Purification and properties of two lectins from the latex of the euphorbiaceous plants Hura crepitans L. (sand-box tree) and Euphorbia characias L. (Mediterranean spurge).

1. From the latex of two members of the plant family Euphorbiaceae, Hura crepitans L. (sand-box tree) and Euphorbia characias L. (Mediterranean spurge), two lectins were purified by affinity chromatography on acid-treated Sepharose 6B followed by elution with D-galactose. 2. The lectin from E. characias is a single molecular species with Mr 80 000, made up of two identical subunits with Mr 40 000, and is a glycoprotein containing 11% carbohydrate. 3. The lectin from H. creptians appears as a mixture of three isolectins with Mr 140 000, consisting of four different subunits with Mr values 37 500, 35 500, 31 000, and 29 000. 4. Both lectins have haemagglutinating activity, with no specificity for human blood groups. The haemagglutinating activity is inhibited by D-galactose and by galactose-containing oligosaccharides. 5. The lectin from H. crepitans is mitogenic to human T-, but not to B-, lymphocytes. The latex of E. characias is mitogenic to T- and, to a lesser extent, to B-, lymphocytes, but the purified E. characias lectin has no mitogenic activity. 6. The lectin from H. crepitans, but not that from E. characias, inhibits protein synthesis by a rabbit reticulocyte lysate.     

Biosynthesis and glycosylation of the insulin receptor. Evidence for a single polypeptide precursor of the two major subunits

The biosynthesis and carbohydrate processing of the insulin receptor were studied in cultured human lymphocytes by means of metabolic and cell surface labeling, immunoprecipitation with anti-receptor autoantibodies, and analysis on sodium dodecyl sulfate-polyacrylamide gels under reducing conditions. In addition to the two major subunits of Mr = 135,000 and Mr = 95,000, two higher molecular weight bands were detected of Mr = 210,000 and Mr = 190,000. The Mr = 210,000 band and the two major subunits were labeled by [3H]mannose, [3H]glucosamine, [3H]galactose, and [3H]fucose, and were bound by immobilized lentil, wheat germ, and ricin I lectins. On the other hand, the Mr = 190,000 band was labeled only by [3H]mannose and [3H]glucosamine and was bound only by lentil lectin. All four components could be labeled with [35S] methionine; however, in contrast with the other three polypeptides, the Mr = 190,000 band was not labeled by cell surface iodination with lactoperoxidase, suggesting that it is not exposed at the outer surface of the plasma membrane. Pulse-chase studies with [3H]mannose showed that the Mr = 190,000 was the earliest labeled component of the receptor; radioactivity in this band reached a maximum 1 h after the pulse, clearly preceded the appearance of the other components, and had a very brief half-life (t1/2 = 2.5 h). The Mr = 210,000, Mr = 135,000, and Mr = 95,000 bands were next in appearance and reached a maximum 6 h in the chase period. Monensin, an ionophore which interferes with maturation of some proteins, blocked both the disappearance of the Mr = 190,000 protein and the appearance of the Mr = 135,000 and Mr = 95,000 subunits. The mannose incorporated in the Mr = 190,000 component was fully sensitive to treatment with endoglycosidase H while that in the Mr = 210,000 band and the two major subunits was only partially sensitive. Tryptic fingerprints of the 125I-labeled Mr = 210,000 band suggested that this component contains peptides of both the Mr = 135,000 and Mr = 95,000 subunits. In conclusion, the Mr = 190,000 component appears to represent the high mannose precursor form of the insulin receptor that undergoes carbohydrate processing and proteolytic cleavage to generate the two major subunits. In addition, the Mr = 210,000 band is probably the fully glycosylated form of the precursor that escapes cleavage and is expressed in the plasma membrane.     

Calcium and lymphocyte activation.

The role of ionized calcium in the early phases of activation of human peripheral blood lymphocytes was evaluated by stimulating the cells with a calcium ionophore A23187 (Lilly) or with mitogenic lections over a broad range of extracellular calcium concentrations (< 1 to > 1000 μM). A number of biochemical parameters shown previously to be altered during stimulation of these cells by mitogenic lectins were studied including: 1) amino acid transport, 2) phosphatidylinositol turnover, 3) cyclic nucleotide accumulation, and 4) calcium uptake. The ionophore (0.1–0.5 μg/ml) was shown to produce stimulatory effects in all of these systems with the changes closely simulating those produced by the lectins themselves both in regard to time course and magnitude. A23187 also produced 5–10 fold increases in DNA synthesis as measured at 48–72 hr after exposure of the cells to this agent. The responses to A23187 were shown to be almost completely dependent on the presence of ionized calcium. Since mitogenic lectins are known to stimulate calcium uptake and DNA synthesis appears to require extracellular calcium, the early responses to A23187 suggested that calcium was important both during the early and later phases of lymphocyte activation. However, short time course studies of amino acid transport, cyclic AMP accumulation, and phosphatidylinositol turnover in calcium deficient media failed to provide convincing evidence of calcium dependency in lectin stimulation since the three responses were well preserved (<25% inhibition) in “calcium free” medium containing 1–3 mM ethylene bis (ethylene oxynitrilo) tetraacetic acid (EGTA) (an estimated final Ca²⁺ concentration of <1 μM). Greater than 50% inhibition of the lectin response was seen only when the cells were incubated in calcium free, EGTA-containing medium for 30 min prior to stimulation with lectin. Thus despite the striking ability of A23187 complexed with calcium to mimic the action of mitogenic lectins, its effects may involve more than simple transport of calcium into the cell. A23187 may also exert a direct membrane action as suggested by its ability to produce rapid increases in cAMP and the occurrence of cytotoxicity at 5–10 fold higher concentrations (2–4 μg/ml). However, these data do not entirely exclude a mechanism of ionophore action whereby: 1) mobilization of intracellular stores of calcium and 2) diminished intracellular transport of ionized calcium at extracellular concentrations less than or equal to 1 μM combine to provide an effective stimulus for cellular activation.     

The protozoan parasite Cryptosporidium parvum possesses two functionally and evolutionarily divergent replication protein A large subunits

Very little is known about protozoan replication protein A (RPA), a heterotrimeric complex critical for DNA replication and repair. We have discovered that in medically and economically important apicomplexan parasites, two unique RPA complexes may exist based on two different types of large subunit RPA1. In this study, we characterized the single-stranded DNA binding features of two distinct types (i.e. short and long) of RPA1 subunits from Cryptosporidium parvum (CpRPA1A and CpRPA1B). These two proteins differ from human RPA1 in their intrinsic single-stranded DNA binding affinity (K) and have significantly lower cooperativity (omega). We also identified the RPA2 and RPA3 subunits from C. parvum, the latter of which had yet to be reported to exist in any protozoan. Using fluorescence resonance energy transfer technology and pull-down assays, we confirmed that these two subunits interact with each other and with CpRPA1A and CpRPA1B. This suggests that the heterotrimeric structure of RPA complexes may be universally conserved from lower to higher eukaryotes. Bioinformatic analyses indicate that multiple types of RPA1 are present in the other apicomplexans Plasmodium and Toxoplasma. Apicomplexan RPA1 proteins are phylogenetically more related to plant homologues and probably arose from a single gene duplication event prior to the expansion of the apicomplexan lineage. Differential expression during the life cycle stages in three apicomplexan parasites suggests that the two RPA1 types exercise specialized biological functions.     

Isolation and characterization of lectins from Vicia villosa. Two distinct carbohydrate binding activities are present in seed extracts

An uncharacterized lectin from Vicia villosa seeds has been reported to bind specifically to mouse cytotoxic T lymphocytes (Kimura, A., Wigzell, H., Holmquist, G., Ersson, B., and Carlsson, P., (1979) J. Exp. Med. 149, 473-484). We have found that V. villosa seeds contain at least three lectins which we have purified by affinity chromatography on a column of immobilized porcine blood group substances eluted with varying concentrations of N-acetylgalactosamine and by anion exchange chromatography. The three lectins are composed of two different subunits with Mr = 35,900 (subunit B) and 33,600 (subunit A), estimated from their mobility on sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Sedimentation equilibrium analysis suggests that the purified lectins are tetramers. They have been designated B4, A4, and A2B2 to indicate their apparent subunit compositions. The purified B4 and A4 lectins contain 6.7-9.8% carbohydrate by weight; in addition, both are rich in the acidic and hydroxylic amino acids and lack cysteine and methionine. The A4 lectin agglutinates A erythrocytes specifically and binds to A1 erythrocytes (273,000 sites/cell) with an association constant of 1.8 X 10(7) M-1. Although a blood group A agglutinating activity was recognized in the original preparation of V. villosa lectins, lectins with this activity were obtained in relatively small amounts from seed extracts. The predominant lectin in V. villosa seeds, B4, does not agglutinate A, B, or O erythrocytes.     

Benzene dioxygenase in Pseudomonas putida. Subunit composition and immuno-cross-reactivity with other aromatic dioxygenases.

The terminal oxygenase component of benzene dioxygenase from Pseudomonas putida strain ML2 was shown to contain two subunits, of Mr 54,500 and 23,500, by SDS/polyacrylamide-gel electrophoresis. The native Mr of the terminal oxygenase was estimated to be 168,000 +/- 4000. Polyclonal antibodies raised against each of the subunits cross-reacted with two polypeptides in cell-free extracts from toluene-grown Pseudomonas putida strain N.C.I.B. 11767. The Mr values of these polypeptides were similar to those reported for the subunits from the terminal dioxygenase component of toluene dioxygenase. These polypeptides were present only when this strain was grown on toluene. No cross-reactivity was observed with subunits of the naphthalene dioxygenase or benzoate dioxygenase systems.     

Characterization of the trimeric, self-recognizing Geodia cydonium lectin I

A D-galactose-specific lectin I was extracted from the sponge Geodia cydonium and purified by affinity chromatography. The molecular weight of lectin I as determined by high-pressure liquid gel chromatography, was found to be 36500 +/- 1300. Disc gel electrophoresis in the presence and in the absence of sodium dodecyl sulfate showed that lectin I is a trimer composed of three different subunits (Mr: 13800, 13000 and 12200); two of the three subunits are linked by one disulfide bond. Isoelectric focusing gave a pI of 5.6 for the native molecule and a pI of 4.4 and of 7.4 for the subunits. The three subunits carry carbohydrate side chains, composed of D-galactose (94%) and of arabinose (5%). Based on experiments with lectins, the terminal D-galactose residues are bound by beta 1 leads to 6 and/or beta 1 leads to 4 glycosidic linkages. The Geodia lectin I contains, besides two carbohydrate recognition sites, at least one receptor site for a second lectin I molecule.     

Galactosyl-binding lectins from the tunicate Didemnum candidum. Purification and physicochemical characterization

The plasma of the ascidian Didemnum candidum possesses lectin activity directed toward galactosyl moieties. We report the purification by affinity chromatography, the physicochemical properties, amino acid composition, and partial N-terminal amino acid sequence of two galactosyl-binding lectins D. candidum lectins I and II (DCL-I and DCL-II) from the plasma of this protochordate species. Both lectins were purified by affinity chromatography (on acid-treated Sepharose 4B and asialofetuin conjugated to Sepharose 4B) to homogeneity as judged by immunoelectrophoresis, size exclusion chromatography on high performance liquid chromatography, and polyacrylamide gel electrophoresis. Isoelectric focusing in polyacrylamide gels revealed that DCL-I focuses as a family of bands at pH 3.8-5.2, while DCL-II focuses at pH 9.2-10.2. Gas chromatography analyses of alditol acetate derivatives indicated that no carbohydrate components are associated with the lectins. Approximate subunit molecular weights estimated by polyacrylamide gel electrophoresis and size exclusion chromatography on high performance liquid chromatography in 6 M guanidine HCl under reducing conditions were 13,400-14,500 for DCL-I and 14,500-15,500 for DCL-II. Native molecular weights estimated by sedimentation equilibrium were 56,600 (DCL-I) and 57,500 (DCL-II), indicating that both species are constituted by four equal-sized subunits. Frictional ratios suggested that both lectins are globular proteins. Using rabbit antisera, the two molecules appeared serologically distinct. The extinction coefficient for DCL-I was E280 mg/ml = 2.52 ml mg-1 cm-1. Circular dichroism analyses of DCL-I suggested 29% alpha-helix and 37% beta-structure in the protein. Excitation/emission fluorescence spectra for DCL-I yielded maximum excitation and emission wavelengths at 288 and 330 nm, respectively. Amino acid compositions of DCL-I and DCL-II differed mainly in the proportions of aspartic and glutamic acids, serine, alanine, cysteine, valine, phenylalanine, and histidine. Amino acid compositions of DCL-I and DCL-II were compared to each other and to immunoglobulins and putative recognition molecules by the parameter S delta Q. DCL-I exhibited similarities in amino acid composition to lectins from the tunicate Halocynthia pyriformis, the lamprey Petromyzon marinus, and the horseshoe crab Carcinoscorpius rotundicauda, rabbit C-reactive protein, and lamprey and carp immunoglobulin mu chains. DCL-II showed amino acid composition and similarities with several fish immunoglobulin light chains, immunoglobulin-related molecules isolated from mouse and marmoset T cells, and carp and goldfish immunoglobulin heavy chains.(ABSTRACT TRUNCATED AT 400 WORDS)     

A novel mannose-specific and sugar specifically aggregatable lectin from the bark of the Japanese pagoda tree (Sophora japonica)

A new D-mannose/D-glucose-specific lectin (B-SJA-II) was isolated from the bark of the Japanese pagoda tree, Sophora japonica. B-SJA-II was separated from a well known D-galactose/N-acetyl-D-galactosamine-specific lectin (B-SJA-I) by affinity chromatography on lactamyl-Sepharose, then purified by affinity chromatography on maltamyl-Sepharose. On sodium dodecyl sulfate-polyacrylamide gel electrophoresis, B-SJA-II gave four bands: subunit a-1 (Mr = 19,400), a-2 (Mr = 18,200), b-1 (Mr = 15,000), and b-2 (Mr = 13,200). Carbohydrate analysis and binding study with horseradish peroxidase-labeled lectins on the bands electroblotted onto polyvinylidene difluoride membrane showed that the three subunits other than b-2 have N-linked oligosaccharides typical of plant glycoproteins. The binding assay with horseradish peroxidase-glycoproteins revealed that all the subunits can bind sugar specifically with fetuin and asialofetuin. Furthermore, B-SJA-II aggregated to form precipitates in the absence of a specific sugar and became soluble upon addition of the specific sugar. The results indicate that each subunit has a sugar-binding site for the mannosyl core of N-linked oligosaccharide chains and recognizes each other sugar specifically to form aggregates. According to the N-terminal amino acid sequences obtained, the subunits are classified into two groups. The first group (a-1 and a-2) has an N-terminal sequence 50% identical with that of other S. japonica lectins (Hankins, C. N., Kindinger, J. I., and Shannon, L. M. (1988) Plant Physiol. 86, 67-70) and the amino acid sequence initiating at position 123 of concanavalin A (Cunningham, B. (1975) J. Biol. Chem. 250, 1503-1512), while the N-terminal sequence of the second group (b-1 and b-2) is homologous to that of concanavalin A, but completely different from that of the first group.     

Isolation and characterization of a lectin from the seeds of Dioclea lehmanni.

Affinity chromatography of the globulin fraction from the seeds of Dioclea lehmanni on Sephacryl S-200 yielded two lectins, one slightly retarded and another strongly bound. The latter, which was a glucose/mannose specific lectin, was purified and the following properties were determined: pI, Mr of subunits, carbohydrate content, A, aminoacid composition, hemagglutination and inhibition patterns, N-terminal sequence and mitogenic activity. These properties of the lectin were very similar to those of the Con A and Dioclea grandiflora lectins.     

Purification and characterization of natural human interferon omega 1. Two alternative cleavage sites for the signal peptidase.

Human interferon omega 1 (IFN-omega 1 = IFN-alpha II1) is a recently discovered protein structurally related to IFN-alpha and -beta; the biological activities of IFN-omega 1 and its physiological role are not known to date. We have purified IFN-omega 1 from preparations of human leukocyte IFN, derived from peripheral blood leukocytes induced with Sendai virus, by two sequential cycles of monoclonal antibody affinity chromatography. The resulting protein was at least 95% pure as determined by reverse-phase high performance liquid chromatography and showed an Mr of 24,500 upon sodium dodecyl sulfate-gel electrophoresis (theoretical Mr, 19,984). Amino acid sequence analysis revealed that only about 40% of the molecules have the NH2 terminus expected on the basis of the sequence similarity to IFN-alpha, whereas the others contain two additional amino acids. This difference probably results from variable cleavage of the pre-protein by the signal peptidase. No evidence for COOH-terminal heterogeneity was found. Essentially all IFN-omega 1 molecules are glycosylated; enzymatic deglycosylation resulted in a reduction of the Mr to 20,500. Experiments using several plant lectins indicated the presence of biantennary complex oligosaccharides containing neuraminic acid. Two major peaks were observed upon chromatofocusing, with isoelectric points of 8.1 and 8.5. The specific antiviral activity of purified IFN-omega 1 assayed on human cells was determined to be 2.7 x 10(8) IU/mg, similar to that of other human class I IFNs; potent antiviral activity was also observed on cells of bovine and ovine but not of equine or murine origin.     

Isolation and partial characterization of an N-acetylgalactosamine-specific lectin from winter-aconite (Eranthis hyemalis) root tubers.

A lectin was isolated from root tubers of winter aconite (Eranthis hyemalis) by affinity chromatography on fetuin-agarose, and it was partially characterized with respect to its biochemical, physicochemical and carbohydrate-binding properties. The Eranthis hyemalis lectin is a dimeric protein (Mr 62000) composed of two different subunits of Mr 30000 and 32000, held together by disulphide bonds. It is especially rich in asparagine/aspartic acid, glutamine/glutamic acid and leucine, and contains 5% covalently bound carbohydrate. Hapten inhibition assays indicated that the winter-aconite lectin is specific for N-acetylgalactosamine. In addition, the lectin exhibits a pronounced specificity towards blood-group-O erythrocytes. The winter-aconite lectin is the first lectin to be isolated from a species belonging to the plant family Ranunculaceae. It appears to be different from all previously described plant lectins.     

Opposite effects on human colon cancer cell proliferation of two dietary Thomsen-Friedenreich antigen-binding lectins

Increased cell surface expression of the Thomsen-Friedenreich antigen (TF antigen, Galbeta1-3GalNAcalpha-) is a common feature in malignant and pre-malignant epithelia. Our previous studies have shown that dietary TF-binding lectins from peanut (Arachis hypogea) and edible mushroom (Agaricus bisporus) produce marked but different effects on human intestinal epithelial cell proliferation. This study investigates the proliferative effects of the other two known dietary TF-binding lectins: jacalin (Artocarpus integrifolia, JAC) and amaranth lectin (Amaranthus caudatus, ACA). JAC produced dose-dependent and non-cytotoxic inhibition of proliferation in HT29 human colon cancer cells with maximal effects of 46 +/- 4% at 20 microg/ml, whereas ACA produced dose-dependent stimulation of proliferation with maximal effects of 22 +/- 3% at 20 microg/ml when assessed both by incorporation of [3H]thymidine into DNA and by cell counting. The lectin-mediated effects were inhibitable by the presence of appropriate Galbeta1-3GalNAc-expressing glycoproteins but differences existed between JAC and ACA in their patterns of inhibition by such substances. Ligand binding equilibrium studies using iodinated lectins revealed different Kd of the two lectins for HT29 cell surface glycoproteins. Lectin blots of cell membrane extracts showed different binding patterns in all the four TF-binding lectins. These results provide further evidence that dietary TF-binding lectins can have marked effects on the proliferation of human malignant gastro-intestinal epithelial cells and hence may play a role in intestinal cancer development, and also show that the biological effects of dietary lectins cannot be predicted solely from their carbohydrate binding properties.     

Purification, characterization and comparison of two non-haem bromoperoxidases from Streptomyces aureofaciens ATCC 10762.

Two non-haem bromoperoxidases (BPO 1 and BPO 2) were purified from the 7-chlorotetracycline-producing strain Streptomyces aureofaciens ATCC 10762. Both enzymes showed azide-insensitive brominating activity, and bromide-dependent peroxidase activity. BPO 1 was a dimer (Mr 65,000) with subunits of identical size (Mr 31,000). The pI was estimated to be 4.5. The enzyme did not cross-react with antibodies raised against the non-haem bromoperoxidase (Mr 90,000) from S. aureofaciens Tü24, a strain that also produces 7-chlorotetracycline. The Mr of BPO 2 was estimated to be 90,000. The enzyme had three identical subunits (Mr 31,000), and its isoelectric point was 3.5, identical with that of the bromoperoxidase from S. aureofaciens Tü24. Moreover, BPO 2 was immunologically identical with the bromoperoxidase from S. aureofaciens Tü24, although both it and BPO 1 could be distinguished electrophoretically from the latter bromoperoxidase.     

Quaternary structure of higher plant glyceraldehyde-3-phosphate dehydrogenases.

1. NAD(P)+-induced changes in the aggregational state of prepurified NADP-linked glyceraldehyde-3-phosphate dehydrogenase (EC 1.2.1.13) were used to isolate the enzyme from Spinacia oleracea, Pisum sativaum and Hordeum vulgare. Each of the three plant species contains two separate isoenzymes. Isoenzyme 1 (fast moving during conventional electrophoresis) precipitates with the ammonium sulfate fraction 55--70% saturation. It shows two separate subunits in dodecylsulfate gels, which are probably arranged as A2B2 in the native enzyme molecule. Isoenzyme 2 (slow moving during conventional electrophoresis) precipitates with the ammonium sulfate fraction 70--95%. It contains a sigle subunit of the same Mr as subunit A in isoenzyme 1 and is apparently a tetramer (A4). The molecular weights of subunits A/B for spinach, peas and barley were determined as 38,000/40,000, 38,000/42,000 and 36,000/39,000 respectively. 2. The NAD-specific glyceraldehyde-3-phosphate dehydrogenase (EC 1.2.1.12) was purified from Spinacia oleracea and Pisum sativum by affinity chromatography on blue Sepharose CL-6B. The enzyme from both plant species is shown to be a tetramer of subunits with Mr 39,000. 3. The present findings contrast with heterogeneous results obtained previously by other authors. These results suggested that there are considerable interspecific differences in the quaternary structure of glyceraldehyde-3-phosphate dehydrogenases from higher plants.     

Apolipoproteins and the association of egg yolk proteins with plasma high density lipoproteins after ovulation and follicular atresia in the rainbow trout (Salmo gairdneri)

The apolipoproteins of trout plasma lipoproteins have been characterized by sodium dodecyl sulfate-glycerol polyacrylamide gel electrophoresis. The high density lipoproteins (HDL) (1.085 less than d less than 1.21 g/ml) contain four apolipoproteins, two major species with Mr 25,000 (apoA-I-like) and Mr 13,000 (apoA-II-like) and two minor species (Mr 55,000 and 40,500). The very low density (d less than 1.015 g/ml) and low density lipoproteins (1.015 less than d less than 1.085 g/ml) contain two high Mr apolipoproteins (apoB-like) with Mr 260,000 and 240,000 (the smaller is the preponderant species in low density lipoproteins), as well as a third apolipoprotein with Mr 76,000. Type A apolipoproteins are present in the very low density lipoproteins, as are a group of apolipoproteins with Mr 9,000-11,000 (apoC-like). Egg yolk proteins appear in the plasma of females about 30 days after natural ovulation or after that induced by salmon gonadotropin and during massive intraovarian atresias, either spontaneous or induced by 17 alpha,20 beta-dihydroxy-4-pregnen-3-one. Two egg yolk proteins intimately associated with HDL have been identified. They may account for as much as 35% of total plasma proteins. Lipovitellin (Mr 112,000) is composed of two subunits in a 1:1 molar ratio (lipovitellin 1 with Mr 92,000 and lipovitellin 2 with Mr 20,000) and is present as a dimer with another yolk protein (Mr 10,000). These results show that resorption of the yolk during follicular atresia in an oviparous vertebrate is correlated with the presence of egg yolk proteins combined with HDL in the plasma.     

Carbohydrate-binding proteins of human serum: isolation of two mannose/fucose-specific lectins

Two lectins with specificities for mannose and fucose have been isolated from human serum by affinity chromatography. One mannose-binding protein (MBP 1) has a native Mr of 700,000 with subunits of Mr 32,000 and has specificities for N-acetylglucosamine, N-acetylmannosamine and glucose as well as for mannose and fucose. The other mannose-binding protein (MBP 2) has a native Mr of 200,000 with subunits of Mr 28,000 and is specific only for mannose and fucose. MBP 2 appears to recognize the core sugars of asparagine-linked oligosaccharides as well as the terminal sugars. Both lectins are calcium-dependent, requiring approx. 0.095 mM calcium for half-maximal binding. MBP 1 binds maximally between pH 7-9, whereas MBP 2 has a pH optimum of 6-7. The binding activity of both proteins decreases rapidly below pH 5. The apparent association constants (Ka) for binding to mannon are 2.1 X 10(8) M-1 for MBP 1 and 1.3 X 10(8) M-1 for MBP 2. These data provide further evidence of the complex nature of mammalian carbohydrate recognition systems.     

Purification and characterization of a novel, oligomeric, plasminogen kringle 4 binding protein from human plasma: tetranectin

Purification of alpha 2-plasmin inhibitor (alpha 2PI) from human plasma by affinity chromatography on plasminogen-Sepharose resulted in copurification of a contaminating protein with Mr 17,000 as judged by sodium dodecyl sulphate/polyacrylamide gel electrophoresis. This contaminating protein could not be removed from the purified alpha 2-PI preparation by several types of gel chromatography applied. The use of the kringle 1-3 part of plasminogen, K(1 + 2 + 3), bound to Sepharose for affinity chromatography, instead of plasminogen-Sepharose, resulted in an alpha 2PI preparation without this contaminant. The contaminating protein was found to interact specifically with the kringle 4 part of plasminogen (K4) and not with K(1 + 2 + 3) or miniplasminogen. The K4-binding protein was purified by ammonium sulphate precipitation, affinity chromatography on K4-Sepharose, ion-exchange chromatography and gel filtration on AcA 34. The relative molecular mass of the protein (Mr 68 000) was estimated by gel filtration. This suggests a tetrameric protein composed of four subunits (Mr 17,000), that are dissociated by 1% sodium dodecyl sulphate. Dissociation into subunits was also demonstrated by gel filtration in the presence of 6 M guanidine hydrochloride. A specific antibody was raised in rabbits against the purified protein and this antibody was shown not to react with any known fibrinolytic components. The pI of the K4-binding protein was found to be 5.8. The first three N-terminal amino acids were determined to be Glu-Pro-Pro. The concentration of the protein in plasma was estimated to be 0.20 +/- 0.03 microM (15 +/- 2 mg/l). The electrophoretic mobility of the K4-binding protein was shown by crossed immunoelectrophoresis to be influenced by the presence of Ca2+, EDTA and heparin. The protein was found to enhance plasminogen activation catalyzed by tissue-type plasminogen activator (t-PA) in the presence of poly(D-lysine). The protein appeared to be a novel plasma protein tentatively called 'tetranectin'.     

The morphology of L-fucose, D-mannose specific lectin (SFL 100-2) produced by Streptomyces No. 100-2

The morphology of an L-fucose specific lectin, SEL 100-2, from a Streptomyces sp. was studied. Electron microscopic observation showed that purified SFL 100-2 preparation consisted of particles homogeneous in size. The diameter was 25 nm. The digitized images of these particles had 2-fold rotation symmetry. The sedimentation coefficient (s020,w) was determined to be 20.6S. The particle weight and the Stokes radius were calculated to be 8.0 X 10(5) daltons and 94 A, respectively, by three independent methods, i.e., gel filtration, sedimentation equilibrium and velocity measurements. The frictional ratio (f/fmin) was estimated to be 1.53. These values are quite similar to those of human alpha 2-macroglobulin. 125I-Labeled peptide mapping indicated that these particles were built up of about twelve identical subunits (Mr = 68,000). The size of SFL 100-2 in culture broth was found to be the same as that of the particles in the purified preparations. The shape and other properties of SFL 100-2 are discussed and compared with those of the tail of lambda phage and type 1 pili of Escherichia coli, whose amino acid compositions were quite similar to that of SFL 100-2 and also those of L-fucose specific plant lectins.     

Isolation and characterization of a lectin from the snail Biomphalaria glabrata and a study of its combining site

The hemagglutinins from the spawn of the water snail Biomphalaria glabrata were isolated by affinity chromatography on hog gastric mucin coupled to Sepharose 4B. The N-acetyl-D-glucosamine eluate (0.1 M) was fractionated further on Bio-Gel P-300, yielding two fractions. Fraction 1 had an Mr of 350 000 and displayed one band in immunoelectrophoresis, but was heterogeneous in discontinuous electrophoresis. It agglutinated human red blood cells with A1 and B specificity at concentrations of 12 and 72 micrograms nitrogen/ml, respectively. Fraction 2 had an Mr on gel filtration of 67 000 and was homogeneous in immuno- and polyacrylamide electrophoresis, and in isoelectrofocusing. It is composed of three subunits with Mr of 17 000 and one smaller subunit of 15 000. This fraction (lectin I) is a glycoprotein containing 6% hexoses and 2.5% hexosamines. For minimal agglutination of human A1 and B red blood cells 2.4 and 72.0 micrograms nitrogen/ml, respectively, of lectin I were required. O red blood cells were not agglutinated. Lectin I precipitated well with a human blood group substance of A1 specificity, moderately with a B- and poorly with an H-substance. Precipitin-inhibition studies revealed that among other sugars N-acetylneuraminic acid was the most potent inhibitor. Immunofluorescence studies confirmed the good interaction of lectin I with receptors of A1 and B erythrocytes and the failure of lectin I to attach to O-erythrocytes. Since N-acetylneuraminic acid is present on the cell surface of all human erythrocytes, it cannot be the dominant part of the receptor for the B. glabrata lectin I, despite its effectiveness as an inhibitor.