Concanavalin A-Sepharose Research Articles

Dimeric Galectin-1 Binds with High Affinity to α2,3-Sialylated and Non-sialylated Terminal N-Acetyllactosamine Units on Surface-bound Extended Glycans

Galectin-1 is a member of the galectin family of glycan-binding proteins and occurs as an approximately 29.5-kDa noncovalent homodimer (dGal-1) that is widely expressed in many tissues. Here, we report that human recombinant dGal-1 bound preferentially and with high affinity (apparent K(d) approximately 2-4 microM) to immobilized extended glycans containing terminal N-acetyllactosamine (LN; Galbeta1-4GlcNAc) sequences on poly-N-acetyllactosamine (PL; (-3Galbeta1-4GlcNAcbeta1-)(n)) sequences, complex-type biantennary N-glycans, or novel chitin-derived glycans modified to contain terminal LN. Although terminal Gal residues are important for dGal-1 recognition, dGal-1 bound similarly to alpha3-sialylated and alpha2-fucosylated terminal LN, but not to alpha6-sialylated and alpha3-fucosylated terminal LN. The binding specificity of human recombinant dGal-1 was similar to that observed with purified bovine heart-derived dGal-1. Unexpectedly, dGal-1 bound free ligands in solution with relatively low affinity and displayed no preference for extended glycans, indicating that dGal-1 preferentially recognizes extended glycans only when they are surface-bound, such as found on cell surfaces. Human dGal-1 also bound to both native and desialylated human promyelocytic HL-60 cells with similar affinity as observed for immobilized long chain PL. Binding to these cells was reduced upon treatment with endo-beta-galactosidase, which cleaves PL sequences, indicating that cell-surface PLs are ligands. To test the role of dimerization in dGal-1 binding, we examined the binding of a mutated form of dGal-1 that weakly dimerizes (monomeric Gal-1 (mGal-1)) and a covalently dimerized (chemically cross-linked) form of mGal-1 (cd-mGal-1). dGal-1 and cd-mGal-1 had similar affinities that were both approximately 3.5-fold higher for immobilized PL than observed for mGal-1, suggesting that dGal-1 acts as a dimer to cross-link terminal LN units on immobilized PL. These results indicate that dGal-1 functions as a dimer to recognize LN units on extended PLs on cell surfaces.

Characterization and properties of acid phosphatases with phytase activity produced by Aspergillus caespitosus

High levels of thermostable acid phosphatases were produced by Aspergillus caespitosus in culture media supplemented with xylan birchwood or agricultural residues, as carbon sources. The optimal culture conditions for production of phosphatases were 40 degrees C and pH 6.0. Extra- and intra-cellular acid phosphatases were purified by chromatography on DEAE-cellulose, followed by concanavalin A-Sepharose affinity separation. Both extra- and intra-cellular enzymes were glycoproteins showing 63.0 and 58.3% of carbohydrate content respectively. Molecular masses estimated on Sepharose CL-6B column were 186 and 190+/-15 kDa, and 84 and 74+/-5 kDa according to SDS/PAGE, for extra- and intra-cellular acid phosphatases respectively. Taken together, these results suggest that both native enzymes were homodimers. Optimum temperature and pH for both phosphatase activities were 80 degrees C and 5.5 respectively. The extra- and intra-cellular acid phosphatases were stable for more than 60 min at 60 degrees C. The extracellular acid phosphatase was slightly inhibited by NaF, in contrast with the significant inhibition of the intracellular form. KH(2)PO(4) inhibited both activities equally. Both extra- and intracellular acid phosphatases were tartarate-resistant. Among several phosphorylated substrates used, the extracellular enzyme preferentially hydrolysed p-nitrophenyl phosphate. Kinetic parameters calculated for the hydrolysis of p-nitrophenyl phosphate by extracellular acid phosphatase were h (Hill coefficient)=1.2, K(0.5)=0.082 mM and V(max)=4.43 units/mg, whereas the intracellular enzyme exhibited Michaelian kinetics with K(m)=0.029 mM and V(max)=0.082 unit/mg. Phytase activity was also observed for both the enzymes, suggesting that they could be useful for biotechnological applications.

The Hydrodynamic Properties of Dark- and Light-activated States of n-Dodecyl β-D-Maltoside-solubilized Bovine Rhodopsin Support the Dimeric Structure of Both Conformations

Rhodopsin (Rho) has been extracted in n-dodecyl beta-D-maltoside (DM) from bovine retinal rod outer segments and purified to homogeneity by affinity chromatography on concanavalin A-Sepharose. Because chemical cross-linking of Rho and photoactivated Rho (Rho*) provided initial evidence for the oligomeric nature of the photoreceptor protein, we carried out a hydrodynamic characterization of the native and activated conformations of detergent-solubilized Rho. The molecular weights of the complexes between dark and photoexcited states of Rho and DM were determined by gel filtration chromatography on Sephacryl S-300, in the presence of 0.1% DM. Subtracting the size of the corresponding detergent micelles resulted in molecular masses of 78 kDa for native Rho and 76 kDa for Rho*. The measured content of 0.97 g of detergent/g of protein resulted in a calculated partial specific volume of 0.765 cm(3)/g for the protein-detergent complex and a molar mass of 64-65 kDa for the protein moiety. The sizes of Rho.DM and Rho*.DM complexes were also evaluated by sedimentation on 10-30% sucrose gradients, in the presence of 0.1% DM, and molecular masses of about 60 kDa were estimated for both the dark- and light-activated states of the photoreceptor protein. The size of Rho was determined to be 65,300 and 69,800 Da, respectively, when the purified Rho.DM complex was either chromatographed on Sephacryl S-300 or ultracentrifuged on sucrose gradients in the absence of DM. All these results were consistent with a dimeric quaternary structure for both conformations of Rho. Additionally, the functional integrity of the purified photoreceptor protein following gel filtration chromatography and ultracentrifugation was demonstrated by three criteria as follows: (i) its characteristic UV-visible absorption spectra, (ii) its capability to photoactivate transducin, and (iii) its ability to serve as a substrate for rhodopsin kinase.

Structural variability of BM-40/SPARC/osteonectin glycosylation: implications for collagen affinity.

We performed a detailed investigation of N-glycan structures on BM-40 purified from different sources including human bone, human platelets, mouse Engelbreth-Holm-Swarm (EHS) tumor, and human BM-40 recombinantly expressed in 293 and osteosarcoma cells. These preparations were digested with endoglycosidases and N-glycans were further characterized by sequential exoglycosidase digestion and high-performance liquid chromatography (HPLC) analyses. Bone BM-40 carries high-mannose structures as well as biantennary complex type N-glycans, whereas the protein from platelets and 293 cells has exclusively bi- and triantennary complex type structures. BM-40 derived from the EHS tumor carries biantennary complex type and additional hybrid structures. Using the osteosarcoma-derived MHH-ES1 cell line we successfully expressed a recombinant BM-40 that bears at least in part the bone-specific high-mannose N-glycosylation in addition to complex type and hybrid structures. Using chromatography on Concanavalin-A Sepharose, we further purified a fraction enriched in high-mannose structures. This array of differentially glycosylated BM-40 proteins was assayed by surface plasmon resonance measurements to investigate the binding to collagen I. BM-40 carrying high-mannose structures binds collagen I with higher affinity, suggesting that differentially glycosylated forms may have different functional roles in vivo.

Properties of a polygalacturonase‐inhibiting protein isolated from ‘Oroblanco’ grapefruit

Polygalacturonase inhibiting protein (PGIP) was extracted from 'Oroblanco' grapefruit type (triploid pummelo-grapefruit) albedo tissue, purified and partially characterized. Extraction was carried out at 4 degrees C with a high ionic strength extraction buffer. After dialysis and concentration by ultrafiltration the extract was chromatographed on concanavalin A-Sepharose. The PGIP activity was bound by the lectin and then eluted using 250 mM alpha-methyl mannopyranoside, resulting in a 17-fold purification of the PGIP and demonstrating its glycoprotein nature. The anion-exchange and size-exclusion chromatography steps that followed gave a PGIP that was 857-fold purified relative to the initial tissue extract, and having a 44 kDa molecular weight, as estimated by SDS-PAGE electrophoresis. PGIP inhibition activity was tested with endo-polygalacturonase (EC 3.2.1.15) produced by Penicillium italicum and Botrytis cinerea. The radial diffusion and reducing sugar assays showed that P. italicum and B. cinerea endo-PGs were affected by PGIP, whereas no endo-PG activity was detected in the culture filtrate of P. digitatum. In vitro tests revealed that PGIP inhibited P. italicum and B. cinerea growth. By contrast, the influence of PGIP on P. digitatum, growth was negligible, perhaps because this fungus does not produce endo-PG. Following heating for 10 min at 65 degrees C the inhibitory activity of PGIP was reduced by 43%. PGIP activity decreased further as heating temperature increased, and was completely suppressed after heating at 100 degrees C for 10 min.

Purification and biochemical characterisation of a membrane-bound α-glucosidase from the parasite Entamoeba histolytica

An α-glucosidase was solubilised from a mixed membrane fraction of Entamoeba histolytica and purified to homogeneity by a two-step procedure consisting of ion exchange chromatography in a Mono Q column and affinity chromatography in concanavalin A-sepharose. Although the enzyme failed to bind the lectin, this step rendered a homogenous and more stable enzyme preparation that resolved into a single polypeptide of 55 kDa after SDS-PAGE. As measured with 4-methylumbelliferyl-α- d-glucopyranoside (MUαGlc) as substrate, glycosidase activity was optimum at pH 6.5 with different buffers and at 45 °C. Although the enzyme preferentially hydrolysed nigerose (α1,3-linked), it also cleaved kojibiose (α1,2-linked), which was the second preferred substrate, and to a lesser extent maltose (α1,4), trehalose (α1,1) and isomaltose (α1,6). Activity on α1,3- and α1,2-linked disaccharides was strongly inhibited by the glycoprotein processing inhibitors 1-deoxynojirimycin and castanospermine but was unaffected by australine. Glucose and particularly 3-deoxy- d-glucose and 6-deoxy- d-glucose were strong inhibitors of activity, whereas 2-deoxy- d-glucose and other monosaccharides had no effect. Enzyme activity on MUαGlc was very sensitive to inhibition by diethylpyrocarbonate suggesting a critical role of histidine residues in enzyme catalysis. Other amino acid modifying reagents such as N-ethylmaleimide and N-(3-dimethylaminopropyl)- N'ethylcarbodiimide showed a moderate effect or none at all, respectively. Results are discussed in terms of the possible involvement of this glycosidase in N-glycan processing.

Functional dissection of the transmembrane domains of the transporter associated with antigen processing (TAP).

The transporter associated with antigen processing (TAP1/2) translocates cytosolic peptides of proteasomal degradation into the endoplasmic reticulum (ER) lumen. A peptide-loading complex of tapasin, major histocompatibility complex class I, and several auxiliary factors is assembled at the transporter to optimize antigen display to cytotoxic T-lymphocytes at the cell surface. The heterodimeric TAP complex has unique N-terminal domains in addition to a 6 + 6-transmembrane segment core common to most ABC transporters. Here we provide direct evidence that this core TAP complex is sufficient for (i) ER targeting, (ii) heterodimeric assembly within the ER membrane, (iii) peptide binding, (iv) peptide transport, and (v) specific inhibition by the herpes simplex virus protein ICP47 and the human cytomegalovirus protein US6. We show for the first time that the translocation pore of the transporter is composed of the predicted TM-(5-10) of TAP1 and TM-(4-9) of TAP2. Moreover, we demonstrate that the N-terminal domains of TAP1 and TAP2 are essential for recruitment of tapasin, consequently mediating assembly of the macromolecular peptide-loading complex.

Identification of paraoxonase 3 in rat liver microsomes: purification and biochemical properties.

Three paraoxonase genes (PON1, PON2 and PON3) have been described so far in mammals. Although considerable information is available regarding PON1, little is known about PON2 and PON3. PON3 has been isolated recently from rabbit serum [Draganov, Stetson, Watson, Billecke and La Du (2000) J. Biol. Chem. 275, 33435-33442] and liver [Ozols (1999) Biochem. J. 338, 265-275]. In the present study, we have identified the presence of PON3 in rat liver microsomes and a method for the purification to homogeneity is presented. PON3 has been purified 177-fold to apparent homogeneity with a final specific activity of 461 units/mg using a method consisting of seven steps: solubilization of the microsomal fraction, hydroxyapatite adsorption, chromatography on DEAE-Sepharose CL-6B, non-specific affinity chromatography on Cibacron Blue 3GA, two DEAE-cellulose steps and a final affinity chromatography on concanavalin A-Sepharose. SDS/PAGE of the final preparation indicated a single protein-staining band with an apparent molecular mass of 43 kDa. The isolated protein was identified by nanoelectrospray MS. Internal amino acid sequences of several peptides were determined and compared with those of human, rabbit and mouse PON3, showing a high similarity. Some biochemical properties of PON3 were also studied, including optimum pH, K(m) and heat and pH stability.

Extracellular alkaline phosphatase from the filamentous fungus Aspergillus caespitosus: purification and biochemical characterization.

Among 30 species of filamentous fungi isolated from Brazilian soil, Aspergillus caespitosus produced and secreted the highest levels of alkaline phosphatase in culture medium supplemented with xylan. The extracellular alkaline phosphatase was purified by DEAE-cellulose and concanavalin A-sepharose chromatography. The enzyme was a glycoprotein containing up to 56% sugar with molar mass of 134.8 kDa, according to gel filtration in Sepharose CL-6B, and 57 kDa according to SDS-PAGE. Nondenaturing electrophoresis (6% PAGE) of the purified enzyme produced a single band, suggesting that the native enzyme was a homodimer. Optima of temperature and pH were 75 degrees C and 8.5, respectively. The enzyme was stable at 50 degrees C and its activity was enhanced by 95% in the presence of Mg2+ (1 mmol/L). 4-Nitrophenyl phosphate was the preferentially hydrolyzed substrate with K(m) and upsilon lim values of 74 mumol/L and 285 mumol/s, in the absence, and 90 mumol/L and 418 mumol/s, in the presence of Mg2+, respectively. The enzyme also hydrolyzed other phosphorylated amino acids (O-phosphothreonine, O-phosphotyrosine, O-phosphoserine).

Binding of the pheromonal steroid, 5α-androst-16-en-3-one, to membrane-enriched fractions of boar and rat olfactory epithelium; preliminary evidence for binding protein being a glycoprotein

A high degree of binding of 5α-[ 3 H ]-androstenone was recorded in membrane-enriched fractions of porcine olfactory tissue. The specific (i.e. high affinity, low capacity) binding had a mean K a approximately 2×10 8 M −1. A Hill plot of the data showed a Hill coefficient of approximately 2, possibly suggesting co-operativity of binding, with binding constants increasing from 8×10 7 to 1.6×10 9 M −1 with increasing substrate concentration. The level of specific binding of 5α-[ 3 H ]-androstenone was nearly 10-fold higher than in corresponding respiratory tissue preparations and was markedly reduced in the presence of excess (approximately 1 μM) unlabelled 5α-androstenone. Corresponding fractions derived from rat olfactory tissue showed only 25% of the binding recorded for the pig. After incubation of 5α-[ 3 H ]-androstenone with solubilised olfactory cilial tissue (porcine), gel filtration and chromatography on a typical “glycoprotein” column (Concanavalin A-Sepharose B) were performed. Specific binding was recorded only in fractions corresponding to glycoproteins with M r of approximately 70–90 kDa. In a third series of experiments, fractions containing high concentrations of cilia, some still attached to the dendritic endings (as shown by electron microscopy) were obtained by a novel method involving stripping them off the nasal epithelium. The basal adenylate cyclase (AC) activity was very significantly ( P<0.01) higher in olfactory, compared with respiratory, cilia; storage at −70 °C for 3 weeks greatly reduced AC activity. When fresh male and female porcine olfactory cilia preparations were incubated with 5α-androstenone plus GTP, AC activity was increased fourfold ( P<0.01). However, responses of porcine respiratory cilia were not significant statistically, neither were changes in basal levels of AC activities in rat olfactory cilia.

Binding of Zona Binding Inhibitory Factor-1 (ZIF-1) from Human Follicular Fluid on Spermatozoa

Previous studies showed that zona binding inhibitory factor-1 (ZIF-1) was the glycoprotein mainly responsible for the spermatozoa zona binding inhibitory activity of human follicular fluid. ZIF-1 has a number of properties similar to glycodelin-A. A binding kinetics experiment in the present study demonstrated the presence of two binding sites of ZIF-1 on human spermatozoa. These binding sites were saturable, reversible, and bound to (125)I-ZIF-1 in a time-, concentration-, and temperature-dependent manner. Glycodelin-A shared one common binding site with ZIF-1 on spermatozoa, and it could displace only 70% of the (125)I-ZIF-1 bound on human spermatozoa. ZIF-1 and glycodelin-A formed complexes with the soluble extract of human spermatozoa. Coincubation of solubilized zona pellucida proteins reduced the binding of ZIF-1 to two complexes of the extract, suggesting that the ZIF-1 binding sites and zona pellucida protein receptors on human spermatozoa were closely related. ZIF-1, but not glycodelin-A, significantly suppressed progesterone-induced acrosome reaction of human spermatozoa. The carbohydrate moieties derived from ZIF-1 reduced the binding of native ZIF-1 on human spermatozoa as well as the zona binding inhibitory activity of the glycoprotein, although the intensity of the effects are lower when compared with the native protein. These effects are not due to the action of the molecules on the motility, viability, and acrosomal status of the treated spermatozoa. Deglycosylated ZIF-1 had no inhibitory effect on both ZIF-1 binding and zona binding capacity of spermatozoa. We concluded that the carbohydrate part of ZIF-1 was critical for the functioning of the glycoprotein.

Purification of angiotensin I converting enzyme from pig lung using concanavalin-A sepharose chromatography

Angiotensin I converting enzyme (ACE) plays a major role in blood pressure regulation, catalyzing the conversion of angiotensin I to the vasoconstrictor angiotensin II. In this report we describe a two-step affinity chromatography method for preparative purification of ACE from pig lung using Concanavalin-A Sepharose 4B and affinity chromatography on Lisinopril Sepharose 6B. The same purification scheme was used to obtain Cobalt-ACE, where zinc ion located at the active site is replaced by cobalt. Cobalt-ACE visible spectrum shows a characteristic broad peak from 500 to 600 nm. The shape and maximum absorptivity of this peak changes in presence of ACE inhibitors that bind at the catalytic site.

ELISA FOR QUANTITATION OF SERUM C-erbB-2 ONCOPROTEIN IN BREAST CANCER PATIENTS

ABSTRACT C-erbB-2 is a protooncogene that is overexpressed in various cancers, either due to its amplification and/or increased transcription, and has been associated with more aggressive disease and a poor clinical prognosis in 20–30% of patients with breast cancer. Besides the prognostic factors such as tumor size, tumor grade, lymph node status, etc., which are significant in the management of breast cancer, C-erbB-2 level might also serve as an additional factor. Immunohistochemistry is the most frequently used method to study the expression of C-erbB-2 in breast cancer. We have generated a panel of monoclonal antibodies against C-erbB-2 oncoprotein with a view to evaluate their application for the diagnosis and therapy of breast cancer. In the present study, a simple, quantitative sandwich ELISA has been developed that uses two monoclonal antibodies directed against the extracellular domain of C-erbB-2 product, designated as CIBCgp185 as the capture antibody and CIBCHER-2 as the detector antibody. C-erbB-2 protein, isolated from BT474 cells, a human breast carcinoma cell line with high expression of C-erbB-2 and purified by Concanavalin A-Sepharose 4B affinity chromatography and HPLC has been used to develop the ELISA procedure. Sera samples of 150 healthy women donors and of 300 breast cancer patients with different histological types of malignancies have been analysed. The control women had serum C-erbB-2 in the range of 4.0–13.2 ng/mL, whereas the 300 breast cancer patients studied had a range of 4.8–75.2 ng/mL with a cut off value of 13.8 ng/mL. Our study showed that 18.6% of breast cancer patients had elevated levels of circulating C-erbB-2. These results might suggest that the serum C-erbB-2 level can be used as a potential tumor marker for breast cancer and that the Sandwich ELISA procedure might serve as an excellent alternative to immunohistochemistry in the near future.

Biochemical and enzymatic characterization of a partially purified casein kinase-1 like activity from Trypanosoma cruzi

Two protein kinase activities that use casein as a substrate, Q-I and Q-II, were identified in the epimastigote stage of Trypanosoma cruzi upon chromatography on Q-Sepharose. Q-I was purified further through concanavalin A-sepharose (Q-I*) to remove any trace of the contaminating protease cruzipain. The optimal activity for Q-I* was obtained at pH 8.0, 25 °C, 5 mM MgCl 2 and 75 mM NaCl. The size and p I of Q-I* were determined to be 33–36 kDa and 9.6, respectively. When two selective peptide substrates for casein kinases (CKs) (P1: RRKDLHDDEEDEAMSITA for CK1 and P2: RRRADDSDDDDD for CK2) were used, Q-I* was shown to specifically phosphorylate P1. Kinetic studies showed that Q-I* has a K m of 5.3±0.34 mg/ml for casein, 157.6±5.3 μM for P1 and 35.9±3.9 μM for ATP. The enzyme was inhibited by N-(2-amino-ethyl)-5-chloroisoquinoline-8-sulfonamide (CKI-7) or 1-(5-chloroisoquinoline-8-sulfonyl) (CKI-8), two inactivators of mammalian CKs. CKI-7 behaved as a competitive inhibitor with respect to ATP, with a K I of 75–100 μM. Treatment with high concentrations of polylysine or heparin also resulted in a significant inhibition of Q-I*. Two well-known activators of mammalian CKs, spermine and spermidine, were also tested. Spermine and spermidine activated Q-I* in a dose-dependent manner. Based on the following characteristics: (1) the ionic strength required for elution from anion-exchange resins; (2) its molecular size and monomeric structure; (3) p I; (4) high level of specificity for P1; (5) inactivation by CKI-7 and CKI-8; and (6) insensitivity to GTP and low concentrations of heparin, we conclude that Q-I* belongs to the CK1 family of protein kinases.

Alpha1-antichymotrypsin-like proteins I and II purified from bovine adrenal medulla are enriched in chromaffin granules and inhibit the proenkephalin processing enzyme "prohormone thiol protease".

Proteolytic processing of inactive proenkephalin and proneuropeptides is essential for the production of biologically active enkephalins and many neuropeptides. The incomplete processing of proenkephalin in adrenal medulla suggests that endogenous protease inhibitors may inhibit proenkephalin processing enzymes. This study demonstrates the isolation and characterization of two isoforms of adrenal medullary alpha1-antichymotrypsin (ACT), referred to as ACT-like proteins I and II, which are colocalized with enkephalin in chromaffin granules and which inhibit the proenkephalin processing enzyme known as prohormone thiol protease (PTP). Subcellular fractionation demonstrated enrichment of 56- and 60-kDa ACT-like proteins I and II, respectively, to enkephalin-containing chromaffin granules (secretory vesicles). Immunofluorescence cytochemistry of chromaffin cells indicated a discrete, punctate pattern of ACT immunostaining that resembles that of [Met]enkephalin that is stored in secretory vesicles. Chromatography of adrenal medullary extracts through DEAE-Sepharose and chromatofocusing resulted in the separation of ACT-like proteins I and II that possess different isoelectric points of 5.5 and 4.0, respectively. The 56-kDa ACT-like protein I was purified to apparent homogeneity by Sephacryl S200 chromatography; the 60-kDa ACT-like protein II was isolated by butyl-Sepharose, Sephacryl S200, and concanavalin A-Sepharose columns. The proenkephalin processing enzyme PTP was potently inhibited by ACT-like protein I, with a K(i,app) of 35 nM, but ACT-like protein II was less effective. ACT-like proteins I and II had little effect on chymotrypsin. These results demonstrate the biochemical identification of two secretory vesicle ACT-like proteins that differentially inhibit PTP. The colocalization of the ACT-like proteins and PTP within chromaffin granules indicates that they could interact in vivo. Results from this study suggest that these ACT-like proteins may be considered as candidate inhibitors of PTP, which could provide a mechanism for limited proenkephalin processing in adrenal medulla.

Purification and characterization of banana fruit acid phosphatase.

An acid phosphatase (APase, EC 3.1.3.2) from ripened banana (Musa cavendishii L. cv. Cavendish) fruit has been purified 1,876-fold to electrophoretic homogeneity and a final p-nitrophenylphosphate (pNPP)-hydrolyzing specific activity of 745 micromol Pi produced (mg protein)(-1) min(-1). Non-denaturing PAGE of the final preparation resolved a single protein-staining band that co-migrated with APase activity. SDS-PAGE and analytical gel filtration demonstrated that the purified enzyme exists as a 40-kDa monomer. That the enzyme is glycosylated was indicated by its tight absorption to Concanavalin A-Sepharose. Banana APase was relatively heat stable, displayed a symmetrical pH/activity profile with maximal activity at pH 5.8, and was activated 180% and 150% by 5 mM Mn2+ and Mg2+, respectively. The enzyme exhibited a broad substrate selectivity, with maximal specificity constants (Vmax/Km) obtained with pNPP, phosphoenolpyruvate, phenyl phosphate, and O-phospho-L-tyrosine. Potent inhibition by Pi, molybdate, vanadate, arsenate, and Zn2+ was observed. Putative metabolic functions of the APase are discussed in relation to maintaining significant Pi mobility during banana fruit ripening.

Thermostable conidial and mycelial alkaline phosphatases from the thermophilic fungus Scytalidium thermophilum.

An extracellular (conidial) and an intracellular (mycelial) alkaline phosphatase from the thermophilic fungus Scytalidium thermophilum were purified by DEAE-cellulose and Concanavalin A-Sepharose chromatography. These enzymes showed allosteric behavior either in the presence or absence of MgCl(2), BaCl(2), CuCl(2), and ZnCl(2). All of these ions increased the maximal velocity of both enzymes. The molecular masses of the conidial and mycelial enzymes, estimated by gel filtration, were 162 and 132 kDa, respectively. Both proteins migrated on SDS-PAGE as a single polypeptide of 63 and 58.5 kDa, respectively, suggesting that these enzymes were dimers of identical subunits. The best substrate for the conidial and mycelial phosphatases was p-nitrophenylphosphate, but beta-glycerophosphate and other phosphorylated compounds also served as substrates. The optimum pH for the conidial and mycelial alkaline phosphatases was 10.0 and 9.5 in the presence of AMPOL buffer, and their carbohydrate contents were about 54% and 63%, respectively. The optimum temperature was 70-75 degrees C for both activities. The enzymes were fully stable up to 1 h at 60 degrees C. These and other properties suggested that the alkaline phosphatases of S. thermophilum might be suitable for biotechnological applications.

A glycoprotein from Schistosoma mansoni eggs binds non-antigen-specific immunoglobulin E and releases interleukin-4 from human basophils.

We have recently shown that soluble extracts from Schistosoma mansoni eggs (SmEA) triggered basophils from nonsensitized donors to rapidly release interleukin (IL)-4. Assuming that this mechanism might play a role in vivo in biasing the immune response towards a Th2 phenotype, we determined basic properties of the IL-4-inducing activity contained in SmEA. Sensitivity to pepsin digestion indicated protein nature. Binding to and specific elution from Concanavalin A-sepharose suggested that this protein contains mannose residues, thus being a glycoprotein. The IL-4-inducing activity was stable for 30 min at room temperature towards shifting the pH between 3 and 10. When incubated at 100 degrees C, it was stable at pH 3, but less stable at neutral and alkaline pH. Electroelution from an SDS-PAGE gel indicated an apparent molecular weight of the IL-4-inducing activity between 31 and 66 kDa. Although binding to purified human immunoglobulin E (IgE) and activating basophils IgE-dependently, SmEA appears to activate basophils in a non-antigen-specific way, since the cells were purified from noninfected donors. Because the IL-4-inducing activity was found to be released from eggs, it could be an important factor in the environment of the eggs skewing the immune response towards the Th2 phenotype.

Monoglucosylated Oligomannosides Are Released during the Degradation Process of Newly Synthesized Glycoproteins

The Chinese hamster ovary mutant MI8-5 is known to synthesize Man(9)GlcNAc(2)-P-P-dolichol rather than the fully glucosylated lipid intermediate Glc(3)Man(9)GlcNAc(2)-P-P-dolichol. This nonglucosylated oligosaccharide lipid precursor is used as donor for N-glycosylation. In this paper we demonstrate that a significant part of the glycans bound to the newly synthesized glycoproteins in MI8-5 cells are monoglucosylated. The presence of monoglucosylated glycans on glycoproteins determines their binding to calnexin as part of the quality control machinery. Furthermore, we point out the presence of Glc(1)Man(5)GlcNAc(1) in the cytosol of MI8-5 cells. This indicates that part of the monoglucosylated glycoproteins can be directed toward a deglycosylation process that occurs in the cytosol. Besides studies on glycoprotein degradation based on the disappearance of protein moieties, MI8-5 cells can be used as a tool to elucidate the various step leading to glycoprotein degradation by studying the fate of the glycan moieties.

Subcellular distribution and glycosylation pattern of androgen receptor from sheep omental adipose tissue.

Sex steroids are known to have an influence on the distribution, metabolism and accretion of adipose tissue. These steroids carry out their function via specific receptors. We have previously reported the presence of oestrogen and progesterone receptors in sheep adipose tissues. In this study, we have tested the subcellular distribution of androgen receptor (AR) in sheep omental adipose tissue. Subcellular fractions - microsomes, plasma membrane and nuclei-cell debris - were isolated by differential and sucrose gradient centrifugation and confirmed by electron microscopy. The AR was determined in each fraction by Western blot analyses. As anticipated, the receptor was found in the cytosolic fraction, but a high concentration was also present in the microsomal fraction, a lesser amount in the plasma membrane fraction, and only a small amount was left in the nuclei-cell debris fraction. Two minor immunostaining bands with approximate molecular weights of 250 and 140 kDa and a major band at 110 kDa were detected in the cytosolic fraction, but only the 110 kDa band was detected in the membrane fractions. A 104 kDa band was observed on occasion and believed to be a degradation product. The cytosolic isoforms were tested for sensitivity to glycosidases. This treatment resulted in a decrease in the amount of the 250 and 140 kDa bands. To substantiate that the 250 and 140 kDa isoforms were glycoproteins, the cytosolic fraction was chromatographed on Concanavalin A-Sepharose. The 110 kDa band was eluted in the 0.4 M KCl salt wash while the 250 and 140 kDa bands were eluted with alpha-methylmannoside. Treatment of the glycoprotein (alpha-methylmannoside) peak with glycosidases converted the 250 and 140 kDa bands to the 110 kDa band. These data confirm the presence of AR in subcellular fractions of adipose tissue and suggest that it exists in various glycosylated isoforms.