nuclear DNA polymerases were estimated by gel filtration on Sephadex G-150. Two peaks of DNA polymerase activity were detected when the Tris-soluble extract was chromatographed. The molecular weights of these peaks of activity were calculated to be 266,000 and 104,000. It was concluded that the first peak of activity represented an aggregate of the second. A single peak of DNA polymerase activity was obtained when the NaCl-soluble nuclear extract was chromatographed on Sephadex G-150. It corresponded to a molecular weight of approximately 40,000. Chromatography on DEAE-cellulose indicated that the two enzymes differed in ionic charge. The bulk of the Tris-soluble DNA polymerase activity eluted with 0.045-0.055 M KCl, while the NaCl-soluble enzyme had a higher affinity for the anion exchange resin and was not eluted until the KCl concentration was 0.165-0.21 M. The partially purified enzymes were very labile. Storage at 4°C, 0°C or -20°C did not increase enzyme stability. The presence of glycerol, which had no effect on enzyme activity, helped maintain the stability of both enzymes for at least 1 month at -20°C. The enzymic properties of the nuclear DNA polymerases differed. In Tris-HCl buffer, a pH of 7.5 was optimal for the polymerase reaction catalyzed by either enzyme, but in phosphate buffer the pH optima were 7.2 and 6.0 for the Tris-soluble and NaCl-soluble enzymes, respectively. The presence of DNA, all 4 deoxynucleoside 5'-triphosphates and Mg²⁺ ions was required for the activity of both crude and partially purified forms of the nuclear DNA polymerases. Substitution of Mn²⁺ or Ca²⁺ for Mg²⁺ resulted in lower enzymic activity. The addition of dithio-threitol greatly enhanced the activity of both enzymes, especially the purified preparations. The presence of thiol reagents, p-hydroxy-mercuribenzoate and N-ethylmaleimide, inhibited both of the nuclear DNA polymerase activities. In the presence of 1 mM nalidixic acid the activity of the Tris-soluble enzyme was abolished whereas the NaCl-soluble DNA polymerase activity was greatly enhanced. The activities of the nuclear DNA polymerases were also affected differently by monovalent cations. The addition of NH₄⁺, K⁺ or Na⁺ to the assay mixture inhibited the activity of the Tris-soluble enzyme but stimulated by 30-170% the activity of the NaCl-soluble DNA polymerase. The two enzymes also differed in template preference. The crude Tris-soluble DNA polymerase functioned equally well with either heat-denatured or native DNA, while the purified form showed a slight preference for native over heat-denatured DNA. The purified NaCl-soluble DNA polymerase, which in crude extracts consistently preferred native DNA as template, showed a strict dependence on native DNA. Differences between the Tris-soluble and NaCl-soluble DNA polymerases in extractability from purified nuclei, molecular weight, ionic charge and in their enzymic properties clearly indicate that two distinct DNA polymerase activities are associated with purified nuclei prepared from rat intestinal mucosa cells. The close association between the NaCl-soluble DNA polymerase and the deoxyribonucleoprotein complex and its absolute dependence on native DNA template support the conclusion that it is a repair enzyme in vivo. The role of the Tris-soluble enzyme is less certain. / Medicine, Faculty of / Biochemistry and Molecular Biology, Department of / Graduate

The effects of epinephrine and electrical stimulation on the activation of glycogen phosphorylase were studied in isolated rat diaphragm and frog sartorius, and rat gastrocnemius in vivo. The resting ratio of phosphorylase, as expressed by the ratio of phosphorylase a to total phosphorylase (-AMP/+AMP ratio) was found to be low of the order of 0.05 in diaphragm and sartorius. In rat gastrocnemius this value was high at 0.26. As measured by the ratio of activity at pH 6.8 to that at pH 8.2 (pH 6.8/8.2 ratio), phosphorylase kinase was essentially in its inactive form under conditions of no stimulation. On treatment with epinephrine, phosphorylase was activated, and there was a significant increase in the activity ratio for phosphorylase kinase, up to 10-fold, indicating that conversion of nonactivated kinase to its activated form had occurred. Epinephrine also produced marked increases, up to 15-fold, in the tissue levels of adenosine 31, 51-monophosphate (cyclic AMP). When muscle was induced to contract by electrical stimulation, phosphorylase was markedly and rapidly activated. In contrast to the effect of epinephrine, electrical stimulation produced no conversion of phosphorylase kinase to its activated form as measured by the pH 6.8/8.2 ratio. This was found for both direct and neural stimulation, at various frequencies, for different times of stimulation, in vitro and in vivo, and in two species. No increase in the tissue levels of cyclic AMP were detected on electrical stimulation. It was concluded that the mechanism of activation of phosphorylase during electrical stimulation is basically different from that produced by adrenergic amines. The data strongly suggests that during muscle contraction phosphorylase is activated by a mechanism which does not involve conversion of phosphorylase kinase to its activated form. In further work, the relationship between phosphorylase activation and muscle contraction was studied. It was found that for any given frequency of stimulation, phosphorylase was activated within 2 sec to a particular ratio for that frequency. On further stimulation, the ratio did not increase. When the temperature was lowered, the steady state phosphorylase ratio for a given frequency was lowered, but activation still occurred rapidly. In experiments in which calcium was removed from the medium by using chelating agents, a correlation was demonstrated between phosphorylase activation and contractile tension. From these results it appears that the mechanism of phosphorylase activation is closely coupled to the contractile mechanism. It is proposed that calcium ion, which is important in excitation-contraction coupling and tension development, is responsible for phosphorylase activation. It is further suggested that calcium ion released into the myoplasm may act with nonactivated phosphorylase kinase to catalyse the conversion of phosphorylase b to phosphorylase a. / Medicine, Faculty of / Anesthesiology, Pharmacology and Therapeutics, Department of / Graduate

This study has examined the expression of the glutathione S-transferases (GSH S-T) in fetal rat livers in order to provide more information about the role played by this important group of enzymes in the fetus. The study commenced with an examination of the subunit composition of adult and fetal rat liver GSH S-T using affinity chromatography followed by polyacrylamide gel electrophoresis in sodium dodecyl sulphate. Adult livers contained four major GSH S-T subunits. An additional and previously unidentified subunit was detected in fetal livers. This subunit, which differed from that found in rat placenta, had a Mᵣ of approximately 25 500. Densitometric measurements suggest that the newly detected subunit accounts for as much as 26% of the GSH S-T in fetal livers. The novel fetal isoenzyme comprising this subunit was purified using a combination of affinity chromatography, carboxymethyl-cellulose column chromatography and chromatofocusing. The six major basic rat liver GSH S-T were purified for reference and comparative purposes. The fetal isoenzyme is composed of two non-identical subunits, namely, subunit Yc (Mᵣ 28 000) and the fetal subunit referred to as 'Yfetus'· The enzyme which I have termed GSH S-transferase Yc Y fetus has an isoelectric point of approximately 8.65 and has GSH S-T activity towards a number of substrates. Significantly, the fetal isoenzyme has one of the highest glutathione peroxidase activities yet described for the purified rat liver GSH S-T towards the model substrate, cumene hydroperoxide. Kinetic studies reveal that the fetal isoenzyme has a catalytic efficiency for the peroxide substrate which is four fold higher than that of the adult rat liver isoenzyme, GSH S-T YcYc. The in vitro effect of the GSH S-T substrate and teratogen, acrolein, on this fetal isoenzyme was investigated and compared with acrolein's effect on some of the adult rat liver GSH S-T isoenzymes in the standard 1-chloro-2,4-dinitrobenzene assay. Surprisingly, acrolein was identified as a non-competitive inhibitor of the GSH S-T. Exposure to acrolein in various guises could therefore result in inhibition of the fetal isoenzyme and its subsequent failure in inhibiting lipid peroxidation. Inhibitor studies were performed to look at the effect of acrolein, as well as other substrate and non-substrate ligands, on the glutathione peroxidase activity of GSH S-T YcY fetus and YcYc. The glutathione peroxidase activity of the fetal isoenzyme was far less susceptible to acrolein inhibition than the YcYc isoenzyme and the fetal isoenzyme was found to retain significant glutathione peroxidase activity despite saturating concentrations of non-substrate ligand. This study suggests that the fetal isoenzyme serves a specific function in protecting fetuses against the possible teratogenic effects of organic peroxides.

One of the main problems in the field of multifunctional proteins such as ligandin is the possibility that multiple forms and isoproteins may exist. Two forms of liver ligandin [ GSH (reduced glutathione) S-transferase B] have been described, a heterodimeric form consisting of equal amounts of Ya (22000 daltons) and Yc (25000 daltons) subunits, and a homodimeric form containing only Ya. Because rat testis ligandin, prepared by the standard technique of anion-exchange and molecular exclusion chromatography, contains more Yc subunit than Ya, it has been claimed that testis and liver ligandin are different entities (Bhargava, Ohmi, Listowsky and Arias (1980) J. Biol. Chem. 255, 724-727). This thesis investigated the nature and character of ligandin in the steroid-producing tissues of the rat. A comparative study was undertaken to establish whether testis ligandin differed from liver ligandin. Different methods of purification were used to investigate testis ligandin and its relationship to other GSH S-transferases in steroidogenic tissues. Testis ligandin purified by immunoaffinity chromatography using anti-liver YaYa ligandin antiserum yielded a product identical with liver preparations (Yc=Ya). This suggests that the differences previously described may be due to contamination of testis ligandin by a closely related species. Testis ligandin prepared by the standard technique was similar to that previously reported, containing more Yc than Ya. Cross-linking studies of standard testis ligandin preparations with dimethylsuberimidate showed more than one band in the 50000 dalton region, further strengthening the view that these testis ligandin preparations may be contaminated. Since this contaminant was likely to be another GSH S-transferase, sodium dodecyl sulphate/ polyacrylamide-gel-electrophoretic analysis was performed on testis GSH S-transferases separated by CM-cellulose chromatography. GSH S-transferase AA which was present in large amounts, was shown to migrate in the same region as Yc subunit. CM-cellulose chromatography of a 'pure' standard testis ligandin preparation revealed significant amounts of GSH S-transferase AA migrating as Yc subunit, in addition to ligandin consisting of equal amounts of Ya and Yc subunits, indicating that testis ligandin is identical with liver ligandin and that previously described differences are due to a contaminant identified as GSH S-transferase AA. Studies on ligandin in other steroid-synthesising tissues showed that ovary and adrenal ligandin prepared by standard techniques also contained more Yc than Ya. Separation of ovary GSH S-transferases on CM-cellulose showed that GSH S-transferase B, the peak reacting with anti-liver YaYa ligandin antisera contained equal amounts of Ya and Y c subunits, suggesting a situation similar to that in the testis exists. Glutathione peroxidase II activity of testis and ovary GSH S-transferases was investigated. Fractions corresponding to GSH S-transferase AA, A and B exhibited activity with cumene hydroperoxide. The considerable glutathione peroxidase activity of GSH S-transferases in testis and ovary suggest a protective function for the cells of gonadal tissue against oxidative damage to essential intracellular components. Further attempts to clarify the function of ligandin in the steroid-synthesising tissues were made. The pattern of gonadal ligandin development during early life, puberty and pregnancy determined by radioimmunoassay was found to parallel serum steroid hormone concentrations. This correlation was not observed in liver or kidney. Ligandin was localised to specific cells of the steroid synthesising tissues using immunocytochemical techniques. These findings suggest that there may be a functional link between steroidogenic cells, or products of their activity and certain GSH S-transferases. Phenobarbital pre-treatment did not have any effect on developing testis, ovary or adrenal ligand in concentrations. Immunocytochemical localisation of ligandin in rat steroid-producing tissues using a peroxidase anti-peroxidase (PAP) technique with anti-liver YaYa ligandin antiserum as the first antibody, showed staining in the testis to be limited to the interstitial (Leydig) cells. Stromal cells of the ovary and the fascicular, glomerular and reticular zones of the adrenal cortex also contained immunoreactive material. PAP staining with anti-testis ligandin antisera (testis ligandin prepared using the standard technique) showed far greater intensity of staining in these tissues, presumably due to reaction with both ligand in and GSH S-transferase AA. This study has clarified the structural aspects of testis ligandin and demonstrated identity with liver ligandin. Ontogeny of ligandin in the steroidogenic tissues and localisation to specific regions in these tissues suggests a functional link between ligandin, GSH S-transferases, GSH peroxidases and activity of steroidogenic tissue.

In this study the purification of human basic and near-neutral liver, and human basic and acidic lung glutathione S-transferases (GSH S-T) was undertaken. Purification of the basic and near-neutral GSH S-T was achieved using a combination of affinity chromatography, chromatofocusing and immunoaffinity chromatography. Affinity and ion exchange chromatography were employed in the purification of the basic and acidic lung forms. The purified proteins had similar physicochemical characteristics to the GSH S-T purified by others. The binding of 1-chloro-2,4-dinitrobenzene (CDNB) to the 3 classes of human GSH S-T, viz. basic, near-neutral and acidic and the effects of such binding, if any, were examined. Human acidic lung GSH S-T is irreversibly inactivated by CDNB in the absence of the co-substrate glutathione (GSH). The time-dependent inactivation is pseudo-first order and demonstrates saturation kinetics, suggesting that inactivation occurs from an EI complex. GSH protects the enzyme against CDNB inactivation. In contrast, the basic and near-neutral GSH S-T are not significantly inactivated by CDNB. Incubation with [¹⁴C]-CDNB indicated covalent binding to all 3 classes of GSH S-T. When the basic and acidic GSH S-T were incubated with [¹⁴C]-CDNB and GSH, cleaved with cyanogen bromide, and chromatographed by HPLC, a single peptide fraction was found to be labelled in both classes. Incubation in the absence of GSH yielded 1 and 2 additional labelled peptide fractions for the basic and acidic transferases, respectively. These results suggest that while CDNB arylates all 3 classes of human GSH S-T, only the acidic GSH S-T possesses a specific GSH-sensitive CDNB binding site, which when occupied leads to time-dependent inactivation of the enzyme. The tissue distribution and localization of the 3 classes of human GSH S-T in normal and tumour tissue was examined. Antibodies to representatives of the 3 classes were raised in rabbits, and radial immunodiffusion employed to quantitate their concentrations in the cytosol of 18 organs from 9 individuals. The data provide the first direct, quantitative evidence for the inter-individual and inter-organ variation suggested by earlier workers. The absence of the near-neutral GSH S-T in 5 of the 9 individuals studied confirms an earlier suggestion of a "null" allele for this transferase. Basic and acidic GSH S-T (apart from in a single liver), were always present. Near-neutral GSH S-T, when present, were found in all tissues examined. The marked inter-organ and inter-individual variation observed in this study may explain individual and organ susceptibility to drugs, toxins and carcinogens. The immunohistochemical localization of the 3 classes of GSH S-T reveals important differences in their localization, and may provide insight into their functions in various organs and tissues.

Note:

Les cellules exposent à leurs surface glycoconjugués qui jouent important dans des événements biologiques importants tels que la communication entre cellules, la croissance de cellules saines ou cancéreuses et les processus d'infection d'agents pathogènes. Certaines structures polysaccharidiques qui contiennent le résidu Galf ont attiré beaucoup d’intérêt au cours des dernières décennies. En effet, le galactofuranose peut être exprimé chez de nombreuses espèces pathogènes, telles que Mycobacterium tuberculosis, Aspergillus et Leishmania, mais il est absent chez les mammifères. Par conséquent, ces glycoconjugués sont considérés comme des cibles intéressantes pour des approches thérapeutiques.Les galactofuranosyltransférases (GalfT) catalysent le transfert des résidus de galactofuranose dans les structures des glycoconjugués. Cependant, ces GalfT sont des enzymes faiblement décrites, malgré leur rôle crucial dans la virulence ainsi que dans la pathogénicité de nombreux micro-organismes. Jusqu'à présent, seule la GalfT2 de Mycobacterium tuberculosis a été entièrement caractérisée.Dans cette thèse, quatre GalfTs de Leishmania major, l'agent responsable de la leishmaniose, ont été caractérisées. Elles ont été d'abord clonées, surexprimées dans E. coli et purifiées. Ensuite, leurs paramètres cinétiques respectifs ont été déterminés. De plus, puisque ces GalfT sont situées dans l'appareil de Golgi de Leishmania, nous avons supposé que leur glycosylation pourrait être un élément important pour leur stabilité etleur activité. Ainsi, des GalfT glycosylés ont été produites à l'aide de Leishmania tarentolae et les résultats préliminaires de leur activité enzymatiques ont été obtenus.Les GalfT leishmaniennes démontrent des résultats prometteurs pour le développement de nouvelles stratégies chimio-enzymatiques pour la synthèse de glycoconjugués contenant du Galf, ainsi que pour la conception de nouveaux médicaments contre la leishmaniose. / Cells are heavily decorated by diverse glycoconjugates that are involved in important biological events such ascell-cell communication, growth of healthy or cancerous cells and pathogens infection process. Among these polysaccharidic structures, Galf-containing glycans have been the subject of increasing interest in the last decades. Indeed, the galactofuranose can be found in many pathogenic species, such as Mycobacteriumtuberculosis, Aspergillus and Leishmania, but is absent in mammals. Therefore, these glycoconjugates are considered as interesting targets for therapeutic approaches.Galactofuranosyltransferases (GalfTs) catalyse the transfer of galactofuranose residues into glycoconjugatesstructures. However, GalfTs are poorly described enzymes despite their crucial role in the virulence and the pathogenicity of numerous microorganisms. Up to date, only one mycobacterial GalfT has been fully characterized.In this thesis, four putative GalfTs of Leishmania major, the causing agent of leishmaniosis diseases, were characterized. They were first cloned, overexpressed in E. coli and purified. Then, their respective kineticparameters were determined. In addition, since these GalfT are located in the Golgi apparatus of Leishmania, we assumed that their glycosylation could be an important element for their stability and activity. So, glycosylatedGalfTs were produced using, Leishmania tarentolae, and preliminary results of their enzymatic activity were obtained.Still, leishmanian GalfTs demonstrate promising results for the development of new chemoenzymatic strategies for Galf-containing glycoconjugates synthesis, as well as the design of new drugs against leishmaniasis.

Diss. (sammanfattning) Stockholm : Karol. inst., 2004. / Härtill 3 uppsatser.

The glutathione S-transferases are a group of enzymes which catalyse the conjugation of reduced glutathione with a variety of electrophilic molecules, and they are therefore thought to play a major role in drug biotransformation and the detoxification of xenobiotics. The cytosolic GSH S-transferase isoenzymes of rat, man and mouse have been assigned to three groups, Alpha, Mu and Pi, based on N-terrninal amino acid sequences, substrate specificities, immunological cross-reactivity and sensitivities to inhibitors. The kinetic mechanism of the GSH S-transferases is controversial, due to the observation of non-Michaelian (non-hyperbolic) substrate-rate saturation curves. The most detailed investigations of the steady-state kinetics of glutathione S-transferase have been performed with isoenzyme 3-3 (class Mu) and the substrate 1,2-dichloro-4-nitrobenzene (DCNB). Explanations for the apparently anomalous non-hyperbolic kinetics have included subunit cooperativity, steady-state mechanisms of differing degrees of complexity and the superimposition of either product inhibition or enzyme memory on these mechanisms. This study has confirmed the biphasic kinetics for isoenzyme 3-3 with DCNB and shown non-hyperbolic kinetics for this isoenzyme with 1-chloro-2,4-dinitrobenzene (CDNB) and for isoenzyme 3-4 with DCNB and CDNB. It is proposed that the basic steady-state random sequential Bi Bi mechanism is the simplest mechanism sufficient to explain the non-hyperbolic kinetics of GSH S-transferases 3-3 and 3-4 under initial rate conditions. Neither more complex steady-state mechanisms nor the superimposition of product inhibition or enzyme memory on the simplest steady-state mechanism are necessary.