Characterization and quantitation of concanavalin a binding by plasma membrane enriched fractions from soybean root

The binding of concanavalin A (Con A) to soybean root membranes in plasma membrane enriched fractions (recovered from the 34/45% interface of simplified discontinuous sucrose density gradients) was studied using a radiochemical assay employing tritiated (³H)-Con A. The effect of lectin concentration, time, and membrane protein concentration on the specific binding of ³H-Con A by the membranes was evaluated. Kinetic analyses showed that Con A will react with membranes in that fraction in a characteristic and predictable manner. The parameters for an optimal and standard binding assay were established. Maximal binding occurred with Con A concentrations in the range of 8 to 16% of the total membrane protein with incubation times greater than 40 min at 22 C. Approximately 10¹⁵ molecules of ³H-Con A were bound per microgram of membrane protein at saturation. Binding was reversible. Greater than 92% of the total Con A bound at saturation was released by addition of α-methyl mannoside.     

Plasma Membrane Glycoproteins of Ciona intestinalis Spermatozoa that Interact with the Egg1

In the ascidian Ciona intestinalis the species-specific interaction between the spermatozoon and the egg occurs between the vitelline coat (VC) of the egg and the plasma membrane of the apical part of the head of the spermatozoa. Concanavalin A (Con A)-binding sites are present on this area of the sperm surface. We used Con A to identify and isolate the spermatozoon plasma membrane components that may be involved in the interaction with the VC. These glycoproteins have been identified on SDS-PAGE of a sperm membrane fraction (SMF) enriched with the extermal proteins, after incubation of the gel with ³H-Con A. Affinity chromatography on Con A-agarose has been used for the purification of sperm plasma membrane proteins with and affinity for the lectin. The biological activity of the Con A-retained fraction was determined with binding and fertilization assays.     

Distribution of electric charges and concanavalin A binding sites on autophagic vacuoles and lysosomes in mouse hepatocytes

The distributions of electric charges and Concanavalin A binding sites in autophagic vacuoles and lysosomes in mouse hepatocytes were studied by utilizing a frozen ultrathin section labeling method with cationized ferritin (CF) or anionized ferritin and ferritin-conjugated Concanavalin A (Con A-F) as visual probes. Our observations revealed that the inner surface of the autophagic vacuole membrane has more anionic sites (CF binding) than other organelle membranes. This suggests that if the limiting membranes of autophagic vacuoles originate from preexisting membranes, such membranes must undergo structural and compositional alternation during the formation of the autophagic vacuoles. In contrast to CF, Con A-F showed no distinct binding to the membranes of autophagic vacuoles, but the contents of vacuoles displayed varying Con A-F binding, depending on the stage of the autophagic process. Increased binding was seen in more mature autophagic vacuoles. Since lysosomes showed a preferential accumulation of Con A-F particles, molecules with Con A-F binding sites in autophagic vacuoles may be of lysosomal origin. Con A-F distribution varied from lysosome to lysosome in the same cell, indicating heterogeneity of lysosomal contents. These results suggest that ferritin-conjugated lectin labeling methods applied to frozen, ultrathin section are a useful new approach in analyzing the natural history of autophagic vacuoles and the heterogeneity of lysosomes.     

The distribution of Concanavalin A binding sites on the intestinal epithelium of the nematodes Ascans suum and Parascaris equorum

Summary Receptors for Concanavalin A (Con A), were localized on the intestinal epithelium of the nematodes Ascaris suum and Parascaris equorum. Fixed tissue incubated in ³H-Con A showed labeling of the microvilli surface and basal membrane. Using Con A coupled with peroxidase, the tips of the microvilli of Ascaris suum and the tips and lateral surfaces of Parascaris equorum were stained. The basal membrane of both species was also labeled. No labeling was observed on control tissue incubated without Con A or on tissue incubated with Con A to which -methyl-D-mannoside was added.     

Interaction of concanavalin A with differentiated and undifferentiated murine neuroblastoma cells.

Tritium-labeled acetyl-concanavalin A (³H-Con A) was used to study its kinetics of binding at 0 °C to murine neuroblastoma cells (clone neuro 2-A) grown in the differentiated (monolayer) and Undifferentiated (spinner) states. The binding of ³H-Con A to both cell types gives sigmoidal saturation curves, suggesting positively cooperative binding of the lectin. The Hill coefficient is 1.75 for differentiated and 1.36 for Undifferentiated cells. The maximal number of ³H-Con A molecules bound per cell is 2.3 × 10⁷ and 3.4 × 10⁷ for differentiated and Undifferentiated cells, respectively, and the apparent rate constants for formation of the lectin-cell complex are 6.13 × 10², m^?1, s^?1 for the Undifferentiated and 6.68 × 10², m^?1, s^?1 for the differentiated cells. The lectin bound to spinner cells does not dissociate spontaneously to any measurable extent over a 60-min period at 0 or 37 °C, but the lectin-cell complex dissociates rapidly after addition of α-methyl-d-mannopyranoside. At 37 °C, this sugar causes virtually complete dissociation of the cell-lectin complex within 30 min. The ³H-Con A dissociated from spinner cells is indistinguishable from the original ³H-Con A by sodium dodecyl sulfate-urea polyacrylamide gel electrophoresis, gel filtration through Bio-Gels P-30 and P-100, and specific binding to spinner cells. Both the original and the dissociated ³H-Con A are dimers at pH 7.4. The sugar-induced dissociation of the labeled lectin from spinner cells is not accompanied by shedding or inactivation of the lectin binding sites of the cell surface.     

Production of monoclonal antibodies against a cell surface concanavalin a binding glycoprotein

Concanavalin A-binding (Con A)-binding cell surface glycoproteins were isolated, via Con A-affinity chromatography, from Triton X-100-solubilized Chinese hamster ovary (CHO) cell plasma membranes. The Con A binding glycoproteins isolated in this manner displayed a significantly different profile on sodium dodecyl sulfate-polyacrylamide gels than did the Tritonsoluble surface components, which were not retarded by the Con A-Sepharose column. [¹²⁵I]-Con A overlays of the pooled column fractions displayed on sodium dodecyl sulfate-polyacrylamide gel electro-phoresis (SDS-PAGE) demonstrated that there were virtually no Con A receptors associated with the unretarded peak released by the Con A-Sepharose column, whereas the material which was bound and specifically eluted from the Con A-Sepharose column with the sugar hapten α-methyl-D-mannopyranoside contained at least 15 prominent bands which bound [¹²⁵I]-Con A. In order to produce monoclonal antibodies against various cell surface Con A receptors, Balb/c mice were immunized with the pooled Con A receptor fraction. Following immunization spleens were excised from the animals and single spleen cell suspensions were fused with mouse myeloma P3/X63-Ag8 cells. Numerous hybridoma clones were subsequently picked on the basis of their ability to secrete antibody which could bind to both live and glutaraldehyde-fixed CHO cells as well as to the Triton-soluble fraction isolated from the CHO plasma membrane fraction. Antibody from two of these clones was able to precipitate a single [¹²⁵I]-labeled CHO surface component of ～265,000 daltons.     

Use of lectins as probes for analyzing embryonic induction

Summary Lectins were used as probes to investigate the mechanism of embryonic induction. Concanavalin (Con A) and gorse agglutinin out of 7 species of lectins tested were found to have strong neural-inducing effect on the presumptive ectoderm of newt gastrulae. Their effects were abolished by the addition of -methyl-D-mannoside and -L-fucose, respectively. Succinyl-Con A had a weak inducing activity in comparison to Con A. Autoradiography of³H-Con A-treated explants revealed that Con A bound to the inner surface, but not to the outer surface of ectoderm and was successively incorporated into cytoplasm.³H-Thymidine incorporation was lower in the first half and higher in the second half of the 60 h cultivation period in Con A-treated explants as compared to controls.Con A-Sepharose had a strong inductive effect. This suggests that neural induction is caused through Con A binding to the plasma membrane, but not through incorporation into the cytoplasm of the ectoderm cells.     

The thermodynamic parameters of the interaction of concanavalin A with glycosyl-free liposomes: a microcalorimetric study

The nonspecific interaction of the mitogenic lectin Concanavalin A (Con A) with glycosyl-free liposomes of various composition has been investigated by microcalorimetric titration measurements. The results obtained show the following features of main interest: (1) the affinity constants (K_a) of the interaction of Con A with liposomal bilayers are in the order of magnitude 10⁵–10⁶M^?1. The reaction enthalpies (ΔH) are positive, and small (approximately 0.1 KJ mol^?1 lipid), compared to the free energy terms (?ΔG = 30–40 KJ mol^?1 lipid). All lectin–lipid interactions are strongly entropy-controlled (ΔH/TΔS < 1.0). These thermodynamic features are characteristic for hydrophobic interaction processes. (2) The liposomal head-group charge does not significantly affect the lipid-affinity of Con A. Electrostatic forces thus appear to play a minor role in lectin–lipid interactions. (3) The lipid affinity of Con A is sensitive to the fluidity of the liposomal bilayers, increasing with increasing fluidity. Below the gel to liquid-crystal phase transition temperature, the lectin binding to liposomal bilayers is inhibited. (4) The binding isotherms, corresponding to the interaction of Con A with liposomes, composed of tightly packed, saturated phospholipids, exhibit pronounced positive cooperativity. This phenomenon is absent in the binding curves, corresponding to the interaction of Con A with more fluid liposomal bilayers. (5) The Con A specific inhibitor α-D -methylmannopyranoside (50 mM) drastically increases the molar reaction enthalpy. The K_a term is significantly reduced in presence of the inhibitor sugar. Urea induces analogous changes in the thermodynamic parameters of the lectin–lipid interaction. The effects of α-D -methylmannopyranoside are thus not Con A specific, but are attributable to solvent effects. (6) It was shown that the binding of one Con A molecule affects a large number (approximately 1000) of phospholipid molecules in the liposomal bilayer. (7) The affinity constants (K_a) of the interaction of Con A with glycosyl-free lipids are smaller by a factor of approximately 10, compared to the K_a terms, reported for Con A binding to biological membranes. The presence of glycosidic receptor groups thus controls the specificity of lectin–membrane interactions, whereas the nonspecific lectin–lipid interactions appear to represent the main driving force for the strong attachment of the lectin to membrane surfaces.     

Gonadotropin and prostaglandins binding sites in nuclei of bovine corpora lutea

Highly purified nuclei isolated from bovine corpora lutea showed marked enrichment of NAD pyrophosphorylase, a marker for this organelle. Rough endoplasmic reticulum and lysosomal markers were undetectable, whereas plasma membrane and Golgi markers were detectable but not enriched in nuclei. These highly purified nuclei exhibited specific binding with ¹²⁵I-labeled human choriogonadotropin, [³H]prostaglandin E₁ and [³H]prostaglandin F_2α. However, these bindings were only 15.4% (human choriogonadotropin), 7.9% (prostaglandin E₁) and 8.9% (prostaglandin F_2α) of the plasma membrane binding observed under the same conditions. Washing of nuclei and plasma membranes twice with buffer containing 0.1% Triton X-100 resulted in gonadotropin and prostaglandin F_2α binding site and 5′-nucleotidase (EC 3.1.3.5) losses from nuclei that were different from those observed for plasma membranes. More importantly, the washed nuclei exhibited 44% (human choriogonadotropin), 21–26% (prostaglandins) of original specific binding despite virtual disappearance of 5′-nucleotidase activity. The nuclear membranes isolated from nuclei, specifically bound ¹²⁵I-labeled human choriogonadotropin and [³H]prostaglandin F_2α to the same extent or significantly more ([³H]prostaglandin E₁, P < 0.05) than nuclei themselves, despite the marked losses of chromatin. In summary, our data suggest that gonadotropin and prostaglandins bind to nuclei and that this binding was intrinsic and was primarily associated with the nuclear membrane.     

Receptors for gonadotropins and prostaglandins in lysosomes of bovine corpora lutea.

The total mitochondrial fraction of bovine corpus luteum specifically bound [³H]prostaglandin (PG) E₁, [³H] PGF_2α, and ¹²⁵I-labeled human lutropin (hLH) despite very little 5′-nucleotidase activity, a marker for plasma membranes. Since the total mitochondrial fraction isolated by conventional centrifugation techniques contains both mitochondria and lysosomes, it was subfractionated into mitochondria and lysosomes to ascertain the relative contribution of these fractions to the binding. Subfractionation resulted in an enrichment of cytochrome c oxidase (a marker for mitochondria) in mitochondria and of acid phosphatase (a marker for lysosomes) in lysosomes. The lysosomes exhibited little or no contamination with Golgi vesicles, rough endoplasmic reticulum, or peroxisomes as assessed by their appropriate marker enzymes. Subfractionation also re ulted in [³H] PGE₁, [³H] PGF_2α, and ¹²⁵I-labeled hLH binding enrichment with respect to homogenate in lysosomes but not in mitochondria. The lysosomal binding enrichment and recovery were, however, lower than in plasma membranes. The ratios of marker enzyme to binding, an index of organelle contamination, revealed that plasma membrane and lysosomal receptors were intrinsic to these organelles. Freezing and thawing had markedly increased lysosomal binding but had no effect on plasma membrane binding. Exposure to 0.05% Triton X-100 resulted in a greater loss of plasma membrane compared to lysosomal binding. In summary, the above results suggest that lysosomes, but not mitochondria, in addition to plasma membranes, intrinsically contain receptors for PGs and gonadotropins. Furthermore, lysosomes overall contain a greater number of PGs and gonadotropin receptors compared to plasma membranes and these receptors are associated with the membrane but not the contents of lysosomes.     

The interaction of calcium and procaine with hepatocyte and hepatoma tissue culture cell plasma membranes studied by fluorescence spectroscopy

The fluorescent probe l-anilinonaphthalene-8-sulfonate (ANS) has been used to investigate the properties of plasma membranes derived from normal hepatocytes and from hepatoma tissue culture (HTC) cells as well as used to study the effects of Ca²⁺ and procaine on these membrane systems. The interaction of ANS with hepatocyte plasma membranes (50 nmol/mg protein; K_D = 120,μM) resulted in a marked enhancement of fluorescence and a 20-nm blue shift. Both Ca²⁺ and procaine further increased the fluorescence intensity. Binding studies showed no alteration in the number of ANS binding sites but a significant decrease in K_D (40–50 μm). Procaine was also shown to completely displace Ca²⁺ from the membrane. The interaction of ANS with HTC cell plasma membranes again resulted in an enhancement in fluorescence intensity but with different binding properties (102 nmol/mg protein; K_D = 74 μM) from the hepatocyte system. The addition of Ca⁺² resulted in the formation of high and low affinity ANS binding sites as shown by Scatchard plot analysis with K_D values of 15 μm and 50 μm. The effect of procaine on ANS fluorescence in the normal and transformed cell membranes was indistinguishable; however, in the latter system procaine only displaced 60% of the bound Ca²⁺. These studies suggest several structural and binding alterations between plasma membranes derived from hepatocytes and HTC cells.     

Electrophoretic comparison of polypeptides from enriched plasma membrane fractions from developing soybean roots

The polypeptide complement of enriched soybean (Glycine max [L.] Merr. cult. wells) root plasma membrane fractions was studied by two-dimensional gel electrophoresis. Good resolution was obtained when polypeptides were solubilized in sodium dodecyl sulfate and when butylated hydroxytoluene was included in the vesicle isolation and solubilization media. The pattern obtained on the two-dimensional slab gel for root plasma membrane was characteristic for that membrane. The polypeptide complements from mitochondrial membranes and from enriched fractions of three other endomembrane components were solubilized and electrophoresed for comparison. Each membrane preparation was identifiable on the basis of its characteristic electrophoretogram. Electrophoresis of protein solubilized from plasma membrane fractions isolated from meristematic and mature root tissue revealed both qualitative and quantitative differences in the respective protein complements.     

Studies on nitrate reductase activity in Laminariadigitata (Huds.) Lamour. I. Longitudinal and transverse profiles of nitrate reductase activity within the thallus

The distribution of the enzyme nitrate reductase (NR) within the thallus of the brown alga Laminaria digitata (Huds.) Lamour is described for plants sampled from the east coast of Scotland in May and June when growth rates are at a maximum. Highest NR activities (≈ 0.2 μmol NO₃^? reduced·g^?1 wet wt·h^?1) occurred in the mature blade. NR activities declined towards the basal meristematic region of the blade. Activities in the stipe and holdfast were also low, being between 0.05 and 0.07 μmol NO₃^? reduced·g^?1 wet wt·h^?1. The activities of the enzyme glutamine synthetase (GS), which is important in the assimilation of NH₄⁺, showed a similar distribution within the blade to those of NR.The transverse profile of NR activity in the stipe exhibited a decline from the outer to the inner tissues. Maximum activities (0.13 μmol NO₃^? reduced·g^?1 wet wt·h^?1) occurred in the meristoderm, while those of the cortex and medulla were 0.04 and 0.01 μmol NO₃^? reduced·g^?1 wet wt·h^?1 respectively.These data indicate that most of the NO₃^? assimilation occurs in the mature blade rather than in the meristematic tissue where there is a high nitrogen demand for growth. The data are consistent with the maintenance of meristematic growth by the internal transport of organic nitrogen from the mature blade.     

Molt-inhibiting hormone stimulates vitellogenesis at advanced ovarian developmental stages in the female blue crab, Callinectes sapidus 1: an ovarian stage dependent involvement

The finding that molt-inhibiting hormone (MIH) regulates vitellogenesis in the hepatopancreas of mature Callinectes sapidus females, raised the need for the characterization of its mode of action. Using classical radioligand binding assays, we located specific, saturable, and non-cooperative binding sites for MIH in the Y-organs of juveniles (J-YO) and in the hepatopancreas of vitellogenic adult females. MIH binding to the hepatopancreas membranes had an affinity 77 times lower than that of juvenile YO membranes (K_D values: 3.22 × 10^-8 and 4.19 × 10^-10 M/mg protein, respectively). The number of maximum binding sites (B_MAX) was approximately two times higher in the hepatopancreas than in the YO (B_MAX values: 9.24 × 10^-9 and 4.8 × 10^-9 M/mg protein, respectively). Furthermore, MIH binding site number in the hepatopancreas was dependent on ovarian stage and was twice as high at stage 3 than at stages 2 and 1. SDS-PAGE separation of [¹²⁵I] MIH or [¹²⁵I] crustacean hyperglycemic hormone (CHH) crosslinked to the specific binding sites in the membranes of the J-YO and hepatopancreas suggests a molecular weight of ~51 kDa for a MIH receptor in both tissues and a molecular weight of ~61 kDa for a CHH receptor in the hepatopancreas. The use of an in vitro incubation of hepatopancreas fragments suggests that MIH probably utilizes cAMP as a second messenger in this tissue, as cAMP levels increased in response to MIH. Additionally, 8-Bromo-cAMP mimicked the effects of MIH on vitellogenin (VtG) mRNA and heterogeneous nuclear (hn) VtG RNA levels. The results imply that the functions of MIH in the regulation of molt and vitellogenesis are mediated through tissue specific receptors with different kinetics and signal transduction. MIH ability to regulate vitellogenesis is associated with the appearance of MIH specific membrane binding sites in the hepatopancreas upon pubertal/final molt.     

Water permeability of thin lipid membranes

The osmotic permeability coefficient, P_f, and the tagged water permeability coefficient, P_d, were determined for thin (<100 A) lipid membranes formed from ox brain lipids plus DL-α-tocopherol; their value of approximately 1 x 10^-3 cm/sec is within the range reported for plasma membranes. It was established that P_f = P_d. Other reports that P_f > P_d can be attributed to the presence of unstirred layers in the experimental determination of P_d. Thus, there is no evidence for the existence of aqueous pores in these thin phospholipid membranes. The adsorption onto the membrane of a protein that lowers its electrical resistance by a factor of 10³ was found not to affect its water permeability; however, glucose and sucrose were found to interact with the membrane to modify P_f. Possible mechanisms of water transport across these films are discussed, together with the implications of data obtained on these structures for plasma membranes.     

Binding of [3H]cytochalasin B and [3H]colchicine to isolated liver plasma membranes

The binding to isolated hepatocyte plasma membranes of radioactively labelled inhibitors of microfilamentous and microtubular protein function ([³H]cytochalasin B and [³H]colchicine, respectively) was studied as one means of assessing the degree of association of these proteins with cell surface membranes. [³H]Cytochalasin B which behaved identically to the unlabelled compound with respect to binding to these membranes was prepared by reduction of cytochalasin A with NaB³H₄. The binding was rapid, readily reversible, proportional to the amount of membrane and relatively insentive to changes of pH or ionic strength. At 10^?6 M [³H]cytochalasin B, glucose or p-chloromercuribenzoate, an inhibitor of glucose transport inhibited binding by about 20%; treatment of membranes with 0.6 M KI which depolymerizes F actin to G actin caused about 60% inhibition of binding. These two types of inhibition were additive indicating two separate classes of binding sites, one associated with sugar transport and one with microfilaments. Filamentous structures with the diameter of microfilaments (50 Å) were seen in electron micrographs of thin sections of the membranes. At concentrations greater than 10^?5 M [³H]cytochalasin B, binding was proportional to drug concentration, characteristic of non-specific adsorption or partitioning. Intracellular membranes of the hepatocyte also bound [³H]cytochalasin B, those of the smooth endoplasmic reticulum to a greater extent than plasma membranes.[³H]Colchicine bound to plasma membranes in proportion to the amount of membrane and at a rate compatible with binding to tubulin. However, other properties of the binding including effects of temperature, drug concentration and antisera against tubulin were different from those of binding to tubulin. Hence, no evidence was obtained for association of microtubular elements with these membranes. Despite this there appeared to be an interdependence between microtubule and microfilament inhibitors: vinblastine sulfate stimulated [³H]cytochalasin B binding and cytochalasin B stimulated ³H colchicine binding. [³H]Colchicine also bound to intracellular membranes, especially smooth microsomes.     

Neurotensin receptors in canine intestinal smooth muscle: preparation of plasma membranes and characterization of (Tyr3−125I)-labelled neurotensin binding

To study the binding of (Tyr³−¹²⁵I)-labelled neurotensin to intestinal muscle, plasma membranes have been purified from dog intestinal circular smooth muscle. Purification was done by differential centrifugation followed by separation on a sucrose gradient. Electron microscopic study revealed that the dissected circular muscles used as the source of membranes were free of myenteric plexus and that the plasma membrane fraction obtained was free of any mitochondria or synaptosomes. The fraction used was obtained at the interface of 14%–33% sucrose density on the gradient and was 25-times enriched in the plasma membrane marker enzyme 5′-nucleotidase activity as compared to post-nuclear supernatant. This fraction contained negligible activity of mitochondrial membrane marker enzyme cytochrome c oxidase and low activity of a putative endoplasmic reticulum marker enzyme NADPH-cytochrome-c reductase. This membrane fraction contained a high density of neurotensin binding sites. This binding was studied by kinetic and by saturation approaches. Analysis of data from saturation binding studies by the computer programs (EBDA and LIGAND) suggested the presence of a two-site model (K_d1 = 0.118 nM, K_d2 = 3.18 nM, B_max1 = 9.73 fmol/mg and B_max2 = 129.8 fmol/mg). A part of specifically bound neurotensin was rapidly dissociated. No cooperativity between the two receptor types could be detected. A kinetic analysis of binding gave the K_d value equal to 0.107 nM. Carboxy terminal amino acid residues 8–13 were found to be essential for the binding activity and replacement of Tyr¹¹ by tryptophan reduced the affinity of the peptide by 10 times in displacement studies. Binding was modulated by sodium ions and a guanine nucleotide Gpp[NH]p. MgCl₂, CaCl₂ and KCl were also found to reduce the specific binding. Evidence was found of a high specific binding to another membrane fraction poor in plasma membranes and rich in synaptosomes. We concluded that plasma membrane of canine intestinal circular muscle contains neurotensin receptors with recognition properties distinct from those obtained in previous studies of neurotensin binding sites in murine tissues. Another neurotensin binding site may be present on neuronal membranes.     

Characterization of ATPase activity associated with corn leaf plasma membranes

A Mg²⁺-dependent, cation-stimulated ATPase was associated with plasma membranes isolated from corn leaf mesophyll protoplasts. Potassium was the preferred monovalent cation for stimulating the ATPase above the Mg²⁺-activated level. The enzyme was substrate-specific for ATP, was inhibited by N,N′-dicyclohexylcarbodiimide, diethylstilbestrol, p-chloromercuribenzoate, and orthovanadate, but was insensitive to oligomycin or sodium azide. A K_m of 0.28 millimolar Mg²⁺-ATP was determined for the K⁺-ATPase, and the principal effect of potassium was on the V_max for ATP hydrolysis. Since potassium stimulation was not saturated at high concentrations, a nonspecific role was proposed for potassium stimulation. A nonspecific phosphatase was also found to be associated with corn leaf plasma membranes. However, it could not be determined positively whether this activity represented a separate enzyme.     

Calmodulin stimulation of unidirectional calcium uptake by the endoplasmic reticulum of barley aleurone

The role of calmodulin (CaM) in gibberellic acid (GA₃)-stimulated Ca²⁺ uptake was investigated in endomembranes isolated from aleurone cells of barley (Hordeum vulgare L.). Unidirectional Ca²⁺ -uptake activity of endoplasmic reticulum (ER) was higher in membranes isolated from aleurone layers treated for 16 h with GA₃ and Ca²⁺ compared with those isolated from layers incubated in Ca²⁺ alone. However, the level of uptake from Ca²⁺-treated tissue could be stimulated to that of the GA₃-treated cells by applying exogenous CaM which increased the V _max of the Ca²⁺ transporter approximately threefold. Calcium uptake in ER from GA₃-treated tissue was inhibited by the CaM antagonist W7 in 50% of experiments, whereas the activity in membranes from non-GA₃-treated tissue was unaffected. Treatment with GA₃ also led to a twofold increase in CaM levels in aleurone layers within 4–6 h, paralleling the time course of the stimulation of Ca²⁺ uptake and preceding the stimulation of α-amylase secretion. We propose that the elevation of Ca²⁺ uptake into the ER induced by GA₃ may be coordinated and regulated by elevated levels of membrane-associated CaM and this may regulate Ca²⁺-dependent α-amylase synthesis in the lumen of the ER.     

Preparation of a human lung purified plasma membrane fraction: Confirmation by enzyme markers,electron microscopy,and histamine H1 receptor binding

Summary A simple and rapid method of isolating plasma membranes from human peripheral lung tissue is described. The method involves homogenization of tissue in 0.25m sucrose-buffered medium followed by differential and sucrose density gradient centrifugation. Enzymatic and morphological characterization of the plasma membrane fraction revealed minimal contamination by nonplasma membrane fragments. The isolated plasma membranes showed an 18-fold purification of 5-nucleotidase activity compared to the original homogenate. Electronmicroscopic studies of the plasma membrane fraction revealed the presence of small membrane vesicles having a trilaminar membrane structure. To further examine the purity of the plasma membrane preparation, the binding of the H₁ receptor antagonist,³H pyrilamine, to the plasma membrane-enriched fraction was compared to the binding to crude membrane preparations. Both the plasma membrane-enriched fraction and the crude membrane preparation had similar K_d's for the histamine antagonist, but the plasma membrane-enriched fraction had a threefold greater binding capacity, reflecting the relative enrichment of plasma membranes of the preparation. Thus, a method has been developed for the isolation of plasma membranes from human peripheral lung which should provide material for a variety of biochemical and pharmacological studies.