Characterisation of sucrose synthase activity in the sugarcane culm

Schafer, Wolfgang Erich (Stellenbosch : University of Stellenbosch, 2004-04)

Dissertation (PhD)--University of Stellenbosch, 2004.

Thesis

ENGLISH ABSTRACT: This study had three main goals: 1. to investigate the occurrence on the protein level of sucrose synthase (SuSy) isoforms in sugarcane sink tissue, 2. to determine the kinetic properties of these isoforms, 3. to establish the tissue localisation of SuSy in the sugarcane culm The results are summarised below: Three SuSy isoforms were obtained from leaf roll tissue. The SuSyA and SuSyB isoforms differed in terms of charge characteristics, with SuSyA not binding to an anion exchange column that bound SuSyB and SuSyC under the same conditions. Both SuSyB and SuSyC isoforms were eluted at 180 mM KCl. The SuSyA and SuSyB isoforms were present during autumn, but during winter only the SuSyC isoform could be isolated. Even though they eluted at the same salt concentration, SuSyB and SuSyC were different isoforms, because they had different kinetic parameters, as well as different immunological properties. SuSyB and SuSyC could not have been mixtures of the same isoforms, since a polyclonal antiserum against SuSyB, which inactivates native SuSyB, did not inactivate SuSyC. All three isoforms had significantly different kinetic parameters, with the SuSyA isoform also having a much lower sucrose breakdown/synthesis ratio than the other two isoforms. Therefore, at least three SuSy isoforms occur in sugarcane leaf roll tissue on the protein level. The SuSyC isoform was subsequently kinetically characterised in detail. Data showed that the enzyme employs an ordered ternary complex mechanism, with UDP binding first and UDP-glucose dissociating last. These experimentally obtained kinetic parameters were then used to extend a kinetic model of sucrose accumulation. Data show that when the experimentally determined SuSy kineticparameters were entered into the model, a 40 % increase in sucrose concentration and 7 times reduction in fructose concentration resulted. These data illustrate the pronounced physiological effects that may result from the presence of different SuSy isoforms. SuSy protein localisation data, obtained by an immunohistochemical approach, indicated that SuSy protein was present in both storage parenchyma and vascular tissue of young, intermediate, and mature internodes. SuSy enzyme activity in different parts of the internodes was similar, except for internode 3, which had much higher activity in the bottom part of the internode, possibly because growth is faster here, hence a higher demand for sucrose cleavage exists here.

AFRIKAANSE OPSOMMING: Hierdie studie het ten doel gehad: 1. om die teenwoordigheid van sukrose sintase (SuSy) isovorme in suikkerriet swelgweefsel te ondersoek 2. om die kinetiese eienskappe van hierdie isovorme te ondersoek 3. om die weefsellokalisering van SuSy in die suikerrietstingel te bepaal Die resultate word hieronder opgesom: Drie SuSy isovorme is gevind in blaarrol weefsel. Die SuSyA en SuSyB isovorme het verskil in terme van ladingseienskappe, met SuSyA wat nie aan ‘n anioonuitruilkolom gebind het nie waaraan SuSyB en SuSyC wel onder dieselfde kondisies gebind het. Beide SuSyB en SuSyC isovorme is geëlueer van die kolom teen 180 mM KCl. Die SuSyA en SuSyB isovorme was teenwoordig gedurende herfs, maar in die winter was slegs SuSyC teenwoordig. Ten spyte van die feit dat SuSyB en SuSyC teen dieselfde soutkonsentrasie geëlueer is, het hulle verskillende isovorme verteenwoordig, aangesien hulle kinetiese en immunologiese eienskappe verskil het. SuSyB en SuSyC kon nie mengsels van dieselfde isovorme gewees het nie, want ‘n poliklonale antiserum teen SuSyB, wat SuSyB geïnaktiveer het, het nie SuSyC geïnaktiveer nie. Al drie isovorme het betekenisvol verskil wat kinetiese eienskappe betref, met die SuSyA isovorm wat ook ‘n baie laer sukrose afbraak/sintese verhouding gehad het as die ander twee isovorme. Daar is dus ten minste drie SuSy isovorme teenwoordig op die proteïen vlak in suikerriet blaarrol weefsel. Die in-detail kinetiese analise van die SuSyC isovorm het getoon dat die ensiem ‘n geordende drietallige kompleks meganisme het, met UDP wat eerste bind en UDP-glukose wat laaste dissosieer. Die eksperimenteel bepaalde kinetiese parameters is toe gebruik om ‘n kinetiese model van sukrose akkumulering uit tebrei. Data het getoon dat wanneer die generiese SuSy kinetiese parameters in die oorspronklike model vervang word met die eksperimenteel bepaalde waardes, die berekende sukrose konsentrasie met ongeveer 40 % toeneem, terwyl die fruktose konsentrasie ongeveer 7 keer afneem. Hierdie resultaat toon die groot fisiologiese effek wat die uitdrukking van verskillende SuSy isovorme op suikermetabolisme kan hê. Die SuSy proteïen lokaliseringsdata, wat met ‘n immunohistochemiese benadering verkry is, het aangedui dat SuSy in beide bergingsparenchiemselle sowel as vaatweefsel teenwoordig is in jong, intermediêre en volwasse internodes. SuSy ensiemaktiwiteit in verskillende dele van die internodes was soortgelyk, behalwe in internode 3, wat baie hoër aktiwiteit gehad het in die onderste deel van die internode as bo, moontlik weens vinniger groei in hierdie deel van die internode, wat afhanklik is van afbraakprodukte van sukrose.

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