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61.
Monoclonal antibodies generated by immunization with a plasma-membrane preparation from suspension-cultured cells of Nicotiana glutinosa L. were used in combination with fluoresceinor rhodamine-labeled goat anti-mouse immunoglobulins to identify heterokaryons in protoplast fusion procedures. Antibody labeling did not inhibit callus formation nor plantlet regeneration. The antibodies are non-invasive and surface labeling provides clear optical discrimination of true heterokaryons from unfused aggregates as well as from parental protoplasts and homokaryons. Labeling is stable throughout fusion and hence by pre-labeling parental protoplast populations the strategy is both versatile and of general applicability. 相似文献
62.
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. 相似文献
63.
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. 相似文献
64.
S. K. Podder A. Chakraborti K. Vijayalakshmi P. Lalit Kumar Singh 《Journal of biosciences》1987,11(1-4):495-502
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, GMn. The Presence of 5% GMn 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% GMn 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. 相似文献
65.
Lawrence G. Palmer 《The Journal of membrane biology》1987,96(2):97-106
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? 相似文献
66.
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. 相似文献
67.
Contribution of solvent drag through intercellular junctions to absorption of nutrients by the small intestine of the rat 总被引:19,自引:0,他引:19
Summary The lumen of the small intestine in anesthetized rats was recirculated with 50 ml perfusion fluid containing normal salts, 25mm glucose and low concentrations of hydrophilic solutes ranging in size from creatinine (mol wt 113) to Inulin (mol wt 5500). Ferrocyanide, a nontoxic, quadrupally charged anion was not absorbed; it could therefore be used as an osmotically active solute with reflection coefficient of 1.0 to adjust rates of fluid absorption,J
v
, and to measure the coefficient of osmotic flow,L
p
. The clearances from the perfusion fluid of all other test solutes were approximately proportional toJ
v
. FromL
p
and rates of clearances as a function ofJ
v
and molecular size we estimate (a) the fraction of fluid absorption which passes paracellularly (approx. 50%), (b) coefficients of solvent drag of various solutes within intercellular junctions, (c) the equivalent pore radius of intercellular junctions (50 Å) and their cross sectional area per unit path length (4.3 cm per cm length of intestine). Glucose absorption also varied as a function ofJ
v
. From this relationship and the clearances of inert markers we calculate the rate of active transport of glucose, the amount of glucose carried paracellularly by solvent drag or back-diffusion at any givenJ
v
and luminal glucose concentration and the concentration of glucose in the absorbate. The results indicate that solvent drag through paracellular channels is the principal route for intestinal transport of glucose or amino acids at physiological rates of fluid absorption and concentration. In the absence of luminal glucose the rate of fluid absorption and the clearances of all inert hydrophilic solutes were greatly reduced. It is proposed that Na-coupled transport of organic solutes from lumen to intercellular spaces provides the principal osmotic force for fluid absorption and triggers widening of intercellular junctions, thus promoting bulk absorption of nutrients by solvent drag. Further evidence for regulation of channel width is provided in accompanying papers on changes in electrical impedance and ultrastructure of junctions during Na-coupled solute transport. 相似文献
68.
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. 相似文献
69.
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. 相似文献
70.
Summary It has previously been shown by Macey and Farmer (Biochim. Biophys. Acta
211:104–106, 1970) that phloretin inhibits urea transport across the human red cell membrane yet has no effect on water transport. Jennings and Solomon (J. Gen. Physiol.
67:381–397, 1976) have shown that there are separate lipid and protein binding sites for phloretin on the red cell membrane. We have now found that urea transport is inhibited by phloretin binding to the lipids with aK
1 of 25±8 m in reason-able agreement with theK
D
of 54±5 m for lipid binding. These experiments show that lipid/protein interactions can alter the conformational state of the urea transport protein. Phloretin binding to the protein site also modulates red cell urea transport, but the modulation is opposed by the specific stilbene anion transport inhibitor, DIDS (4,4-diisothiocyano-2,2-stilbene disulfonate), suggesting a linkage between the urea transport protein and band 3. Neither the lipid nor the protein phloretin binding site has any significant effect on water transport. Water transport is, however, inhibited by up to 30% in a pH-dependent manner by DIDS binding, which suggests that the DIDS/band 3 complex can modulate water transport. 相似文献