Arabinogalactan proteins constitute a class of plant cell surface proteoglycans with widespread occurrence and suggested functions in various aspects of plant growth and development, including cell proliferation, expansion, marking, and death. Previous investigations of subcellular fractions from suspension-cultured cells of "Paul's Scarlet" rose (Rosa sp.) have revealed extensive structural similarity between some soluble arabinogalactan proteins from the cell wall space and some plasma membrane-associated arabinogalactan proteins, thus inspiring the present investigation of the mechanism through which these inherently water-soluble molecules are held on the plasma membrane. Several lines of evidence gained through a combination of methods including reversed-phase chromatography, treatment with phosphatidylinositol-specific phospholipase C, and chemical structural analysis now show that some rose arabinogalactan proteins carry a ceramide class glycosylphosphatidylinositol lipid anchor. The predominant form of the ceramide is composed of tetracosanoic acid and 4-hydroxysphinganine. Plasma membrane vesicles readily shed arabinogalactan proteins by an inherent mechanism that appears to involve a phospholipase. This finding has significance toward understanding the biosynthesis, localization, and function of arabinogalactan proteins and toward stimulating other studies that may expand the currently very short list of higher plant proteins found to carry such membrane lipid anchors.

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The effect of aluminum on the ordering and dynamics of lipid molecules in plasmalemma was studied by electron paramagnetic resonance via the membrane‐inserted reporter spin label molecules (methyl ester of 5‐doxyl‐hexadecanoic acid) in situ in mycelia of the ectomycorrhizal fungus Amanita muscaria. It was found, first, that the plasmalemma is structured into coexistent regions of lipids with different ordering and dynamics, and second, that aluminum stress is accompanied by the corresponding relative decrease of the proportion of the less ordered membrane domains. This effect is opposite to that found previously for Lactarius piperatus, where the membrane responded by an increase of the portion of the less ordered membrane domains. Such qualitatively diverse effects of aluminum at the membrane level is of interest as it coincides with the opposite effect on growth, i. e. inhibition of Amanita muscaria and stimulation of Lactarius piperatus.

tected in either type of domain in the lipid ordering of the Lateral heterogeneity in terms of co-existing domains with a distinct molecular organization is an area of increasing inter-bilayer as characterized by order parameter S. However, the relative population of domains in the bilayer exhibited est in membrane biology. The structural and dynamic aspects of the in-plane domain organization of lipids are becoming stronger temperature dependence. Typically, the relative proportion of disordered domains with less molecular order well documented, especially for model membrane systems. Potato (Solanum tuberosum L. cv. Desirée) callus cells and (smaller S) was larger in the membranes of callus cells roots of plantlets from stem node culture were doped with a compared to those of root cells, indicating higher fluidity throughout the measured temperature range (5-35°C). The spin-labeled analog of the methyl ester of palmitic acid bear-Arrhenius activation energies for rearrangement of lipid ing the paramagnetic nitroxide group at position C 5 of the acyl chain, which serves as a monitor of membrane fluidity of molecules within the bilayer were found to be higher for root tissue membranes, indicating the ability of root cells to oppose the region close to the polar phospholipid head groups of the bilayer. Model reconstruction of the line-shapes of the experi-actively any drastic changes of membrane structuring under mental spectra revealed the co-existence of two types of temperature stress. The distinctions in organization of lateral membrane domains with different ordering and dynamics of domains between the callus and root cell membranes may be correlated with differences in growth rate and metabolic activ-lipids in the membranes of both callus and root cells. With changes in temperature, relatively small differences were de-ity between these two types of tissue.

Translational diffusion of a fluorescent sterol probe was measured in the plasma membranes of protoplasts isolated from cortical cells of the primary root of maize seedlings. The apparent lateral diffusion coefficient was typically observed to be nearly insensitive to temperature, while the mobile fraction increased with increasing temperature. These fluorescence photobleaching recovery (FPR) measurements were compared with the electron paramagnetic resonance (EPR) spectra of the methyl ester of 13-doxyl palmitic acid in membranes of corn root tissue in situ. The complex spectra observed with this probe were analyzed as weighted sums of simpler spectra of various order parameters and rotational correlation times. The reconstituted spectra calculated from the model show that EPR also detects a mobile (less ordered, fluid) fraction, distinguished by the order parameter S = 0.1 to 0.2, which becomes more abundant as temperature increases and is qualitatively comparable to the mobile fraction determined by the FPR method. The observed results on the mobile fractions and the diffusion rates for translational (FPR) as well as rotational (EPR) motions are interpreted in terms of membrane organization, thus providing information on the population and structural patterns of the coexisting domains with a special emphasis on the response of the membrane to temperature changes.

Electron paramagnetic resonance (EPR) spectra were measured for the spin labelled phorbol-12,13-diester [5,6]PA bound to membranes of the particulate fraction of mouse brain tissue rich in PKC receptors. [5,6]PA is a bioactive derivative of the potent tumor promoter 12-O-tetradecanoylphorbol-13-acetate (TPA), carrying the spin label in a doxyl group in position 7' of the 12-O-tetradecanoyl residue. A mathematical model based on special algorithms (Griffith, O.H. and Jost, P.C. Spin labeling: theory and applications, 1976) allows a satisfactory reconstruction of the experimental spectrum. It reveals that in the experimental spectrum the signal from the [5,6]PA molecules bound non-specifically to the lipid bilayer of the membranes is superimposed by the signal of [5,6]PA molecules bound specifically, i.e. to the active site of PKC (approximately 10% of total EPR signal intensity). Moreover, interpretation of spectral parameters indicates that in [5,6]PA molecules bound specifically the tetradecanoyl chain exhibits a larger motional freedom compared to that in [5,6]PA bound non-specifically. These new findings are in accordance with views featured independently by two recent molecular models of interaction of PKC with cellular membranes (8,9).

The relatively small concentrations required for in vivo bioactivity of diterpene ester skin irritants and promoters (approximately 10 nmol per animal; approximately 10 nM in cell cultures) has discouraged studies of EPR spectra of bioactive, TPA-analogous, spin-labeled phorbol-12,13-diesters [(n,m)PA] bound to their membrane receptors, protein kinases C (PKC). To meet the requirements of present EPR spectrometers, particulate fraction from mouse brain containing at least 25 x 10(-12) mol of receptors/mg protein (PKC species) were employed together with certain (n,m)PA selected to give an optimal ratio of specific to non-specific binding. For selection and optimization of experimental conditions, a theoretical model was developed that considers all characteristic parameters of the system. By fitting the model calculations to the experimental data of competitive agonist displacement from the particulate fraction of tritium-labeled TPA, the dissociation constants Kd for four selected (n,m)PA used as antagonists were determined. Optimal experimental conditions are met by (5,6)PA and by (5,8)PA, in that for both compounds the relative amount of displaced (n,m)PA is in accordance with the predictions derived from the model. Moreover, the model turned out also to be reliable for samples containing either small or large amounts of membranes. To obtain an EPR spectrum of an agonist bound to brain particulate fraction, the (5,6)PA was used. It shows a broad EPR spectrum typical for an immobilized molecule. The spectrum changes if an excess of TPA is added to the system; the slight differences in shape are due to displacement of (5,6)PA from specific receptor sites by non-labeled TPA and show up as a decreased central peak amplitude. This is the first time that the agonist/receptor interaction of a diterpene ester type irritant and tumor promoter has been demonstrated by direct spectroscopic measurement.