Isolation of plasma membranes from leaf protoplasts : a search for difinitive markers

Jackson, Robert Thomas (1986) Isolation of plasma membranes from leaf protoplasts : a search for difinitive markers. University of Southampton, Doctoral Thesis.

Record type: Thesis (Doctoral)

Abstract

A search has been performed for definitive markers for the plasma membrane of leaf protoplasts to enable the positive identification of this membrane in isolated cell fractions. Preliminary investigations with maize leaf protoplasts demonstrated that a plasma membrane-enriched fraction could not be obtained by a conventional method, involving fractionation of a protoplast lysate by differential and sucrose gradient centrifugation. The yield of protoplasts from the leaves of maize was low. A much higher yield was obtained from the leaves of barley. Attempts were made to label specifically the surface of the plasma membrane of barley leaf protoplasts that had been aged overnight. Iodination, using Na¹²⁵I and the solid phase reagent Iodogen, was unsuccessful because of penetration of the membrane by the Na¹²⁵I and resultant indiscriminate labelling. When protoplasts were stained with the electron dense and luminescent lanthanides, terbium and europium, these ions did not appear to penetrate the plasma membrane. Similar results were obtained when whole tissue segments were stained. A spectrofluorometric method was developed for the assay of trace amounts of terbium in membrane fractions derived from prelabelled protoplasts. Fluorescence measurements were subject to interference from a number of sources. Europium-152 labelling studies were performed. The lanthanide dissociated from the membrane at protoplast lysis and redistributed, resulting in indiscriminate labelling. Protoplasts were labelled with a β-cyclodextrin-dansyl chloride complex and 4,4'-diisothiocyano-2,2'-disulphonic acid stilbene (DIDS). A low level of incorporation of these fluorescent labels into protoplast membrane fractions was observed. Experiments performed with the diazonium salt of the auxin affinity label [^3H]Chloramben (2,5-dichloro-3-aminobenzoic acid) suggest that it could be used as a surface label for the plasma membrane in intact barley leaf protoplasts. The label was unable to penetrate and binding was permanent. The binding of a structurally similar molecule, diazo-p-amino[^14C]benzoic acid, was less specific. Various cell fractionation procedures were employed in order to separate the labelled (presumptive) plasma membranes. Optimal protoplasts lysis conditions were selected. A 500g supernatant fraction, derived from the lysate, was fractionated on gradients of sucrose, Nycodenz and Percoll. Membrane fractions were also partitioned in aqueous polymer (dextran-polyethylene glycol) two-phase systems. By these separation procedures it was not possible to obtain a plasma membran fraction of sufficient purity to enable further characterization. (D72218/87)

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