Showing papers on "Bovine serum albumin published in 1980"

Radioimmunoassay for the vitamin K-dependent protein of bone and its discovery in plasma.

Abstract: The vitamin K-dependent protein of bone has been detected in human plasma by radioimmunoassay at 4.5 ng per ml. The plasma protein has the same apparent molecular weight as the pure bone Gla protein (BGP) and other studies indicate the plasma protein is probably the intact bone protein. BGP also has been detected in bovine serum by radioimmunoassay. The bovine serum levels of BGP decrease with developmental age from 200 ng per ml in fetal calves to 26 ng per ml in adult cows. The implications of the discovery of BGP in plasma to the function of this unique protein are discussed. This assay employs rabbit antibody directed against calf BGP and has a sensitivity of 0.1 ng. The antibody crossreacts with purified human BGP but not with BGP from rat or rabbit bone. Studies with peptides of known structure derived from enzymatic digests of BGP indicate that the rabbit antibody recognizes the COOH-terminal region of the 49-residue calf bone protein.

Antibodies specific for the carboxy- and amino-terminal regions of simian virus 40 large tumor antigen.

Abstract: Antibodies specific for the amino- and carboxy-terminal portions of simian virus 40 large tumor (T) antigen were obtained by immunization of rabbits with synthetic peptides corresponding to these regions. The amino-terminal synthetic peptide has the sequence Ac-Met-Asp-Lys-Val-Leu-Asn-Arg-(Tyr). The tyrosine residue was introduced in order to couple the peptide to bovine serum albumin with bis-diazotized benzidine. The carboxy-terminal peptide has the sequence Lys-Pro-Pro-Thr-Pro-Pro-Pro-Glu-Pro-Glu-Thr. It was coupled to bovine serum albumin with glutaraldehyde. The antisera against both peptides reacted with large T antigen. The specificity of the immune reaction was demonstrated by inhibition experiments using excess synthetic peptides. Furthermore, fragments of T antigen encoded by the nondefective adenovirus 2-simian virus 40 hybrid viruses Ad2+ND2 and Ad2+ND4, which contain the carboxy terminus and lack the amino terminus of large T antigen, were precipitated only with antiserum to the carboxy-terminal peptide. Small T antigen was not precipitated with either serum, suggesting that the amino terminus of small T antigen has a conformation different from that of large T antigen or that it is sterically hindered by a host protein. The procedures used here are of general importance for identification and characterization of gene product.

Studies of protein adsorption on polystyrene latex surfaces

Abstract: Adsorption isotherms of bovine serum albumin and y-globulin on polystyrene latices are reported Under certain conditions, for each protein, the isotherms exhibit bimodal adsorption characteristics in confirmation of earlier reports The maximal adsorbance values are near expectation for a close-packed monolayer It is noted that the ratio of the lower to upper plateau adsorbance values are 040 for the γ-globulin case and 056 for the serum albumin examples The hydrodynamic thickness of the adsorbed layer as a function of the amount of adsorbed protein has been characterized by photon correlation spectroscopy analysis No evidence for multilayer adsorption at higher coverages or conformational “flattening” of the adsorbed protein in the lower plateau regime could be established Based on this information, we propose a mechanism for bimodal adsorption which does not require major conformational changes, and which postulates the existence of a critical supersaturation ratio for homogeneous nucleation of a close-packed two-dimensional surface “crystal” of protein Below this concentration, the development of the ordered structure is kinetically limited by the finite configurational relaxation time of the protein at the surface In this regime, a less dense partially organized “glassy” monolayer is found The lower plateau regime involves random independent adsorption of isolated molecules

The interaction of plasma proteins with polymers. I. Relationship between polymer surface energy and protein adsorption/desorption

Abstract: Adsorption of bovine albumin, gamma-globulin and fibrinogen from phosphate buffer solution (pH = 7.5) onto several polymer films was studied using the radioiodinated proteins (125I). The kinetics of desorption of the proteinated polymer films in bovine plasma was determined. Contact angle measurements on these same polymers allowed the calculation of dispersive (WA d) and polar (Ip) components of the polymer-protein solution system. Results from these measurements show that the nondispersive-dispersive force balance at the polymer-protein solution interface, expressed by the Ip/WA d ratio, is an important factor for binding of proteins on polymer surfaces. The purity of fibrinogen and the cleaning procedures for the polymer surfaces influence the absolute values of proteins adsorbed on polymer surfaces.

Increased levels of cyclic adenosine-3',5'-monophosphate in human polymorphonuclear leukocytes after surface stimulation.

Abstract: Levels of cyclic AMP (cAMP) (but not cyclic GMP) in suspensions of human polymorphonuclear leukocytes (PMN) increased promptly after exposure of the cells to stimuli such as the chemotactic peptide N-formyl methionyl leucyl phenylalanine, the immune complex bovine serum albumin/anti-bovine serum albumin and calcium ionophore A23187 cAMP increased rapidly, reaching a maximum of twice the basal level 10--45 s after stimulation; after 2--5 min the amount of cAMP had subsided to basal levels Elevations in cAMP levels were concurrent with, or followed, membrane hyperpolarization (measured by uptake of the lipophilic cation triphenylmethyl phosphonium) and always preceded lysosomal enzyme release and superoxide anion (O2) production Elevated cAMP levels could be uncoupled from these later events by removal of extracellular divalent cations, replacement of extracellular Na+ with K+ or choline+, and by use of low concentrations of stimulus; each of these conditions virtually abolished lysosomal enzyme release and O2 generation, while leaving the stimulated elevation of cAMP levels unimpaired Calcium ionophore A23187 did not provoke membrane hyperpolarization, thus uncoupling changes in membrane potential from changes in cAMP levels These data suggested that cAMP is not a critical component in the earliest steps of stimulus-secretion coupling Surface stimulation of cells pretreated with prostaglandins E1 or I2 yielded very high levels of cAMP; these high levels may be an important part of the mechanism by which stable prostaglandins inhibit lysosomal enzyme release and O2 generation

Increased Glycosylation of Serum Albumin in Diabetes Mellitus

Abstract: The level of glycosylated albumin has been determined in the serum of normal and diabetic subjects after purification of the albumin to apparent homogeneity. The sugar was released from the albumin preparations as 5-hydroxymethylfurfural (HMF) after 24 h of hydrolysis in 2 N acetic acid at 92°C, and it was assayed by the thiobarbituric acid reaction. The mean value for glycosylated albumin, expressed as picomoles of HMF per nanomole of albumin, obtained from 10 normal control and 65 diabetic subjects, was 64 and 124, respectively. The level of glycosylated albumin correlates with the mean blood glucose concentration (N = 55, r = 0.715), but not with the fasting blood sugar concentrations. Moreover, a linear relationship was observed between the amounts of glycosylated hemoglobin (HbAla−c) and glycosyl-albumin (n = 74, r = 0.88). In an insulin-treated diabetic patient, there was a different temporal relationship between blood glucose concentrations and glycosylated hemoglobin and albumin levels. While HbAla−c was lowered by only 15% after 20 days, glycosylated albumin had dropped by more than 50% during the same time. Our results indicate that glycosylated albumin might provide a valuable tool to assess the average blood sugar levels between shorter intervals, since the turnover of serum albumin is considerably faster than that of HbAla−c.

Binding to Cellular Macromolecules as a Possible Mechanism for the Cytotoxicity of Misonidazole

Abstract: Reduction of the nitro group occurred when [14C]misonidazole was treated with zinc dust in aqueous solution in the presence of ammonium chloride. When the reduction mixture was allowed to react with calf thymus DNA or bovine albumin, radioactivity was bound to both DNA and protein. Under the same conditions, misonidazole did not bind to these macromolecules. Analysis of the reduction mixture indicated that the hydroxylamine, amine, and hydrazo derivatives of mizonidazole were the major products. In a number of tissues of C3H mice after administration of [14C]misonidazole, radioactivity was detected in the DNA, RNA, and protein fractions. Similar results were also obtained with Chinese hamster ovary cells incubated with the drug in the absence of oxygen. It is postulated that nitroreduction and binding of the nitroreduction products to macromolecules is a probable mechanism for the mutagenic and cytotoxic properties of misonidazole.

A radioimmunoassay for 1,25-dihydroxycholecalciferol.

Abstract: We describe a radioimmunoassay for 1,25-dihydroxycholecalciferol in human serum. We raised antisera in rabbits to 1,25-dihydroxycholecalciferol-3-hemisuccinate coupled to bovine serum albumin, and obtained sensitive, high-titer antibodies. These antibodies had a high affinity for 1,25-dihydroxycholecalciferol and cross reacted mainly with 25-hydroxycholecalciferol and 24,25-dihydroxycholecalciferol. Addition of 1 mL of normal rabbit serum per liter reduced this interference to 5 and 4%, respectively. However, these interfering steroids are present in large excess, so extensive purification of 1,25-dihydroxycholecalciferol from serum is necessary. The steroid was extracted with ethyl acetate/cyclohexane, purified on Sephadex LH-20, and then chromatographed on a column of silicic acid. The radioimmunoassay is sensitive to 5 pg/tube (3 ng/L of serum). The between-assay CV was 14%. The mean concentration of 1,25-dihydroxycholecalciferol in the serum of 54 healthy adults was 38 (SD 12) ng/L, with no sex-related difference. The assay was further validated by the finding of low or undetectable concentrations in patients with chronic renal failure and of increased concentrations in the serum of patients with primary hyperparathyroidism. In comparison with previously described methods, the major advantage of the present assay is the use of stable gamma-globulins, which are available in large amounts, as binding protein.

Effects of bile salts on the plasma membranes of isolated rat hepatocytes

Abstract: The conjugated trihydroxy bile salts glycocholate and taurocholate removed approx. 20--30% of the plasma-membrane enzymes 5'-nucleotidase, alkaline phosphatase and alkaline phosphodiesterase I from isolated hepatocytes before the onset of lysis, as judged by release of the cytosolic enzyme lactate dehydrogenase. The conjugated dihydroxy bile salt glycodeoxycholate similarly removed 10--20% of the 5'-nucleotidase and alkaline phosphatase activities, but not alkaline phosphodiesterase activity; this bile salt caused lysis of hepatocytes at approx. 10-fold lower concentrations (1.5--2.0mM) than either glycocholate or taurocholate (12--16mM). At low concentrations (7 mM), glycocholate released these enzymes in a predominantly particulate form, whereas at higher concentrations (15 mM) glycocholate further released these components in a predominantly 'soluble' form. Inclusion of 1% (w/v) bovine serum albumin in the incubations had a small protective effect on the release of enzymes from hepatocytes by glycodeoxycholate, but not by glycocholate. These observations are discussed in relation to the possible role of bile salts in the origin of some biliary proteins.

The content and composition of protein in creamery milks in south-west Scotland

Abstract: The content and composition of protein in milk samples from creameries in south-west Scotland were determined over a period of 12 months. The composition of the whole casein was expressed in terms of αsl-, β-, κ-, αs2- and γ-caseins, and that of the total milk serum protein in terms of β-lactoglobulins (β-lg), α-lactalbumins, bovine serum albumin, and a mixture of immunoglobulins, proteose-peptone component 3 and lactoferrin (IPL). Concentrations of the individual caseins varied appreciably and for most, concentration was closely correlated with and showed the same seasonal pattern as total casein concentration. Concentrations of the milk serum proteins also varied but only those of β-lg and the IPL fraction were closely correlated with that of total milk serum protein and seasonal trends were not marked. Relative amounts of the individual proteins, on the other hand, showed smaller variations and so throughout the experimental period the milks contained a protein complex of comparatively constant composition. Because of this comparative constancy it would appear that seasonal variations in milk properties in south-west Scotland are unlikely to be determined to a major extent by milk protein composition, but could be more affected by protein concentration.

Immunological Quantification by High-Affinity Antibodies of O6-Ethyldeoxyguanosine in DNA Exposed to N-Ethyl-N-nitrosourea

Abstract: Three immunological methods [radioimmunoassay (RIA), enzyme-linked immunosorbent assay, and radioimmunosorbent technique] were established for quantification of the potentially mutagenic O6-ethyldeoxyguanosine (O6-EtdGuo) in DNA treated with the carcinogen ethylnitrosourea in vivo or in vitro. To obtain high-affinity antibodies for specific detection of low levels of O6-EtdGuo in small amounts of DNA (cells), different schemes were applied for immunization of rabbits with the hapten O6-ethylguanosine coupled to various carrier proteins (rat serum albumin, bovine serum albumin, keyhold limpet hemocyanin). Low-dose immunization with the hapten-keyhold limpet hemocyanin conjugate resulted in antibodies with an affinity constant of 1 to 2 X 10(10) liters/mol and very low levels of cross-reactivity with normal as well as other alkylated DNA components. The RIA (the most sensitive of the three assays) detects 0.05 pmol of O6-EtdGuo at 50% inhibition of tracer (O6-ethyl[8,5'-3H]-3'-deoxyguanosine)-antibody binding. This permits quantification by RIA of O6-EtdGuo at an O6-EtdGuo:2'-deoxyguanosine molar ratio of approximately 3 X 10(-7) in a hydrolysate of 100 micrograms of ethylated DNA. By chromatographic separation of O6-EtdGuo prior to the RIA, this value can be lowered to less than 5 X 10(-8).

Inhibition of immune precipitation by complement.

Abstract: Immune precipitation of bovine serum albumin (BSA) by rabbit anti-BSA antibody (Ab) was studied at 37 degrees C in the presence of normal human serum, normal rabbit serum, decomplemented serum and reagents permitting complement activation either by the classical pathway or the alternative pathway. Inhibition of precipitation occurred in the presence of complement. Antibody-excess complexes were kept soluble more easily than complexes formed at equivalence. Human serum was a better inhibitor than rabbit serum. Analysis of the phenomenon showed that during the first minutes of the reaction immune complexes were maintained in solution by the classical pathway only, but at later stages the alternative pathway was essential. Such soluble immune complexes precipitated after further incubation for 24 hr. The subsequent addition of ethylenediamine tetra-acetate (EDTA) to serum at 37 degrees C holding immune complexes in solution led to a slow aggregation of the complexes. Their size was found to be approximately 25S as assessed by sucrose density-gradient ultracentrifugation and they bore C3 and C4 fragments.

Demonstration of specific binding sites for human serum albumin in group C and G streptococci.

Abstract: A total of 297 bacterial strains belonging to 27 species was tested for quantitative uptake of radiolabeled human serum albumin. Specific binding sites with high affinity for human serum albumin were found exclusively in group C and G streptococci. The albumin binding was found to be a time-dependent, saturable, and displaceable process which obeyed simple kinetic equations. Scatchard analysis revealed that human serum albumin bound to a homogeneous population of receptors with an affinity in the order ot 10(7) liters/mol and that the average bacterial cell carried more than 80,000 binding sites. The albumin receptor is a heat-stable component susceptible to proteolytic digestion. It has a surface localization separate from the receptors for immunolgobulin G, fibrinogen, aggregated beta 2-microglobulin, and haptoglobin. In individual strains, albumin reactivity was also detected independently of these other types of interactions with human proteins.

Molecule selective sensor for industrial use and procedure for its preparation

Abstract: A molecule selective sensor having two permeable membranes in contacting relationship with each other, one membrane being a protein membrane found in nature, as for example, hog intestine membrane, air bladder of fish, epidermal section of mammals and reptiles, etc. and the other membrane being the reaction product of acrylamide, polyvinylalcohol, an enzyme, bovine albumin and glutaric aldehyde.

The role of commercial bovine serum albumin preparations in the culture of one-cell rabbit embryos to blastocysts

Abstract: Summary. Normal bovine serum albumin (BSA) in a complete medium without energy substrates promoted growth of 1-cell embryos to hatched blastocysts. Defatted charcoal-treated BSA did not promote growth to the blastocyst stage but the addition of pyruvate or palmitic and oleic acids allowed blastocyst growth but not blastocyst hatching. Sodium dodecyl sulphate-gel electrophoresis showed that both the normal and defatted BSA samples were heavily contaminated by proteins other than albumin. Fractionation of the normal BSA on Sephadex G-200 indicated that the property of promoting complete blastocyst hatching was not due to the albumin but was associated with the higher mol. wt fraction of the BSA. Extraction of normal BSA with chloroform appeared to destroy the hatching-promoting ability as neither the residue after extraction nor defatted BSA to which the organic extractate had been added promoted complete blastocyst hatching. It is concluded that commercial BSA may have at least two effects on blastocyst growth: (1) energy provision via albumin-bound fatty acids, and (2) promotion of blastocyst hatching by a non-albumin component.

Anion channel blockers inhibit lysosomal enzyme secretion from human neutrophils without affecting generation of superoxide anion

Abstract: The role of permeant anions in lysosomal enzyme secretion from human neutrophils was investigated by means of anion-channel-blocking agents: 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid (DIDS), 4-acetamido-4'-isothiocyanostilbene-2,2'-disulfonic acid (SITS), and pyridoxal phosphate. Lysosomal enzyme release from cytochalasin B-treated human neutrophils stimulated by immune complexes (bovine serum albumin and IgG anti-bovine serum albumin) was inhibited by DIDS, SITS, and pyridoxal phosphate at concentrations that inhibited sulfate fluxes. Enzyme secretion triggered by calcium ionophore A23187 was also inhibited by DIDS and SITS; these agents acted on secretory events subsequent to Ca2+ influx. Neither the species of permeant anion(s) nor the role of anion fluxes in degranulation was identified, although influxes of chloride, hydroxide, or phosphate ions were not critical. In contrast to degranulation, generation of superoxide anions (O2.-) stimulated by immune complex or A23187 was not inhibited by these agents. Ultrastructural cytochemical studies demonstrated that, although lysosomal contents were not discharged from stimulated cells, vacuole formation and lysosome-lysosome fusion were unaffected by SITS or DIDS. Data suggest that anion channel blockers specifically inhibit fusion of lysosomes with the plasma membrane or its invaginations.

Interaction of Lipids with the Plasma Membrane of Sperm Cells. I. The Antifertilization Action of Cholesterol

Abstract: The inhibitory action of cholesterol-containing suspensions on fertilizing capacity in uterine-capacitated rabbit sperm cells showed a direct dependence on the concentration of sterol. Dispersion with synthetic phosphatidylcholine as a nonsonicated suspension or as liposomes did not alter this antifertilization effect. Esterification of the sterol, however, caused a complete loss of inhibitory activity. Cholesterol inhibited induction of the acrosome reaction among epididymal rat spermatozoa incubated under chemically defined conditions. Other agents with a negative effect on the acrosome reaction were seminal plasma membrane vesicles and palmitic acid. Egg lecithin-liposomes and bovine serum albumin, especially after being delipidated, facilitated it. These results corroborate the viewpoint that changes in the lipid bilayer of sperm plasma membrane significantly influence fertilizing capacity among mammalian spermatozoa.

Quantitation of aflatoxin B1 and aflatoxin B1 antibody by an enzyme-linked immunosorbent microassay.

Abstract: A specific microtest plate enzyme immunoassay has been developed for the rapid quantitation of aflatoxin B1 at levels as low as 25 pg per assay Multiple-site injection of rabbits with an aflatoxin B1 carboxymethyloxime-bovine serum albumin conjugate was used for the production of hyperimmune sera Dilutions of the purified antibody were air dried onto microplates previously treated with bovine serum albumin and glutaraldehyde and then incubated with an aflatoxin B1 carboxymethyloxime-horseradish peroxidase conjugate The amount of enzyme bound to antibody was determined by monitoring the change in absorbance at 414 nm after the addition of a substrate solution consisting of hydrogen peroxide and 2,2'-azino-di-3-ethyl-benzthiazoline-6-sulfonate Antibody titers determined in this manner closely correlated with those determined by radioimmunoassay Competition assays as performed by incubation of different aflatoxin analogs with the peroxidase conjugate showed that aflatoxins B1 and B2 and aflatoxicol caused the most inhibition of conjugate binding to antibody Aflatoxins G1 and G2 inhibited the conjugate binding to a lesser degree, whereas aflatoxins M1 and B2a had no effect of the assay

Sequence of residues 400--403 of bovine serum albumin.

Abstract: A large tryptic peptide of bovine serum albumin (residues 377--582) was subjected to 32 cycles of Edman degradation to determine the sequence of the last remaining unknown segment of this protein. Residues 400--403 were identified as gly-Phe-Gln-Asn. Amide assignments were also made at positions 388 (glutamine), 389 (asparagine), 391 (aspartic acid) and 392 (glutamine).