Showing papers on "Bovine serum albumin published in 1983"

Taurine, hypotaurine, epinephrine and albumin inhibit lipid peroxidation in rabbit spermatozoa and protect against loss of motility.

Abstract: Loss of forward motility of rabbit epididymal spermatozoa in high K phosphate buffer is in- hibited by taurine, hypotaurine, epinephrine and bovine serum albumin. Pyruvate and lactate also show this effect. The rate of lipid peroxidation in these spermatozoa, as measured by rate of formation of malonaldialdehyde, is also inhibited by these agents. A close linear correlation be- tween percent inert spermatozoa and malondialdehyde was found, which was independent of the rate of peroxidation. Complete cessation of motility was observed at 0.5 nmol malondialdehyde/ iocells in the absence or presence of these agents, which is the same value found in other sus- pending media in a previous study LAlvarez and Storey (1982) Biol. Reprod. 27:1102-11081. Albumin was the most effective agent in preventing loss of motility and inhibiting lipid peroxida- tion. Hypotaurine was the next most effective, followed by taurine, epinephrine, pyruvate and lactate. Hypotaurine reduces the amount of rate of superoxide production, as measured by the rate of reduction of acetylated ferricytochrome c by 0, from rabbit sperm under these conditions and concomitantly reduces inactivation of the superoxide dismutase in these cells. Since superoxide seems to be the major inducer of lipid peroxidation in rabbit sperm, the protective effect of hypo- taurine, which should be readily permeant to the plasma membrane, may be ascribed to scavenging of intracellular superoxide. The mechanism of the protective action of albumin is not known. Rabbit epididymal spermatozoa lose motility over time if Ca2� or Mg2 are omitted from the suspending medium. This loss is not correlated with the rate of lipid peroxidation, which is un- affected by the absence or presence of these ions. The linear correlation between loss of motility and lipid peroxidation no longer holds in the absence of Ca2* and Mg2�, implying motility loss by a different mechanism.

Evidence that pituitary cation-sensitive neutral endopeptidase is a multicatalytic protease complex.

Abstract: Pituitary cation-sensitive neutral endopeptidase splits peptide bonds on the carboxyl side of hydrophobic amino acids (chymotrypsin-like activity), basic amino acids (trypsin-like activity), and acidic amino acids (peptidyl-glutamyl-peptide bond hydrolyzing activity). All three activities copurify, are inhibited by cations, and reside in a single high-molecular weight soluble protein complex. Treatment with sodium dodecylsulfate and 2-mercaptoethanol dissociates this complex into five low-molecular weight components. Incubation of the complex at 37 degrees C in buffers of high ionic strength produces aggregation and progressive loss of all three activities. Experiments with inhibitors and activators indicate that the three activities are catalyzed by distinct components. Benzyloxycarbonyl-glycyl-glycyl-leucinal, a peptide aldehyde transition state analog of the substrate used to measure the chymotrypsin-like activity, exclusively inhibits that activity (Ki = 2.5 x 10(-4) M), while markedly activating the trypsin-like activity. The trypsin-like activity is inhibited by leupeptin (Ki = 1.2 x 10(-6) M) and by sulfhydryl blocking agents, and activated by thiols, suggesting that this activity is due to a thiol protease. The peptidylglutamyl-peptide hydrolyzing activity is activated almost 10-fold by low concentrations of sodium dodecylsulfate, inhibited by bovine serum albumin, and suppressed at high enzyme concentrations, suggesting that this component readily interacts with other proteins, including the complex itself. The results indicate that cation-sensitive neutral endopeptidase is a multicatalytic protease complex whose distinct proteolytic activities are associated with separate components of this high-molecular weight protein.

Microbial degradation of dissolved proteins in seawater1

Abstract: An experimental protocol using radiolabeled proteins was developed to investigate the rates and mechanisms whereby dissolved proteins are degraded in natural marine plankton communities. The results of field observations and laboratory experiments indicate that proteins are degraded by a particle-bound, thermolabile system, presumably bacteria-associated enzymes, with an apparent half-saturation constant of ca. 25 ..mu..g bovine serum albumin (BSA) per liter. Gel permeation chromatography indicated that peptides of chain length intermediate between BSA and the final products of degradation (MW<700) do not accumulate in the medium. Competition experiments indicate that the system is relatively nonspecific. Turnover rates for the protein pool in samples collected in the Southern California Bight were of the same order of magnitude as the turnover rate of the L-leucine pool and were correlated with primary productivity, chlorophyll a concentrations, bacterial abundance and biomass, and L-leucine turnover rate. These data suggest that amino acids derived from proteins are utilized preferentially and do not completely mix with the amino acids in the bulk phase.

Protein adsorption on crosslinked polydimethylsiloxane using total internal reflection fluorescence

Abstract: Total internal reflection fluorescence (TIRF) is used to examine the adsorption of bovine serum albumin (BSA) and bovine fibrinogen on crosslinked polydimethylsiloxane (silicone rubber) surfaces from flowing solutions. By comparing experimentally observed adsorption rates with the predictions of a convection/diffusion model, it is shown that the adsorption of both BSA and fibrinogen on silicone rubber is diffusion-controlled over the range of solution concentrations (0.01

Characteristics of the metabolism-induced binding of misonidazole to hypoxic mammalian cells.

Abstract: [14C]Misonidazole (MISO) becomes bound to macromolecules of mammalian cells upon hypoxic incubation. Intracellular enzyme processes are implicated since the temperature dependence for this process showed an activation energy of 33.5 kcal/mol. The sensitizer bound to both hypoxic and aerobic cells was associated with the macromolecular fraction and the soluble fraction in the proportion, 23 and 77%, respectively. The initial rate of binding of [14C]MISO to the macromolecular (acid-insoluble) fraction of hypoxic EMT-6 mouse tumor and V-79 hamster cells increased proportionally with the square root of extracellular concentration of MISO up to at least 5mM. High concentrations of dimethyl sulfoxide (an effective OH radical scavenger), allopurinol (an effective inhibitor of xanthine oxidase), and diamide (a chemical which can deplete cellular levels of glutathione) had little or no effect on this metabolism-induced binding process. The addition of high concentrations of exogenous cysteamine to hypoxic cell cultures resulted in almost complete inhibition of binding. Extracellular bovine albumin at high concentration in hypoxic cell cultures had little effect on the production of adducts to cell macromolecules and only small amounts of [14C]MISO were found to bind to the extra-cellular bovine albumin. This result suggests that MISO preferentially binds to molecules within the cell in which it is metabolically activated. In experiments where cells labeled under hypoxic conditions with [14C]MISO were subsequently permitted to proliferate in aerobic monolayers, a half-life of the acid-insoluble addition products of approximately 55 hr was measured. A large number of [14C]MISO adducts (approximately 10(9)/cell) can be generated in hypoxic cells without any evidence of cytotoxicity, and they are slowly cleared from cells. These are favorable characteristics as regards the development of this technique as a marker for hypoxic cells in solid tumors.

Identification of the serum binding proteins for iron, zinc, cadmium, nickel, and calcium.

Abstract: The binding of five biologically important metals to serum proteins has been studied. After suitable radioactive isotopes were added to serum proteins separated and precipitated by two-dimensional immunoelectrophoresis, the sample plates were exposed to roentgenogram film. 59Fe bound to transferrin alone; 65Zn bound mostly to albumin, but also to another 12 proteins; 109Cd was mostly associated with alpha 2-macroglobulin, but was also present on albumin, immunoglobulins G and A, and prealbumin; 63Ni, added in high concentration, was associated with an alpha 2-mobility protein and albumin; and, finally, 45Ca was mostly bound to albumin, but seven other binding proteins were also identified, with transferrin predominant. The results are not quantitative, but the technique is simple and specific, and the information gained can direct further studies on isolated proteins.

High efficiency plating method for Leishmania promastigotes in semidefined or completely-defined medium.

Abstract: A simple technique, developed for the isolation of clones derived from single, promastigote cells of Leishmania donovani and Leishmania tropica, involved the use of semisolid agar. Both species of Leishmania promastigotes formed discrete colonies at high efficiency either in semidefined medium containing 10% fetal calf serum or in completely-defined medium lacking serum. Visible colonies appeared between 8 and 14 days in growth medium containing 10% fetal calf serum. Replacement of the fetal calf serum with bovine serum albumin and Tween-80 increased the time of colony formation by 50% but did not affect the cloning efficiency. Viability of colonies transferred from semisolid agar to liquid suspension culture was 100%.

Facilitated calcium diffusion by intestinal calcium-binding protein

Abstract: The calcium flux through an aqueous compartment was determined using a flow-through dialysis cell in which two dialysis membranes isolated the aqueous compartment. Addition of the intestinal vitamin D-dependent calcium-binding protein (CaBP) significantly enhanced the calcium flux at near physiological calcium concentrations (1 X 10(-6) M). Bovine serum albumin had no effect on the calcium flux. CaBP appears to be one of a class of low molecular weight, soluble binding proteins that enhance ligand diffusion.

Changes in the emulsifying and foaming properties of proteins during heat denaturation

Abstract: Changes in the emulsifying and foaming properties of ovalbumin, 7S globulin, κ-casein, β-lactoglobuHn and bovine serum albumin were followed during heat denaturation, and these surface properties were correlated with the corresponding surface hydrophobicity, in order to investigate the role of surface hydrophobicity in the surface properties of proteins. The surface hydrophobicity of ovalbumin, 7S globulin and jc-casein increased with heat denaturation, while that of β-lactoglobuHn and bovine serum albumin decreased. The emulsifying activity and emulsion stability of proteins correlated linearly with surface hydrophobicity, although protein structure changed greatly during heat denaturation. On the other hand, the foaming power of proteins correlated curvilinearly with surface hydrophobicity during heat denaturation. No significant correlation was observed between the foam stability and the surface hydrophobicity of proteins.On the basis of these results, the relationships between the surface properties a...

Fibrinogen and fibronectin as substrates for epidermal cell migration during wound closure

Abstract: Pieces of glass coverslip coated with human fibronectin or human fibrinogen were implanted under one margin of a skin wound on adult newt (Notophthalmus viridescens) hind limbs. In contrast to uncoated glass or glass coated with nest serum, bovine serum or bovine serum albumin, glass treated with either fibronectin or fibrinogen supported considerable epidermal cell migration. When optimal amounts of each protein were used, the amount of migration on fibrinogen-coated glass did not differ from the amount on fibronectin-coated glass or from the amount on the wound bed. Migration on a fibronectin substrate, could be blocked by treating the substrate with an antiserum against fibronectin just prior to implantation. Similarly, migration on a fibrinogen substrate could be blocked by exposing it to an antiserum against fibrinogen. While we have yet to determine it fibrinogen and fibronectin are interacting directly with the cell surface, our observations suggest that these two proteins may play an important role in wound closure by providing a suitable substrate for epithelial cell migration.

Principles, problems, and strategies in the use of antigenic mixtures for the enzyme-linked immunosorbent assay.

Abstract: Competition between proteins and other macromolecules for adsorption sites on plastic was studied with the enzyme-linked immunosorbent assay (ELISA) to determine effects of the use of antigenic mixtures or extracts of organisms on assays of antibodies and antigens by ELISA. A comparison of a number of different polystyrene microplates with bovine albumin and human immunoglobulin G (IgG) as antigens showed two major classes of plates; those which adsorbed albumin poorly and those which adsorbed albumin well. IgG adsorbed well on all plates, but plates which adsorbed albumin best also gave significant background levels of nonspecific binding of conjugate. When mixtures of IgG and bovine serum albumin were used as coating antigens, significant competition was observed; the component present at 1% or less in the mixture was essentially undetectable unless excessive amounts of conjugate were used. The important factor was the ratio of competitor to antigen, not the absolute amount. Other proteins (ovalbumin, rabbit albumin, human albumin, and gelatin) were equally effective competitors for adsorption sites on plastic. Nonionic detergents (Tween 20, and Triton X-100) were strong competitors even at 10:1 competitor-to-antigen ratios. In antigen capture assays, normal serum components blocked attachment of antigen-specific IgG, but this competition could be lessened to a degree by the use of strongly binding polystyrene plates. In indirect ELISA for measurement of serum antibody, the use of antigenic mixtures gave significantly lower antibody titers when the desired antigen was less than 1% of the total protein coated. Therefore, the use of mixed or crude antigens in ELISA presents significant problems concerning the sensitivity and specificity of tests.

Variability in different lots of commercial bovine serum albumin affects cell multiplication and hatching of rabbit blastocysts in culture

Abstract: Rabbit morulae were cultured in vitro for 4 days in a synthetic culture medium supplemented with two different lots of commercial bovine serum albumin (BSA) and two different amino acid formulations in a factorial 2 x 2 arrangement. One lot of BSA caused complete hatching of a proportion of blastocysts and formation of more than twice as many cells per blastocyst (hatched and unhatched) as that of the second BSA lot which did not cause complete hatching of any blastocysts. The mean cell numbers of hatched blastocysts were more than twice those of non-hatched blastocysts. There was no significant effect of amino acid formulation.

Enzyme immunoassay for urinary albumin.

Abstract: We describe an enzyme-linked immunosorbent assay for urinary albumin, performed on microtiter plates with use of commercially available antisera and peroxidase conjugate. The assay range is 3-1000 micrograms/L, the sensitivity 625 pg. The method is suitable for measurement of albumin excretion in either normal or pathological urine. For 20 normal children, the range of urinary albumin excretion was 1.7-22.9 mg/24 h.

Long-term culture of human endothelial cells.

Abstract: Human umbilical vein endothelial cells can be grown in vitro for 28 passages (CPDL 58) in Medium 199 supplemented with newborn bovine serum and a partially purified growth factor derived from bovine brain. Newborn bovine serum is superior to fetal bovine serum for the proliferation of human umbilical vein endothelial cells seeded at low density in the presence of the growth factor. The endothelial cells, which can be passaged every 7 to 10 d at a 1-to-5 split ratio, retain their morphological and biochemical characteristics. The proliferation of cells seeded at low density (10(3)/cm2) is proportional to the concentration of the growth factor present in the medium. The growth factor, which has an isoelectric point between 5.0 and 5.5, can support cell proliferation at reduced serum concentrations; half-maximal growth is achieved in medium containing the growth factor and 3% serum. The brain endothelial cell growth factor does not stimulate DNA synthesis significantly in cultures of human skin fibroblasts.

Heat-Induced Changes in the Proteins of Whey Protein Concentrate

Abstract: Three-level fractional factorial experiments were used to study effects of heating conditions (pH, time, temperature, solids content, calcium addition) on whey protein concentrate. Increasing pH and temperature led to lower solubility at pH 4.6 and 7.0, lower sulfhydryl content, higher hydroxymethylfurfural, generally darker color, lower DNBS-available lysine and altered pepsin pancreatin digestion profiles. Mercaptoethanol and SDS demonstrated relative importance of disulfide and hydrophobic bonds on solubility loss. Polyacrylamide gel electrophoresis indicated heat stability of proteose peptones; susceptibility was greatest at pH 8.0, 95°C for β-lactoglobulin and α-lactalbumin, and pH 4.6, 95°C for bovine serum albumin. HPLC gel filtration showed that heating rendered a high molecular weight fraction undissociable by mercaptoethanol.

Multiple mechanisms of dissociated epidermal cell spreading.

Abstract: To test the possibility that epidermal cells use a common basement membrane protein whenever they spread, in vitro experiments were conducted using trypsin-dissociated guinea pig epidermal cells and the following proteins: human serum, bovine serum albumin, serum fibronectin, Type IV collagen, laminin, and epibolin (a recently described serum glycoprotein which supports epidermal cell spreading; Stenn, K.S., 1981, Proc. Natl. Acad. Sci. U.S.A. 78:6907.). When the cells were added to media containing the specific proteins, all the tested proteins, except for serum albumin, supported cell spreading. Added to protein-coated substrates in defined media, the cells spread on fibronectin, epibolin, and laminin-Type IV collagen, but not on albumin or whole serum. In none of these experiments were the results qualitatively affected by the presence of cycloheximide. Antibodies to a specific protein blocked cell spreading on that protein but not on the other active proteins, e.g. whereas antibodies to epibolin blocked cell spreading on epibolin, they did not affect spreading on fibronectin, collagen, or laminin. In a second assay in which the cells were allowed to adhere to tissue culture plastic before the protein-containing medium was added, the cells spread only if the medium contained epibolin. Moreover, under these conditions the spreading activity of whole serum and plasma was neutralized by antiepibolin antibodies. These results support the conclusion that dissociated epidermal cells possess multiple spreading modes which depend, in part, on the proteins of the substrate, proteins of the medium, and the sequence of cell adhesion and protein exposure.