Purification and properties of neutral β-galactosidases from human liver

Two neutral β-galactosidase isozymes were purified from human liver. The initial step of purification was removal of the acidic β-galactosidases by adsorption on concanavalin A-Sepharose 4B conjugate. Subsequent purification steps included ammonium sulfate precipitation, diethylaminoethyl cellulose column chromatography, Sephadex G-100 gel filtration, and preparative polyacrylamide-gel isoelectric focusing. The final step of purification was affinity chromatography of the separated isoelectric forms on ?-aminocaproyl-β-d-galactosylamine-Sepharose 4B conjugate. The purified β-galactosidase isozymes had activity toward both β-d-galactoside and β-d-glucoside derivatives of 4-methylumbelliferone and p-nitrophenol with a pH optimum around 6.2. These enzyme forms were also found to possess lactosylceramidase II activity with a pH optimum in the range of 5.4 to 5.6, but not lactosylceramidase I activity and no activity toward galactosylceramide or GM₁-ganglioside. The molecular weight was found to be in the range of 37,500–39,500 for the two neutral isozymes and they had similar K_m and V values; the more acidic form (designated β-galactosidase N₁) was more heat stable than the other form (designated β-galactosidase N₂). Antibodies evoked against the N₁ and N₂ β-galactosidases gave identical precipitin lines retaining enzymatic activity. No cross-reactivity was observed between the neutral and the acidic isozymes when examined with the respective antisera.     

Stimulated rat T cell-derived inhibitory factor for cellular DNA synthesis (STIF). I. Isolation and characterization

A lymphokine inhibitory for cellular DNA synthesis (termed STIF) was isolated from the culture supernatants of concanavalin A (Con A)-stimulated SD rat spleen cells. STIF inhibited the DNA synthesis of mouse bone marrow cells as well as mouse leukemia cells. STIF has an apparent m.w. of 45,000 to 50,000 and is separable from IL 2, m.w. 20,000 to 25,000, by Sephacryl S-200 gel filtration, but not from immune interferon (IFN) having the same m.w. as STIF. Con A-Sepharose chromatography of the fraction containing STIF and IFN could separate these lymphokines into Con A-unbound and Con A-bound fractions, respectively. Further fractionation of the STIF fraction by DEAE-Sephadex A-50 or Mono Q-FPLC anion exchange chromatography indicated that the STIF fraction contained two components of STIF activity, both showing the same pI value (5.1 to 5.6) on flat-bed isoelectric focusing. STIF was characterized as a sugar-free lymphokine of trypsin-sensitive protein nature.     

I-Cell disease: isoelectric focusing, concanavalin A-Sepharose 4B binding and kinetic properties of human liver acid beta-D-galactosidases.

Isoelectric focusing of the acid beta-D-galactosidases (beta-D-galactoside galactohydrolase, EC 3.2.1.23) in normal crude liver supernatant fluids demonstrated multiple isoelectric forms in the pH range 4.58-5.15, while corresponding I-cell disease samples showed an absence of isoelectric forms in the pH range 4.99-5.15. Concanavalin A-Sepharose 4B chromatography of the I-cell disease mutant C.A. demonstrated a 31% and 37% decrease in the binding of 4-methyl-umbelliferyl-beta-D-galactosidase and GM1 beta-D-galactosidase activities, respectively, when compared to normal samples. Isoelectric focusing profiles of the concanavalin A-Sepharose 4B alpha-methyl-D-mannoside effluents containing normal and I-cell disease acid beta-D-galactosidase were generally similar, but the unadsorbed I-cell disease enzyme from concanavalin A-Sepharose 4B demonstrated more activity in the pH range 4.21-4.49 than normals. Normal and I-cell disease acid beta-D-galactosidase "A" and "B", separated by gel column chromatography were found to have similar properties with respect to apparent molecular weights pH vs. activity profiles and apparent Km values for the 4 methylumbelliferyl-beta-D-galactopyranoside, GM1-ganglioside and asialofetuin (ASF) substrates. However, the apparent V values for the ICD samples were consistently reduced when compared to the results obtained with the corresponding normal fractions. The greatest decreases in apparent V were obtained for acid beta-D-galactosidase activities in I-cell disease crude supernatant fluids, and for the separated I-cell disease "B" enzyme. The differences in the isoelectric focusing profiles, the altered binding to concanavalin A-Sepharose 4B, and the reduced V values with natural and synthetic substrates may be related to changes in carbohydrate composition of I-cell disease acid beta-D-galactosidase.     

Entamoeba histolytica: cytopathogenicity, including serum effects on contact-dependent and toxin-induced lysis of hamster kidney cell monolayers

Some properties of toxin, isolated from extracts of axenically cultivated Entamoeba histolytica or excreted by intact amoebae, were investigated using a toxicity assay in microplates with monolayers of baby hamster kidney cells. Preparative isoelectric focusing showed that the highest cytotoxic activity was present in a fraction of antigen containing protein bands with an isoelectric point between 4.5 and 5. Activity of the toxin was stable between pH 4 and 10. Nonimmune rabbit serum and concanavalin A, coupled to Sepharose beads, were able to bind a large amount of toxin. Cytotoxicity of antigen was inhibited by specific immune IgG and by unknown factors in nonimmune serum with a molecular weight between 50,000 and 100,000. The toxin was inactivated by trypsin, but not by trypsin inhibitor. Its activity was thiol dependent. Serum also had a marked inhibitory effect on contact lysis of BHK cells induced by intact trophozoites. A considerable reduction of both contactdependent and toxin-induced Cytopathogenicity was observed when Diamond's TP-S-1 medium was used in the assay, in which the TP broth had been autoclaved. It is suggested that Entamoeba histolytica exocytozes toxin, which acts on adjacent cells during close contact.     

Phosphodiesterase from cultured tobacco cells. Physical and chemical properties.

Phosphodiesterase isolated from suspension cultures of tobacco cells showed high affinity for concanavalin A-Sepharose and gave single superimposed bands of protein and carbohydrates on disc gel electrophoresis, suggesting that it is a glycoprotein. It contains 14% carbohydrate by weight, and has relatively high contents of basic and aromatic amino acids. Its isoelectric point is at pH 8.8, and the molecular weight of its subunits was estimated as 72 000 from a plot of the retardation coefficient on sodium dodecyl sulfate gel electrophoresis versus the molecular weight. The enzyme was catalytically active in an immobilized state on a concanavalin A-Sepharose column.     

Trehalase from the cellular slime mold Dictyostelium discoideum: Purification and characterization of the homogeneous enzyme from myxamoebae

Trehalase (α-α′-trehalose 1-d-glucohydrolase, EC 3.2.1.28) was solubilized from myxamoebae of the cellular slime mold Dictyostelium discoideum by a freeze-thaw cycle and was subsequently purified to homogeneity using the techniques of ethanol fractionation, molecular sieve chromatography, DEAE-cellulose ion-exchange chromatography, chromatofocusing, and preparative polyacrylamide disc gel electrophoresis. The 1000-fold purified enzyme had a specific activity of about 104 units/mg, which was accompanied by a net recovery of 5 to 7% of the original activity. The purified enzyme was maximally active at pH 5.5, showed high specificity for trehalose, and exhibited a typical hyperbolic response as a function of trehalose concentration with a K_m of 1.2 mm. The enzyme was maximally active at 50 °C and had an energy of activation of 12–13 kcal/mol. Thermal stability studies demonstrated that full enzymatic activity was recovered following a 5-min incubation of trehalase at temperatures up to 45–50 °C. Analysis of various compounds for inhibitory effects indicated that Tris and urea were slightly effective, reducing enzymatic activity by 28 and 6% at concentrations of 100 and 10 mm, respectively. Of five heavy metals tested, HgCl₂ was the most inhibitory, reducing activity by 58% when present at a final concentration of 1.0 mm. Enzymatic activity was not affected by any adenine derivative examined (e.g., ATP, ADP, AMP, cAMP, adenosine, and adenine). The molecular weight of the native enzyme was determined by molecular sieve chromatography, pore gradient electrophoresis, and electrophoresis as a function of acrylamide concentration. All three methods yielded a value of about 10⁵ ± 5 × 10³. Estimation of the subunit or monomer molecular weight by sodium dodecyl sulfate-gel electrophoresis indicated a value of 95–100 × 10³. The isoelectric point as determined in 7.5% polyacrylamide gels with pH 3–10 ampholytes was 7.2–7.3. The purified enzyme adsorbed to concanavalin A-Sepharose in the presence of KCl (0.1 m) and was eluted with α-methylmannoside, thereby suggesting an association between trehalase and carbohydrate. In agreement with this conclusion was the observation that trehalase could be specifically stained for carbohydrate with the Alcian blue and periodic acid-Schiff's reagents following polyacrylamide disc gel electrophoresis.     

Fractionation of murine macrophage inhibitory factor into two distinct species

Murine migration inhibitory factor (MIF) produced by concanavalin A-stimulated lymph node cells from C57BL/6 mice was fractionated by Sephadex G-100 gel filtration, density gradient electrophoresis, and isoelectrofocusing in a sucrose density gradient and assayed on in vitro-cultivated bone marrow macrophages from C57BL/6 mice. Two major MIF species, pH3-MIF with an isoelectric point of 3.0–4.3 and pH5-MIF with an isoelectric point of 4.6 to 5.2, were obtained. The similarity of murine MIF to guinea pig and human MIF is discussed.     

gp110—A major sialoglycoprotein of human cells: Isolation and partial characterization from a malignant melanoma cell line

A major sialoglycoprotein (gp110) was isolated from NP-40 extracts of the human melanoma cell line SK-MEL-37 by concanavalin A-Sepharose and wheat germ agglutinin-Sepharose affinity chromatography, and preparative sodium dodecyl sulfate-polyacrylamide gel electrophoresis. A rabbit antiserum was prepared to this concanavalin A- and wheat germ agglutinin-binding glycoprotein and used to study the biochemical properties and distribution of gp110 in human cells. gp110 is highly acidic (pI ~ 3.8–4.0) and located on the cell surface in melanoma cells. It contains sialylated, N-linked complex chains as well as sialylated, O-linked carbohydrate chains. gp110 was detected as a major glycoprotein on all human cell lines tested (except erythrocytes), although its apparent molecular weight varied from cell line to cell line. The pI of gp110 from normal and malignant human kidney epithelial cells was identical, indicating that gp110's from two cell types do not substantially differ in their sialylated carbohydrate moieties.     

Glucosinolate Biosynthesis: Sulfation of Desulfobenzylglucosinolate by Cell-Free Extracts of Cress (Lepidium sativum L.) Seedlings

After removal of myrosinase activity by concanavalin A-Sepharose 4B chromatography, cell-free extracts of light-grown cress (Lepidium sativum L.) seedlings, catalyzed the sulfation of desulfobenzylglucosinolate (K_m, 0.23 millimolar) to benzylglucosinolate using PAPS (K_m, 1 millimolar) as sulfur donor. Sulfotransferase activity, which was optimal at pH 9.0, was stimulated by MgCl₂, MnCl₂, β-mercaptoethanol, and dithiothreitol and was inhibited by ZnSO₄ and SH-reagents. The enzyme also sulfated desulfoallyglucosinolate to allylglucosinolate (sinigrin) but was inactive towards all phenylpropanoids and flavonoids tested.     

Studies on pituitary follitropin. I. An improved procedure for the isolation of highly potent ovine hormone.

An improved method of isolation of ovine pituitary follitropin has been described. The method involves extraction of frozen glands at pH 9.0 in presence of an enzyme inhibitor, metaphosphoric acid precipitation at two stages, ammonium sulfate fractionation, glycoprotein fractionation, ion exchange chromatography on SP-C50 followed by affinity chromatography on concanavalin A-Sepharose, and filtrations on Sephadex G-100. A highly active preparation with a yield of about 11 mg/kg is obtained and has an activity of about 110 × NIH-FSH-S10 standard in the human chorionic gonadotropin (HCG)-augmentation assay in rats. Its lutropic activity as estimated by specific in vitro bioassay and radioreceptor assay was about 0.005–0.01 unit (NIH-LH-S19)/mg. It is more acidic than lutropin and thyrotropin. Its amino acid composition and carbohydrate content has been analyzed. Its isoelectric point is approximately pH 4.9.     

Partial Purification and Characterization of a 3'- Phosphoadenosine 5' -Phosphosulfate: Desulfoglucosinolate Sulfotransferase from Cress (Lepidium sativum)

A 3′ -phosphoadenosine 5′ -phosphosulfate (PAPS):desulfoglucosinolate sulfotransferase (EC 2.8.2-) was extensively purified from light-grown cress (Lepidium sativum L.) seedlings by gel filtration and concanavalin A-Sepharose 4B, Matrex Gel Green A, and Mono Q fast protein liquid chromatography. The purified enzyme, which required bovine serum albumin for stabilization, had a native molecular weight of 31,000 ± 5,000 and an apparent isoelectric point of 5.2. Using PAPS (K_m 60 micromolar) as sulfur donor, it catalyzed the sulfation of desulfobenzylglucosinolate (K_m 82 micromolar), desulfo-p-hydroxybenzylglucosinolate (K_m 670 micromolar), and desulfoallylglucosinolate (K_m 6.5 millimolar) at an optimal pH of 9.0. All other potential substrates tested, including flavonoids, flavonoid glycosides, cinnamic acids, and phenylacetaldoxime, were not sulfated. Sulfotransferase activity was stimulated by MgCl₂, MnCl₂ and reducing agents and inhibited by ZnCl₂, PbNO₃ NiCl₂ and the reaction product PAP. The thiol reagents N-ethylmaleimide, p-chloromercuriphenylsulfonic acid, and 5,5′ -dithio-bis-(2-nitrobenzoic acid) were also potent inhibitors, but the enzyme was protected from covalent modification by β-mercaptoethanol. The kinetics of desulfobenzylglucosinolate sulfation were consistent with a rapid equilibrium ordered mechanism with desulfobenzylglucosinolate binding first and PAPS second.     

Affinity Chromatography of B-Glucosidase and Endo-B-Glucanase from Aspergillus Niger on Concanavalin A-Sepharose: Implications for Cellulase Component Purification and Immobilization

Affinity chromatography of a commercial preparation of 3-glu-cosidase from Aspergillus niger using concanavalin A-Sepharose (CAS) was employed as a means of purifying this glycoprotein. However, mannose (up to 1.08 M) was ineffective as an eluent of this enzyme from CAS, as were several other sugars and their derivatives, including 0.5 M glucose. Also, washing the CAS:8-glucosidase complex with buffer at pH 3.5 in the absence of MnCl₂ and CaCl₂ (required to preserve the binding activity of concanavalin A below pH 5.0) did not result in elution of this enzyme. On the contrary, endo-glucanase activity present in a crude cellulase complex (A. niger) which bound to CAS could be eluted by mannose (0.5–0.7 M) and was fractionated Into at least two components. The CAS:β-glucosidase complex hydrolyzed cellobiose to glucose and possessed an activity of 2, 158 units/g dry CAS. It could be used, therefore, for continuous cellobiose hydrolysis without leakage of enzyme from the support.     

Isolation and partial characterization of a proteinase inhibitor from human colorectal adenocarcinoma

A proteinase inhibitor has been isolated from human colorectal adenocarcinomas by extraction with a low-ionic-strength buffer and a combination of Con A-Sepharose, Sephadex G-200, DEAE-cellulose and chromatofocusing steps. The preparation appeared to be homogeneous upon gel exclusion chromatography and SDS-polyacrylamide gel electrophoresis and had an estimated molecular weight of 66 000. The inhibitor was able to bind and inhibit urokinase, plasmin, trypsin, tissue plasminogen activator and thrombin. The binding appeared to be stoichiometric and relatively fast. The isoelectric point of the protein was 4.6–4.7. The inhibitor did not crossreact with antisera elicited against α₂-macroglobulin, α₂-antiplasmin, antithrombin III or C₁-inhibitor, but it did crossreact with an antiserum against α₁-antitrypsin in double immunodiffusion. The antiserum only partially attenuated the activity of the inhibitor. Whereas α₁-antitrypsin completely inhibited the amidolytic activity of elastase, the tumor inhibitor had no effect on elastase under the same conditions.     

Design and synthesis of novel dasatinib derivatives as inhibitors of leukemia stem cells

We used the concept of bioisosteres to design and synthesize a novel series of dasatinib derivatives for the treatment of leukemia. Unfortunately, most of the dasatinib derivatives did not show appreciable inhibition against leukemia cell lines K562 and HL60. However, acrylamide compound 2c had comparable inhibitory activity with dasatinib against K562 cells (IC₅₀?=?0.039?nM vs. 0.069?nM). And amide compound 2a and acrylamide compound 2c also had comparable inhibitory activity with dasatinib against the leukemia cell line HL60 (IC₅₀?=?0.25?nM and 0.26?nM vs. 0.11?nM). Against the leukemia progenitor cell line KG1a, triazole compounds 15a and 15d–15f and oxadiazole compounds 24a–24d were more potent than dasatinib. In particular, the hydroxyl compounds 15a and 24a were about 64 and 180 fold more potent than dasatinib against KG1a cells (IC₅₀?=?0.14?μM and 0.05?μM vs. 8.98?μM). Compounds 15a and 24a also inhibited colony formation in MCF-7 cells and inhibited cell migration in the cell wound scratch assay in B16BL6 cells. Moreover, hydroxyl compounds 15a and 24a had low toxicity in vivo.     

Carbohydrate heterogeneity of vesicular stomatitis virus G glycoprotein allows localization of the defect in a glycosylation mutant of CHO cells

The carbohydrate portion of the G glycoprotein of vesicular stomatitis virus (VSV) grown in CHO cells (CHO/VSV) has been fractionated on BioGelP6, concanavalin A-Sepharose, and pea lectin-agarose. The results suggest that, in addition to sialic acid and fucose heterogeneity, the asparagine-linked complex carbohydrate moieties of CHO/VSV also display branching heterogeneity. Although the majority of the glycopeptides bind to concanavalin A-Sepharose in a manner typical of certain biantennary carbohydrate structures, a significant proportion do not bind to the lectin. The latter behavior is typical of tri- or tetraantennary (branched) carbohydrate structures. The CHO/VSV glycopeptides which do not bind to concanavalin A-Sepharose separate into bound and unbound fractions on pea lectin-agarose suggesting that they include at least two different types of (branched) carbohydrate structures. Glycopeptides from the G glycoprotein of VSV grown in two, independently derived CHO glycosylation mutants which belong to complementation group 4 (Lec4 mutants) were examined in the same manner. In contrast to glycopeptides from CHO/VSV, glycopeptides from Lec4/VSV which passed through concanavalin A-Sepharose did not contain a component which subsequently bound to pea lectin-agarose. A glycopeptide fraction with these lectin-binding properties was also missing from cell surface glycopeptides derived from Lec4 cells. The combined results are consistent with the hypothesis that Lec4 CHO glycosylation mutants lack a glycosyltransferase activity responsible for the addition of a (branch) N-acetylglucosamine residue linked β1,6 to mannose.     

Purification and chemical characterization of polysaccharides obtained from Lampteromyces japonicus by concanavalin A-sepharose affinity chromatography

Two classes of neutral polysaccharide which could not be separated from each other by conventional methods were isolated from the fungus, Lampteromyces japonicus, by affinity chromatography using concanavalin A-Sepharose. The polysaccharide retained on the concanavalin A-Sepharose column was eluted with 0.05 M methyl α-d-mannopyranoside and appeared to be α-mannan, while that which passed through the column was virtually all β-glucan.Both polysaccharides were subjected to Smith-type degradation, methylation, acetolysis and glucosidase treatment. The results indicated that the α-mannan contained predominantly α-(1 → 2)-linked side chains branching from an α-(1 → 6)-linked backbone at the (1 → 2,6)-linked mannopyranosyl residues. Galactose was attached to approximately one-quarter of the non-reducing mannose terminals. The β-glucan seemed to contain mainly (1 → 6)-linked side chains branching from a (1 → 3)-linked backbone at the (1 → 3,6)-linked glucopyranosyl residues.     

Cytokinin Oxidase from Phaseolus vulgaris Callus Cultures : Affinity for Concanavalin

Cytokinin oxidase activity from Phaseolus vulgaris cv Great Northern callus cultures exhibited affinity for the lectin concanavalin A. Over 80% of the activity extracted from the callus tissue bound to a concanavalin A-Sepharose 4B column. The bound activity was eluted from the column by the addition of methylmannose to the eluting buffer. On the basis of this result, it appears that most of the cyokinin oxidase activity present in Great Northern callus cultures exists in the form of a glycoprotein. The apparent pI of this enzyme, as estimated by chromatofocusing, is approximately 5.0.     

Prunus serotina Amygdalin Hydrolase and Prunasin Hydrolase : Purification, N-Terminal Sequencing, and Antibody Production

In black cherry (Prunus serotina Ehrh.) seed homogenates, amygdalin hydrolase (AH) participates with prunasin hydrolase (PH) and mandelonitrile lyase in the sequential degradation of (R)-amygdalin to HCN, benzaldehyde, and glucose. Four isozymes of AH (designated AH I, I′, II, II′) were purified from mature cherry seeds by concanavalin A-Sepharose 4B chromatography, ion-exchange chromatography, and chromatofocusing. All isozymes were monomeric glycoproteins with native molecular masses of 52 kD. They showed similar kinetic properties (pH optima, K_m, V_max) but differed in their isoelectric points and N-terminal amino acid sequences. Analytical isoelectric focusing revealed the presence of subisozymes of each isozyme. The relative abundance of these isozymes and/or subisozymes varied from seed to seed. Three isozymes of PH (designated PH I, IIa, and IIb) were purified to apparent homogeneity by affinity, ion-exchange, and hydroxyapatite chromatography and by nondenaturing polyacrylamide gel electrophoresis. PH I and PH IIb are 68-kD monomeric glycoproteins, whereas PH IIa is dimeric (140 kD). The N-terminal sequences of all PH and AH isozymes showed considerable similarity. Polyclonal antisera raised in rabbits against deglycosylated AH I or a mixture of the three deglycosylated PH isozymes were not monospecific as judged by immunoblotting analysis, but also cross-reacted with the opposing glucosidase. Monospecific antisera deemed suitable for immunocytochemistry and screening of expression libraries were obtained by affinity chromatography. Each antiserum recognized all known isozymes of the specific glucosidase used as antigen.     

Concanavalin A-Sepharose Chromatography of Hexosaminidase Isozymes Secreted By Tetrahymena*

Tetrahymena pyriformis strain HSM secretes 4 isozymes of hexosaminidase. Purified isozymes B₁ and B₂ are eluted from the void volume of a concanavalin A-Sepharose column, suggesting that they are not glycosylated. Purified isozymes A₁ and A₂ bind to the column and are eluted at ～0.1 M α-methylmannoside, suggesting that these isozymes are glycoproteins. In agreement with earlier deductions based on a differential kinetic assay for the A and B isozymes, the elution pattern of hexosaminidase activity from material secreted by cells grown to early and late stationary phase was consistent with these secretions containing primarily the B and the A isozymes, respectively.     

Partial Purification and Characterization of Naegleria fowleriβ-Glucosidase1

Naegleria fowleri cells, grown axenically, contain high levels of β-D-glucosidase which catalyzes the hydrolysis of 4-methylumbelliferyl-β-D-glucopyranoside (4MUGlc) (K_m, 0.9 mM), octyl-β-D-glucoside (K_m, 0.17 mM), and p-nitrophenyl-β-D-glucopyranoside at relative rates of 1.00, 2.88, and 1.16, respectively (substrate concentration, 3.0 mM). When the amebae are subjected to freeze-thawing, sonication, and centrifugation (100,000 g, 1 h), 85% of the β-glucosidase activity appears in the supernatant fraction. The β-glucosidase was purified 40-fold (34% yield) using a combination of chromatographic steps involving DE-52 cellulose, concanavalin A-Sepharose, and hydroxylapatite followed by isoelectric focusing. The predominant soluble β-D-galactosidase activity in the Naegleria extract copurifies with the β-D-glucosidase; the two activities have the same isoelectric point (pI, 6.9), similar heat stabilities, are both inhibited by lactobionic acid (K_i, 0.40 mM), and exhibit optima at pH 4.5, indicating that they are probably the same enzyme. The Naegleriaβ-D-glucosidase has an apparent molecular weight of 66,000, a Stokes radius of 25 Å, and a sedimentation coefficient of 4.2S. The β-glucosidase is not inhibited by conduritol β-epoxide or galactosylsphingosine but is completely inhibited by 1.25 mM bromo conduritol β-epoxide. The latter compound, when present in the growth medium, inhibits the growth of the organism and profoundly alters its ultrastructure, the main effect being the apparent inhibition of cytokinesis and the generation of multinucleate cells. The issue of the role of the β-glucosidase in the metabolism of the ameba and its possible role in pathogenic mechanisms are discussed.