Wheat Germ Lectin-agarose Research Articles

Improved lectin-mediated immobilization of human red blood cells in superporous agarose beads

A new type of agarose bead, superporous agarose, was used as a gel support for immobilization of human red blood cells (RBCs) mediated by wheat germ lectin. The number of immobilized cells was similar to that obtained with commercial wheat germ lectin–agarose but the cell stability appeared to be superior. This allowed improved frontal affinity chromatographic analyses of cytochalasin B (CB)-binding to the glucose transporter GLUT1 which established a ratio of one CB-binding site per GLUT1 dimer for both plain RBCs or those treated with different poly amino acids. The measured dissociation constants, 70±14 n M for CB and 12±3 m M for glucose binding to GLUT1, are similar to those reported earlier.

Chromatography on cells: analyses of solute interactions with the glucose transporter Glut1 in human red cells adsorbed on lectin-gel beads

The affinities of the human red cell glucose transporter Glut1 for D-glucose and cytochalasin B (CB) and the stoichiometry of CB binding vary with the Glut1 environment. In order to study the native state of Glut1 we adsorbed human red cells to wheat germ lectin agarose gel beads for frontal affinity chromatographic analyses. Glut1 showed relatively high affinities for D-glucose ( K d 12±1 m M) and CB ( K d 59±17 n M). The number of CB-binding sites per Glut1 monomer, 0.46±0.16, was approximately doubled upon coating the cells with polylysine, which induced cell association.

Isolation of the [3H]Gabapentin-Binding Protein/α2δ Ca2+Channel Subunit from Porcine Brain: Development of a Radioligand Binding Assay for α2δ Subunits Using [3H]Leucine

The novel antiepileptic agent gabapentin (Neurontin) binds with high affinity to the α2δ subunit of a voltage-dependent Ca2+channel. We report here a simple purification scheme for detergent-solubilized α2δ subunits from porcine brain. This involves sequential chromatography on Q-Sepharose, Cu2+-charged iminodiacetic acid–Sepharose, wheat germ lectin–agarose, and Mono Q. The purified protein was essentially homogeneous by SDS–polyacrylamide gel electrophoresis with a subunitMrof 145,000. Using [3H]gabapentin as the radiolabeled tracer and (S)-3-isobutyl γ-aminobutyric acid to define nonspecific binding, the overall purification factor was 2760-fold and the apparent yield 26.6%. We also developed and validated a novel binding assay for α2δ Ca2+channel subunits using the ligand pairl-[3H]leucine/l-isoleucine. Evenin binding assays of crude brain membrane fractions, [3H]leucine proved to be remarkably stable and specific for the α2δ Ca2+channel subunit. [3H]Leucine offers several advantages over custom-labeled [3H]gabapentin: it has a higher specific activity, is relatively inexpensive, and is available from commercial sources.

Novel Affinity Support Prepared by Crosslinking Purified Insulin Receptors to Agarose

The insulin receptor interacts with other membrane proteins and possibly also a cytoplasmic second messenger(s) of insulin action. As an approach to identify and purify these ligands of the insulin receptor, we have developed an insulin receptor affinity support which retains the properties of the membrane associated insulin receptors. Insulin receptors first were purified from rat liver by affinity chromatography on wheat germ lectin agarose and insulin agarose. The purified insulin receptors then were crosslinked to agarose. The agarose immobilised insulin receptors had an affinity for insulin indistinguishable from the native receptors. In addition, insulin enhanced the autophosphorylation of the β-subunit of these agarose immobilised insulin receptors. The insulin binding and autophosphorylation activities were stable for at least one week when this novel affinity support was stored at 4°C.

Solubilization and affinity purification of the Y2 receptor for neuropeptide Y and peptide YY from rabbit kidney.

Neuropeptide Y (NPY) is an important neuropeptide in both central and peripheral neurones whereas peptide YY (PYY) is a gut hormone present in endocrine cells in the lower bowel. Both peptides interact with multiple binding sites that have been further classified into Y1 and Y2 receptors. We have solubilized native Y2 receptors both from basolateral membranes of proximal convoluted tubules from rabbit kidney and from rat hippocampal membranes. Solubilization of functional Y2 receptors was obtained with both 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate (CHAPS) and digitonin and resulted in each case in a single class of high affinity binding sites. The soluble receptor retained the binding specificity for different peptides and long C-terminal fragments of NPY exhibited by membrane preparations. Gel filtration of solubilized receptors resulted in a single peak of specific PYY binding activity corresponding to Mr = 350,000 whereas affinity labeling revealed a major band of Mr = 60,000. Since this binding activity was inhibited by guanosine 5'-3-O-(thio)triphosphate (GTP gamma S) the Y2 receptor is probably solubilized as a receptor complex containing a G-protein along with the ligand binding protein. Y2 receptor binding sites from kidney tubular membranes were purified to homogeneity by a three-step procedure employing Mono S cation-exchange adsorption, affinity chromatography on wheat germ lectin-agarose beads, and affinity chromatography on NPY-Affi-Gel. Electrophoresis and silver staining of the final receptor preparation revealed a single protein with Mr = 60,000 whereas gel filtration showed a single peak at approximately Mr = 60,000. The purified protein can be affinity labeled with [125I-Tyr36]PYY, indicating that the Mr = 60,000 protein contains the ligand binding site of the Y2 receptor, and this binding is not affected by GTP gamma S. Scatchard transformation of binding data for the purified Y2 receptors was compatible with a single class of binding sites with Kd = 76 pM. The purified Y2 receptors retain their binding properties with regard to affinity and specificity for different members of the pancreatic polypeptide-fold peptide family. The specific activity of purified Y2 receptors was calculated to approximately 14.7 nmol of ligand binding/mg of receptor protein, which is consistent with the theoretical value (16.6 nmol/mg) for a pure Mr = 60,000 protein binding one PYY molecule. Purification to homogeneity thus reveals the Y2 receptor as an Mr = 60,000 glycoprotein.

Application of high-performance liquid chromatography to the purification of the putative intestinal peptide transporter

A membrane protein of relative molecular mass ( M r) 127 000 was identified by photoaffinity labelling as (a component of) the uptake system for small peptides and β-lactam antibiotics in rabbit small intestine. This binding protein is a microheterogeneous glycosylated integral membrane protein which could be solubilized with non-ionic detergents and enriched by lectin affinity chromatography on wheat germ lectin agarose. For the final purification of this protein and separation from aminopeptidase N of M r 127 000, fast protein liquid chromatography (FPLC) was used. Gel permeation, hydroxyapatite and hydrophobic interaction chromatography were not successful for the purification of the 127 000-dalton binding protein. By anion-exchange chromatography on a Mono Q column with either Triton X-100 or n-octylglucoside as detergent, a partial separation of the 127 000-dalton binding protein from aminopeptidase N was achieved. By cation-exchange chromatography on a Mono S HR 5 5 column at pH 4.5 using Triton X-100 as detergent also only a partial separation from aminopeptidase N could be achieved. If, however, Triton X-100 was replaced with n-octylglucoside, the binding protein for β-lactam antibiotics and small peptides of M r 127 000 could be completely separated from aminopeptidase N. These results indicate that Triton X-100 should be avoided for the purification of integral membrane proteins because mixed protein-detergent micelles of high molecular weight prevent a separation into the individual membrane proteins. The putative peptide transport protein was finally purified by rechromatography on Mono S and was obtained more than 95% pure as determined densitometrically after sodium dodecyl sulphate gel electrophoresis. By application of FPLC even microheterogeneous membrane glycoproteins from the intestinal mucosa can be purified to such an extent that a sequence analysis and immunohistochemical localization with antibodies prepared from the purified protein is possible.

Identification of a novel glycoprotein (AGp110) involved in interactions of rat liver parenchymal cells with fibronectin.

We have identified an integral membrane glycoprotein in rat liver that mediates adhesion of cultured hepatocytes on fibronectin substrata. The protein was isolated by affinity chromatography of detergent extracts on wheat germ lectin-Agarose followed by chromatography of the WGA binding fraction on fibronectin-Sepharose. The glycoprotein (AGp110), eluted at high salt concentrations from the fibronectin column, has a molecular mass of 110 kD and a pI of 4.2. Binding of immobilized AGp110 to soluble rat plasma fibronectin required Ca2+ ions but was not inhibited by RGD peptides. Fab' fragments of immunoglobulins raised in rabbits against AGp110 reversed the spreading of primary hepatocytes attached onto fibronectin-coated substrata, but had no effect on cells spread on type IV collagen or laminin substrata. The effect of the antiserum on cell spreading was reversible. AGp110 was detected by immunofluorescence around the periphery of the ventral surface of substratum attached hepatocytes, and scattered on the dorsal surface. Immunohistochemical evidence and Western blotting of fractionated liver plasma membranes indicated a bile canalicular (apical) localization of AGp110 in the liver parenchyma. Expression of AGp110 is tissue specific: it was found mainly in liver, kidney, pancreas, and small intestine but was not detected in stomach, skeletal muscle, heart, and large intestine. AGp110 could be labeled by lactoperoxidase-catalyzed surface iodination of intact liver cells and, after phase partitioning of liver plasma membranes with the detergent Triton X-114, it was preferentially distributed in the hydrophobic phase. Treatment with glycosidases indicated extensive sialic acid substitution in at least 10 O-linked carbohydrate chains and 1-2 N-linked glycans. Immunological comparisons suggest that AGp110, the integrin fibronectin receptor and dipeptidyl peptidase IV, an enzyme involved in fibronectin-mediated adhesion of hepatocytes on collagen, are distinct proteins.

The thyroid stimulating hormone receptor in human disease

The initial step in the action of thyrotropin (TSH) is its binding to the TSH receptor. TSH receptor antibodies are detected in up to 90% of patients with Graves' disease. Serial measurements of TSH receptor antibodies in patients with Graves' hyperthyroidism are helpful in predicting relapse. The TSH receptor was purified using affinity chromatography on wheat germ lectin agarose and TSH-agarose. Using an immunoblotting technique to characterize the TSH receptor, it was found to be an oligomeric glycoprotein consisting of three noncovalently bound subunits of Mr approximately 70,000, approximately 50,000 and approximately 35,000 which on reduction yield a single subunit of Mr approximately 25,000.

Isolation of a protein fraction from Bordetella pertussis that facilitates entry of the calmodulin-sensitive adenylate cyclase into animal cells

Bordetella pertussis, the pathogen responsible for whooping cough, releases a soluble calmodulin-sensitive adenylate cyclase into its culture medium. Several investigators have shown that the partially purified adenylate cyclase is capable of entering animal cells and elevating intracellular cAMP levels [Confer, D. L., & Eaton, J. W. (1982) Science 217, 948-950; Shattuck, R. L., & Storm, D. R. (1985) Biochemistry 24,6323-6328]. However, the mechanism for entry of the catalytic subunit of the adenylate cyclase into animal cells is unknown. Recently, it was determined that the purified catalytic subunit of the enzyme is unable to enter animal cells [Masure, H. R., Oldenburg, D. J., Donovan, M. G., Shattuck, R. L., & Storm, D. R. (1988) J. Biol. Chem. 263, 6933-6940]. On the basis of these data and other observations, we hypothesized that the culture medium of B. pertussis contains one or more additional polypeptides which facilitate entry of the adenylate cyclase catalytic subunit into animal cells. In this study, we report that a cell-invasive preparation of B. pertussis adenylate cyclase was rendered noninvasive after passage through a wheat germ lectin-agarose column. A fraction was eluted from the wheat germ lectin-agarose column with N-acetyl-D-glucosamine. This fraction, when combined with the noninvasive adenylate cyclase, was able to restore the ability of the adenylate cyclase preparation to enter neuroblastoma cells and increase intracellular cAMP levels. Furthermore, the fraction eluted from the wheat germ lectin-agarose column was found to be trypsin and chymotrypsin sensitive, suggesting that this material was proteinaceous.(ABSTRACT TRUNCATED AT 250 WORDS)

Stabilization of soluble active rat liver glucagon receptor

Active glucagon receptor was solubilized with 3-(3-cholamidopropyl)dimethylammonio-1-propanesulfonate (Chaps) from rat liver plasma membranes but rapidly (<8 h) lost activity. Either inclusion of 1× Hanks' balanced salt solution in the 3 m m Chaps solubilization buffer or its addition after solubilization increased the percentage of total binding attributable to specific glucagon binding from ~10 to >80%; of great importance, it increased the stability from near zero binding at 8 h to 50% binding at 48 h (4 °C). Of the Hanks' solution components, either NaCl (137 m m) or CaCl 2 (1.26 m m) was effective in increasing specific binding to ~70 and 60% respectively: Mg salts were ineffective. Soluble receptor binding activity was assayed by dextran-coated charcoal adsorption of free hormone. The assay is rapid, simple, and reproducible. It is suitable for monitoring receptor activity during purification and molecular characterization. Competition binding studies gave an IC 50 value of 10–20 n m (slope factor ~1), with or without GTP. Dissociation assays revealed GTP sensitivity when receptors were solubilized either as glucagon-receptor complexes or free receptor. Active glucagon-receptor complexes could be eluted from wheat germ lectin-agarose: neither concanavalin A-agarose nor soybean agglutinin-agarose bind receptor. A glucagon degrading activity which cosolubilized with the receptor but did not require detergent for extraction was distinguishable from the soluble receptor not only by solubility but also by its heat stability (30 °C), its inhibition by bacitracin, its affinity for glucagon, its retention of activity for at least 1 week at 4 °C, and its size.

Human Thyrotropin Receptor Subunits Characterized by Thyrotropin Affinity Purification and Western Blotting*

We studied the subunit structure of the human TSH receptor in thyroid tissue from patients with Graves' disease and multinodular goiter by TSH affinity chromatography, immunoprecipitation with Graves' immunoglobulins (Igs), and a modified technique of Western blotting. Human TSH receptor-binding activity was purified about 1,270-fold by sequential affinity chromatography on wheat germ lectin-agarose and TSH-agarose. Analysis by sodium dodecyl sulfate-polyacrylamide gel electrophoresis of nonreduced affinity-purified receptors eluted in sodium dodecyl sulfate sample buffer revealed three noncovalently linked subunits of 70,000, 50,000, and 35,000 mol wt. When reduced, a major subunit of 25,000 mol wt was identified. When 3 mol/L NaCl was used to elute affinity-purified receptors only the 50,000 mol wt nonreduced subunit was detected. This subunit bound [125I]bovine TSH and was precipitated by Graves' Igs. Modifications to the conventional Western blotting technique enabled thyroglobulin components (approximately 220,000 mol wt), thyroid microsomal antigen (a doublet of approximately 110,000 mol wt), and putative TSH receptor subunits of 70,000 and 50,000 mol wt to be identified in thyroid particulate membranes by Graves' Igs. Blotting of affinity-purified receptors eluted in sodium dodecyl sulfate sample buffer revealed subunits of either 70,000 or 50,000 mol wt, with a minority of Graves' serum samples. We conclude that the nonreduced human TSH receptor is an oligomeric complex comprising three different subunits of 70,000, 50,000, and 35,000 mol wt. The reduced receptor exists as a single subunit of 25,000 mol wt, which may be disulfide linked to form the higher mol wt forms. The 70,000 and 50,000 mol wt subunits contain epitopes that bind Graves' Igs in modified Western blots, thus directly confirming that the human TSH receptor is a target for Graves' Igs.

Affinity chromatography with the immobilized agonist N-0434 yields an active and highly purified preparation of the dopamine D-2 receptor from bovine striatum

Partial purification of the dopamine D-2 receptor from bovine striatum, solubilized in the presence of 1% digitonin, was obtained by chromatography on wheat germ lectin agarose. The preparation was purified approximately 10-fold. The stability of the receptor preparation was considerably improved and non-specific protein absorption on the affinity gel used later was decreased. Further purification was achieved on a column containing a D-2-selective agonist, N-0434. Approximately 90% of the receptor activity was bound to the gel and 20–40% of the activity could be eluted by pH shock. The total purification factor after one affinity chromatogtaphy step was estimated to be at least 1500. An active preparation of at least 20% purity was obtained after a second cycle of affinity chromatography. This corresponds to an enrichment of more than 5000 times compared to the solubilized receptor preparation.

Further characterization of serum alkaline phosphatase from male and female beagle dogs.

Alkaline phosphatase (AP) from the sera of both male and female beagle dogs was partially purified and then analyzed for the presence of AP isoenzymes having intestinal or osseous characteristics as detected by bromotetramisole inhibition or wheat germ lectin agarose electrophoresis, respectively. The sera from both sexes were similar in regard to the presence of AP isoenzymes with intestinal (16 vs. 20%) or osseous (19 vs. 23%) characteristics, but serum AP from the male had a greater sialic acid content and only the male serum contained a detectable constitutive acidic (pI = 3.4) AP isoenzyme. This was similar to a serum AP isoenzyme previously found elevated in the sera of dogs afflicted with hyperadrenocorticalism or of dogs treated with certain corticosteroids.

Glycoproteins synthesized by cultured cardiac valve endothelial cells: Unique absence of fibronectin production

We have previously reported that cultured porcine cardiac valve endothelial cells released less fibronectin into the culture supernatant when compared to other porcine endothelial cells. In this report we compared the spectrum of glycoproteins synthesized by cardiac valve endothelial cells to glycoproteins synthesized by comparison endothelial cells derived from the ascending thoracic aorta. The cells were endogenously radiolabeled and extracted with detergent. Glycoproteins in the cell extracts were then isolated on wheat germ lectin-agarose and compared using autoradiography following polyacrylamide gel electrophoresis. Fibronectin was identified by immunoblotting with specific antibody. The results showed that the outstanding difference between the endothelial cell types was the virtual absence of fibronectin in the cardiac valve endothelial cell extract.

Characterization of a high-molecular-weight form of epidermal growth factor in an extract of human urine

We purified from a side fraction of the commercial preparation of urokinase from large volumes of human urine a high-molecular-weight (HMW) form of human epidermal growth factor (hEGF). Sequence analysis of the amino terminus of the intact molecule and of two tryptic fragments and carboxypeptidase Y analysis revealed the molecule to correspond to residues 828–1023 of the hEGF precursor predicted by the nucleotide sequence of human renal hEGF mRNA, with hEGF forming its carboxyl terminus. HMW hEGF bound poorly to concanavalin A-agarose, quite avidly to wheat germ lectinagarose, and completely to phenyl boronate-agarose, suggesting that it was O-glyco-sylated. Sephacryl S-200 chromatography of freshly-voided urine revealed mostly hEGF, with smaller amounts of a much higher molecular weight hEGF, but little material that was the size of the HMW hEGF we characterized. The large fragment we characterized presumably is cleaved from the larger form by enzyme(s) present in urine during the collection, storage, and processing of urine. We have confirmed that hEGF is synthesized as a large precursor molecule, as predicted by the nucleotide sequence of hEGF mRNA.

An atypical insulin receptor with high affinity for insulin-like growth factors copurified with placental insulin receptors.

Insulin receptors purified from human placenta by sequential affinity chromatography on wheat germ lectin-agarose and insulin coupled to 1,1'-carbonyldiimidazole-activated agarose (CDI-agarose) retained full binding activity but bound a greater than predicted amount of 125I-labeled insulin-like growth factor I (IGF-I). IGF-I and multiplication-stimulating activity (MSA; the rat homologue of IGF-II) were equipotent in displacing either 125I-labeled IGF-I or 125I-labeled MSA from the purified receptors; insulin was 5-15 times more potent. Competitive binding studies indicated that this IGF binding activity could not be explained by cross-reaction with classical insulin receptors or by coelution of IGF-I or IGF-II receptors. Instead, it was due to a minor population of discrete atypical insulin receptors (6-18% total insulin receptors) with moderately high affinity (Kd = 2-4 X 10(-9) M) for IGF-I and MSA. These receptors were not an artifact of insulin-CDI-agarose chromatography, since they were present in wheat germ lectin-agarose-purified preparations and could also be purified from insulin-succinyldiaminodipropylamino-agarose. Affinity labeling with 125I-labeled MSA revealed that these atypical receptors had the same binding subunit (Mr 140,000) as classical insulin and IGF-I receptors. They displayed intermediate reactivity with polyclonal and monoclonal antibodies to the insulin and IGF-I receptors. It is therefore likely that insulin receptors purified by immunoadsorption would also contain atypical insulin receptors. The finding of more than one type of insulin receptor might relate to the slight variations in the cDNA nucleotide sequences and the multiple mRNA species reported for the insulin receptor [Ebina, Y., Ellis, L., Jarnagin, K., Edery, M., Graf, L., Clauser, E., Ou, J.-H., Masiarz, F., Kan, Y. W., Goldfine, I. D., Roth, R. A. & Rutter, W. J. (1985) Cell 40, 747-758].

Homogeneous bivalent insulin receptor: Purification using insulin coupled to 1,1′-carbonyldiimidazole activated-agarose

A novel affinity gel, consisting of insulin coupled to 1,1′-carbonyldiimidazole-activated agarose (CDI-agarose), was used to purify insulin receptors from human placenta to homogeneity. This affinity gel is reproducibly prepared and is reported to have a number of advantages over the standard cyanogen bromide activated supports, such as ease and simplicity of coupling and minimal ligand leakage and non-specific binding. Insulin receptors in Triton X-100-solubilized microsomal membranes were purified 2,000-fold by sequential affinity chromatography on wheat germ lectin-agarose and insulin-CDI-activated agarose. They have one of the highest specific insulin-binding capacities (6 nmol/mg protein) reported and can be calculated to have a binding valence of two on the basis that the molecular weight of the oligomeric receptor is 300–350,000.