Showing papers on "Bovine serum albumin published in 1987"

Glucose autoxidation and protein modification. The potential role of 'autoxidative glycosylation' in diabetes.

Abstract: Monosaccharide autoxidation (a transition metal-catalysed process that generates H2O2 and ketoaldehydes) appears to contribute to protein modification by glucose in vitro The metal-chelating agent diethylenetriaminepenta-acetic acid (DETAPAC), which inhibits glucose autoxidation, also reduces the covalent attachment of glucose to bovine serum albumin A maximal 45% inhibition of covalent attachment was observed, but this varied with glucose and DETAPAC concentrations in a complex fashion, suggesting at least two modes of attachment The extent of inhibition of the metal-catalysed pathway correlated with the extent of inhibition of glycosylation-associated chromo- and fluorophore development DETAPAC also inhibited tryptophan fluorescence quenching associated with glycosylation Conversely, ketoaldehydes analogous to those produced by glucose autoxidation, but generated by 60Co irradiation, bound avidly to albumin and accelerated browning reactions It is therefore suggested that a component of protein glycosylation is dependent upon glucose autoxidation and subsequent covalent attachment of ketoaldehydes The process of glucose autoxidation, or ketoaldehydes derived therefrom, appear to be important in chromophoric and fluorophoric alterations It is noted, consistent with these observations, that the chemical evidence for the currently accepted ‘Amadori’ product derived from the reaction of glucose with protein amino groups is consistent also with the structure expected for the attachment of a glucose-derived ketoaldehyde to protein The concept of ‘autoxidative glycosylation’ is briefly discussed in relation to oxidative stress in diabetes mellitus

Production of hybridoma growth factor by human monocytes.

Abstract: Human mononuclear leukocytes produce a growth factor (HGF) for hybridoma and plasmacytoma cells. HGF has recently been proven to be identical to IFN-β2, 26-kDa protein and BSF-2. HGF can be quantitated in a proliferation assay with the HGF-dependent hybridoma cell line B13.29. By selection of an extremely sensitive variant of this cell line, we were able to measure HGF production of single cells. Limiting dilution analysis of the producing cells in combination with size, density and adherence characteristics showed that HGF is produced by monocytes and not by lymphocytes. There was no need for the monocytes to be stimulated but the cells did require the presence of serum. This serum requirement could be met by purified bovine serum albumin, but not by other proteins like ovalbumin or human γ-globulin. HGF production in vitro by monocytes starts after 2 h of incubation and is completed within 24 h.

Protein adsorption to polymer particles: Role of surface properties

Abstract: Adsorption isotherms of four plasma proteins (fibrinogen, IgG, human serum albumin, and bovine serum albumin) using four different types of small particles as substrates (siliconized glass, Teflon, polyvinylchloride, and Nylon-6, 6) are reported. The suspending liquid medium was phosphate-buffered saline, with a surface tension higher than that of any of the proteins. In keeping with the thermodynamic expectations for these systems, protein adsorption decreases for all solids in sequence from fibrinogen (the most hydrophobic) to IgG, human serum albumin, and bovine serum albumin (the most hydrophilic). Furthermore, the extent of protein adsorption also decreases from the low surface tension (hydrophobic) to the higher surface tension solids, again as expected on thermodynamic grounds. There is one minor yet interesting exception to the thermodynamic pattern: In spite of the slightly lower surface tension of siliconized glass, the extent of protein adsorption is slightly higher to Teflon than to siliconized glass. This result is attributed to the theoretically well known phenomenon of “screening”.

Bovine brain cytosol contains three immunologically distinct forms of inositolphospholipid-specific phospholipase C.

Abstract: We previously reported that cytosolic fractions of bovine brain contain two immunologically distinct forms of phospholipase C (PLC), PLC-I and PLC-II. We now report the purification of another form of inositolphospholipid-specific phospholipase C from bovine brain cytosol, designated PLC-III, and the comparison of the catalytic properties of the three isozymes. Approximately 450 micrograms of pure PLC-III was obtained from 36 bovine brains, and it had a final specific activity of 30-40 mumol of phosphatidylinositol hydrolyzed per min per mg of enzyme in the presence of 0.1% deoxycholate. PLC-III exhibited an apparent Mr of 85,000 in NaDodSO4/PAGE, which is considerably smaller than the Mr of 150,000 for PLC-I and 145,000 for PLC-II. Neither of the two mixtures of monoclonal antibodies nor the rabbit polyclonal antibodies directed against either PLC-I or PLC-II cross-reacted with PLC-III. The catalytic properties of the three isozymes were studied by using small unilamellar vesicles prepared from either phosphatidylinositol (PtdIns) or phosphatidylinositol 4,5-bisphosphate (PtdInsP2) as substrates. Hydrolysis of both PtdIns and PtdInsP2 by the three enzymes was dependent on Ca2+. However, at low Ca2+ concentration, PtdInsP2 was the preferred substrate for all three enzymes. When PtdIns was the substrate, the three enzymes exhibited similar specific activities at their optimum pH, which was 4.8 for PLC-I, 5.0 for PLC-II, and 5.5 for PLC-III. But at neutral pH, the order of specific activity was PLC-III greater than PLC-II greater than PLC-I. In contrast, the order of specific activity was PLC-I greater than PLC-III greater than PLC-II for PtdInsP2 hydrolysis, which means that PLC-I is the most specific for PtdInsP2. The three enzymes were affected differently by bovine serum albumin: inhibition of PLC-I and activation of PLC-III were observed, whereas PLC-II was unaffected. This observation suggests that any putative protein effectors for PLC should be critically scrutinized.

Covalent Binding of Acetaldehyde to Proteins: Participation of Lysine Residues

Abstract: The results of this study demonstrate that lysine is the major amino acid participating in the binding of acetaldehyde to proteins. The formation of both stable and unstable acetaldehyde-albumin adducts was shown to occur via the reaction of acetaldehyde with lysine residues. This conclusion was based on the following experimental evidence: (a) the ratio of stable to unstable adducts of bovine serum albumin was similar to that observed for polylysine; (b) acetylation of albumin markedly reduced acetaldehyde binding; (c) the radio-activity profiles (obtained by high-performance liquid chromatographic analysis) of [14C]acetaldehyde modified amino acids hydrolyzed from total and stable adducts of albumin were nearly identical to those of polylysine or alpha-t-boc-lysine. Analysis of stable adducts of albumin indicated two major modified lysine residues; one residue was much more acidic and the other more basic than unmodified lysine. Unstable adducts were shown to be Schiff bases since NaBH4 treatment resulted in the formation of N-ethyllysine residues. The reducing agents, NaCNBH3 and ascorbic acid, both increased stable adduct formation via increased binding to lysine residues; however, a different elution profile of modified lysine residues was observed for these reducing agents. NaCNBH3 increased the formation of N-ethyllysine residues exclusively, whereas ascorbate increased the formation of the acidic adduct of lysine and also caused the formation of an additional modified lysine residue which was present only in the ascorbate-treated polypeptides. In addition to their detection by radioactivity measurements, the acetaldehyde-lysine adducts could also be detected by the fluorescence of their ophthalaldehyde derivatives.(ABSTRACT TRUNCATED AT 250 WORDS)

Orosomucoid as one of the serum components contributing to normal capillary permselectivity in rat skeletal muscle

Abstract: The effects of human serum orosomucoid (normal serum concentration 0.7-1.0 g l-1) on capillary permeability were investigated in 12 isolated maximally vasodilated rat hindquarters perfused with bovine serum albumin (50 g l-1) in modified Tyrode. Measurements were made of capillary filtration coefficient (CFC), permeability surface area product (PS) for vitamin B12 and isogravimetric clearance of radiolabelled albumin (Cl alb). The results were compared with those obtained using perfusates without addition of orosomucoid ('albumin group') or perfusates containing horse serum ('serum group'). Clearance of albumin was almost four-fold higher in the albumin than in the serum group, 0.0895 +/- 0.0066 (n = 12) and 0.0252 +/- 0.016 ml min-1 per 100 g (n = 18), respectively, while intermediate Cl alb values were obtained with human orosomucoid in the perfusate (greater than 0.1 g l-1), 0.0436 +/- 0.0034 ml min-1 per 100 g) (n = 8). These changes in Cl alb were not accompanied by any differences in CFC or PS. We conclude that orosomucoid is one of the components in serum (besides albumin) needed for the maintenance of normal permselectivity of the capillary walls of rat skeletal muscle. Alternatively, human orosomucoid is structurally related to other substances exerting this 'serum effect'.

Cationization of protein antigens. I. Alteration of immunogenic properties.

Abstract: We have shown that a cationized form of bovine serum albumin (BSA) produced by substituting anionic side chain carboxylic groups with aminoethylamide groups possesses unique immunologic properties. The two forms of antigen, native (nBSA) and cationized (cBSA), cross-react at the level of the B cell, as evidenced by the ability of antibody raised against one form to react with the other and by inhibition assays using ELISA. T cell cross-reactivity was also observed in proliferation assays, but the amount of cBSA required for stimulation was 500 times less than the amount of native protein needed. In vivo, cBSA produced responses which, at their optimal levels, were at least double the response to nBSA and which showed a different kinetic pattern, peaking later and lasting longer than the response to the native molecule. Moreover, antibodies were produced in response to administration of cBSA but not nBSA when given i.v. in saline, without an adjuvant. Although a mechanism for these phenomena is not yet clear, we speculate that the cBSA may have a greater affinity for antigen-presenting cells or for the T cell receptor, or that the altered structure may enhance recognition of the molecule by APC and/or helper T cells.

Evaluation of a simplified blood pepsinogen assay

Abstract: Blood pepsinogen concentrations in cattle were determined, using a simple, direct method based on the hydrolysing effect of serum on buffered bovine albumin substrate (2 g/dl). The amount of tyrosine released was measured at 680 nm, using the Folin and Ciocalteu's colorimetric method. To ensure an optimal digestion in vitro, a glycine-hydrochloric acid buffer (0.1M) was evaluated at various pH. Serum samples with medium (2,591 mU of tyrosine) and high (6,222 mU of tyrosine) pepsinogen concentrations had the highest proteolytic activity at pH 2.5. Substituting albumin by hemoglobin substrate resulted in almost double the amount of hydrolyzed products. For preparation of standard pepsin or pepsinogen curves, the incorporation of serum (activated or inactivated) diminished the tyrosine released to only half the amount obtained without serum. The use of serum or plasma did not significantly affect the results of the test, nor did prolonged storage at -70 C result in marked differences in the results.

Thermal Denaturation and Aggregation of Whey Proteins

Abstract: The denaturation of whey protein in a rennet casein whey, as measured by protein rendered insoluble at pH 4.5 and reactive thiol groups, increased slowly on heating at 60 and 70?C and increased very quickly on heating at 80 and 90?C. This shows that whey protein denaturation is a co-operative process with a large thermal coefficient. Electrophoresis showed that the relative denaturation rates of the individual whey proteins differed/The order of heat resistance found was proteose peptone >a-lactalbumin>B-lactoglobuIin>bovine serum albumin immunoglobulin. The contribution of the individual whey proteins to the total protein denaturation is shown. The thermodenaturation of the total whey protein increased with increasing pH in the range 4.5-7.0 and electrophoresis showed that the thermal response of the individual whey proteins to pH differed. Greatest denaturation of B-lactoglobulin occurred at high pH, bovine serum albumin was most resistant at high pH while the thermal denaturation of a-lactalbumin was relatively independent of pH. Thermal precipitation was also found to be pH dependent with greatest aggregation occurring in the pH range 4.5-5.5. That thermal precipitation of whey proteins is influenced mainly by electrostatic charge is shown by the increased precipitation at high pH on the neutralization of the high negative charge of denatured protein molecules by the addition of Ca^+

Receptor-specific agglutination tests for detection of bacteria that bind globoseries glycolipids.

Abstract: Specific binding to the globoseries of glycolipid receptors explains the adherence of uropathogenic Escherichia coli to host cells. The minimal receptor disaccharide Gal alpha 1----4Gal beta [galactose alpha (1----4)galactose beta] is recognized by most attaching clinical isolates. However, wild-type isolates can express adhesins with several different receptor specificities. Bioassays do not permit separate analysis of each receptor specificity, since the target cells contain multiple potentially receptor-active molecules. In this study, bacterial adhesins were analyzed by using receptors immobilized into latex beads in one of two ways. In one way, di- and trisaccharides were covalently linked via a spacer arm to latex beads coupled with bovine serum albumin. In the other way, receptor-active glycolipids were coated onto the bovine serum albumin-latex beads. The latex beads were subsequently used for agglutination by using type strains with known receptor specificity. The composition was optimized regarding receptor structure and size of latex beads. Gal alpha 1----4Gal beta was as active as the trisaccharide derivative Gal alpha 1----4Gal beta 1----3glucose or Gal alpha 1----4Gal beta 1----3glucosamine. Among the natural glycolipids tested, globotetraosylceramide was the most active. Subsequently, the sensitivity and specificity of the Gal alpha 1----4Gal beta-latex and globotetraosylceramide-latex reagents were compared for 733 E. coli urinary isolates. Hemagglutination of human erythrocytes was used as the positive standard. No significant difference in the specificity or sensitivity of the latex reagents was found; the sensitivity ranged from 86%, when isolates agglutinating human erythrocytes of blood groups P1 and p were included, to 93%, when those isolates agglutinating erythrocytes of blood group p were excluded. These reagents provide tools for bacterial identification in patients with urinary tract infection.

Specific antisera against the catecholamines: L-3,4-dihydroxyphenylalanine, dopamine, noradrenaline, and octopamine tested by an enzyme-linked immunosorbent assay.

Abstract: Antisera were raised against L-3,4-dihydroxyphe-nylalanine (L-DOPA), dopamine (DA), noradrenaline (NA), and octopamine (OA). This was achieved by coupling each molecule to bovine serum albumin or human serum albumin using glutaraldehyde. The conjugated aromatic amines were kept in a reducing medium containing sodium metabisulfite. Antiserum specificity was tested using an enzyme-linked immunosorbent assay method for catecholamines. Competition experiments were done between the immunogen coated on the well plates and each catechol-amine, either in the free state or in conjugated form, previously incubated with an antiserum. In each case, the non-conjugated compound was poorly recognized. The nonre-duced conjugates of L-DOPA and DA were well recognized, whereas those of NA and OA were poorly immunoreactive. The cross-reactivity ratios established in the competition experiments allowed the specificity of the immune response to be defined. In each case, it was found to be high. The results suggest that the antibodies of L-DOPA and DA antisera recognize preferentially the catechol moiety, whereas for the anti-NA and anti-OA antibodies, the lateral chain is important.

Purification and Properties of an Extracellular Protease Produced by the Entomopathogenic Fungus Beauveria bassiana

Abstract: Beauveria bassiana GK2016 grown in a medium with gelatin as the sole carbon and nitrogen source produced an extracellular protease. The protease production was highest when the fungus was grown on a semiliquid medium and was purified about 18-fold, with a recovery of 21%. The protease molecular weight was estimated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis to be about 35,000. It had an optimum activity at pH 8.5 and 37 degrees C and was rapidly inactivated at 50 degrees C. Its enzymatic activity was that of an endopeptidase which hydrolyzed elastin, casein, and gelatin but was much less active on bovine serum albumin and collagen. No trypsinlike activity was detected on N-alpha-benzoyl-dl-arginine-p-nitroanilide. It was, however, inhibited by phenylmethylsulfonyl fluoride, indicating that a serine residue is present in the active site. The protease was unaffected by metal-chelating agents, sulfhydryl reagents, trypsin inhibitor, and chymotrypsin inhibitor.

Purification and characterization of a protease from Bacteroides gingivalis 381.

Abstract: An intracellular membrane-free, trypsinlike protease was isolated from cells of Bacteroides gingivalis 381. The protease was extracted from the cells by ultrasonic treatment and was purified about 250-fold with a recovery of 2% by sequential procedures. The properties of the protease were as follows: its optimal pH was 8.5; its activity was almost completely lost on incubation at 50 degrees C for 15 min; its activity was inhibited by diisopropylfluorophosphate, p-toluenesulfonyl-L-lysine chloromethyl ketone hydrochloride, leupeptin, Mn2+, Cu2+, and Zn2+; it hydrolyzed casein, azocasein, N-alpha-benzoyl-DL-arginine-p-nitroanilide (BAPNA), bovine serum albumin, azocoll, and gelatin, but not N-alpha-benzoyl-DL-lysine-p-nitroanilide or human serum immunoglobulin A; its molecular weight was estimated as 45,000 by gel filtration and 50,000 by sodium dodecyl sulfate-polyacrylamide gel electrophoresis; and its Km values for azocasein and BAPNA were 1.11% and 0.19 mM, respectively.

Isolation and characterization of a mannose-specific endocytosis receptor from rabbit alveolar macrophages.

Abstract: Rabbit alveolar macrophages express a plasma-membrane receptor that recognizes glycoprotein ligands bearing terminal mannose, fucose or N-acetylglucosamine residues. Macrophage membranes were washed extensively with buffers containing high salt and mannose or EDTA to remove endogenously bound ligand, before Triton X-100 extraction. The extracts were chromatographed on mannose-Sepharose. Elution with mannose, followed by dialysis and a second mannose-Sepharose step with EDTA elution, produced a preparation that migrated as single protein band of Mr 175,000 on SDS/polyacrylamide-gel electrophoresis. The purified protein binds mannose-BSA (bovine serum albumin) with a dissociation constant of 1.9 X 10(-8) M. Ligand binding is Ca2+ and pH-dependent, with maximal binding at neutral pH and low binding below pH 6.0. The binding of 125I-mannose-BSA is inhibited by ligands bearing high-mannose oligosaccharides, such as mannan or beta-glucuronidase, as well as the monosaccharides mannose, fucose and N-acetylglucosamine. Galactose, galactosylated BSA, glucose and mannose 6-phosphate are non-inhibitory. Amino acid compositional analyses indicate that the receptor contains high concentrations of aspartate/asparagine and glutamate/glutamine, and low amounts of methionine. The carbohydrate composition was studied by lectin overlays of electrophoretically transferred receptor, and the results indicate the presence of N-linked complex and O-linked sialylated oligosaccharides. A protein of Mr 175,000 was immunoprecipitated from radio-iodinated macrophage membranes with an antibody generated against purified rabbit lung mannose receptor.

Abnormal solubility behavior of .beta.-lactoglobulin: salting-in by glycine and sodium chloride

Abstract: The causes of the salting-in of beta-lactoglobulin by glycine and NaCl, a solubility behavior contrary to expectations, were probed by a detailed study of the interactions between these solvent components and the protein. The preferential interactions of beta-lactoglobulin with solvent components in aqueous glycine and NaCl systems have been compared with those of bovine serum albumin and lysozyme. At neutral pH, beta-lactoglobulin exhibited insignificant preferential interactions in glycine and NaCl at low cosolvent concentrations and an increasing preferential hydration at higher concentrations, the levels approaching the values expected from the other two proteins. These results indicate considerable binding of the electrolytes to beta-lactoglobulin, sufficient to compensate for the exclusion due to perturbation of the solvent surface tension. The difference between the preferential interactions of beta-lactoglobulin and the other proteins with these two solvent additives was shown to be the cause of the increase of beta-lactoglobulin solubility even at high concentrations of the additives, at which they have salting-out effects on the other proteins. The preferential interactions of NaCl with the three proteins were examined as a function of pH. The results showed no pH dependence of the preferential hydration for bovine serum albumin and lysozyme, while this parameter increased significantly for beta-lactoglobulin at lower pH. This suggests that the binding of electrolytes to beta-lactoglobulin is due to a unique charge distribution on the surface of the protein around neutral pH, which imparts to this protein a large dipole moment.

Bovine serum albumin density gradient isolation of rat pancreatic islets.

Abstract: The use of a bovine serum albumin (BSA) density gradient for isolation of rat pancreatic Islets of Langerhans after collagenase digestion has been compared with the standard Ficoll separation technique. The criteria studied were islet yield (insulin extraction of the islet interfaces and pellet), purity of preparation (amylase content of the islet preparation), insulin release characteristics, and the result of isologous transplantation in diabetic rats. The islet interface of the BSA gradient contained 62.2% of the total insulin, whereas the corresponding interface of the Ficoll gradient contained only 36.6% (P less than 0.001). The amylase content of the Ficoll-separated islet preparation was 6133 U/L, as opposed to 1230 U/L with BSA (P less than 0.001). BSA-isolated islets gave similar insulin release characteristics to non-density-gradient-isolated islets, whereas Ficoll-separated islets showed suboptimal insulin release. Single-donor-single-recipient transplantation was successfully performed with BSA-isolated islets whereas multiple donors were required with Ficoll-separated islets. Thus significantly improved results were found with the bovine serum albumin density gradient separation in all criteria and consequently the use of this gradient represents an advance in islet isolation techniques.

Quantitative Estimation of α-Helix Coil Content in Bovine Serum Albumin by Fourier Transform-Infrared Spectroscopy

Abstract: Heat denaturation of albumin aqueous solution enables us to control the α-helix content to a reasonable level, and denatured albumin, whose conformation is different, can be obtained. Fourier transform-infrared absorption spectra in the amide I, II, and III band regions of these albumins have been measured and compared with each other. As a result, the characteristic frequencies associated with each conformation of α-helix, β-sheet, and random structure have been observed; moreover it has been newly proved that the intensity ratio of the amide II band to the amide I band depends on the α-helix content in albumin. In this paper, we demonstrate that this intensity ratio is very useful for quantitative estimation of α-helix content in albumin and apply this method to analysis of ATR spectra of albumin adsorbed on polyethylene surface.