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PubMed1988

Characterization of rat heart plasma membrane Ca2+/Mg2+ ATPase.

The Ca2+/Mg2+ ATPase of rat heart plasma membrane was activated by millimolar concentrations of Ca2+ or Mg2+; other divalent cations also activated the enzyme but to a lesser extent. Sodium azide at high concentrations inhibited the enzyme by about 20%; oligomycin at high concentrations also inhibited the enzyme slightly. Trifluoperazine at high concentrations was found inhibitory whereas trypsin treatment had no significant influence on the enzyme. The rate of ATP hydrolysis by the Ca2+/Mg2+ ATPase decayed exponentially; the first-order rate constants were 0.14-0.18 min-1 for Ca2+ ATPase activity and 0.15-0.30 min-1 for Mg2+ ATPase at 37 degrees C. The inactivation of the enzyme depended upon the presence of ATP or other high energy nucleotides but was not due to the accumulation of products of ATP hydrolysis. Furthermore, the inactivation of the enzyme was independent of temperature below 37 degrees C. Con A when added into the incubation medium before ATP blocked the ATP-dependent inactivation; this effect was prevented by alpha-methylmannoside. In the presence of low concentrations of detergent, the rate of ATP hydrolysis was reduced while the ATP-dependent inactivation was accelerated markedly. Both Con A and glutaraldehyde decreased the susceptibility of Ca2+/Mg2+ ATPase to the detergent. These results suggest that the Ca2+/Mg2+ ATPase is an intrinsic membrane protein which may be regulated by ATP.

Adenosine Triphosphate↗
PubMed1990

Characterization of molecular species of liver microsomal carboxylesterases of several animal species and humans.

Seven carboxylesterase isozymes were purified to electrophoretic homogeneity from liver microsomes of mouse, hamster, guinea pig, rabbit, and monkey by the same procedure used previously to obtain three isozymes from the rat, and their physical, enzymological, and immunological properties were compared with those of the rat isozymes. The substrate specificity and immunological reactivity of liver microsomal carboxylesterases from pig, cow, beagle dog, and human were also examined for comparison, though these enzymes were not purified. The ten purified preparations have similar subunit weight (57,000-64,000), but their isoelectric points differ widely (4.7-6.5). The purification procedure of all isozymes included concanavalin A-Sepharose column chromatography. The isozymes were not eluted from the column with a high concentration of sodium chloride, but were efficiently eluted with alpha-methylmannoside. This observation suggested that the carboxylesterases studied are glycoproteins. All the isozymes except rat RL1 and RL2 possess a high hydrolytic activity toward all the substrates examined. Long-chain monoglyceride was hydrolyzed by the purified carboxylesterase isozymes. Anti-rat RH1 immunoglobulin G was found to possess high cross-reactivity with all isozymes tested, except monkey MK2, by immunoblotting analysis. The amino acid compositions of carboxylesterase isozymes showed considerable similarities, except for monkey MK2. The amino-terminal amino acid sequences showed a striking homology, except for monkey MK2, though the amino-terminal amino acid itself was different in every isozyme. Hepatic microsomal carboxylesterases in mammals play an important role in drug and lipid metabolism in the endoplasmic reticulum, and it is noteworthy that the isozymes from various species examined here showed considerable similarities in physical, enzymatic, and immunochemical properties.

Amino Acid Sequence↗
PubMed1985

Rapid, high-yield purification of cell surface membrane using colloidal magnetite coated with polyvinylamine: sedimentation versus magnetic isolation.

A new technique for the magnetic isolation of external plasma membrane from Dictyostelium discoideum is described and compared to a previously published procedure employing sedimentation of silica-coated plasma membrane. The magnetic isolation technique involves coating intact cells with a polyvinylamine-magnetite colloid and overcoating with polyacrylate to form a dense pellicle. The magnetite pellicle totally coated the cells and was not internalized. Coated cells were lysed and membrane fragments retrieved from the cell homogenate using a diverging field electromagnet. The membrane obtained in such a manner was analyzed for marker enzyme activity and cell surface label. The plasma membrane was obtained in high yield (42%) with an average purification of 8-fold. The polyvinylamine-magnetite pellicle shielded the external plasma membrane face to proteolysis by papain and pronase. It also acted as a barrier to alpha-methylmannoside in concanavalin A-carbohydrate competition studies.

Binding Sites↗
PubMed1985

Interaction of human erythrocyte Band 3 with Ricinus communis agglutinin and other lectins.

Agglutination and competition studies suggest that human erythrocyte Band 3 can interact with both mannose/glucose- and galactose-specific lectins. Purified Band 3 reconstituted into lipid vesicles binds concanavalin A, but the nonspecific binding component, measured in the presence of alpha-methylmannoside, is very high. This glycoprotein also carries binding sites for the galactose-specific lectin Ricinus communis agglutinin. Binding was inhibited poorly by lactose, but much more effectively by desialylated fetuin glycopeptides, suggesting that the lectin recognizes a complex oligosaccharide sequence on Band 3. The glycoprotein bears two separate classes of binding sites for R. communis agglutinin. High-affinity binding sites exist which show strong positive cooperativity and correspond in number to the outward-facing Band 3 molecules. A low-affinity binding mode is abolished by 40% ethyleneglycol, suggesting the involvement of hydrophobic lectin-glycoprotein interactions. Studies on binding of R. communis agglutinin to human erythrocytes indicate positively cooperative binding to 7 X 10(5) very-high-affinity sites per cell, and lectin binding is completely inhibitable by lactose. Based on its binding characteristics in vesicles, it seems likely that Band 3 forms the major receptor for this lectin in human erythrocytes. Properties such as positive cooperativity thus appear to be a common feature of the interaction of Band 3 with a variety of lectins of different specificity, both in erythrocytes and lipid bilayers.

Anion Exchange Protein 1, Erythrocyte↗
PubMed1986

The staining of type V collagen obtained from FBJ virus-induced osteosarcoma with concanavalin A.

Type V collagen from FBJ virus-induced osteosarcoma of mice has a high content of saccharide as has been noted for type V collagens from different sources. In the present study, this collagen was found to contain significant amounts of mannose and hexosamine. Three alpha chains of this collagen were electrophoretically separated and cleaved by cyanogen bromide. The cyanogen bromide peptides, following their separation by SDS-polyacrylamide gel electrophoresis, were transferred onto nitrocellulose paper and stained with concanavalin A. Several bands derived only from alpha 3(V) stained positively, but this was inhibited by the presence of alpha-methylmannoside. Thus, at least one of these three chains appears to have an asparagine-linked oligosaccharide.

Animals↗
PubMed1989

Changes in polyphosphoinositide metabolism during mediator release from stimulated rat mast cells.

The metabolism of the polyphosphoinositides, diphosphoinositide (DPI) and triphosphoinositide (TPI), was studied during mediator release from rat mast cells. Serosal mast cells were purified by density gradient centrifugation and prelabeled with 32PO4. Incorporation of 32PO4 into DPI and TPI was determined by thin-layer chromatography on oxalic acid impregnated silica gel plates. 32PO4 incorporation into DPI and TPI was increased by Concanavalin A (ConA) or compound 48/80. The concentration of ConA causing a half-maximal increase in DPI labeling was less than that required for a comparable change in histamine release. The increases in DPI labeling and histamine release in response to ConA were enhanced by phosphatidylserine. The addition of alpha-methylmannoside to mast cells after challenge with ConA rapidly halted DPI and TPI labeling. The results of these studies indicate that changes in the metabolism of polyphosphoinositides may be an intrinsic part of the biochemical mechanisms that control mediator release from mast cells.

Animals↗
PubMed1983

Inhibition of human NK-induced cell lysis and soluble cell-lytic molecules with anti-human LT antisera and various saccharides.

The present study examines and compares the cytolysis of K-562 and MOLT-4 cells mediated by human natural killer (NK) cells from fresh peripheral blood and lymphotoxins (LT) derived from human lymphoid cell populations after lectin stimulation in vitro. Lymphotoxins were obtained from 5-hr concanavalin A (Con A)-restimulated human peripheral blood lymphocytes (PBL) which were precultured for 5 days in medium and fetal calf serum or with allogeneic human B-lymphoid cell lines. Two classes of probes were employed in both direct (cell) and indirect (supernatant) induced target-cell lysis: (a) various saccharides and (b) antibodies reactive with human LT forms. Two sugars, N-acetylglucosamine and alpha-methylmannoside, were able to inhibit direct cell lysis of both MOLT-4 and K-562 target cells. However, saccharide inhibition was distinct for each type of target even when effector cells were obtained from the same donor. These same saccharides were also able to inhibit 20-30% of the total LT activity in a supernatant for L-929 cells and 50-90% of the lytic activity on MOLT-4 cells. Anti-human F(ab')2 (IgG) and rabbit anti-alpha 2 LT sera blocked direct cell lysis of MOLT-4 and K-562 targets in 50% of the experiments. The anti-alpha 2 LT serum only recognizes a portion of the LT forms in these supernatants. These results reveal that, while both direct and indirect cell lysis are complex phenomena, they may both occur in some cases by a common mechanism(s).

Animals↗
PubMed1977

Separation of human renin substrate from renin and a major contaminating albumin using a concanavalin A-sepharose column.

Human plasma renin substrate was purified and separated from renin by a concanavalin A-Sepharose affinity column. Human renin substrate as well as renin were bound to concanavalin A. Renin substrate was eluted with 0.1 M D-glucose in 20 mM Tris/HCl buffer, pH 8.0. The specific activity increased from 38.5 to 653 ng of angiotensin-I equivalents per mg of protein (17-fold) and the recovery was 85%. Renin was eluted completely with 0.2 M alpha-D-methylglucoside and 0.2 M alpha-D-methylmannoside.

Angiotensin II↗
PubMed1980

Characterization of human serum gamma-glutamyltranspeptidase.

The contribution of proteolysis to leakage of gamma-glutamyltranspeptidase from the liver into the serum was examined by comparing the properties of human serum gamma-glutamyltranspeptidase with those of partially purified enzyme preparations solubilized from human liver with Triton X-100 and with papain. The Triton-solubilized enzyme was hydrophobic, whereas the papain-solubilized enzyme was hydrophilic. Serum gamma-glutamyltranspeptidase was found to be hydrophilic, since it could be extracted without addition of detergent after acetone treatment to remove bound cholate. The affinity of serum-gamma-glutamyltranspeptidase to concanavalin A-Sepharose was compared with those of the Triton-solubilized and papain-solubilized enzymes. All three enzymes were adsorbed to concanavalin A-Sepharose in 50 mmol/l imidazole buffer, pH 7.2. The serum enzyme and papain-solubilized enzyme were eluted with 100 mmol/l alpha-methylmannoside in 50 mmol/l imidazole buffer containing 1% Triton X-100. The molecular weights of the Triton-solubilized, papain-solubilized and serum-gamma-glutamyltranspeptidases were estimated as 98 000 +/- 2000, 90 000 +/- 2000 and 90 000 +/- 2000, respectively, by electrophoresis. These results show chemical similarities between the serum-gamma-glutamyltranspeptidase and papain-solubilized gamma-glutamyltranspeptidase.

Adult↗
PubMed1986

Steroid hormone-induced changes in secreted proteins in the filamentous fungus Achlya.

In the fungus Achlya, sexual development in the male strain E87 is mediated by the steroid hormone antheridiol. Treatment of vegetatively growing cells of E87 with antheridiol resulted in changes in the [35S]methionine labeling of several secreted proteins. The most heavily labeled group of proteins observed in the secreted fraction from control cells appeared on one-dimensional gels as a prominent wide band which could be resolved into three closely spaced components with relative molecular weights (MWs) of 57, 54, and 50 kD, respectively. After hormone treatment the two lower MW proteins of the group were barely detectable. Concomitant with the observed reductions in the labeling of the 54 and 50 kD proteins was the increased labeling of a doublet of very prominent proteins with relative MWs of 44.4 and 43 kD, respectively. The results of experiments with Endoglycosidase H suggested that the 44.4 and 43 kD proteins seen in hormone-treated cultures, might result from the removal or reduced synthesis of high mannose oligosaccharide groups found on the 54 and 50 kD proteins normally seen in control cultures. Additional support for this suggestion was provided by the observation that the 54 and 50 kD proteins from control cultures appeared to bind to conA columns and to be eluted with alpha-methylmannoside, while there was little or no binding of the 44.4 and 43 kD proteins from hormone-treated cells. Although other possibilities are not excluded, the results are suggestive of a steroid hormone-induced alteration in glycoprotein processing. The functions of the observed hormonally-responsive secreted proteins as well as those of the secreted proteins in non-hormone-treated cultures, are not known at this time.

Acetylglucosaminidase↗
PubMed1988

Occurrence of glucose polymer in undifferentiated PC12 cells.

A large glucose polymer was found, following pronase digestion, in PC12 pheochromocytoma cells metabolically labeled with [1-3H]galactose. The polymer was excluded from a Bio-Gel A-0.5 m column and adsorbed by immobilized concanavalin A-Sepharose from which it was eluted with 10 mM alpha-methylmannoside. Glucose was found to be the sole component monosaccharide. Except for those capable of degrading glycogen, no exo- or endo-glycosidases cleaved the polymer. This is the first report on the occurrence of a glucose polymer in undifferentiated PC12 cells.

Adrenal Gland Neoplasms↗
PubMed1991

Purification and electrophoretic analysis of glycosylated chicken growth hormone (G-cGH): evidence of G-cGH isoforms.

It has been shown that chicken growth hormone (cGH) exhibits functional and molecular heterogeneity. Mass and charge variants have been described in fresh pituitary extracts and in pure preparations of the hormone. In an attempt to further study the molecular heterogeneity of cGH we have purified the glycosylated variant of this hormone by affinity chromatography and analyzed it by different electrophoretic methods. Purification was achieved by homogeneizing chicken pituitaries in a protease inhibitor solution (0.5 mM PMSF and aprotinin, 50 KIU/ml); the supernatant of the alkaline extract (pH 9.5) was precipitated with 0.15 M ammonium sulfate and metaphosphoric acid, pH 4.0. The supernatant from this step was further precipitated with 80% ammonium sulfate, pH 6.5. After dialysis and lyophilization, the extract was chromatographed in a Con A-Sepharose column. The fraction eluted with 10 mM alpha-methylmannoside (which contained the glycoproteins) was passed through an immunoaffinity column (anticGH). Glycosylated cGH (G-cGH) was obtained pure after this step. Pure G-cGH was analyzed by nondenaturing electrophoresis (ND-PAGE), SDS-PAGE, isoelectrofocusing (IEF), and bidimensional electrophoresis (2D-PAGE) followed by Western blot and staining either with a specific antibody or with peroxidated Con A. Results showed that monomeric G-cGH has a MW of 29 kDa (under reducing conditions) and is heterogeneous, showing at least three important charge variants with pIs 6.5, 6.7, and 7.2. Mass variants of G-cGH were also detected under nonreducing conditions. Bidimensional analysis revealed that the charge variants had a similar MW (29 kDa).

Animals↗
PubMed1995

The presence of galactofuranose and ribose units in asparagine-linked oligosaccharides of the digenetic trypanosomatid Endotrypanum schaudinni.

It was found that the digenetic trypanosomatid Endotrypanum schaudinni transferred Man7GlcNAc2 in protein N-glycosylation. Endo-beta-N-acetylglucosaminidase H-sensitive oligosaccharides were identified as Man7GlcNAc2, Man6GlcNAc2, Rib1Man6GlcNAc2 and/or Gal1Man6GlcNAc2, Man5GlcNAc containing two galactose or ribose units or one of each residues, Rib1Man5GlcNAc2 and Gal1Man5GlcNAc2. The galactoses were in the furanose configuration. Endo-beta-N-acetylglucosaminidase H-resistant glycopeptides that were retained by concanavalin A-Sepharose and eluted with alpha-methylmannoside were found to contain mannose, galactofuranose and ribose units. The presence of galactofuranoses in N-glycoproteins has been reported previously in several monogenetic trypanosomatids but only in one digenetic parasite (Trypanosoma cruzi). This and a recent publication on the structure of Blastocrithidia culicis N-linked oligosaccharides are the first reports on the presence of ribose in eukaryotic glycoconjugates.

Animals↗
PubMed1983

Role of membrane integral proteins in the modulation of red cell shape by albumin, dinitrophenol and the glass effect.

Defatted serum albumin is found to induce a cup shape in erythrocytes. At 40 mg/ml of albumin, approx. 80% of washed erythrocytes possess this morphology, which can be reversed to disc shape by dinitrophenol. Erythrocytes treated with trypsin, papain, pronase or neuraminidase show enhanced susceptibility to cup formation by albumin; however, chymotrypsinized erythrocytes exhibit a normal response. Red cells treated with concanavalin A (but not its succinylated derivative) show resistance to the cupping effect of albumin as well as the crenating effects of dinitrophenol and glass. The resistance develops after about 20 min following the exposure of cells to the lectin, and is rapidly abrogated on removal of the bound lectin by alpha-methylmannoside. Incubation of the concanavalin A-exposed cells at low temperature leads to prolongation of the time required to achieve the resistance. These results indicate an involvement of membrane integral proteins in mediating the shape modulating effects of albumin, dinitrophenol and exposure to glass.

2,4-Dinitrophenol↗
PubMed1975

Preparation and properties of concanavalin A-binding glycopeptides derived from rat brain glycoproteins.

Mannose-rich glycopeptides derived from brain glycoproteins were recovered by affinity chromatography on Concanavalin A-Sepharose. These glycopeptides, which adsorb to the lectin and are eluted with alpha-methylmannoside, constitute about 25--30% of the total glycopeptide material recovered from rat brain glycoproteins. They contain predominately mannose and N-acetylglucosamine (mannose/N-acetylglucosamine = 3), as well as small amounts of galactose and fucose. Approx. 65% of the Concanavalin A-binding glycopeptide carbohydrate was recovered after treatment with leucine aminopeptidase, gel filtration on Biogel P-4, and ion-exchange chromatography on coupled Dowex 50-hydrogen and Dowex 1-chloride columns. The purified glycopeptide fraction contained six mannose and two N-acetylglucosamine residues per aspartic acid and possessed an apparent molecular weight of about 2000 as assessed by gel filtration and amino acid analysis. Galactose and fucose were absent. Treatment of the purified glycopeptides with alpha-mannosidase drastically reduced their affinity for Concanavalin A, suggesting the presence of one or more terminal mannose residues.

Amino Acids↗
PubMed1991

ATPase-ADPase activities of rat placental tissue.

1. Calcium-stimulated ATPase-ADPase activities were studied in a microsomal fraction of rat placental tissue. 2. The kinetic characteristics correspond to those of ATP-diphosphohydrolase, also known as apyrase (E.C. 3.6.1.5). 3. These characteristics include the lack of specificity towards nucleoside di- and triphosphates, activation by Ca2+, Mg2+ or Mn2+, insensitivity to specific inhibitors of some ATPase and absence of an effect of sulphydryl reagents. 4. Chemical modification of tyrosine, tryptophan, arginine and carboxylic residues decreases both ATPase and ADPase activities. 5. The substrate analogue, 5'-(beta, gamma-methylene)triphosphate, protected both enzyme activities against all the modifying amino acid reagents tested. 6. Placental fractions (homogenate and microsomes) inhibit ADP-dependent platelet aggregation. 7. The solubilized microsomal enzyme has a molecular mass of 67 kDa by size-exclusion chromatography; the pI is 9.36. 8. A differential effect is observed on the activation produced by Concanavalin A on microsomal and solubilized fractions when treated in the presence and absence of alpha-methylmannoside.

Adenosine Triphosphatases↗
PubMed2005

Characterisation of glycoprotein ligands synthesised using solid-phase combinatorial chemistry.

A combination of rational design based on mimicking natural protein-carbohydrate interactions and solid-phase combinatorial chemistry has led to the identification of an affinity ligand which displays selectivity for the mannose moiety of glycoproteins. The ligand was initially identified as 32/18, a triazine scaffold substituted with 2-acetylpyrrole (32) and 5-aminoindan (18). However, characterisation of the immobilised ligand by release from the matrix via a cleavable linker, (4s,5s)-4,5-di(aminomethyl)-2,2-dimethyldioxolane, and using a non-destructive on-resin method, 13C NMR spectroscopy, confirmed that the putative ligand 32/18 was, in fact, 18/18, the disubstituted 5-aminoindan. 1H NMR studies on the interaction of alpha-D-methylmannoside with the ligand 18/18 in solution confirm the involvement of the hydroxyl group in the C-2 position.

Combinatorial Chemistry Techniques↗
PubMed2000

Effects of lectins on calcification by vesicles isolated from aortas of cholesterol-fed rabbits.

Advanced vascular calcification in atherosclerosis weakens arterial walls, thereby imposing a serious rupturing effect. However, the mechanism of dystrophic calcification remains unknown. Although accumulating morphological and biochemical evidence reveals a role for calcifiable vesicles in plaque calcification, the mechanism of vesicle-mediated calcification has not been fully explored. To study whether vesicles' membrane components, such as carbohydrates, may have a role in vesicle-mediated calcification, the effect of sugar-binding lectins on calcification was investigated. Atherosclerosis was developed by feeding rabbits with a diet supplemented with 0.5% cholesterol and 2% peanut oil for 4 months. Calcifiable vesicles were then isolated from thoracic aortas by collagenase digestion. The histological examination of aortas with hematoxylin counter-staining indicated abnormal formation of large plaques enriched with macrophage-derived foam cells. Fourier transform spectroscopy revealed mild calcification in aortas indicating that advanced stages of heavy calcification have yet to be reached. However, vesicles isolated from the aortas were capable of calcification in the presence of physiological levels of Ca(2+), Pi, and ATP. Thus, at this stage of atherosclerosis, aortas may start to produce calcifiable vesicles, but at a level insufficient for substantial formation of mineral in aortas. The assessments by FT-IR analysis and Alizarin red staining indicated that concanavalin A (Con A) substantially increased mineral formation by isolated vesicles. Con A also exerted a marked stimulatory effect on (45)Ca and (32)Pi deposition in a dose-dependent fashion with a half-maximal effect at 6-10 microg/ml. Either alpha-methylmannoside or alpha-methylglucoside, but not mannitol, at 10 mM abolished the stimulation. Con A stimulation was abolished after Con A was removed from calcifying media, suggesting that covalent binding may not be involved in the effect. Galactosides appear to also be implicated in (45)Ca and (32)Pi deposition since Abrus precartorius agglutinin, which specifically binds galactosides, enhanced the deposition. Neither wheat-germ agglutinin that binds N-acetylglucoside nor N-acetylgalactoside-specific Helix pomatia agglutinin was effective, suggesting that the acetylated forms of carbohydrate moieties are either absent in vesicles or may not be involved in calcification. None of these lectins exerted an effect on ATPase. Thus, the effects of lectins appeared to be mediated through interactions with carbohydrate moieties of calcifiable vesicles. Whether stimulation of vesicle-calcification by lectins is of pathological significance in atherosclerotic calcification requires further investigation.

Animals↗