Further Biochemical Characterization of a Cell Fusion Hormone from the Red Alga, Griffithsia pacifica

In the marine red alga, Griffithsia pacifica, the repair processinitiated upon death of an intercalary cell is mediated by aglycoprotein hormone, rhodomorphin. In this paper we show thatthe glycosidic portion of the molecule has terminal -D-mannosylresidues which are at least in part responsible for the bindingof rhodomorphin by concanavalin A. The protein portion of themolecule contains disulfide bridges. These bridges must be intactfor biologically active hormone to be recovered from a denaturedstate. Isolation of active rhodomorphin from SDS-PAGE indicatesa molecular weight of 15,000–17,500. This agrees wellwith our previously published [Watson and Waaland (1983) PlantPhysiol. 71: 327] molecular weight from gel filtration of 14,000. (Received February 26, 1986; Accepted June 2, 1986)     

Isolation of a cell-fusion hormone from Griffithsia pacifica kylin,a red alga

Filaments of Griffithsia pacifica replace dead cells by the process of cell repair. When an intercalary cell is killed, but its cell wall remains intact holding the two halves of the plant together, the cell above it produces a repair rhizoid cell; the cell below it produces a specialized, rhizoid-like repair shoot cell. The repair rhizoid and shoot grow towards each other, meet, and fuse to form a single shoot cell. Evidence from observations of cell repair in vivo has indicated that the repair rhizoid produces a hormone or hormones which induce the production of the repair shoot, maintain the rhizoid-like morphology and growth of the repair shoot, and attract it to the repair rhizoid for fusion. This hormone has been named rhodomorphin. Using an artificial cell-fusion system we show that repair rhizoids and normal rhizoids, but no shoot cell, can induce decapitated filaments to form repair shoot cells. Decapitated filaments form repair shoot cells only when they are exposed to the hormone within 4–6 h after decapitation; after this time they lose their sensitivity to the hormone. A method has been developed for isolating, and assaying for, the cell-fusion hormone. Rhodomorphin retains its activity for several days at room temperature and for at least two years at-16° C.     

Purification and properties of Acid phosphatase-1 from a nematode resistant tomato cultivar

In tomato the acid phosphatase-1 isozyme (Apase-1) is inherited as a single locus linked to the nematode resistance gene (Mi). The Apase-1¹ electrophoretic variant has been purified from a tomato cell suspension culture using ion exchange and concanavalin A sepharose affinity chromatography. A cellulose acetate electrophoresis method was used to distinguish Apase-1¹ rapidly from other Apase isozymes in tomato. The subunit molecular weight of the purified enzyme was estimated to be 31,000 by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The native size of the enzyme, which is reported to be a dimer, was determined to be approximately 51,000 by high performance liquid chromatography gel filtration. Apase-1¹ has a lower pH optimum and a distinct substrate specificity as compared to Apases extracted from tomato fruit or from other plant species. The amino acid composition of Apase-1¹ is similar to that of a potato Apase.     

A SIGNAL GLYCOPROTEIN WITH α-d-MANNOSYL RESIDUES IS INVOLVED IN THE WOUND-HEALING RESPONSE OF ANTITHAMNION SPARSUM (CERAMIALES,RHODOPHYTA)1

A variety of fluorescein isothiocyanate-labeled lectins specific for different sugar moieties were examined as probes for the wound-healing response in the filamentous red alga Antithamnion sparsum Tokida. Among them, only concanavalin A (ConA) and Lens culrinaris agglutinin (LCA), which have specificity to α-D-mannosyl residues, bound specifically to repair cells during the wound-healing process. When ConA or LCA was added at various time intervals after wounding, it first bound (3 h post-wounding) as a thin layer at the tips of the adjacent cells. Later (4–5 h post-wounding) labeling also appeared at the tips of the repair cells. Intense labeling at these sites continued throughout the healing process until repair cell fusion, at which time the lectin labeling was reduced to a narrow ring around the area of fusion. When added to plants prior to wounding and continually monitored, these same lectins acted as inhibitors to the wound-healing response. Other control lectins showed no inhibitory effects. A crude extract solution obtained from decapitated filaments stimulated the wound-healing response, and a lectin-binding component bound strongly to a protein-binding transfer membrane. These results suggest that the labeled compound is a glycoprotein that has α-D-mannosyl residues and is similar to the repair hormone rhodomorphin found in Griffithsia pacifica Kylin.     

Glycans of synaptosomal plasma membrane glycoproteins from adult rat forebrain: Characterization after fractionation by concanavalin A-Sepharose affinity chromatography

Glycopeptides obtained by exhaustive proteolytic digestion of synaptosomal plasma membranes from adult rat forebraini were separated by affinity chromatography on concanavalin A-Sepharoe. Concanavalin A-binding glycopeptides are essentially made up of mannose and N-acetylglucosamine in a molar ration of 3.45:1, whereas glycopeptides not bound to concanavalin A have a complex monosaccharide composition. By gel filtration on Bio-Gel P-30, concanavalin A-binding glycopeptides appear as low-molecular-weight glycopeptides (migrating like ovalbumin glycopeptides), whereas glycopeptides not bound to concanavalin A behave as high-molecular-weight glycopeptides (migrating like fetuin glycopeptides). Comparison of concanavalin A-binding glycopeptides from rat brain synaptosomal plasma membranes with concanavalin A-binding glycoproteins isolated from the same membrane fraction shows clear differences in monosccharide composition. We demonstrate here that this discrepancy is due to the presence on most concanavalin A-binding glycoprotein subunits of at least two different types of glycan: in addition to the concanavalin A-binding glycans, these glycoprotein subunits carry other glycans which do not interact with concanavalin A. Biological implications of the presence of two (or more) types of glycan on the same polypeptide are discussed.     

Purification of a proteinase and proteinase inhibitor from Tetrahymen a pyriformis

• 1.1. A cysteine proteinase and cysteine proteinase inhibitor have been purified from Tetrahymena.
• 2.2. The proteinase was purified by ammonium sulphate fractionation, gel filtration, ion exchange chromatography and affinity chromatography, and appeared homogeneous by gel filtration and electrophoresis (mol. wt approx 28,000). It hydrolysed BAPNA, degraded azocasein, and converted 80S ribosomes to subunits. Thiol reagents inhibited these activities.
• 3.3. The inhibitor was purified by heat treatment, ammonium sulphate fractionation and ion exchange chromatography, and appeared homogeneous by gel filtration and electrophoresis (mol. wt approx 12.500). The inhibitor was heat stable and it inhibited papain, as well as the Tetrahymena proteinase.

     

Effect of attached carbohydrates on the activity of Trichoderma viride cellulases

Trichoderma viride 1,4-β-d-glucan cellobiohydrolase (exo-cellobiohydrolase, 1,4-β-d-glucan cellobiohydrolase, EC 3.2.1.91) purified from a commercial product to electrophoretic homogeneity by a procedure including affinity and DEAE-Sephadex chromatography, has attached carbohydrates in addition to the glycoprotein constituents. These carbohydrates are lost by consecutive gel filtration steps in Sephadex G-25 columns, whereupon there is a rapid increase in enzymatic activity. A single gel filtration step can eliminate d-glucose or cellobiose added to a solution of this enzyme, but not the carbohydrates attached during incubation with Avicel.After free carbohydrate elimination from crude cellulase complexes by Sephadex G-25 chromatography, liberation of d-glucose following incubation at 50°C and pH 4.8 was observed. This indicates that some carbohydrates remain bound after gel filtration. The elimination of carbohydrate from whole cellulase complex [see 1,4-(1,3;1,4)-β-d-glucan 4-glucanohydrolase, EC 3.2.1.4] was favoured by a yeast treatment, with a simultaneous increase in activity, but the process is not reproducible, as a secondary inactivation process exists.     

Rapid purification of a prekallikrein from rat pancreas

A prekallikrein from rat pancreas was purified 1500-fold with an overall yield of 20% using a rapid, simple procedure. DEAE-Sephadex A-50 chromatography permitted the separation of two prekallikrein components present in rat pancreatic homogenates; the major fraction was further purified by Sephadex G-100 gel filtration and immunoadsorption chromatography. The zymogen is a single-chain molecule with pI 4·35. Apparent M_r values of 38,000 and 37,000 were determined by sodium dodecyl sulfate-polyacrylamide gradient electrophoresis and gel filtration, respectively.     

An investigation of the role of transcellular ion currents in morphogenesis ofGriffithsia pacifica Kylin

Summary Transcellular ion currents are thought to play a role in the induction and maintenance of localized growth in plant cells. In the marine red algaGriffithsia pacifica, two types of cells elongate by localized tip growth, rhizoidal and repair shoot cells. The pattern of growth and morphogenesis in these cells can be altered by environmental and hormonal parameters. We examined the role of localized currents in four developmental processes inG. pacifica: 1. normal elongation of rhizoids, 2. the phototropic response of rhizoids, 3. the re-initiation of growth in dark-starved rhizoids, and 4. morphogenesis of repair shoot cells in the presence and absence of rhodomorphin, an endogenous hormone which regulates growth of these cells.We have found that there is a localized region of inflowing current at the growing tips of both rhizoids and repair shoot cells. The current density at these apices, measured approx. 20 m from the cell surface, fluctuates in the range of 0.6 to 8 A cm^–2 with occasional periods of either very large current (> 20 A cm^–2) or no measurable current; however, the current density is not correlated with the rate of elongation. In addition, currents of similar magnitudes are found at the tips of non-growing cells. Rhizoids which have lost their cytoplasmic polarity and have stopped elongating, following prolonged periods in total darkness, can reestablish a polar distribution of organelles and restart localized growth in the absence of any measurable current at their tips. Thus, it appears that inG. pacifica localized transcellular currents are neither sufficient or necessary for the maintenance or reinitation of sites of localized growth and organelle accumulation.     

Characterization of a nicotinic acetylcholine receptor from rabbit skeletal muscle and reconstitution in planar phospholipid bilayers.

The nicotinic acetylcholine receptor from rabbit skeletal muscle was isolated by affinity chromatography and characterized by ¹²⁵I- α -Bungarotoxin binding and gel filtration chromatography. Quantal conductance events were observed when this material was added to planar phospholipid bilayers. These changes were stimulated by carbamylcholine and antagonized by curare, Butx, dithiothreitol and concanavalin A. The receptor preparation was found to bind 0.2 nMoles ¹²⁵I- α-Bungarotoxin per mg protein and the molecular weight was estimated to be 390,000.     

Nebrodeolysin,a novel hemolytic protein from mushroom Pleurotus nebrodensis with apoptosis-inducing and anti-HIV-1 effects

A novel hemolysin was isolated from the edible mushroom Pleurotus nebrodensis by ion exchange and gel filtration chromatography on DEAE-Sepharose and Sephacryl S-100. The hemolysin from Pleurotus nebrodensis hemolysin (nebrodeolysin) is a monomeric protein with a molecular weight of approximately 27 kDa as determined by gel filtration and SDS-PAGE. Nebrodeolysin exhibited remarkable hemolytic activity towards rabbit erythrocytes and caused efflux of potassium ions from erythrocytes. Subsequently, this hemolysin showed strong cytotoxicity against Lu-04, Bre-04, HepG2, L929, and HeLa cells. It was also found that this hemolysin induced apoptosis in L929 and HeLa cells as evidenced by microscopic observations and DNA ladder, respectively. Moreover, this hemolysin was shown to possess anti-HIV-1 activity in CEM cell culture.     

Purification and properties of a high affinity guanosine 3′: 5′-cyclic monophosphate-binding phosphatase from silver beet leaves

A cyclic nucleotide-binding phosphatase was purified from silver beet leaves by a procedure involving chromatography on CM-Sepharose CL-6B, DEAE-Sephacel, casein-Sepharose 4B, concanavalin A-agarose and Ultrogel AcA44. The enzyme is eluted from concanavalin A-agarose by 0.5 M α-methylglucoside at high ionic strength. The enzyme is monomeric, having a subunit molecular weight (M_r) of 28 000; the native M_r is 31 000 as determined from gel filtration. The enzyme catalyzes the hydrolysis of a range of phosphomonoesters including various nucleotides and O-phosphotyrosine but not O-phosphoserine or O-phosphothreonine. The leaf phosphatase is competitively inhited by guanosine 3′ : 5′-cyclic monphosphate (cGMP) and adenosine 3′ : 5′-cyclic monophosphate (cAMP) (K_i-values: 0.4 μM and 3.3 μM, respectively). The leaf phosphotase has the highest affinity for cGMP yet reported for a plant protein.     

The isolation and characterization of a concanavalin A receptor from boar spermatozoa surface

Boar spermatozoa were radioactively labeled by either lactoperoxidase-catalysed iodination or galactose oxidase oxidation followed by reduction with tritiated sodium borohydride. Plasma membrane glycoproteins were solubilized with the non-ionic detergent Nonidet P40 and separated by affinity chromatography on concanavalin A-Sepharose. A major water-soluble concanavalin A receptor of molecular weight greater than 160 000 was isolated by gel filtration and ion-exchange chromatography. Its amino acid and carbohydrate composition were determined. This glycoprotein is susceptible to digestion by trypsin or chymotrypsin.     

A novel pigment protein in the ovary of echinothurid sea urchins,Asthenosoma ijimai and Araeosoma owstoni

• 1.1. A protein conjugated with a pigment, which showed a peak of absorbance at 385 nm, was identified and partially purified from the ovary of Asthenosoma ijimai and Araeosoma owstoni by butanol extraction, gel filtration, ion-exchange chromatography and adsorption chromatography. This protein was observed only in ovaries, but not in testes.
• 2.2. This protein of A. ijimai showed a molecular weight of 600 kDa on gel filtration. The isoelectric point of the protein was 4.7.
• 3.3. The possible presence of this protein was examined by gel filtration chromatography in the ovaries of 11 other species of sea urchins, Glyptocidaris crenularis, Diadema setosum, Temnopleurus hardwicki, Toxopneustes pileolus, Pseudocentrotus depressus, Hemicentrotus pulcherrimus, Strongylocentrotus intermedius, S. nudus, Echinostrephus aciculatus, Anthocidaris crassispina, Echinometra mathaei and Echinocardium cordatum. However, it was not detected.

     

The wound-healing responses of Antithamnion nipponicum and Griffithsia pacifica (Ceramiales, Rhodophyta) monitored by lectins

The binding of FITC labeled lectins to repair cells of Antithamnion nipponicum Yamada et Inagaki and Griffithsia pacifica Kylin, and their physiological effects on somatic cell fusion have been studied. Results indicate that repair cells strongly bind the lectins ConA and LCA, whereas other lectins did not bind to the cell, The binding of these lectins to the dead cell wall shows ConA and LCA specific substances are secreted from the tip of the repair cells. When fluorescently labeled ConA or LCA was added at various time intervals after wounding, it firstly bound (3 h post-wounding) as a thin layer at the tips of the adjacent cells. Later (4–5 h post-wounding) labeling also appeared at the tips of the repair ceils. Intense labeling at these sites continued throughout the wound-healing process until repair cell fusion, at which time the lectin labeling was reduced to a narrow ring around the area of fusion, When added to plants prior to wounding and with continued monitoring, these same lectins were found to act as inhibitors to the wound-healing response. Other control lectins showed no inhibitory effects. These results suggest that a signal glycoprotein with α-D-mannosyl residues is involved in the wound-healing process of Antithamnion nipponicum. Lectins conjugated with visible tags can be used as a very fast and useful tool to monitor these signal substances.     

Partial purification and characterization of platelet factors stimulating the multiplication of normal human glial cells

Multiplication-stimulating activity for human glial cells was purified from human outdated platelets. By ion exchange chromatography anionic activity was separated from cationic activity. The former could be further separated by Sephadex G-200 gel chromatography into two peaks, whose molecular weights were 40 000 and < 10 000. The cationic activity was partially purified by concanavalin A (ConA) Sepharose chromatography, hydroxylapatite chromatography and SDS-polyacrylamide gel electrophoresis. The cationic activity was heterogeneous as demonstrated by isoelectric focusing (Ip 9.5–10.4), gel filtration on Bio-Gel P-150 and SDS-polyacrylamide gel electrophoresis (mol. wt 26 000–33 000). Less than 50 ng/ml was required of the factor to give a glial cell stimulation corresponding to that afforded by 1 % of human serum. A thymidine-degrading enzyme, present in human platelets and to a low degree also in human serum, was found to interfere with the assay for multiplication-stimulating activity. The enzyme (probably a thymidine phosphorylase) converted [³H]thymidine to [³H]thymine, causing a reduced incorporation of ³H into cellular DNA. This difficulty was circumvented by use of an autoradiographic estimation (per cent labelled nuclei) of the multiplication-stimulating activity.     

Purification and properties of the major sialoglycoprotein of the milk fat globule membrane

The major periodate-Schiff positive component (glycoprotein-2) of bovine milk fat globule membranes (MFGM) has been purified by extraction of washed cream with chloroform/methanol followed by chromatography on Sephadex G-200 in sodium dodecyl sulfate. The glycoprotein is > 95% pure by polyacrylamide electrophoresis in dodecyl sulfate and shows the same prominent component at gel percentages of from 5 to 12.5. The molecular weight obtained by extrapolation of the apparent molecular weights on these gels to higher gel percentages was 70,000. An apparent molecular weight of 105,000 was obtained by gel filtration in 1% dodecyl sulfate on Sepharose 4B. The glycoprotein contains 50% carbohydrate by weight, with sialic acid (30.5%), N-acetylglucosamine (22.3%), galactose (15.9%), N-acetylgalactosamine (14.0%), mannose (11.1%), and fucose (5.8%) being the major monosaccharides. Leucine, glutamic acid, and glycine are the major amino acids. Affinity chromatography of deoxycholate-solubilized MFGM indicates that glycoprotein-2 is not the major concanavalin A receptor of these membranes.     

The concanavalin a receptor from human erythrocytes in lipid bilayer membranes. Interaction with concanavalin A and succinyl-concanavalin A

The concanavalin A receptor from human erythrocyte membranes has been isolated by affinity chromatography using the mild, readily-dialyzable detergent dodecyltrimethylammonium bromide. The purified protein has been reincorporated into large unilamellar phospholipid vesicles using a detergent dialysis technique. The mean diameter of these vesicles increases as the lipid: protein ratio decreases. Binding of succinyl-concanavalin A to these vesicles was quantitated using ¹²⁵I-labelled lectin in a filtration assay. The concanavalin A receptor in lipid bilayer vesicles provides specific high affinity binding sites for succinyl-concanavalin A with an association constant of 2.13·10⁶ M^?1. Scatchard plots indicate positive cooperativity of binding at very low lectin concentrations, a characteristic also seen in concanavalin A binding to intact human erythrocytes. The presence of bovine serum albumin has little effect on lectin binding and is not required for expression of cooperativity. Concanavalin A effectively competes with succinyl-concanavalin A for binding to the vesicles with an association constant of 4.83·10⁶ M^?1. Receptor-bearing vesicles are readily agglutinated by concanavalin A but not by its succinylated derivative. The kinetics of vesicle agglutination are biphasic, with an initial rapid phase followed by a pseudo-first order process. We suggest that studies on reassembled receptor proteins in lipid bilayers can provide valuable insight into receptor involvement in transmembrane signalling events and the factors involved in cell membrane behaviour and cell agglutination.     

Concanavalin A and wheat germ agglutinin receptor activity of sialoglycopeptides isolated from the surface of Novikoff hepatoma cells

Novikoff ascites hepatoma cells were highly agglutinable by the plant lectins concanavalin A and wheat germ agglutinin. Treatment of the intact cells with papain released from the cell surface a glycopeptide fraction which possessed concanavalin A and wheat germ agglutinin receptor activity, as judged by its ability to inhibit lectin-induced hemagglutination. A component of the cell-surface glycopeptide fraction, excluded from Sephadex G-50, possessed lectin receptor activities reflecting the cytoagglutination properties of the intact cells from which it was derived. Further resolution of this component by pronase digestion, gel filtration, and ion-exchange chromatography resulted in the isolation of sialoglycopeptides which exhibited potent and specific concanavalin A receptor activity.     

A copper-binding immunoglobulin from a myeloma patient. Purification, identification, and physical characterization.

A copper.protein complex present in the serum of a hypercupremic myeloma patient has been purified to homogeneity using gel filtration, DEAE-cellulose chromatography, and concanavalin A/Sepharose affinity chromatography. Immunoelectrophoresis and hemagglutination inhibition tests showed the copper-bound protein to be an IgG1-type immunoglobulin with lambda light chains. The immunoglobulin is of normal molecular weight (150,000) with normal size light and heavy chains (28,000 and 56,000, respectively). The carbohydrate portion of the molecule appears to be abnormal in that it interacts with concanavalin A, whereas most immunoglobulins of the gammaG-type do not. The copper in the native copper.IgG complex is in an EPR-indeterminable valence state. Copper was efficiently removed from the copper.IgG complex by dialysis against 0.1 M potassium cyanide. The apo-IgG was separated from the copper.cyanide complex by gel filtration. The copper complex was reconstituted by equilibrating the apo-IgG with 7.7 muM cupric ions.