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    Acetyl-CoA carboxylase in the photosynthetic tissue of maize :a thesis presented in partial fulfilment of the requirement for the degree of Master of Science in biochemistry at Massey University
    (Massey University, 1988) Rutherfurd, Shane McArtney
    The aim of this study was, a). to examine further, aspects of the role of acetyl-CoA carboxylase in the regulation of fatty acid synthesis in the provision of acyl lipid for plastid development, and b}. to purify acetyl-CoA carboxylase from maize leaves using the affinity methods which have been used successfully to purify the enzyme from animal tissues. In a constant weight of tissue, carboxylase activity decreased 7.6-fold over the period of 4 to 12 days after sowing, while total acetyl-CoA carboxylase activity increased 9-fold in maize seedlings over the period of 4 to 8 days with no further increase up to day 12. Protein levels decreased 3-fold over the growth period examined, while specific activity was constant at 27.2 to 28.3nmol/min/mg of protein between 4 and 6 days, before increasing to a maximum of 33.2nmol/min/mg of protein at day 7, then decreasing to one third of the maximum value on day 12. Chlorophyll levels in a constant weight of tissue increased 260-fold over the period of 4 to 11 days. The changes in the level of acetyl-CoA carboxylase activity paralleled changes in fatty acid levels in tissue along the length of the 9-day-old maize leaf. The levels of both biochemical parameters increased in the region from the leaf base to 15mm along the leaf. After which they both decreased to a minimum at 25-30mm along the leaf before increasing to a maximum at 60mm along the leaf, and finally decreasing towards the leaf tip. A 5-fold increase in acetyl-CoA carboxylase activity was observed from the least favourable chloroplast stromal concentrations of ATP, ADP, Mg2+ and tt+ in the dark, to the most favourable concentrations of these metabolites present in the chloroplast stroma during light periods. These findings are consistent with, 1). a role for acetyl-CoA carboxylase in the regulation of fatty acid synthesis in maize photosynthetic tissue and, 2). control of acetyl-CoA carboxylase activity via light-dependent changes in the pH and concentrations of ATP, ADP and Mg2+ found in the stroma of chloroplasts. Several attempts were made to purify acetyl-CoA carboxylase using avidin-affinity chromatography. However, after the initial, apparently successful attempt, active enzyme could not be recovered from the avidin-affinity column upon elution with biotin. Changes were made to several chromatographic conditions, and although ionic strength in the range of 0.1 to l.0M KCl, did not affect the elution of active acetyl CoA carboxylase from the column; lowering the column flow rates from l.5ml/hr/ml of gel to 0.15-0.3ml/hr/ml of gel did appear to enhance the binding of the enzyme to the column. Using this flow rate, a 62 000 dalton protein and a 54 500 dalton protein were eluted in a fraction found to contain biotin-containing proteins. Since it is feasible that the 62 000 dalton is biotin-containing and since this protein has a similar molecular weight to 60 000-62 000 dalton biotin-containing subunit of maize leaf acetyl-CoA carboxylase, the potential for purifying acetyl-CoA carboxylase from maize leaves using avidin-affinity chromatography seems to exist. However, further investigation is necessary in order to facilitate the recovery of active carboxylase from the avidin-affinity column.
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    A preliminary investigation : plant cyanogenecity as a possible co-factor in a possum specific toxin : a thesis presented in partial fulfilment of the requirements for the degree of Master of Science in Biochemistry, Massey University, New Zealand
    (Massey University, 1998) Gorrie, Elana Maree
    Since the introduction of thirty Australian brushtail possums into New Zealand in 1858 to start a fur trade industry the possum population has grown considerably. New Zealand is now 'home' to approximately 70 million possums which wreak devastation on our native forests and wildlife. Current effective strategies for the control of possums in uninhabited areas include the use of 1080, brodifacoum, cholecalciferol, cyanide, and trapping or shooting. However these strategies are relatively non-specific in their mode of action and as such cause non-target species, including native wildlife, to die. The use of non-specific toxins and traps is also inappropriate for situations where people, livestock, or pets are present. There is therefore a demand for alternative strategies of possum control that affect only the target species. Methods presently being researched include the development of contraceptive vaccines, possum repellents and possum specific bait stations. This research investigates the feasibility of developing a cyanogenic bait that is activated by a co-factor within the possum diet. The fast acting hydrogen cyanide poison is present in some plants species in an inactive glycoside form. Upon tissue injury the inactive cyanogenic glycoside is exposed to and hydrolysed by catabolic enzymes within the plant thereby releasing the toxin, hydrogen cyanide, at potentially lethal levels for possums. Some plant varieties within cyanogenic species however, have evolved to be acyanogenic due to the absence of either the cyanogenic substrate, the enzyme, or both. The occurrence of these acyanogenic plants which contain either the substrate or the appropriate enzymes are the target of this research. It is these plants that may provide the necessary co-factor for a cyanogenic possum bait to become lethal. Preliminary analyses involved measuring and maximising the cyanide release from plant species known to be highly cyanogenic. Clover leaves (Trifolium repens), cherries (Prumus avium), and almonds (Prumus amygdalus) were the plant tissues analysed to determine whether levels of cyanide toxic to possums could be liberated. All three plant varieties underwent in vitro analyses in which they were exposed to surplus substrate and/or enzymes at varied temperatures and acidities. The maximum cyanide release was determined for each plant variety and in the case of almonds (Prumus amygdalus) a further in vivo study was performed. Although the clover, cherries and almonds all liberated cyanide after addition of either cyanogenic substrate or enzymes, the almonds were the only plant tissue to liberate sufficient levels of cyanide from the in vitro analyses to be considered toxic to possums. The almonds were found to contain high levels of active β-glucosidase enzymes which when incubated with the cyanogenic substrate, amygdalin, released high levels of cyanide. The in vivo analyses of almond macerates administered with amygdalin however were inconclusive in showing almonds as an effective co-factor for the hydrolysis of amygdalin. Nevertheless, two possums did die from cyanide poisoning after the administration of amygdalin with and without added enzymes. A third possum displayed signs of severe cyanide poisoning after it was gavaged with amygdalin and β- glucosidase enzymes but it later made a full recovery. As a result of the limitation imposed by the small size of the in vivo sample group further experimental trials are recommended to possibly obtain a more accurate set of results.
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    Some observations on the ecology and phytochemistry of nickel-accumulating alyssum species from the Iberian peninsula : a thesis presented in partial fulfilment of the requirements for the degree of Master of Science in Chemistry at Massey University
    (Massey University, 1980) Shaw, Sugirthamani
    Experiments were carried out on the tolerance to, and uptake of nickel by Iberian subspecies of A. serpyllifolium. Two of these subspecies, the serpentinic-endemics s.sp. lusitanicum (from Bragança, Portugal) and s.sp. malacitanum (from Málaga, Spain) were hyperaccumulators (>1,000 μg/g in dried leaves) of nickel. Their precursor, s.sp. serpyllifolium (from Granada, Spain) was a non-accumulator of this element. Seeds of the two serpentine-endemics germinated extensively in nickel concentrations up to 12,000μg/g (1.2%) whereas s.sp. serpyllifoliurn only germinated in nickel concentrations of up to 60 μg/ml. Tolerance tests involving measurement of new root lengths of excised seedlings placed in varying nickel concentrations again showed much greater tolerance of the two serpentinophytes. In both series of experiments, the order of tolerance was: s.sp. lusitanicum > s.sp. malacitanum > s.sp. serpyllifolium. In pot trials involving seedlings of s.sp. malacitanum grown in mixtures containing varying amounts of calcium, magnesium and nickel, the most important findings were that nickel uptake is somewhat stimulated by an excess of calcium in the substrate. This relationship was confirmed by interspecies and intra-species analyses of naturally-occurring plants. Enhanced calcium uptake concomitant with nickel uptake by hyperaccumulators results in a higher (more favourable) Ca/Mg ratio and thereby counteracts one of the unfavourable edaphic effects of serpentine soils. The form of nickel in leaves of the three Iberian subspecies was investigated. Nickel existed mainly as a water-soluble polar complex in the vacuoles. Small concentrations of nickel did however exist in cell fractions particularly in the mitochondria where enzyme systems are located. GLC studies on the purified nickel complexes showed that this element is associated principally with malic and malonic acids which are present in high concentrations in the hyperaccumulators but not in s.sp. serpyllifolium. It is suggested that production of malic acid is a mechanism whereby hyperaccumulators can tolerate unfavourable edaphic factors such as nickel-rich soils. Presence of nickel in the mitochondria blocks the citric acid cycle by deactivating malic dehydrogenase leading to build-up of malic acid in the vacuoles which then absorbs excess nickel by a complexing reaction and leads to its diffusion back into the vacuoles from the mitochondria, hence unblocking the citric acid cycle. Malonic acid also blocks the cycle and leads to a reduced level of malic acid and hence lesser tolerance to nickel. This is shown to be the case for s.sp. malacitanum which contains more malonic acid than s.sp. lusitanicum and is also less tolerant to nickel. It is postulated that the chemical evidence suggests that s.sp. lusitanicum and s.sp. malacitanum are sufficiently different chemically to lend weight to the argument that the latter should be promoted to full specific rank as has already been done for s.sp. lusitanicum.
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    Acetyl-CoA carboxylase in photosynthetic tissue : a thesis presented in partial fulfilment of the requirements for the degree of Doctor of Philosophy in Biochemistry at Massey University
    (Massey University, 1981) Nikolau, Basil John
    Acetyl-CoA carboxylase catalyses the rate-limiting reaction in de novo fatty acid biosynthesis in a wide variety of organisms. In plants however, the significance of this enzyme in regulating de_ novo fatty acid biosynthesis is unknown. In this investigation acetyl-CoA carboxylase of maize, barley and spinach leaves has been studied in order to compare some features of this enzyme in the three plants. In maize, acetyl-CoA carboxylase is located in chloroplasts, where it occurs as a soluble enzyme in the stromal fraction. Both mesophyll and bundle sheath cells also exhibit acetyl-CoA carboxylase activity. Activities of acetyl-CoA carboxylase in isolated chloroplasts have been compared to rates of lipid synthesis from acetate in order to investigate the role of acetyl-CoA carboxylase in regulating de novo fatty acid biosynthesis from acetate. Although acetyl-CoA carboxylase activity was higher than that expected from the rate of acetate incorporation into lipids of isolated chloroplasts of maize and barley, the opposite was found for chloroplasts isolated from spinach. In chloroplasts from the emerging leaves of maize seedlings, lipid synthesis was maximal with chloroplasts isolated from the leaf segment corresponding to leaf greening. However, a relationship between leaf development and acetyl-CoA carboxylase activity was less apparent. Consequently a regulatory role for acetyl-CoA carboxylase in de novo fatty acid biosynthesis could not be directly established from a comparison of the rates of lipid synthesis and acetyl-CoA carboxylase activities. An alternative approach to the establishment of a regulatory role for acetyl-CoA carboxylase was to purify the enzyme from maize and barley leaves, and study its response to variable concentrations of substrates, products and certain cellular metabolites. Purification of both enzymes was performed by identical procedures, including polyethylene glycol fractionation, hydrophobic chromatography and gel filtration. Acetyl-CoA carboxylase from both maize and barley leaves appeared to be an integral enzyme, as no evidence for its dissociation was found, contrary to the findings of the bacterial enzyme. The kinetic properties of the partially purified enzyme from maize and barley were very similar. The apparent Michaelis constants for the substrates, acetyl-CoA and HCO-3, were about 0.lmM and 2mM, respectively for both enzymes. The enzymically active form of the substrate, ATP, was found to be Mg.ATP. Furthermore, free ATP inhibited enzymic activity, while free Mg2+, activated the enzyme from both plant sources. Monovalent cations, particularly K+, were positive effectors of acetyl-CoA carboxylase, on the other hand, the products of the acetyl-CoA carboxylase reaction, malonyl-CoA and ADP were inhibitors. ADP inhibition was competitive with respect to ATP, but uncompetitive with respect to acetyl-CoA. Inhibition of acetyl-CoA carboxylase activity by CoA was noncompetitive with respect to acetyl-CoA, while palmitoyl-CoA inhibition was uncompetitive with respect to acetyl-CoA. From the view point of regulation of acetyl-CoA carboxylase activity, the observed response of activity to changes in pH, and the concentrations of Mg2+, K+, ATP and ADP in vitro, may be of significance. Changes in the chloroplastic levels of these effectors of acetyl-CoA carboxylase activity have been reported to occur during light-dark transition of chloroplasts, and would be consistent in regulating acetyl-CoA carboxylase activity in the light and dark.
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    The oxidation of [alpha]-farnesene : a thesis presented in partial fulfilment of the requirements for the degree of Doctor of Philosophy in Chemistry at Massey University
    (Massey University, 1994) Spicer, Julie Ann
    Autoxidation products of the sesquiterpene α-farnesene 1 increase rapidly in apples during cold storage and are believed to play an important role in the production of the cold storage disorder, superficial scald. The site selectivity of the oxidation of α-farnesene was investigated with a variety of useful reagents for photochemical allylic oxidation, hydroxylation and epoxidation. Oxidation products 33-47 were isolated and characterised. The synthesis of conjugated trienes and related oxidation products of α-farnesene 1, principally from the epoxides of α-farnesene, is described. Base-promoted ring opening of 6,7-epoxide 44 by the mixed base potassium tert-butoxide / lithium disopropylamide afforded the conjugated triene 3 whilst the 3,4-epoxide 45 afforded triene 50. In contrast, 10,11-epoxide 43 failed to undergo epoxide ring opening; rearranging instead to the conjugated triene epoxide 51. Base-promoted ring opening of bis-epoxide 46 afforded trienol epoxide 56 at -30°C, whilst cyclisation to tetrahydrofurans 55a and 55b occurred at room temperature. Photosensitised oxidation of 10,11-epoxide 43 followed by in situ treatment with acid gave the cyclic peroxide 4 and upon reduction, tetrahydrofurans 55a and 55b. Bisallylic alcohol 61 was prepared by alkylation of 3-methylsulpholene 29 with geranial 59 followed by thermolysis. Trienes 3 and 4 have been isolated previously as autoxidation products of α-farnesene 1 and are implicated as the causal agents of the superficial scald of stored apples. The asymmetric dihydroxylation of α-farnesene 1 using the Sharpless ligands (DHQ)2- PHAL and (DHQD)2-PHAL was investigated. The isolation and characterisation of the 3,4-, 6,7- and 10,11-diols 41,42 and 114 as well as the tetraol 115 is described. High enantioselectivity and preferential addition to the 6,7-olefin was observed. The isomeric β-farnesene 2 showed a preference for reaction at the 10,11-position. The enantioselective synthesis of an apple aroma constituent, bicyclicacetal 17, is described. Asymmetric dihydroxylation of 6-methylhept-5-en-2-one 15 was carried out using the ligands (DHQ)2-PHAL and (DHQD)2-PHAL according to the method of Sharpless. Acid-catalysed cyclisation then afforded the required acetal 17 in high enantiomeric excess. Enantiomeric excesses were measured using chiral solvating agent 113 and/or synthesis of the corresponding Mosher ester derivatives, followed by 1H or 19F nmr.