Wheat Germ Agglutinin Binding Research Articles

Primary sequence and heterologous expression of nuclear pore glycoprotein p62.

The major nuclear pore protein p62 is modified by O-linked N-acetylglucosamine and functions in nuclear transport. We have cloned, sequenced, and expressed the full-length rat p62 cDNA. The rat p62 mRNA is 2,941 nucleotides long and encodes a protein of 525 amino acids containing 30% serine and threonine residues. The amino acid sequence near the amino-terminus contains unique tetrapeptide repeats while the carboxy-terminus consists of a series of predicted alpha-helical regions with hydrophobic heptad repeats. Heterologous expression of rat p62 in African Green Monkey Kidney COS-1 cells and CV-1 cells was detected using a species-specific antipeptide serum. When transiently expressed in COS-1 cells, rat p62 binds wheat germ agglutinin and concentrates at the spindle poles during mitosis. In CV-1 cells cotransfected with rat p62 cDNA and SV40 viral DNA, rat p62 associates with the nuclear membrane without interfering with the nuclear transport of SV40 large T antigen. The ability to express p62 in tissue culture cells will facilitate analysis of the role of this pore protein in nuclear transport.

Wheat germ agglutinin binding in rat primary sensory neurons: a histochemical study

The binding of wheat germ agglutinin (WGA) to L5 dorsal root ganglion (DRG) cells in the rat was studied with the WGA-FITC conjugate. These cells were also examined with regard to overlap between WGA staining and choleragenoid-like immunoreactivity. The DRG cells showed varying intensity of the staining, which was confined to the cytoplasm. The majority of the small cells were heavily stained, whereas the large cells showed less or occasionally no staining. Lectin binding was observed along nerve fibres in the ganglion, and appeared to be localized to the Schwann cells and to the nodes of Ranvier. Strong staining was also observed in the area surrounding the ganglion cells and seemed to be confined to the satellite cells. A subpopulation of the ganglion cells showed both WGA-staining and choleragenoid-like immunoreactivity. These results indicate a nonuniform affinity of WGA for different subpopulations of DRG neurons.

Purification and characterization of a chemoattractant from electric shock-induced earthworm secretion, its receptor binding, and signal transduction through the vomeronasal system of garter snakes.

Following shocks with low voltage electric current, earthworms, Lumbricus terrestris, secrete a yellow mucus that has alarm properties for conspecifics and chemoattractive properties for garter snakes, Thamnophis sirtalis. A proteinaceous chemoattractant for garter snakes has been isolated and purified to homogeneity from such secretions by means of permeation chromatography and semipreparative nondenaturing polyacrylamide gel electrophoresis. The purified protein is highly attractive to garter snakes; it loses its activity after proteolytic digestion. It is a glycoprotein consisting of a single polypeptide chain with an NH2-terminal alanine. This chemoattractant has a minimum molecular mass of 15.4 kDa calculated from its amino acid and carbohydrate contents and an apparent molecular mass of about 20 kDa as estimated from sodium dodecyl sulfate-polyacrylamide gel electrophoresis. It has a pI of about 4.0, and it binds wheat germ agglutinin but not concanavalin A. This chemoattractant shows a protein to carbohydrate ratio of 2.0 +/- 0.08 (n = 5) and a ratio of total sugar to amino sugar of 1.9 +/- 0.08 (n = 3). The sequence of its NH2-terminal 15 amino acid residues has been determined. Studies were also conducted on the chemosignal transduction through the vomeronasal sensory system of the garter snake. Dot blot analysis showed that the purified chemoattractant bound to snake vomeronasal sensory epithelial membrane fractions. It did not bind to membrane extracts of the nonsensory epithelium of the vomeronasal mushroom body. The chemoattractant also bound specifically to vomeronasal sensory epithelial membrane in a reversible and saturable fashion with Kd and Bmax values of about 0.3 microM and 0.4 nmol/mg of protein, respectively. In electrophysiological studies, the chemoattractant applied to the vomeronasal epithelium caused an increase in firing rate of individual neurons in the accessory olfactory bulb of garter snakes, the projection site for vomeronasal neurons. The present results are the first clear biochemical and electrophysiological evidence for a vomeronasal epithelium response to a purified nonvolatile odorant, and this makes the garter snake vomeronasal system ideal for studying the mechanisms of chemosignal transduction.

Pores of annulate lamellae and nuclei bind wheat germ agglutinin and monoclonal antibody similarly

Both a monoclonal antibody against rat liver nuclear pore complex proteins and a wheat germ agglutinin (WGA) bind to the pore regions of nuclear envelopes and annulate lamellae from extracts of P3J, a cultured cell line derived from ascites of a Burkitt's lymphoma. In this system it is possible to induce large numbers of annulate lamellae whose functions are unknown but may include providing a source of nuclear envelopes. The antibody and the lectin bind fibrous material at pore margins on both sides of the nuclear envelope and cisternae of the annulate lamellae. Treating extracts of PU cells with protease removes the fibrous material from pore regions and prevents binding by WGA. Extracts treated with RNAse still possess fibrous material at the pore regions of both organelles that binds WGA. The similarity of the binding of the two types of pores and the specificity of binding argue that annulate lamellae may be a reserve form of nuclear envelopes. Binding of WGA is prevented by preincubating the extracts in N-acetylglucosamine or N,N,N-triacetylchitotriose, suggesting that both pore complexes include one or more N-acetylglucosamine-containing proteins.

A stalk-specific wheat germ agglutinin binding protein, wst34, in Dictyostelium discoideum can be detected with antiserum raised against Dictyostelium mucoroides stalk

A new stalk-specific wheat germ agglutinin (WGA) binding protein, wst34, has been identified in Dictyostelium discoideum and purified by the use of preparative sodium dodecyl sulfate - polyacrylamide gel electrophoresis and a WGA-affinity column. In normal development, wst34 appears during culmination and is maintained in stalk cells. It has a molecular mass of 34 kilodaltons and a pI value of 5.5-6.5. A polyclonal antiserum raised against stalk cell proteins of Dictyostelium mucoroides recognizes wst34 in western blots of D. discoideum proteins.

Endocytosis of wheat germ agglutinin binding sites from the cell surface into a tubular endosomal network

By using fluorescence and electron microscopy, the endocytic pathway encountered by cell surface components after they had bound wheat germ agglutinin (WGA) was visualized. The majority of these components are thought to consist of sialylated glycoproteins (HMWAG) that represent a subpopulation of the total cell surface proteins but most of the externally disposed plasma membrane proteins of the cell. Examination of semi-thin sections by medium- and high-voltage electron microscopy revealed the three-dimensional organization of vesicular and tubular endosomes. Binding of either fluorescein isothiocyanate-, horseradish peroxidase-, or ferritin-conjugated WGA to cells at 4 degrees C showed that the HMWAG were distributed uniformly over the cell surface. Warming of surface-labeled cells to 37 degrees C resulted in the endocytosis of WGA into peripheral endosomes via invagination of regions of both coated and uncoated membrane. The peripheral endosome appeared as isolated complexes comprising a vesicular element (300-400 nm diam.) surrounded by and continuous with tubular cisternae (45-60 nm diam.), which did not interconnect the endosomes. After 30 min or more label also became localized in a network of anastomosing tubules (45-60 nm diam.) that were located in the centrosomal region of the cell. Endocytosed WGA-HMWAG complexes did not become associated with cisternae of the Golgi apparatus, although tubular and vesicular endosomes were noted in the vicinity of the trans-Golgi region. The accumulation of WGA-HMWAG in the endosomes within the centrosomal region was inhibited when cells were incubated at 18 degrees C. None of these compartments contained acid phosphatase activity, a result that is consistent with other data that the HMWAG do not pass through lysosomes initially. The kinetics of labeling were consistent with the interpretation that recycling of most of the WGA binding surface glycoproteins occurred rapidly from early peripheral endosomes followed by the late trans-Golgi compartment. In conclusion, a portion of cell surface glycoproteins are routed to a complex arrangement of tubular and vesicular compartments following endocytosis that includes a putative post-endosomal, tubular reticulum that appears to be separate from the trans-most Golgi saccule.

Immune Competence of Aedes trivittatus Hemocytes as Assessed by Lectin Binding

Hemocytes were perfused from uninoculated, saline-inoculated, and Dirofilaria immitis microfilariae-inoculated Aedes trivittatus and assessed for their capacity to bind wheat germ agglutinin (WGA). Approximately one-fourth of perfused hemocytes were WGA positive in all groups of mosquitoes at all time periods tested. Results suggest A. trivittatus has a larger inherent population of immune-competent hemocytes as compared with Aedes aegypti and, therefore, is faster and more effective in killing microfilariae by melanotic encapsulation reactions.

WGA binding to the surface of two autologous human melanoma cell lines: Different expression of sialyl and N-acetylglucosaminyl residues

Two autologous human melanoma cell lines were studied to determine their capacities to bind wheat germ agglutinin (WGA). Both cell lines were derived from the same patient, the first, IGR 39, originated from the primary tumor, the second, IGR 37, was established from a metastatic lymph node. WGA binding sites on the surface of these cell lines were compared before and after sialidase and/or tunicamycin treatments. IGR 39 cells exhibited two classes of WGA binding sites with high and low affinities, whereas IGR 37 cells had only one class of high affinity binding sites. After tunicamycin treatment, the capacity of IGR 39 cells to bind WGA was markedly altered, since only one class of WGA binding sites with high affinity was observed under these conditions, whereas tunicamycin did not induce significant changes in the lectin binding of IGR 37 cells. The low affinity WGA binding sites, which were only found on IGR 39 cells, corresponded to sialyl residues present in N-linked glycoproteins. The high affinity binding sites present on both cell lines probably involved sialyl and N-acetylglucosaminyl residues associated with O-linked glycoproteins and/or glycolipids. No direct correlation could be drawn between the number of WGA binding sites and the overall sialic acid levels exposed to sialidase treatment. The 3-fold increase in the amount of cell surface glycopeptides obtained after pronase digestion and specifically binding to WGA-Sepharose was in good agreement with the overall higher number of WGA binding sites on IGR 39 compared to IGR 37 cells. Thus, subtle carbohydrate changes of cell surface glycoconjugates might account for the differences between the biological properties of human melanoma cell lines of low and high tumorigenicity.

Detection of hemicelluloses specific to the cell wall of tracheary elements and phloem cells by fluorescein-conjugated lectins

Binding of fluorescein-conjugated wheat-germ agglutinin (F-WGA) and some other lectins to tissues from various plants were examined by epifluorescence microscopy. F-WGA bound specifically to the walls of tracheary elements (TEs) and phloem cells of pea roots. The binding sites in TEs were localized only in the secondary thickening and became evident at very early stages of differentiation. Fluorescein-conjugated derivatives ofSolanum tuberosum lectin,Lycopersicon esculentum lectin, andDatura stramonium lectin, which bind N-acetylglucosamine residues as WGA, also bound to the secondary thickening of TEs of pea roots. The binding sites for F-WGA were not removed by extraction with hot EDTA and proteinase K, but removed by extraction with an alkali solution. The alkali-extracted binding sites from the roots were precipitated together with hemicelluloses by 80% ethanol. These results indicate that the binding sites are not present on pectins, proteins, or cellulose, but hemicelluloses. Localized distribution of the binding sites for F-WGA in TEs was found also in a variety of angiosperm plants.

Location of the carbohydrates present in the hydrogen ion-potassium ATPase vesicles isolated from hog gastric mucosa

The glycosylation of H+K(+)-ATPase vesicles isolated from hog gastric mucosa was investigated by various methods. Following protein separation on sodium dodecyl sulfate reducing gels and transfer to poly(vinyl difluoride) membranes, binding of concanavalin A was confined to the 94-kDa band which corresponds to the catalytic subunit. In contrast, wheat germ agglutinin binding occurred in a region below the 94-kDa subunit, corresponding to the 60-85-kDa region, and also to protein just above the catalytic subunit. Treatment with glycopeptidase F removed most of the concanavalin A staining and also the wheat germ agglutinin staining found below the 94-kDa region, but spared the higher molecular weight wheat germ agglutinin reactive material. During the deglycosylation experiments a protein of 35-kDa was produced. Sequencing analysis of V8 protease generated peptide fragments of the 35-kDa protein show at least 30% homology with the Na+K(+)-ATPase beta-subunits. Labeling of the carbohydrates by galactosyltransferase and [3H]uridine diphosphate-galactose showed that the sites of labeling were extracellular and were confined to the wheat germ agglutinin staining regions. Two molecular weight regions, below the 94-kDa region, of 60 and 85 kDa were identified. Electron microscopy using postembedding staining techniques showed that both concanavalin A and wheat germ agglutinin staining occurred on the extracellular face of the gastric vesicles.(ABSTRACT TRUNCATED AT 250 WORDS)

Effect of Malnutrition and Hormone Treatments on Intestinal Microvillus Membrane Glycosylation in Suckling Rats

Imposition of undernutrition in suckling rats significantly enhanced (p less than 0.001) sialic acid and reduced (p less than 0.001) fucose contents of intestinal microvillus membranes (MVM) compared to the controls. Administration of cortisone or insulin to undernourished (UN) pups reversed the sialylation and fucosylation pattern of the membranes. The binding of wheat germ agglutinin (WGA) to membranes was increased (p less than 0.001) and that of Ulex europeus agglutinin (UEA) was decreased in nutritionally deprived pups. Cortisone and insulin treatments reduced WGA and enhanced UEA bindings to UN membranes. The binding of peanut agglutinin (PNA) to UN membranes was 60% higher compared to that in the control rats. Cortisone and insulin treatments further enhanced PNA binding to the brush borders in UN pups. The incorporation of 14C-GlcNAc, 14C-mannose and 3H-fucose into MVM was reduced by 32-57% in UN suckling rats compared to the controls. The incorporation of labelled sugars into membranes from 21- and 50- to 60-day-old rats was essentially similar. Cortisone administration to UN pups augmented the incorporation of labelled GlcNAc and mannose but had no effect on fucose incorporation. Insulin treatment of UN pups insignificantly elevated GlcNAc incorporation into the membranes. These findings suggest that imposition of UN during early suckling period delays the maturational development of intestinal glycosylation. Cortisone and insulin induced adult-type glycosylation pattern of the brush borders in nutritionally deprived pups.

The role of carbohydrate moieties of cholecystokinin receptors in cholecystokinin octapeptide binding: Alteration of binding data by specific lectins

Cholecystokinin (CCK) receptors on rat pancreatic acini have been demonstrated to be glycoproteins. In order to study whether their carbohydrate moieties play a role in ligand binding, membrane preparations (adjusted to 0.2 mg protein me) were incubated with 20 pM 125-I-CCK octapeptide ( 125I-CCK 8) for 4 h at 30°C in the presence of lectins with different sugar specificities. Concanavalin A, soy-bean agglutinin, and peanut agglutinin in concentrations up to 1 mM did not alter specific 125I-CCK 8 binding. Ulex europeus lectin I showed a dose-dependent enhancement of CCK binding up to 150% of controls at a concentration of 1 mM. Wheat-germ agglutinin (WGA) was the only lectin found to have an inhibitory effect. Inhibition was dose-dependent, with maximal reduction attained at 42 nM, but CCK binding was only partially inhibited to 66.2 ± 4.4%. Inhibition by WGA was prevented by the presence of N- acetyl- d-glucosamine or N, N′, N″-triacetylchitotriose, sugars that are specific for WGA. The inhibitory effect of WGA was not due to an increase in non-specific binding, increased CCK degradation, or CCK binding to WGA. Binding data indicated that the presence of WGA resulted in a decrease in receptor affinity ( K d = 567 ± 191 v. 299 ± 50 pM). No significant change in the number of available binding sites was observed. This suggests that WGA is not binding to the active binding site. It is conceivable that binding of WGA to N- acetyl- d-glucosamine or its polymers can lead to a conformational change in the receptor protein, and that this carbohydrate moiety is essential for optimal receptor-ligand interaction.

Tumor progression- and metastasis-associated proteins identified using a model of locally recurrent rat mammary adenocarcinomas

A recently established model for local breast cancer recurrence using the 13762NF rat mammary adenocarcinoma was used to evaluate biologic and biochemical properties related to clinical outcome for this class of tumors. Sublines isolated from local tumor regrowths following surgical resection differed from each other and from the 'parental' cell lines for multiple phenotypes, including metastatic propensity. Local recurrence- and primary tumor-derived sublines were examined by two-dimensional polyacrylamide gel electrophoresis (2D-PAGE), lectin binding to electrophoretically separated proteins, and lactoperoxidase-catalyzed cell surface iodination; and differential protein patterns were compared to tumor progression and metastatic potential. 2D-PAGE revealed several quantitatively different spots which correlated with lung colonization potential. In particular, quantities of an apparently unique, non-cell-surface protein, P50.9 (Mr approximately 50,900, pI approximately 7.3) correlated inversely with metastatic propensity, suggesting that it may be associated with, among other possibilities, the negative regulation of the metastatic phenotype. P50.9 was unrelated to four similarly sized metastasis-associated proteins--tumor autocrine motility factor; the rat analog of tumor suppressor, p53; rat cytokeratin 14 or procathepsin D--as determined by amino acid analysis. A major wheat germ agglutinin binding sialoglycoprotein, gp93 (Mr approximately 93,000), was present in smaller amounts as cells were passaged in vivo and re-established as in vitro cultures [MTF7 greater than 'primary' tumor-derived lines (sc1, sc3) much greater than local recurrence-derived lines (LR1, LR1a, LR3, LR4, LR5, LR6)]. Besides cell surface glycoprotein losses, two of six local recurrence-derived sublines expressed a wheat germ agglutinin-binding sialoglycoprotein, gp110 (Mr approximately 110,000), previously undetected on any of the other cell lines including the parental populations. gp110 was found in LR3 and LR6 which were relatively highly metastatic; however, correlation with metastatic potential failed because gp110 was not present on the metastatic parental cell line, MTF7. These results demonstrate specific quantitative and qualitative protein differences associated with the selection of locally recurrent mammary tumors.

Immunohistological distribution of 5T4 antigen in normal and malignant tissues

A trophoblast cell surface antigen has been characterised by a monoclonal antibody (mAb) 5T4, raised following immunisation with solubilised wheat germ agglutinin binding glycoproteins from human syncytiotrophoblast plasma membrane (StMPM). The expression of the 72 kDa glycoprotein was assessed on cryostat sections of a range of neoplastic and non-neoplastic tissues, using an avidin-biotin immunoperoxidase technique. In products of conception, intense reactions were noted with villous syncytiotrophoblast membrane in normal early and term placenta, with weaker positivity of placental site trophoblast. Most normal or non-neoplastic tissues were negative, including liver, kidney, spleen, small intestine, ovary and testis. Faint or moderate positive reactions were present in some specialised epithelia. Of 115 neoplasms examined, 76 showed reactions with tumour cells including carcinomas of the bladder, breast, cervix, endometrium, lung, oesophagus, ovary, pancreas, stomach and testicular non-seminomatous germ cell tumours. Choriocarcinomas and placental site trophoblastic tumours were also positive. Most adenocarcinomas of colon and seminomas were negative as were all malignant melanomas and malignant lymphomas. A radioimmunoassay did not detect the antigen in either normal or pregnancy serum. The relatively low level of expression in normal tissues and reactivity with a wide range of carcinomas suggested that the antibody may be useful in diagnostic or targeting studies.

Toxicity of protease-resistant domains from the delta-endotoxin of Bacillus thuringiensis subsp. israelensis in Culex quinquefasciatus and Aedes aegypti bioassays

The mosquitocidal glycoprotein endotoxin of Bacillus thuringiensis subsp. israelensis was digested with chymotrypsin to yield protease-resistant domains which were then separated from smaller protease digestion products by high-performance liquid chromatography. Once purified, the domains no longer bound wheat germ agglutinin, a lectin which binds N-acetylglucosamine (GlcNAc) and GlcNAc oligomers. Purified protease-resistant domains were as toxic for Culex quinquefasciatus larvae as intact solubilized toxin. In separate experiments, the toxicity of chymotrypsin-digested endotoxin for Aedes aegypti larvae was reduced fivefold or more. A model is presented in which GlcNAc-containing oligosaccharides are required for toxicity for A. aegypti larvae but not C. quinquefasciatus larvae.

The lateral organization of components of the membrane skeleton and superoxide generation in the plasma membrane of stimulated human neutrophils

Studies were performed to examine the lateral organization of the NADPH oxidase system in the plasma membrane of human neutrophils. Analysis of the subcellular fractionation of human neutrophils by isopycnic sedimentation of cavitated cell lysates suggested that there may be more than one population of plasma membrane vesicles formed upon cell disruption. One population (30-32% sucrose) contained surface accessible wheat germ agglutinin binding sites, alkaline phosphatase activity, and cytochrome b. Another population (34-36% sucrose) contained membrane-bound flavin and, when the cells were prestimulated with phorbol myristate acetate (PMA), NADPH-dependent superoxide generating activity. Approximately 25% of the neutrophil cytochrome b cosedimented with the heavy population, confirming our previous hypothesis (Parkos et al. (1985) J. Biol. Chem. 260, 6541-6547) that only a fraction of the total cellular cytochrome b is involved in superoxide production. The heavy plasma membrane fraction was also enriched in membrane associated actin and fodrin as detected by Western blot analysis. After extraction of the plasma membrane vesicles with detergent cocktails, the majority of superoxide generating activity remained associated with the detergent insoluble pellet. Western blot analysis demonstrated that the pellets were also enriched in actin. Further analysis of these pellets using rate-zonal detergent-containing sucrose density gradients indicated that the superoxide generating complex had an approximate sedimentation coefficient of 80 S, suggesting that the neutrophil superoxide generating system may form a complex on the plasma membrane which is associated with or somehow organized by the membrane skeletal matrix. This organization may be of functional relevance not only to the actual production of superoxide, but also to the targeting of microbicidal oxidants.

Wheat germ agglutinin but not concanavalin A modulates protein kinase C-mediated phosphorylation of red cell skeletal proteins

Human red blood cells contain protein kinase C (PKC) which acts exclusively on the membrane skeletal proteins band 4.1, band 4.9 and adducin. PKC activity can be stimulated by the addition of the phorbol ester 12- O-tetradecanoyl phorbol 13-acetate to intact cells. Phosphorylation of band 4.1 by PKC in vitro results in a dramatic reduction in band 4.1 binding to spectrin and actin, as well as to the cytoplasmic domain of band 3. Here we show that the lectin wheat germ agglutinin (WGA), which binds to the extracellular domain of glycophorin results in the inhibition of PKC catalyzed phosphorylation of band 4.1, band 4.9 and likely adducin as well. The lectin concanavalin A, which binds to band 3 was without effect. Our results suggest that the binding of WGA to glycophorin results in a major rearrangement of the membrane skeletal network which correlates with reduced phosphorylation of membrane skeletal proteins by PKC.

Cytochemical and biochemical characterization of glycoproteins in forming and maturing enamel of the rat incisor.

Biochemical and histochemical studies have shown the presence of various carbohydrates in enamel. Using lectin-gold cytochemistry, we have examined the distribution of glycoconjugates containing N-acetyl-D-galactosamine (GalNAc) and/or N-acetyl-glucosamine (GlcNAc)/N-acetyl-neuraminic acid (NeuNAc) residues in rat incisor ameloblasts and in forming and maturing enamel embedded in Lowicryl K4M, LR Gold, and LR White resins. The enamel proteins that contain these carbohydrate moieties were further characterized by lectin blotting. All three resins allowed, albeit to a variable degree, detection of the binding sites for Helix pomatia agglutinin (HPA) and wheat germ agglutinin (WGA) GalNAc, and GlcNAc/NeuNAc, respectively. In general, Lowicryl K4M permitted more intense reactions with both lectins. Lectin binding was observed over the rough endoplasmic reticulum (weak labeling with WGA), the Golgi apparatus, lysosomes, secretory granules, and the enamel matrix. These compartments were shown by double labeling with WGA and anti-amelogenin antibody, and by previous immunocytochemical studies, to contain enamel proteins. Furthermore, WGA binding was more concentrated at the growth sites of enamel. Lectin blotting showed that several proteins in the amelogenin group were glycosylated and contained the sugars GalNAc and GlcNAc/NeuNAc. Fewer proteins were stained by HPA than by WGA, and the staining pattern suggested that the extracellular proteins recognized by these two lectins are processed differently. The HPA-reactive proteins were lost by or during the early maturation stage, whereas many of the WGA-reactive proteins persisted into the mid maturation stage. The heterogeneous staining of certain protein bands observed with WGA suggests that they contain more than one component. Two distinct glycoproteins containing GlcNAc/NeuNAc also appeared during the maturation stage. These results are consistent with the notion that ameloblasts produce an extracellular matrix composed mainly of glycosylated amelogenins which are differently processed throughout amelogenesis.

Glycoproteins in Citrus Foliage and Callus Tissue as Affected by Cold-hardening Temperature and Abscisic Acid

Soluble proteins were extracted from the foliage of several citrus genotypes that widely differed in cold hardiness after 4 weeks exposure to cold-hardening (16 °/5 °C) and noncold-hardening (30 °/21 °C) temperature regimes. The electrophoretic profiles of glycoproteins were analyzed after transfer to nitrocellulose filters by binding of wheat germ and soybean agglutinin as well as lectin from Lotus tetragonolobus. All three lectins detected a pattern of polypeptides that was highly conserved among citrus genotypes. Many of these polypeptides increased during cold-hardening; one exception was an M r 36,000 (Daltons) polypeptide which disappeared in Valencia orange grafted on rough lemon rootstock. Nearly the same glycoprotein profile was detected in callus lines derived from various tissues of Valencia orange. Polypeptides increased with the addition of abscisic acid ( 10 μm) in the medium and at low-temperature (10 °C). A concomitant reduction of freeze damage as assessed by electrolyte leakage was observed.

Bimodal redistribution of surface transmembrane glycoproteins during Ca2+-dependent secretion (acrosome reaction) in boar spermatozoa

We used the acrosome reaction of boar sperm cells to study the dynamics of surface transmembrane glycoproteins (TMG) during a secretory process. The acrosome reaction is the Ca2+-dependent fusion of a large cytoplasmic vesicle (the acrosome) with the overlying segment of the plasma membrane (acrosomal cap) that leads to the release of the acrosomal enzymes. After triggering the acrosome reaction in vitro (2 mM-CaCl2 in the presence of 10 microM-A23187), we used freeze-fracture electron microscopy to follow the topographical rearrangement of a population of acrosomal-cap large intramembrane particles that correspond to transmembrane proteins that bind wheat germ agglutinin. We found that these TMG move in the direction of either one of two opposite poles, proximal and distal, of the acrosomal cap. This bimodal movement of the TMG reorganizes the acrosomal cap into three extensive domains. The first two, on the apical rim and on the equator, are membrane domains to which the TMG are directed and where they accumulate. The third, a large in-between area of protein clearing, corresponds to the region from which TMG were preferentially located before displacement induced by the Ca2+ effect. The topography of these new membrane domains of the acrosomal cap becomes coincident with that of the structural domains of the subjacent acrosomal membrane. Mirroring of the acrosomal membrane by the plasma membrane is followed by fusion between the two membranes, formation of an exquisite labyrinth of hybrid-membrane tubules, followed by fission and release of the acrosomal contents through intertubular fenestrae.