Analysis of labeling of plant protoplast surface by fluorophore-conjugated lectins

Summary Fluorescein or rhodamine conjugates of seventeen different lectins were tested for their ability to label the plasma membrane of live plant protoplasts. During the investigation, a strong effect of calcium was observed on the binding of several lectins to protoplasts derived from suspension cultured rose cells (Rosa sp. Paul's Scarlet). The binding of these lectins was increased by elevating the calcium concentration from 1 to 10 mM in the buffer. Other divalent cations had variable, but similar, effects on lectin binding. The mechanism of this effect appeared to involve the protoplast surface rather than the lectins. Although the cell wall-degrading enzymes used to isolate protoplasts had generally no effect on lectin binding, one clear exception was observed. Binding ofArachis hypogaea agglutinin was markedly reduced on protoplasts isolated with Driselase as compared to protoplasts isolated with a combination of Cellulysin and Pectolyase Y-23. Although most of the lectins that labeled protoplasts derived from cultured rose cells or from corn root cortex (Zea mays L. WF9 × Mo17) had specificities for galactose or N-acetylgalactosamine, some differences in protoplast labeling between lectins of the same saccharide specificity were observed. Two different analyses of the interaction betweenRicinus communis agglutinin and rose protoplasts showed that binding was cooperative with an apparent association constant of 7.2 × 10⁵M^–1 or 9.8 × 10⁵M^–1 with a maximum of approximately 10⁸ lectin molecules bound per protoplast. Treatment of protoplasts with glycosidases which hydrolyze either N- or O-glycosidic linkages of glycoproteins slightly enhanced labeling of protoplasts byRicinus communis agglutinin. Interpretation of these results are discussed.Abbreviations MPR medium, minimal organic medium (Nothnagel andLyon 1986) - APA Abrus precatorius agglutinin - CSA Cytisus sessilifolius agglutinin - ECA Erythrina cristagalli agglutinin - GS-I Griffonia simplicifolia agglutinin - LcH Lens culinarus agglutinin - PNA Arachis hypogaea agglutinin - SBA Glycine max agglutinin - VAA Viscum album agglutinin - VFA Vicia faba agglutinin - WGA Triticum vulgaris agglutinin - Con A Canavalia ensiformis agglutinin - HPA Helix pomatia agglutinin - TPA Tetragonolobus purpureas agglutinin - RCA Ricinus communis agglutinin - DBA Dolichos biflorus agglutinin - SJA Sophora japonica agglutinin - BPA Bauhinia purpurea agglutinin - FITC fluorescein isothiocyanate - Ga1NAc N-acetylgalactosamine - FDA fluorescein diacetate - 2-O-Me-D-Fuc 2-O-methyl-D-fucose Parts of the work presented here are also submitted in partial fulfillment of requirements for the Ph.D. degree.     

Further evidence from sectioned material in support of the existence of a linear terminal complex in cellulose synthesis

Summary Transmembrane linear terminal complexes considered to be involved in the synthesis of cellulose microfibrils have been described in the plasma membrane ofBoergesenia forbesii. Evidence for the existence of these structures has been obtained almost exlusively using the freeze etching technique. In the present study an attempt has been made to complete these studies using conventional fixation, staining, and sectioning procedures. In developing cells ofBoergesenia forbesii, strongly stained structures traversing the plasma membrane and averaging 598.9 nm ± 171.3 nm in length, 28.7 nm ± 4.2 nm in width, and 35.2 nm ± 6.6 nm in depth have been demonstrated. These structures are considered to be linear terminal complexes. At their distal (cell wall) surface, they appear to be closely associated with cellulose microfibrils. At the proximal (cytoplasmic) surface, they are associated with microtubules and polysomes. A model of the possible interrelation of the terminal complexes and microtubules leading to the generation of cell wall microfibrils is proposed.     

A method for the estimation of gene flow parameters from a population structure caused by restricted gene flow and genetic drift

Summary A method has been developed which enables the estimation of the plant gene flow parameters _p (pollen dispersal), _s (seed dispersal) and t (outcrossing rate) from a selection-free continuously structured population in equilibrium. The method uses Wright's F-coefficients and introduces a new F-function which describes the genetic similarity as a function of the spatial distance. The method has been elaborated for wind pollinated plant species but can be modified for insect pollination and for animal species. In practice allozymes will provide for the necessary neutral genetic variation. The more loci used and the more intermediate the gene frequencies, the more reliable the results. For the estimation of _p and t together (when the outcrossing rate is not known) at least two chromosomally unlinked loci are required. The method for estimating _s depends on whether the plant species is annual or perennial. The mechanism of selfing has been analysed by the explanation of the value of t by three components: population density (d), pollen flow (_p) and relative fertilization potential of own pollen (Z). The concepts of neighbourhood size and isolation by distance, developed by Wright, who used a single gene flow parameter , have been extended to the situation which is realistic for seed plants, using all three parameters _p, _s and t. When _p is large with respect to _s, _s largely determines the value of the neighbourhood size, whereas _p is the most dominating factor in isolation by distance. The use of local effective population size and mean gene transport per generation instead of neighbourhood size and neighbourhood area, respectively, is proposed to avoid confusion. Computer simulations have been carried out to check the validity and the reliability of the method. Populations of 200 plants, using two or three loci with intermediate allele frequencies, gave good results in the calculation of _p with known value of t and of _s and N_e. With unknown t, especially with lower values of t, larger populations of at least 1,000 plants are necessary to obtain reasonably accurate results for _p and mean gene transport per generation M.Grassland Species Research Group Publication No. 81     

Reactivity of glycoconjugate in membrane system II: Can a neutral glycolipid function as lectin receptor in the presence of gangliosides in plasma membrane?

To examine how surface Potential controls the reactivity of glycoconjugates at cell surface, the interaction of galactose-sPecific lectinse.g. peanut agglutinin,Ricinus cummunis agglutinin with liPosomes bearing asialo GM₁ were studied in the Presence of varying amount of ganglioside mixture, GM_n. The Presence of 5% GM_n causes comPlete slowing down of PreciPitin reaction and thereby make carbohydrate moiety of asialo GM₁ comPletely inaccessiblei.e. ‘cryPtic’. In contrast the Presence of 1–2% GM_n enhances the aPParent rate and amPlitude of the PreciPitin reaction as surface Potential becomes more negative. The relevance of the findings has been discussed in relation to the exPression and involvement of the cell-surface sialic acid residues during develoPment and differentiation.     

Ion selectivity of epithelial Na channels

Epithelial Na channels are apparently pore-forming membrane proteins which conduct Na much better than any other biologically abundant ion. The conductance to Na can be 100 to 1000 times higher than that to K. The only other ions that can readily get through this channel are protons and Li. Small organic cations cannot pass through the channel, and water may also be impermeant. The selectivity properties of epithelial Na channels appear to be determined by at least three factors: A high field-strength anionic site, most likely a carboxyl residue of glutamic or aspartic acid residues on the channel protein, probably accounts for the high conductance through these channels of Na and Li and to the low conductance of K, Rb and Cs. A restriction in the size of the pore at its narrowest point probably accounts for the low conductance of organic cations as well as the possible exclusion of water molecules. The outer mouth of the channel appears to be negatively charged and may control access to the region of highest selectivity and may serve as a preliminary selectivity filter, attracting cations over anions. These conclusions are illustrated by the cartoon of the channel in Fig. 3. This picture is obviously both fanciful and simplified, but its general points will hopefully be testable. It leaves open a number of important questions, including: does amiloride block the channel by binding within the outer mouth? what does the inner mouth of the channel look like, and does this part of the channel contribute to selectivity? and what, if any, are the interactions between the features of the channel that impart selectivity and those that control the regulation of the channel by hormonal and other factors?     

Demonstration and characterization of Ca2+ channel in tonoplast-free cells ofNitellopsis obtusa

Summary The presence of a Ca²⁺ channel in the plasmalemma of tonoplast-freeNitellopsis obtusa cells was demonstrated and its characteristics were studied using current- and voltage-clamp techniques. A long-lasting inward membrane current (I _m ), recorded using a step voltage clamp, consisted of a single component without time-dependent inactivation. Increasing either [Ca²⁺] _o or [Cl^–] _o 1) enhanced the maximum amplitude of inwardI _m ((I _m ) _p ) and 2) shifted the peak voltage ((V _m ) _p ) at(I _m ) _p to more positive values under ramp-shaped voltage clamping and 3) depolarized the peak value of action potentials. This behavior is consistent with predictions based on the Nernst equation for Ca²⁺ but not for Cl^–. DIDS (4,4-diisothiocyano-2,2-disulfonic acid stilbene) did not suppress(I _m ) _p in tonoplast-free cells, in contrast with its effect on normal cells. La³⁺ and nifedipine blocked(I _m ) _p irreversibly. On the other hand, Ca²⁺ channel agonist, BAY K 8644 irreversibly enhanced(I _m ) _p . Both Sr²⁺ influx and K⁺ efflux increased upon excitation. The charge carried by Sr²⁺ influx was compensated for by K⁺ efflux. It is concluded that only the Ca²⁺ channel is activated during plasmalemma excitation in tonoplast-free cells. In terms of the magnitude of(I _m ) _p , Sr²⁺ could replace Ca²⁺, but Mn²⁺, Mg²⁺ and Ba²⁺ could not. External pH affected(I _m ) _p and the membrane conductance (g _m ) at(I _m ) _p ((g _m ) _p ). Increasing the external ionic strength caused increases in both(I _m ) _p and(g _m ) _p , and shifted(V _m ) _p to positive values. At the same time, Sr²⁺ influx increased. Thus Ca²⁺ channel activation seems to be enhanced by increasing external ionic strength. The possible involvement of surface potential is discussed.     

Voltage gating in VDAC is markedly inhibited by micromolar quantities of aluminum

Summary The mitochondrial outer membrane contains voltagegated channels called VDAC that are responsible for the flux of metabolic substrates and metal ions across this membrane. The addition of micromolar quantities of aluminum chloride to phospholipid membranes containing VDAC channels greatly inhibits the voltage dependence of the channels' permeability. The channels remain in their high conducting (open) state even at high membrane potentials. An analysis of the change in the voltage-dependence parameters revealed that the steepness of the voltage dependence decreased while the voltage needed to close half the channels increased. The energy difference between the open and closed states in the absence of an applied potential did not change. Therefore, the results are consistent with aluminum neutralizing the voltage sensor of the channel. pH shift experiments showed that positively charged aluminum species in solution were not involved. The active form was identified as being either (or both) the aluminum hydroxide or the tetrahydroxoaluminate form. Both of these could reasonably be expected to neutralize a positively charged voltage sensor. Aluminum had no detectable effect of either single-channel conductance or selectivity, indicating that the sensor is probably not located in the channel proper and is distinct from the selectivity filter.     

The Na+-independentd-glucose transporter in the enterocyte basolateral membrane: Orientation and cytochalasin B binding characteristics

Summary Phloridzin-insensitive, Na⁺-independentd-glucose uptake into isolated small intestinal epithelial cells was shown to be only partially inhibited by trypsin treatment (maximum 20%). In contrast, chymotrypsin almost completely abolished hexose transport. Basolateral membrane vesicles prepared from rat small intestine by a Percoll^® gradient procedure showed almost identical susceptibility to treatment by these proteolytic enzymes, indicating that the vesicles are predominantly oriented outside-out. These vesicles with a known orientation were employed to investigate the kinetics of transport in both directions across the membrane. Uptake data (i.e. movement into the cell) showed aK _t of 48mm and aV _max of 1.14 nmol glucose/mg membrane protein/sec. Efflux data (exit from the cell) showed a lowerK _t of 23mm and aV _max of 0.20 nmol glucose/mg protein/sec.d-glucose uptake into these vesicles was found to be sodium independent and could be inhibited by cytochalasin B. TheK _t for cytochalasin B as an inhibitor of glucose transport was 0.11 m and theK _D for binding to the carrier was 0.08 m.d-glucose-sensitive binding of cytochalasin B to the membrane preparation was maximized withl- andd-glucose concentrations of 1.25m. Scatchard plots of the binding data indicated that these membranes have a binding site density of 8.3 pmol/mg membrane protein. These results indicate that the Na⁺-independent glucose transporter in the intestinal basolateral membrane is functionally and chemically asymmetric. There is an outward-facing chymotrypsin-sensitive site, and theK _t for efflux from the cell is smaller than that for entry. These characteristics would tend to favor movement of glucose from the cell towards the bloodstream.     

Effects of membrane physical parameters on hematoporphyrin-derivative binding to liposomes: A spectroscopic study

Summary Physical parameters of membrane bilayers were studied for their effect on the binding of hematoporphyrin derivative (Hpd), which is used as a sensitizer in photodynamic therapy of cancerous tissues. The purpose of this study was to clarify which parameters were relevant, under physiological conditions, to the selectivity of Hpd binding to cancer cells. Fluorescence spectroscopy was used to measure the relative partitioning of the dye between the lipid and aqueous media. Increasing the microviscosity of the liposomes' membranes by various bilayer additives results in a strong reduction of Hpd binding, to an extent independent of the specific additive. The effect of temperature near the physiological value as well as the effect of cross membrane potential are small. Surface potential does not affect the binding constant, indicating that the binding species does not carry a net electric charge.     

l-Alanine uptake in brush border membrane vesicles from the gill of a marine bivalve

Summary Brush border membrane vesicles were prepared from mussel gills using differential and sucrose density gradient centrifugation. These vesicles contained both the maximal Na⁺-dependent alanine transport activity found in the gradient and the maximal activities of -glutamyl transpeptidase and alkaline phosphatase. Electron micrographs showed closed vesicles of approximately 0.1–0.5 m diameter. Transport experiments using these vesicles demonstrated a transient 18-fold overshoot in intravesicular alanine concentration in the presence of an inwardly directed Na⁺ gradient, but not under Na⁺ equilibrium conditions. A reduced overshoot (10-fold) was seen with an inwardly directed K⁺ gradient. Further studies revealed a broad cation selectivity, with preference for Na⁺, which was characteristic of alanine transport but not glucose transport in these membranes. The apparent amino acid specificity of the uptake pathway(s) was similar to that of intact gills and supported the idea of at least four separate pathways for amino acid transport in mussel gill brush border membranes. The apparent Michaelis constant for alanine uptake was approximately 7m, consistent with values forK _t determined with intact tissue.