Th.--Sci. phys.--Lille 1, 1977. N°: 398.

No description available.

M.Sc. (Biochemistry) / An Acinetobacter sp. was isolated which had the ability to metabolise both organic nitriles and inorganic cyanide salts. The enzyme responsible for the degradation of the nitrile groups, was found to be an extra-cellular complex. This complex was partially purified and was shown to consist of not only a number of protein fractions, but also a definite lipid fraction which was identified as being fatty acids. The entire complex had a molecular weight of about 80 000 Da. The enzyme complex exhibited a high degree of stability in the crude form, but rapidly lost its activity on further purification. The complex had a Km of 0.154 ug/ml and a Vmax of 0.534 ug/ml/min for KCN as substrate. This Km value indicates that the complex has a high affinity for KCN and may be of use in the removal of cyanide at low levels. The temperature optimum was shown to be 20·C and the pH-optimum 6.5. Fatty acids were produced both in the presence and absence of a nitrile substrate, and it is unclear whether carbon from CN degradation can be channeled into fatty acid synthesis by this particular bacterium. This bacterium was found to be very effective in the degradation of nitrile compounds. The removal of cyanide from mine effluents is of particular interest in South Africa today and no effective biological method is currently in use.

Sorghum is a staple food in the semi-arid tropics of Asia and Africa, sustaining the lives of the poorest rural people. This project set out to improve the potential economic value of Sorghum bicolor as a crop. The task was undertaken by screening for selected enzymes in the plant that would have a potential market for use in industrial applications and in biotransformations, specifically proteases, polyphenol oxidases and peroxidases. Asurveywas conducted using standard enzyme assays and crude plant extracts, to determine whether the selected enzymes were present. Grain tissue did not appear to have significant protease or polyphenoloxidase activity, but high levels of peroxidases were detected, withthe young grain extracts showing more activity(4.63U/mL)thanripegrain extracts (0.62 U/mL). Leaf tissue extracts contained low levels of protease activity, a considerable amount of polyphenol oxidase (0.127 U/mL), and peroxidase (4.7 U/mL) activities comparable with that found in grain tissue. Root tissue extract was found to contain the highest levels of peroxidase activity (7.8 U/mL) compared to the other extracts. Therefore, sorghum peroxidase from the root was isolated, purified, characterized and applied to biotransformation reactions. Different sorghum strains,withvaryinggraincolour, (Zimbabwe - bronze, Seredo - brown and Epurpur - cream/white) were investigated for the presence of polyphenol oxidase and peroxidase activities. Results of spectrophotometric analysis showed that the enzymes did not appear to be strain specific. However, gel electrophoresis analysis revealed differences in band patterns among the strains. Partial purification of sorghum root peroxidase was achieved after centrifugation, extraction with polyvinylpolypyrrolidone (PVPP), ultrafiltration, and hydrophobic chromatography with phenyl Sepharose, followed by polyacrylamidegelelectrophoresis (PAGE). The specific activity of the 5-fold purified enzyme was found to be 122.3 U/mg. After PAGE analysis, two bands with molecular weights of approximately 30 000 and 40 000 were detected, which compares well with horse radish peroxidase (HRP) which has a molecular weight of approximately 44 000. The colour intensity of the bands in the activity gels indicated that sorghum root peroxidase had apparently higher levels of peroxidase activity than commercial horseradish peroxidase (HRP). Characterizationexperiments revealed that sorghumroot peroxidase is active over a broad temperature range and remains active at temperatures up to 100°C. It also has a broad substrate range. The optimum pH of the enzyme was found to be pH 5 - 6. Under standardized assay conditions, the optimal substrate concentration, using o-dianisidine as substrate, was 50 mM, and the optimal H2O2 concentration under these conditions was found to be 100 mM. Sorghum root peroxidase was applied in a preliminary investigation into the oxidative biotransformationof a number of aromatic compounds. The products obtained were comparable withthose whenthe compounds are reacted with HRP which is the most commonly used commercial peroxidase and has been extensively studied. However, HRP is relatively costly, and the use of peroxidase from sorghum roots as an alternative source, appears to be promising. A patent has been provisionally registered, covering application of sorghum root peroxidase for biotransformations.

Earlier work on aconitase has resulted in the development of the concept that a single enzyme catalyzed the reactions: citrate ⇌ cis-aconitate ⇌ isocitrate. Preliminary work with Pseudomonas aeruginosa, indicated that in this organism two or more enzymes are required for the conversion of citrate to isocitrate. The present study was undertaken in an effort to clarify the status of aconitase in this organism. Methods for the accurate determination of citrate, cis-aconitate, and isocitrate were investigated. Citrate was readily determined by a pentabroraoacetone procedure. No satisfactory method for cis-aconitate was found because of the interference of cysteine and Fe⁺⁺ present in the reaction mixture. Isocitrate was determined with pig heart isocitrate dehydrogenase. Estimation of these three tricarboxylic acids by column and paper chromatography was not achieved. Column chromatography usually resulted in separation, but not in quantitative recovery of these acids, whereas no separation was obtained with paper chromatography. Purification must be effected in order that the characteristics of aconitase may be studied more accurately. Ammonium sulphate precipitation and protamine sulphate treatment were used in attempts to purify a cell-free extract of P. aeruginosa. Little success was attained in these purifications because of the instability of the enzymes, especially that fraction catalyzing isocitrate formation from cis-aconitate. No activity of this reaction was maintained beyond the preliminary purification, whereas activity of citrate formation from cis-aconitate was maintained through several steps. Studies on the rate of formation of citrate and isocitrate from cis-aeonitate by a partially purified system were not conclusive .because of the presence of other enzymes causing the anaerobic disappearance of the three tricarboxylic acids. However, these rate studies indicated that cysteine and Fe⁺⁺ are necessary for optimum formation of citrate from cis-aconitate, but not for isocitrate formation. It appears that a second factor is necessary for optimum isocitrate formation. A more complete purification is necessary to verify these preliminary conclusions and to ascertain the status of the number of enzymes present in the aconitase enzyme system. / Land and Food Systems, Faculty of / Graduate

Pyruvate has been determined at 16, 28 and 40 hours in a culture of Pseudomonas aeruginosa when grown in a glucose medium. Since throughout this entire period the organism possessed the enzyme system capable of rapidly oxidizing pyruvate, it was concluded that pyruvate was being formed and dissimilated continuously and was therefore an intermediate in the oxidation of glucose. It was found that glucose oxidation was not inhibited by 0.02M sodium fluoride and that pyruvate formation and utilization continued unimpaired in the presence of the inhibitor. This would indicate that enolase is not concerned in the formation of pyruvate by P. aeruginosa and therefore the Embden-Meyerhof scheme of glucose degradation does not function in this organism. To study the initial reaction in the degradation of glucose by glucose oxidase of P. aeruginosa a preparation of dried cells was employed using a 24 hour old culture and drying them over phosphorus pentoxide. The dried cells although capable of oxidizing glucose to 2 ketogluconic at pH7.2, were unable to oxidize glucose further than gluconic acid at pH7.5. It was found that the enzyme was not inhibited by malonate, iodoacetate or arsenate but that the system was impaired by cyanide and sodium azide. The inhibition by cyanide would indicate that cytochrome oxidase or a second cyanide sensitive carrier is involved. The action of proteolytic enzymes, light and temperature were determined. The enzyme trypsin was found to destroy the glucose oxidase activity while the effect of pepsin and papain could not be established. Strong light was found to interfere with the full enzyme activity and a temperature of 55°C. completely destroyed the enzyme. Temperatures of 37°C. and 45°C. after 60 minutes reduced the oxygen uptake for the enzyme by as much as 50%. The separation of the enzyme into two fractions, co-enzyme and apo-enzyme, was effected by ammonium sulphate precipitation and dialysis against distilled water. It was found that these two fractions alone, or when combined, could not mediate the reaction glucose to gluconic acid. The co-enzyme was therefore concluded to be dialysable. Addition of known compounds to the fraction showed magnesium and manganese as ions capable of restoring the activity of the enzyme. Magnesium was found to be the more active of the two substances in restoring the enzymes activity. This would indicate that magnesium was the co-enzyme and was capable of being replaced by manganese. / Land and Food Systems, Faculty of / Graduate

This investigation describes the isolation and purification of a deacetylase from trout testes (Salmo gairdnerii) and its initial characterization, the results of which are summarized as follows; - Histone deacetylase activity was demonstrated in the 230,000 x g supernatant fraction of trout testes by a new and sensitive assay. - The deacetylase was purified by salt precipitation, molecular seive chromatography and subsequent ion exchange chromatography. - Two major fractions of enzyme activity were demonstratable with their approximate molecular sizes estimated. - The specificity of the enzyme towards different histone fractions appeared to undergo dramatic changes, depending on prior treatments. - Preliminary results as these furnished some basis for speculation regarding (a) the possible significance of deacetylation not only as a histone modification process but also in the role of gene regulation and (b) the logical prediction as to the existence of enzyme species exhibiting varying substrate specificities to cope with the distinct classes of histones unmasked in trout testes. / Medicine, Faculty of / Biochemistry and Molecular Biology, Department of / Graduate

Bacteroides amylophilus produces a proteolytic enzyme of which 20% is liberated into the medium and 80% is bound.to the cell. Treatment .of the .cells with toluene or mechanical disinteration does not increase the proteolytic activity, indicating that all the protease is superficially located at the bacterial surface. Less than 1% of the total protease activity is released from the cells by osmotic shock procedures which indicated that the protease is not free in the periplasmic space. Speroplast formation liberates 33% of the cell bound protease 40% of which is sedimentable by prolonged high speed centrifugation. Sonic disruption of spheroplasts releases 72% of the protease. After gentl osmotic rupture 48% of the enzyme activity, remained bound to the spheroplast envelope. Prolonged high speed centrifugation results in the sedimentation of all but 16% of the total enzyme. The results give further evidence to the particle bound nature of the protease of Bacteroides amylophilus. Bacteroides amylophilus has a faster rate of growth, with a reduced lag phase and produces a greater cell yield when tryptic peptides are included in the basal medium. Radioactive amino acids are incorporated into cells in significant amounts, indicating that they were not excluded from the cell by a permeability barrier. The amount of incorporation of ¹⁴C amino acids is found to vary for different amino acids and is shown to be concentration dependent. The exogenous ¹⁴C amino acids were incorporated into the cell protein either directly or after an interconversion step. The inhibition of ¹⁴C amino acids uptake by peptides and the direct uptake of ¹⁴C olig-peptides demonstrated peptide uptake by Bacteroides amylophilus. The results suggest that organic nitrogen contributes to the nutrition of Bacteroides amylophilus. / Science, Faculty of / Microbiology and Immunology, Department of / Graduate

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

Nous avons étudié la colonisation végétale de sols de friches industrielles pollués par des hydrocarbures aromatiques polycycliques et des métaux lourds. Différentes approches ont été choisies dans le but de caractériser les premières étapes de la dynamique successionnelle végétale mais aussi de cerner les mécanismes cellulaires impliqués dans le phénomène de tolérance des plantes aux polluants. Des expérimentations en parcelles utilisant le sol de l ancienne cokerie de Neuves- Maisons ont permis de mettre en évidence l importance des banques de graines des sols et des pluies de graines dans l établissement d une communauté pionnière sur sol pollué. Le suivi de la flore des parcelles a permis d établir que la communauté pionnière, constituée de plantes annuelles et bisannuelles lors de la première année, est progressivement colonisée par les plantes pérennes et clonales dominant majoritairement lors de la seconde année. Par ailleurs, la comparaison de communautés se développant sur des sols témoin et pollué suggère une phytotoxicité du sol industriel conduisant à des pertes de richesse et diversité spécifiques. Néanmoins, ces différences semblent s estomper au fur et à mesure du processus de colonisation. Chez les plantes qui parviennent à survivre et croître sur sol pollué, les réponses anti-oxydantes sont sollicitées, confortant l hypothèse de leur rôle crucial dans le succès colonisateur des espèces étudiées. Toutefois, nous avons montré également que le succès de la colonisation pouvait résider également dans une production de graines plus tolérantes vis-àvis de la contamination.Par ailleurs, l analyse de transects au sein même de la friche industrielle d Homécourt, site d une ancienne cokerie, a apporté des éléments de réponse supplémentaires. Les résultats font apparaître une relation entre les descripteurs des communautés (richesse spécifique et indice de diversité de Shannon-Weaver) et les concentrations en Cd et Hg du sol. Par ailleurs, chez les espèces présentes le long des transects (Arrhenaterum elatius, Euphorbia cyparissias ou Tanacetum vulgare), les mesures des défenses antioxydantes et des teneurs en phytochélatines attestent d un stress lié à l exposition métallique. Toutefois, l abondance de ces espèces ne s est pas révélée être liée aux niveaux de contamination du sol. Il en résulte de des perspectives intéressantes en ce qui concerne les applications environnementales de ces résultats / We studied the revegetation of an industrial wasteland soil polluted by polycyclic aromatic hydrocarbons and heavy metals. Different approaches were chosen in order to characterize the first steps of a plant succession and to elucidate the cellular mechanisms involved in plant metal tolerance. Experiments on plots filled with a soil collected from the former coke factory site of Neuves-Maisons (54, France) highlighted the importance of soil seed banks and seed rains in the establishment of a pioneer community on a polluted soil. The study of the plots flora allowed us to prove that the pioneer community, essentially composed of annuals and biannuals during the first year, was gradually colonized by perennials and clonal plants which dominated the second successionnal year. In addition, the comparison of communities established on control and polluted soil suggested a soil phytotoxicity leading to losses of species richness and diversity. Nevertheless, such differences seemed to decrease progressively during the succession process. At last, in the species that survived and grew in the polluted soil, leaf antioxidant enzymes responded, confirming their putative crucial role in the colonization success of these species. However, we showed that this success could also be due to a production of seeds more tolerant toward soil contaminants. Moreover, the study of transects in the industrial wasteland of Homecourt (54, France), a former coke factory site, gave supplementary data, highlighting the relationship between community descriptors (species richness, Shannon-Weaver s diversity indice) and soil Cd and Hg concentrations. Moreover, for the species present along the transects (Arrhenaterum elatius, Euphorbia cyparissias or Tanacetum vulgare), the measurements of antioxidants defences and phytochelatin levels revealed a metallic stress. Nevertheless, the abundance of the species was not related to soil pollutant concentrations. The possible environmental applications of these results will be also discussed