Dolamore, Barbara A.2010-05-262010-05-261988https://hdl.handle.net/10182/1941In order to investigate the biochemical nature of the plasma membrane (PM) of plants, preparations of high purity are necessary. In this study methods have been investigated and developed to achieve this aim. The method of tissue homogenization affects the level of contamination in subsequent plasma membrane-enriched preparations. The use of a Braun homogenizer, modified to allow the attachment of replaceable Schick razor blades, was compared to the mortar and pestle, and shown to be superior. Its use resulted in substantially less organelle damage, gave significantly higher yields and considerable savings in time. The controversy surrounding the specificity of the PTA staining procedure for PM has been clarified. It was found that barley (but not wheat and cauliflower) PM was specifically stained, both in intact tissue and protoplasts. Specificity was retained after homogenization, or protoplast lysis. PTA staining was found to be a reliable method to determine relative purities of PM preparations from barley leaves. Three methods for the isolation of PM from the leaves of eight day-old barley and wheat have been compared. One method utilized differential centrifugation to prepare a general microsomal membrane pellet. These membranes were then fractionated using sucrose buoyant density gradient techniques. The other methods involved partitioning the microsomal membranes in either of two dextran-polyethylene glycol two-phase systems. The composition of the established system is 6.2% (w /w) Dextrap T500, 6.2% (w /w) polyethylene glycol 3350, 0.3M sucrose, 3mM KCl, 5mM potassium phosphate, pH 7.8. The performance of this phase mixture was compared to that of a new system composed of 6.2% (w /w) Dextran T500, 6.2% (w/w) polyethylene glycol 3350, 0.25 M sucrose, 30mM NaCl, 10mM potassium phosphate, pH 7.8. Results of this study demonstrated that phase partitioning is superior to sucrose gradient separation for the isolation of PM from microsomal membranes. Sucrose gradient separated membranes contained only 32% PTA stained vesicles, as revealed by morphometric analysis. Thylakoid membranes (as measured by chlorophyll), mitochondrial fragments (cytochrome C oxidase) and endoplasmic reticulum (NADPH-cytochrome C reductase) were serious contaminants. The membranes separated using both phase partitioning systems contained 66% PTA stained vesicles. Chlorophyll was not detected in the PM preparation isolated by the new phase system, and in the established phase system it was present only in very low quantities. The levels of mitochondrial membranes and endoplasmic reticulum were also very low in both phase systems. The new phase system was found to be significantly more rapid and much more cost effective than the established system, yielding PM of a high degree of purity. Protoplasts were used as a source of PM as cell contents could be liberated gently, and this was found to result in a cleaner microsomal membrane preparation. This may reduce contamination of the plasma membrane-enriched fraction. The yield of wheat protoplasts was increased 7.8 fold by stripping off the abaxial epidermis rather than dicing the leaves, prior to incubation with cellulysin (2%) and macerozyme (0.5%). The inclusion of hemicellulase did not increase the yield. To assist PM identification in the various membrane preparations, several surface labelling techniques were investigated. Fluorescein isothiocyanate (FITC), was successfully used to label the PM of viable protoplasts. The effectiveness of FITC was dependent on the pH of the incubation medium. A decrease in pH from 8.5 to 6.5 resulted in reduced specificity of FITC for the PM and intense labelling of cellular contents. FITC was also conjugated to concanavalin A, and it selectively bound to mannosll and glucosyl residues on the PM. High levels of cellular autofluorescence prevented the use of FITC as an effective PM marker in membrane preparations, although it has much potential as a tool in plasma membrane surface studies. A technique reported to radiolabel protoplast surfaces with tritium, involving oxidization of surface galactose-containing molecules with galactose oxidase and then reduction with NaB[³H]₄, was investigated. Electron microscopic autoradiography revealed that specific surface labelling had not occurred. Only 38% of the total label was found on the surface of the protoplast. The new phase partitioning system, designed originally for purification of PM from microsomal membranes from intact barley and wheat leaves, was also effective in obtaining a highly enriched plasma membrane preparation from barley and wheat protoplasts.enhttps://researcharchive.lincoln.ac.nz/pages/rightsplasma membraneprotoplastsbarleywheatelectron microscopyphase partitionautoradiographysurface labelisolationThesisDigital thesis can be viewed by current staff and students of Lincoln University only. If you are the author of this item, please contact us if you wish to discuss making the full text publicly available.ANZSRC::0601 Biochemistry and Cell BiologyANZSRC::060702 Plant Cell and Molecular BiologyANZSRC::060705 Plant PhysiologyQ112846656