Five alpha-D-galactopyranosyl-binding isolectins from Bandeiraea simplicifolia seeds.

The alpha-D-galactopyranosyl-binding lectin previously purified from Bandeiraea simplicifolia seeds (Hayes, C.H., and Goldstein, I.J. (1974) J. Biol. Chem. 249, 1904) is shown to consist of five isolectins separable on polyacrylamide gel electrophoresis at pH 9.5. The isolectins are tetrameric structures composed of various combinations of two different glycoprotein subunits designated A and B. The A and B subunits appear to be immunochemically indistinguishable against rabbit antisera prepared from the isolectin mixture. The A subunit contains no methionine, whereas the B subunit contains 1 residue. The subunits migrate differently on polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate and, although each subunit contains 1 residue of cysteine, they react differently toward 5,5'-dithiobis(2-nitrobenzoic acid). The carbohydrate binding specificity of the two subunits differs significantly: the A subunit exhibits a primary specificity for alpha-D-GalNAcp but also reacts with alpha-D-Galp units, whereas the B subunit shows a sharp specificity toward alpha-D-Galp residues. The differences in carbohydrate binding specificity were exploited in separating the isolectins. B. simplicifolia I isolectins (A4) and (A3B) were purified on a Bio-Gel melibionate column, and (A2B2), (AB3), and (B4) were separated on a column of insolubilized blood group A substance.     

Isolation of a gastrin releasing peptide receptor from normal rat pancreas.

The presence of a putative GRP receptor on rat pancreatic particulate membranes was demonstrated by covalent cross-linking to 125I-gastrin releasing peptide (GRP), which revealed a radioactive band with Mr = 80-90 kDa on reduced SDS-PAGE. Fresh rat pancreatic membranes contained a GRP receptor which was solubilized with Triton X-100 as assessed by its failure to sediment at 100,000 x g for one hour and its ability to pass through a 0.22 mu filter. When 125I-GRP binding was studied using Sephadex G50 gel filtration chromatography to separate bound from unbound ligand, substantial amounts of 125I-GRP binding were observed in rat crude solubilized pancreatic membranes, but essentially no specific binding was observed until the crude solubilized membranes were fractionated by ammonium sulfate precipitation. Specific 125I-GRP binding was 500, 700 and 1400 fmol/mg protein, respectively, in the 0-25%, 25-50% and 50-80% saturated ammonium sulfate fractions (125I-GRP concentration = 1 nM). Specific binding was temperature dependent, saturable and of high affinity, (KD = 2.3 nM). A unique 70 kDa band was visualized by silver staining of the SDS-PAGE of eluates of GRP(14-27) affinity gel compared with eluates of control affinity gels incubated with the 25-50% (NH4)2SO4 fraction. The lower Mr than that observed with covalent cross-linking may represent the binding subunit of a larger receptor protein. This ligand-affinity isolated protein is thus a good candidate for the GRP receptor, or the binding subunit of it, from normal rat pancreas.     

A structural comparison of the A and B subunits of Griffonia simplicifolia I isolectins

A structural comparison between the A and B subunits of the five tetrameric Griffonia simplicifolia I isolectins (A4, A3B, A2B2, AB3, B4) was undertaken to determine the extent of homology between the subunits. The first 25 N-terminal amino acids of both A and B subunits were determined following the enzymatic removal of N-terminal pyroglutamate blocking groups with pyroglutamate aminopeptidase. Although 21 amino acids were common to both subunits, there were four unique amino acids in the N-terminal sequence of A and B. Residues 8, 9, 17, and 19 were asparagine, leucine, lysine, and asparagine in subunit A and threonine, phenylalanine, glutamic acid, and serine in subunit B. The last six C-terminal amino acids, released by digestion with carboxypeptidase Y, were the same for both subunits: Arg-(Phe, Val)-Leu-Thr-Ser-COOH. Subunit B, which contains one methionyl residue, was cleaved by cyanogen bromide into two fragments, a large (Mr = 31,000) and a small (Mr = 2700) polypeptide. Failure of the small fragment to undergo manual Edman degradation indicated an N-terminal blocking group, presumably pyroglutamate. Both subunits were digested with trypsin and the tryptic peptides were analyzed using reverse-phase HPLC. Tryptic glycopeptides were identified by labeling the carbohydrate moiety of the A and B subunit using sodium [3H] borohydride. Cysteine-containing tryptic peptides were similarly identified by using [1-14C]iodoacetamide. Approximately 30% of the tryptic peptides were common to both subunits. Thus, although the N- and C-terminal regions of A and B are similar, the subunits each possess unique sequences.     

Solubilization of human placental tumor necrosis factor receptors as a complex with a guanine nucleotide-binding protein.

Human placental membranes exhibited high-affinity receptors for tumor necrosis factor (TNF) (Kd = 5.6 x 10(-10) M) with a density of 1.2-1.7 x 10(10) sites/mg protein. The receptors were solubilized from these membranes with 1% Nonidet P-40, and the solubilized receptor was adsorbed to Con A-Sepharose and wheat germ agglutinin agarose columns, indicating that the TNF receptor derived from human placenta contains carbohydrate chains recognized by these lectins. TNF binding activity was eluted from a column of Sephacryl S-300 as a single peak of Mr 300 kDa. The solubilized receptor was further purified by TNF-Sepharose prepared by coupling of TNF to tresyl-activated Sepharose 4B. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis of the purified sample resolved five major bands of Mr 90, 78, 41, 35, and 11 kDa, suggesting that these polypeptides constitute a multimeric complex with a molecular mass of 300 kDa, as observed in gel filtration study. Furthermore, the TNF-Sepharose-bound fraction demonstrated GTP gamma S binding and GTPase activity. Immunoblot analysis showed that the 41- and 35-kDa polypeptides were recognized by antisera against alpha subunits and beta subunit of GTP-binding proteins, respectively. These results suggest that the native TNF receptor couples to a guanine nucleotide-binding protein to form a large complex structure in human placental membranes.     

Isolectins from Solanum tuberosum with different detailed carbohydrate binding specificities: unexpected recognition of lactosylceramide by N-acetyllactosamine-binding lectins

Glycosphingolipid recognition by two isolectins from Solanum tuberosum was compared by the chromatogram binding assay. One lectin (PL-I) was isolated from potato tubers by affinity chromatography, and identified by MALDI-TOF mass spectrometry as a homodimer with a subunit molecular mass of 63,000. The other (PL-II) was a commercial lectin, characterized as two homodimeric isolectins with subunit molecular masses of 52,000 and 55,000, respectively. Both lectins recognized N-acetyllactosamine-containing glycosphingolipids, but the fine details of their carbohydrate binding specificities differed. PL-II preferentially bound to glycosphingolipids with N-acetyllactosamine branches, as Galbeta4GlcNAcbeta6(Galbeta4GlcNAcbeta3)Galbeta4Glcbeta1C er. PL-I also recognized this glycosphingolipid, but bound equally well to the linear glycosphingolipid Galbeta4GlcNAcbeta3Galbeta4GlcNAcbeta3Galbeta4Glcbeta1Cer. Neolactotetraosylceramide and the B5 pentaglycosylceramide were also bound by PL-I, while other glycosphingolipids with only one N-acetyllactosamine unit were non-binding. Surprisingly, both lectins also bound to lactosylceramide, with an absolute requirement for sphingosine and non-hydroxy fatty acids. The inhibition of binding to both lactosylceramide and N-acetyllactosamine-containing glycosphingolipids by N-acetylchitotetraose suggests that lactosylceramide is also accomodated within the N-acetylchitotetraose/N-acetyllactosamine-binding sites of the lectins. Through docking of glycosphingolipids onto a three-dimensional model of the PL-I hevein binding domain, a Galbeta4GlcNAcbeta3Galbeta4 binding epitope was defined. Furthermore, direct involvement of the ceramide in the binding of lactosylceramide was suggested.     

Purification of the inducible murine macrophage nitric oxide synthase. Identification as a flavoprotein.

The synthesis of nitric oxide (.NO) from L-arginine has been demonstrated in a number of cell types and functions either as a cell signaling agent or as a key component of the cell-mediated immune response. Both constitutive and inducible activities have been described. Herein we report the purification of inducible .NO synthase (EC 1.14.23) from activated murine macrophages using a two-column procedure. Crude 100,000 x g supernatant was passed through a 2'-5'-ADP-Sepharose 4B affinity column followed by a DEAE-Bio-Gel A anion exchange column. The .NO synthase ran as a band of Mr = 130,000 on sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Gel filtration experiments using a Superose 6 HR 10/30 column estimated the native molecular weight to be 260 +/- 30 kDa, indicating that the native enzyme exists as a dimer. Activity was dependent upon L-arginine (Km = 16 +/- 1 microM at 37 degrees C and pH 7.5) and NADPH. Both (6R)-tetrahydro-L-biopterin and FAD enhanced activity, whereas Mg2+ and FMN had no effect on activity. Fluorescence studies demonstrated the presence of one bound FAD and one bound FMN per subunit.     

Characterization of cholecystokinin receptor subunits on pancreatic plasma membranes

To determine the size and subunit structure of the pancreatic cholecystokinin (CCK) receptor, 125I-CCK33 was covalently cross-linked to its receptor on mouse pancreatic acinar plasma membranes utilizing the bifunctional cross-linker disuccinimidyl suberate. When CCK was cross-linked at pH 7.4 to either purified plasma membranes or to isolated pancreatic acini and then followed by preparation of plasma membranes, the major labeled protein band revealed by polyacrylamide gel electrophoresis was Mr = 120,000 in the absence of reducing agent and Mr = 80,000 in the presence of reducing agent. A similar banding pattern was also observed when different cross-linkers, ethylene glycol bis(succinimidyl succinate) or dithiobis (succinimidyl propionate), were employed. At pH 6.0, where CCK binding to its receptors is optimal, the labeling pattern was similar to that seen at 7.4, although the two bands were more heavily labeled. Both the binding of CCK to its receptors on plasma membranes and the appearance of the two cross-linked proteins on gels were inhibited in a parallel manner by increasing concentrations of unlabeled CCK8; similar results were observed with dibutyryl cyclic GMP, a competitive inhibitor of CCK binding and action. The data indicate, therefore, that the CCK receptor possesses subunit structure whereby an Mr = 76,000 binding subunit is linked to an Mr = 40,000 nonbinding subunit by a disulfide bond.     

Purification and preliminary characterization of guinea pig testicular proteinase inhibitors

Three guinea pig testicular, low-molecular-weight, acid-stable inhibitors specific for trypsin-like proteinases were isolated, purified, and characterized. The procedure comprised acid extraction of testicular acetone powder, pH precipitation of the extract, gel filtration of the supernatant on Sephadex G-100 and G-50, ion-exchange chromatography on SP-Sephadex, followed by QAE-Sephadex. Final purification was by rechromatography on Sephadex G-50 superfine gel. The three proteinase inhibitors were labeled A, B, and Cnb, the latter to denote nonbinding of Cnb to the QAE-Sephadex. Components A and Cnb showed competitive, whereas B showed noncompetitive, inhibition against trypsin. All three inhibitors were active against trypsin but were ineffective against chymotrypsin. The inhibition constants, Ki, were obtained using trypsin-catalyzed hydrolysis of the N-benzyloxycarbonyl-L-arginyl amide of 7-amino-4-trifluoro-methylcoumarin (CBZ-Arg-AFC) at pH 8.0. The values were calculated to be, for A, 1.5 x 10(-8) M; for B, 1.5 x 10(-8) M; and, for Cnb, 2.2 x 10(-7) M. The Ki values calculated from inhibition of trypsin-catalyzed hydrolysis of the active site titrant 4-methylumbelliferyl-p-guanidinobenzoate (MUGB) using Easson-Stedman plots were, for A, 7.7 x 10(-9) M; for B, 6.7 x 10(-9) M; and, for Cnb, 1.4 x 10(-7) M. The Mrs as determined by active site titration with MUGB were A, 11.2 kDa; B, 10.5 kDa; Cnb, 17.0 kDa. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis gave Mr values for A of 11 kDa, for B of 4 kDa, and for Cnb of 19 kDa. The discrepancy in Mr values for B indicates that it may function as a dimer or trimer in the active state.     

Lectins from<Emphasis Type="Italic">Griffonia simplicifolia</Emphasis> seeds

The physical-chemical and carbohydrate binding specificity ofGriffonia simplicifolia I (GS I) isolectins, one of the 4 lectins isolated fromGriffonia simplicifolia seeds, are described.Association constants for the binding of methyl α- and β-D-galactopyranoside and methyl 2-acetamido-2-deoxy-α-D-galactopyranoside to the A₄, A₂ B₂ and B₄ isolectins are reported.Precipitation reactions of theGriffonia simplicifolia isolectins with guaran and type B blood group substance are described.The hypothesis that subunit B is a precursor of subunit A, a process involving proteolytic cleavage of the B subunit, was tested by conducting structural studies on the 2 subunits. The results indicated that the A and B subunits are probably products of 2 separate but closely related, possibly contiguous genes.     

Probing the topography of lectins with site-specific spin-labeled glycosides

Three new spin-labeled glycosides, spin-label I [1-[4-(beta-D-galactopyranosyloxy)phenyl]-3-(2,2,6,6-tetramethyl-1 -oxypiperidin-4-yl)-2-thiourea], spin-label II (2,2,6,6-tetramethyl-1-oxypiperidin-4-yl alpha-D-galactopyranoside), and spin-label III [1-(methyl 2-deoxy-alpha-D-galactopyranosid-2-yl)-3-(2,2,6,6- tetramethyl-1-oxypiperidin-4-yl)-2-thiourea], were investigated as structural probes of Griffonia simplicifolia I isolectins (GS I) A4 and B4, respectively, by electron spin resonance (ESR) and inhibition of guaran isolectin precipitation. The p-aminophenyl beta-galactoside spin-label I was strongly immobilized by the B4 isolectin (Kd = 0.42 mM; 2T parallel = 54.0 +/- 0.3 G), while binding to the A4 isolectin was so weak (KI congruent to 2 mM) that binding was undetectable by ESR. The preference for the B4 isolectin was indicative of a more extended hydrophobic binding locus adjacent to the carbohydrate-specific binding site. The alpha-galactosyl spin-label II bound slightly more strongly to the A4 than to the B4 isolectin, as evidenced in both Kd values and particularly by differences in the degree of immobilization (2T parallel = 53.5 vs. 51.5 G, respectively). The 2-N-substituted methyl galactoside spin-label III was so poor an inhibitor of both isolectins (KI congruent to 1-2 mM) that ESR detection of the bound complex was not feasible. In all cases above, the spin-labels were displaced by specific monosaccharide haptens.     

Purification and characterization of a cystatin from the leaves of methyl jasmonate treated tomato plants

A multidomain cystatin was purified from the leaves of mature and seedling tomato plants (Lycopersicon esculentum, cv Bonnie Best) that had been sprayed with methyl jasmonate. For seedlings, cystatin purification was accomplished using EDTA washing, KCI extraction, 70 degrees C heat treatment, ammonium sulfate fractionation and gel filtration chromatography. For mature plants, DEAE chromatography was also needed to separate a protease, hydrolysis products of cystatin and serine proteinase inhibitors from the intact cystatin. Purified tomato cystatin has a molecular weight (Mr) of 88 kDa, eight papain binding domains, is a non-competitive inhibitor of papain with K1 of 1.4 nM and is not a glycoprotein. Tryptic peptides (Mr 26, 13 kDa) and most chymotryptic peptides (Mr 33, 13 kDa) of tomato cystatin retain inhibitor activity. Amino acid analysis revealed no Cys; Asx, Glx, Gly, Ser accounted for almost half the residues and there was some homology with potato multicystatin. Activity is stable at pH 4-11 at 4 degrees C, but unstable at neutral pH at > 60 degrees C (Ea = 92.5 kJ/mole). Extracts of mature plants treated with methyl jasmonate contain lower Mr cystatins that appear to result from the action of an endogenous 26 kDa protease on the 88 kDa inhibitor.     

Studies of functional domains of the regulatory subunit from cAMP-dependent protein kinase isozyme I

Homogenous regulatory subunit from rabbit skeletal muscle cAMP-dependent protein kinase (isozyme I) was partially hydrolyzed with low (1 g/1300 g) or high (1 g/6 g) concentrations of trypsin. After treatment with low trypsin two main peptides (Mr = 35,000 and 12,000) were produced. The cAMP-binding activity (2 mol cAMP/mol of subunit monomer) was recovered in the monomeric Mr = 35,000 peptide. The ability of either fragment to inhibit catalytic subunit activity was lost. Treatment of the regulatory subunit with a high concentration of trypsin yielded three main fragments (Mr = 32,000, 16,000, and 6,000) which could be resolved by Sephadex G-75 and purified further on DEAE-cellulose columns. One of the peptides (Mr = 32,000) bound 2 mol cAMP/mol fragment. The Mr = 16,000 fragment was very labile and bound cAMP with an undetermined stoichiometry. Cyclic AMP dissociation curves for the native regulatory subunit and its Mr = 32,000 component were similar and suggested the presence of two nonidentical binding sites in each monomer. Using the same procedure, the Mr = 16,000 fragment or homogenous cGMP-dependent protein kinase appeared to contain a single type of binding site. Purified Mr = 32,000 fragment was readily converted to the Mr = 16,000 fragment using high trypsin as assessed by protein bands on SDS-disc gels or by following transfer of radioactivity from Mr = 32,000 peptide covalently labeled with 8-N3-[32P] cAMP to radiolabeled Mr = 16,000 fragment. The smallest regulatory subunit fragment (Mr = 6,000) did not bind cAMP, but was dimeric and could be part of the dimerization domain in the native protein. A model is presented to explain the possible structural-functional relationships of the regulatory subunit.     

Quaternary structure of the giant extracellular hemoglobin of the leech Macrobdella decora

The molecular dimensions of the extracellular hemoglobin of the leech Macrobdella decora, determined by scanning transmission electron microscopy were 29.8 nm x 19.5 nm (diameter x height) for negatively stained specimens. Measurements of molecular mass (Mm) of unstained specimens with the microscope gave Mm = 3560 +/- 160 kDa. Small-angle X-ray scattering measurements gave a diameter of 28.0(+/- 0.5) nm, radius of gyration 10.5(+/- 0.2) nm and volume 7500(+/- 300) nm3. The hemoglobin had no carbohydrate and its iron content was found to be 0.23(+/- 0.02)% (w/w), corresponding to a minimum Mm of 24,000(+/- 1300) kDa. SDS/polyacrylamide gel electrophoresis of the unreduced hemoglobin showed that it consisted of three subunits, which have apparent Mm values of 12 (1), 25 (2) and 29 kDa (3). The reduced hemoglobin consisted of four subunits, I (12 kDa), II (14 kDa), III (26 kDa) and IV (30 kDa). Subunit 1 corresponded to subunit I, subunit 2 to subunits III and IV and subunit 3 to subunit II. Partial N-terminal sequences were obtained for subunit 1, the two chains of subunit 2 and one of the two chains of subunit 3, suggesting that the hemoglobin consists of at least five different polypeptide chains. The percentage fraction of the three unreduced subunits was determined by densitometry of SDS/polyacrylamide gel patterns and quantitative determination of Coomassie R-250 dye bound to the individual bands in reduced and unreduced patterns to be, monomer (subunit I) : non-reducible subunit (subunit 2) : reducible dimer (subunit 3) = 0.35 : 0.29 : 0.35 (S.D. = +/- 0.05). This corresponded to a stoichiometry of 74 +/- 11 : 37 +/- 5 : 38 +/- 6, assuming the molecular masses to be 17 kDa, 30 kDa and 34 kDa, taking into account the anomalously high mobility of annelid globins in SDS-containing gels. The stoichiometry calculated from the amino acid compositions of the hemoglobin and the three subunits was 82 +/- 12 : 29 +/- 4 : 40 +/- 8. Gel filtration of the hemoglobin at pH 9.8, at neutral pH subsequent to dissociation at pH 4 and at neutral pH in the presence of urea and Gu.HCl provided no evidence for the existence of a putative 1/12 of the whole molecule (Mm approx. 300 kDa). Furthermore, the largest subunits obtained had Mm of 60 to 100 kDa and had a much decreased content of subunit 2, suggesting that the hemoglobin was not a simple multimeric protein. Three-dimensional reconstruction from microscope images provided a model of Macrobdella hemoglobin that is very similar to the reconstruction of Lumbricus hemoglobin: the radial mass distribution curves are virtually superimposable.(ABSTRACT TRUNCATED AT 400 WORDS)     

The quaternary structure of the plasma membrane b-type cytochrome of human granulocytes

Hydrodynamic, crosslinking and immunoprecipitation studies were performed on detergent solubilized cytochrome b to demonstrate that the two copurifying polypeptides of molecular weight 91,000 (glycosylated) and 22,000 [1,2] formed a molecular complex. The hydrodynamic studies indicated that the cytochrome b/detergent complex had a sedimentation coefficient, partial specific volume and Stokes radius of 5.25 S, 0.82 cm3/g and 6.2 nm in Triton X-100 and 6.05 S, 0.80 cm3/g and 5.6 nm in octylglucoside, respectively. These studies also indicated that the detergent-protein complex has a molecular mass of 202 and 188 kDa in Triton X-100 and octylglucoside, respectively, is asymmetric in shape with a frictional coefficient of 1.3-1.4 and binds significant amounts of detergent. The molecular mass of the protein portion of the detergent-cytochrome complex was estimated to be between 100 and 127 kDa. Crosslinking studies with disuccinimidyl suberate and alkaline cleavable bis[2-(succinimidooxy-carbonyloxy)ethyl]sulfone revealed that the Mr = 91,000 and Mr = 22,000 components of purified cytochrome b are closely associated and can be covalently bound to form a polypeptide which, by SDS-polyacrylamide gel electrophoresis, has Mr values of 110,000-120,000 and 120,000-135,000 on 8% and 11% (w/v) SDS-polyacrylamide gels, respectively. Cleavage of the crosslinked species resulted in the reappearance of the Mr = 91,000 and Mr = 22,000 species. Sedimentation profiles of crosslinked cytochrome b in linear sucrose density gradients made up in H2O were identical to those of non-crosslinked controls. A close association of the two protein species was further confirmed by the ability of antibody specific for the smaller subunit to immunoprecipitate the larger one also. Experiments aimed at identifying the heme-carrying subunit(s) were inconclusive, since dissociation of the complex resulted in loss of cytochrome b spectrum. These results, in combination with our SDS-polyacrylamide gel electrophoresis molecular-weight estimates, provide strong evidence for the cytochrome b being an alpha-beta-type heterodimer composed of a glycosylated Mr = 91,000 and non-glycosylated Mr = 22,000 polypeptide.     

Purification,characterization and molecular cloning of a monocot mannose-binding lectin from <Emphasis Type="Italic">Remusatia vivipara</Emphasis> with nematicidal activity

A mannose-binding lectin (RVL) was purified from the tubers of Remusatia vivipara, a monocot plant by single-step affinity chromatography on asialofetuin-Sepharose 4B. RVL agglutinated only rabbit erythrocytes and was inhibited by mucin, asialomucin, asialofetuin and thyroglobulin. Lectin activity was stable up to 80°C and under wide range of pH (2.0–9.3). SDS-PAGE and gel filtration results showed the lectin is a homotetramer of Mr 49.5 kDa, but MALDI analysis showed two distinct peaks corresponding to subunit mass of 12 kDa and 12.7 kDa. Also the N-terminal sequencing gave two different sequences indicating presence of two polypeptide chains. Cloning of RVL gene indicated posttranslational cleavage of RVL precursor into two mature polypeptides of 116 and 117 amino-acid residues. Dynamic light scattering (DLS) and gel filtration studies together confirmed the homogeneity of the purified lectin and supported RVL as a dimer with Mr 49.5 kDa derived from single polypeptide precursor of 233 amino acids. Purified RVL exerts potent nematicidal activity on Meloidogyne incognita, a root knot nematode. Fluorescent confocal microscopic studies demonstrated the binding of RVL to specific regions of the alimentary-tract and exhibited a potent toxic effect on M. incognita. RVL-mucin complex failed to interact with the gut confirming the receptor mediated lectin interaction. Very high mortality (88%) rate was observed at lectin concentration as low as 30 μg/ml, suggesting its potential application in the development of nematode resistant transgenic-crops.     

Physical-chemical characterization and carbohydrate-binding activity of the A and B subunits of the Bandeiraea simplificolia I isolectins.

Bandeiraea simplicifolia I plant seed isolectins comprise a family of tetrameric alpha-D-galactopyranosyl-binding glycoproteins composed of various combinations of teo different kinds of subunits designated A and B. Subtypes of the A (Aa, Ab, Ac, Ad, and Ae) and B (Ba, Bb, Bc, Bd, and Be) subunits were demotypes varies from seed to seed (e.g., some seeds contain only B subunits, others only A subunits), subtypes Ac and Bc predominate in a natural mixture of the isolectins. Two-dimensional agar gel diffusion studies indicate that, in addition to common structural features, each subunit contains its own distinct antigenic determinants. Although the A and B subunits have closely similar amino acid compositions, they differ markedly in one respect: the B subunit has one methionine residue whereas the A subunit contains no methionine. The neutral carbohydrate content of both subunits is identical. The ability of biopolymers and synthetic glycoproteins to precipitate A4 and B4, as well as the capacity of sugars and oligosaccharides to inhibit precipitate formation, was examined. On the basis of these studies, it is suggested that hydrogen bonding occurs between the hydrogen atoms of the C-3 and C-4 hydroxyl groups of alpha-D-GalNAcp and alpha-D-galp units and the A and B subunits, respectively.     

江苏菜豆同工凝集素的分离纯化及性质研究

江苏菜豆经酸水(PH2.0)抽提,硫酸铵分级沉淀,分离植物血球凝集素(PHA-P),分子量为128000的糖蛋白,活性回收率在80％以上,PHA-P经SP-sephadexc-50离子交换层析,分成L_4,L_3E_1,L_2E_2,L_1E_3,和E_4同工凝集素。 L_4和E_4等电点为5.4和6.5。亚基分子量分别是31000和33000,并有类似的氨基酸组成。PAGE分析为单一蛋白带。红细胞凝集活性随电泳迁移速度的加快而增强,促淋性细胞分裂活性则减弱。E_4血凝活性受CalNAc,EDTA抑制和Zn~(++)的促进。     

Lectin-like binding of pertussis toxin to a 165-kilodalton Chinese hamster ovary cell glycoprotein

Chinese hamster ovary (CHO) cells cluster in the presence of pertussis toxin, a response that is correlated with the ADP-ribosylation of a Mr = 41,000 membrane protein by the toxin. A ricin-resistant line of CHO cells (CHO-15B) which specifically lacks the terminal NeuAc----Gal beta 4GlcNAc oligosaccharide sequence on glycoproteins did not cluster in response to pertussis toxin. These cells do contain the Mr = 41,000 protein substrate for the enzymatic activity of the toxin which suggests that pertussis toxin, like certain plant lectins, does not bind to or is not internalized by the CHO-15B cells. There was no evidence of pertussis toxin binding to gangliosides or neutral glycolipids isolated from CHO cells but the toxin bound to a Mr = 165,000 component in N-octyglucoside extracts of CHO cells that had been separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and electroblotted to nitrocellulose. Plant lectins from Ricinus communis and Erythina cristagalli detected a similar size band in CHO cells and also did not react with CHO-15B cells. Unlike pertussis toxin, these plant lectins recognized two other major bands in CHO cell extracts and reacted best after sialidase treatment of nitrocellulose transfers containing CHO cell extracts. Conversely, sialidase treatment abolished binding a pertussis toxin and wheat germ agglutinin, a plant lectin that reacts with multivalent sialic acid residues on glycoproteins, to the Mr = 165,000 band. Purified B oligomer of pertussis toxin also uniquely detected a Mr = 165,000 component in CHO cell extracts while the A subunit of pertussis toxin was unreactive. These results indicate that pertussis toxin binds to a CHO cell glycoprotein with N-linked oligosaccharides and that sialic acid contributes to the complementary receptor site for the toxin. In addition, they suggest that a glycoprotein may serve as a cell surface receptor for pertussis toxin and that this interaction is mediated by a lectin-like binding site located on the B oligomer.     

Purification, subunit structure and properties of a high-molecular-mass protein phosphatase capable of dephosphorylating mRNP of the brine shrimp Artemia sp

A phosphoprotein phosphatase active towards casein, phosphorylase a and mRNP proteins has been detected in the cytosol of cryptobiotic gastrulae of Artemia sp. This phosphatase has a relative molecular mass (Mr) of 225,000 as measured by gel filtration on Sephadex G-200 and has been purified to near homogeneity by ion-exchange chromatography on different DEAE-substituted matrices, affinity chromatography on polylysine-agarose, histone-Sepharose 4B and protamine-agarose, hydrophobic chromatography on phenyl-Sepharose 4B and gel filtration on Sephadex G-200. Sodium dodecyl sulphate gel electrophoresis of the final purification step revealed that the enzyme contains two types of subunits, alpha and beta, with Mr of 40,000 and 75,000, respectively. These values, in conjunction with the native Mr and the molar ratios of the subunits estimated by densitometric analysis of the gel, suggested that the subunit composition of the enzyme is alpha 2 beta 2. When treated with 1.7% (v/v) 2-mercaptoethanol at -20 degrees C or with ethanol, the enzyme released the catalytic alpha subunit of Mr 40,000. The protein phosphatase was activated by basic proteins e.g. protamine (A 0.5 = 1 microM), histone H1 (A 0.5 = 1.6 microM) and polylysine (A 0.5 = 0.2 microM) and inhibited by ATP (I 0.5 = 12 microM), NaF (I 0.5 = 3.1 mM) and pyrophosphate (I 0.5 = 0.6 mM). The enzyme is a polycation-stimulated protein phosphatase. Purified mRNP proteins, phosphorylated by the mRNP-associated casein kinase type II, are among the substrates used by the enzyme. The function of reversible phosphorylation-dephosphorylation of mRNP as a regulatory mechanism in mRNP metabolism is discussed.     

A disulfide-bonded trimer of myoglobin-like chains is the principal subunit of the extracellular hemoglobin of Lumbricus terrestris

The extracellular hemoglobin of Lumbricus terrestris (3900 kDa) consists of at least six different polypeptide chains: I through IV (16-19 kDa), V (31 kDa) and IV (37 kDa) (Vinogradov, S.N., Shlom, J.M., Hall, B.C., Kapp, O.H. and Mizukami, H. (1977) Biochim. Biophys. Acta 492, 136-155). SDS-polyacrylamide gel electrophoresis of the unreduced hemoglobin shows that chains II, III and IV form a disulfide-bonded 50 kDa subunit. This subunit was isolated by gel filtration of the hemoglobin on Sephacryl S-200 (a) at neutral pH in 0.1% SDS and (b) in 0.1 M sodium acetate buffer (pH 4.0); in the latter case it retains heme. The 50 kDa subunit obtained by method (b) was reduced and subjected to chromatofocusing on PBE 94 column: the elution pattern obtained with Polybuffer 74 (pH 4.5) and monitored at 280 nm, consisted of three peaks A, B and C; peaks A and B but not C, had absorbance at 410 nm. SDS-polyacrylamide gel electrophoresis showed that peaks A, B and C corresponded to chains II, IV and III, respectively. Amino acid analyses and N-terminal sequence determinations identified chain II as the whose primary structure had been determined (Garlick, R. and Riggs, A. (1982) J. Biol. Chem. 257, 9005-9015). Carbohydrate analysis of the native hemoglobin shows it to contain 2.0 +/- 0.5% carbohydrate consisting of mannose and N-acetylglucosamine in a mole ratio of about 9:1. The carbohydrate content of the 50 kDa subunit is 1.8 +/- 0.5%; it consists of mannose and N-acetylglucosamine in the same ratio and it appears to be associated with chain IV. Rabbit polyclonal antisera to 50 kDa subunit, prepared by method (a), and to the native hemoglobin were shown to cross-react with the hemoglobin and the 50 kDa subunit, respectively, by immunodiffusion. One of eight mouse monoclonal antibodies directed against the native hemoglobin reacted strongly with the 50 kDa subunit prepared by methods (a) and (b) in an enzyme-linked immunosorbent assay (ELISA). Another monoclonal antibody reacted strongly with the 50 kDa subunit obtained by method (b). Neither of the two hybridomas exhibited a strong reaction with any of the three constituent chains of the 50 kDa subunit. These results suggest that the unusual disulfide-bonded 50 kDa subunit, consisting of three myoglobin-like polypeptide chains of which only two have heme, is an integral part of the native Lumbricus hemoglobin molecule.