Showing papers on "Bovine serum albumin published in 1981"

A candidate reference method for determination of total protein in serum. I. Development and validation.

Abstract: We developed a candidate Reference Method for measuring total serum protein by use of the biuret reaction. The method involves a previously described biuret reagent (Clin. Chem. 21: 1159, 1975) and Standard Reference Material (SRM) 927 bovine albumin (National Bureau of Standards) as the standard. At 25 degrees C, color development for 30 or 60 min provides identical serum protein values. Glucose (up to 10 g/L) and bilirubin (up to 300 mg/L) do not interfere. Hemoglobin, at 3 g/L, increases apparent serum protein by 0.4 g/L. The presence of dextran in serum causes easily detected turbidity, but this interference can be eliminated by centrifuging the reaction mixture. Therapeutic concentrations of ampicillin, carbenicillin, penicillin, oxacillin, nafcillin, chloramphenicol, cephalothin, and methicillin in blood do not interfere, nor do triglycerides up to 10 g/L. Within-run and day-to-day standard deviations of the method are 0.1 and 0.4 g/L, respectively.

The osmotic pressure of concentrated protein solutions: Effect of concentration and ph in saline solutions of bovine serum albumin

Abstract: Osmotic pressure measurements are reported as a function of bovine serum albumin (BSA) concentration in 0.15 M sodium chloride at pH 4.5, 5.4, and 7.4. The measured values increased markedly with increasing BSA concentration and with increasing pH (and therefore increasing macroion charge). At a concentration of 450 g/liter solution and a pH of 7.4, osmotic pressure was nearly five atmospheres, which is more than four times the value measured at the same concentration and a pH of 4.5 and about 30 times the value expected for an ideal solution. A semi-empirical analytical expression was developed which gave good agreement between prediction and the experimental data of this and other studies. The data were also compared to the prediction of a three-term virial equation wherein the second and third virial coefficients were calculated by using McMillan-Mayer solution theory. The expression for the potential of mean force was obtained by comparing various contributions to the potential energy of interaction. The terms for electrostatic repulsion and dispersion attraction are the same as those used in the DLVO theory of colloid stability. The predicted curves are of the correct order of magnitude and follow the correct qualitative trend with pH, but they fail to display the strong pH-dependence of the data. The factors responsible for this deficiency are assessed and opportunities for developing a more realistic potential function are identified.

Effect of macromolecular crowding upon the structure and function of an enzyme: glyceraldehyde-3-phosphate dehydrogenase

Abstract: The specific activity of rabbit muscle glyceraldehyde-3-phosphate dehydrogenase (GAPD) has been measured as a function of GAPD concentration in the absence and presence of 18 g/dL ribonuclease A. The specific activity of GAPD at fixed concentration has been measured as a function of the concentration of added ribonuclease A, beta-lactoglobulin, bovine serum albumin, and poly(ethylene glycol) (Mr 20000) at additive concentrations of up to 30 g/dL. All of the data may be semiquantitatively accounted for by a simple model based upon the following qualitative assumptions: (1) Under the conditions of the reported experiments, GAPD exists primarily as an equilibrium mixture of monomers and tetramers of GAPD subunits. (2) The monomers have a much larger specific activity than do the tetramers. (3) The addition of high concentrations of unrelated globular proteins does not affect the activity of either monomer or tetramer but does promote the formation of tetramer due to space-filling properties of the added species, as proposed by Minton [Minton, A. P. (1981) Biopolymers (in press)].

Intracellular pH measurements by 31P nuclear magnetic resonance. Influence of factors other than pH on 31P chemical shifts.

Abstract: Titration curves plotting chemical shift vs. pH for inorganic phosphate and glucose 6-phosphate in solutions of various composition are presented. Physiological concentrations of K+ (0.1 M) and Mg2+ (5 mM) are shown to significantly shift the titration curve. The Mg2+ effect can be partly or completely reversed by addition of sufficient quantities of adenosine triphosphate or organic acids. The acidic protein bovine serum albumin and soluble maize root tip protein have no noticeable effect on the titration curves, whereas the basic protein protamine exerts a profound effect. The results clearly indicate that knowledge of intracellular ionic strength and free Mg2+ concentrations in the sample are required if the determination of intracellular pH by 31P NMR is to be considered accurate within +/- 0.05-01 pH unit.

L-Fucose-terminated glycoconjugates are recognized by pinocytosis receptors on macrophages

Abstract: 125I-Labeled L-fucose-albumin complex and rat preputial beta-glucuronidase are rapidly cleared from plasma after intravenous infusion. L-Fucose-albumin retards the plasma clearance of beta-glucuronidase whereas D-fucose-albumin is inactive. In vitro, 125I-labeled L-fucose-albumin is taken up into rat or rabbit alveolar macrophages by receptor-mediated pinocytosis. Uptake (37 degrees C) is time-dependent, is saturable with increasing ligand concentration (Kuptake = 4.4 X 10(-8) M), and requires Ca2+. 125I-labeled D-fucose-albumin is poorly taken up. Binding (4 degrees C) is saturable and Ca2+ dependent. Binding and uptake are fully inhibited by yeast mannan. A series of neoglycoproteins, including L-fucose-albumin, were tested as inhibitors of uptake of 125I-labeled beta-glucuronidase into macrophages. The following order of potency was observed: L-Fuc = D-Man greater than GlcNAc approximately D-Glc greater than D-Xyl much greater than than D-Gal = L-Ara = D-Fuc. L-Fucose-terminated oligosaccharides coupled to bovine serum albumin also block 125I-labeled beta-glucuronidase uptake into macrophages.

The role of the visceral yolk sac in mediating protein utilization by rat embryos cultured in vitro.

Abstract: Conceptuses from 9·5-day pregnant rats have been cultured for 48 h in heat-inactivated homologous serum. Embryonic development was normal. The protein contents of embryos and visceral yolk sacs after different periods of culture were recorded. When 125-labelled polyvinylpyrrolidone or [3H]dextran were added to the culture serum, radioactivity was accumulated by the yolk sac, but only background levels were detected in the embryo itself. The amount of radioactivity found in the yolk sac varied with the length of the interval before harvesting during which 125 I-labelled PVP or [3H]dextran was present. When formaldehyde-denatured 125 I-labelled bovine serum albumin was added to the culture serum, little radioactivity accumulated in the yolk sac and only background levels were found in the embryo. Trichloroacetic acid-soluble radioactivity steadily appeared in the culture serum, however. When conceptuses were cultured in glucose- and vitamin-supplemented dialysed serum from rats injected 2 h previously with [3H]leucine, radioactivity was found in both embryos and yolk sacs. The amount of radioactivity in these tissues increased with duration of exposure to 3H-labelled serum proteins. After short exposures little of the yolk sac and embryonic radioactivity was acid-insoluble, but this proportion increased with duration of exposure. These results are interpreted as follows. Intact macromolecules cannot enter the cells of the embryo itself, but are captured by pinocytosis into the cells of the visceral yolk-sac endoderm. Indigestible macromolecules such as 125 I-labelled polyvinylpyrrolidone and [3H]- dextran accumulate within the yolk-sac lysosomes, but proteins are digested there by the lysosomal enzymes. The radiolabelled digestion product of 125 I-labelled bovine albumin is [125 I]iodotyrosine, which cells cannot utilize and so is excreted into the culture serum. The labelled digestion product of the 3H-labelled rat serum proteins is [3H]leucine, which is used for protein synthesis in both embryo and yolk sac. The experiments provide direct evidence for the long-suspected role of the yolk sac in mediating embryonic nutrition in the period of development prior to the establishment of a functional chorioallantoic placenta.

Diffusion of injected macromolecules within the cytoplasm of living cells

Abstract: The diffusion of macromolecules introduced into the cytoplasm of human fibroblasts by erythrocyte-mediated microinjection was measured by the fluorescence recovery after photobleaching technique. The apparent diffusion coefficients for fluorescein-labeled IgG and fluorescein-labeled bovine serum albumin were approximately 10(-8) cm2/sec at 22 degrees C, consistent with the kinetics of spreading of the fluorescent probe following microinjection and approximately 1/70 the values in aqueous buffer. The diffusion of labeled bovine serum albumin was shown to be strongly dependent on temperature and, in fact, similar to that expected in a 61% aqueous sucrose solution. However, the marked reduction in diffusion at 5 degrees C could be fully reversed by incubation with 0.1 mM colchicine. These findings suggest that cytoplasmic diffusion rates are reduced relative to rates in aqueous media as a result of increased aqueous phase viscosity or the impedence provided by structural elements. Several simple models to account for the data are presented.

Inhibition of human natural cytotoxicity by macromolecular antiproteases

Abstract: Human natural cell-mediated cytotoxicity is inhibited by macromolecular protease inhibitors. Human plasma alpha 1 antiproteases are more effective than the plant antiproteases lima bean trypsin inhibitor and soybean trypsin inhibitor for reduction of cytotoxicity to the "slow" targets T24 human bladder carcinoma and NKI-1 melanoma. This inhibition of natural cytotoxicity is more readily demonstrable in serum-free medium containing crystalline bovine serum albumin than in medium containing fetal calf serum. Although electrophoretically homogeneous plasma alpha 1 antitrypsin inhibits natural cytotoxicity, partially purified alpha 1 antitrypsin preparations that contain several apha 1 proteins are more inhibitory at equivalent trypsin inhibitory capacities. Partially purified alpha 1 antichymotrypsin with no antitrypsin activity is an extremely potent inhibitor. Thus, it seems likely that several of the plasma antiproteases, including alpha 1 antitrypsin and alpha 1 antichymotrypsin, are capable of influencing natural cytotoxicity. These data indicate that serine-dependent proteases hae a critical role in triggering and/or effecting cell-mediated cytolysis. Furthermore, since alpha 1 antichymotrypsin and alpha 1 antitrypsin are acute phase proteins, the increase in plasma concentration or turnover rates of the proteins could influence natural killer cell activity in vivo.

Substitution of a synthetic polymer for protein in a mammalian gamete culture system.

Abstract: Polyvinylalcohol (PVA) was tested as a replacement for protein (bovine serum albumin, BSA) in supporting motility, acrosome reactions, and fertilizing ability of hamster spermatozoa in vitro. Bovine serum albumin is normally required for all of these processes. After incubation for 5--6 hours in a simple culture medium containing BSA and PVA (0.1 mg/ml) and essential low molecular weight factors from blood serum, 85% of motile spermatozoa had undergone acrosome reactions. Sperm motility was equally well maintained by PVA in the absence of BSA but virtually no spermatozoa showed acrosome reactions even after prolonged incubation. Serum factors were later replaced by hypotaurine (10 microM), isoproterenol (1 microM), and penicillamine (20 microM). Spermatozoa incubated in this defined medium with BSA alone or with BSA and PVA fertilized more than 90% of oocytes. No oocytes were penetrated when BSA was replaced by PVA although vigorous sperm motility was maintained. Polyvinylalcohol may help elucidate the mechanism of the acrosome reaction by permitting effects of protein and other substances to be studied without loss of sperm motility (viability). Polyvinylalcohol could also replace BSA in solutions used for manipulation of zona pellucida-free oocytes. It is suggested that PVA may find general application in cell culture media.

Specific quantitation by HPLC of protein (lysine) bound glucose in human serum albumin and other glycosylated proteins

Abstract: A specific and sensitive method for quantification of the fructose-lysine linkages present in non-enzymatically glycosylated albumin and other proteins is described. Protein is hydrolyzed for 18 h in 6 mol/l HCl at 95 degrees C to yield furosine (epsilon-N-(2-furoylmethyl)-L-lysine) known as a specific degradation product of fructose-lysine. Furosine is then separated on HPLC and quantified by its UV-absorbance against a prepared fructose-lysine standard. The method has been successfully used for the determination of glycosyl-albumin in diabetic patients starting from 100 microliter serum or less, as well as for various other proteins. Unlike the usually employed thiobarbituric acid assay the present procedure is truly specific for the detection of ketoamine linkages of glycosylated proteins.

Magnetic modulation of release of macromolecules from polymers

Abstract: Sustained-release systems were made by incorporating bovine serum albumin and magnetic steel beads in an ethylene-vinyl acetate copolymer matrix. When exposed to aqueous medium, the polymer matrix released the albumin slowly and continuously. Application of an oscillating magnetic field increased the release rate by as much as 100%. Intervals of 6-hr periods of magnetic exposure and nonexposure were alternated over a 5-day period, resulting in corresponding increases and decreases in release and establishing a pattern of modulated sustained release.

Blood compatibility of hydrophilic polymers.

Abstract: Blood compatibility has been studied for hydrophilic polymers such as poly(vinyl alcohol) (PVA), its derivative, and polyethylene grafted with water-soluble monomers. The surfaces in contact with electrolyte solutions have been characterized by measuring the zeta potentials. The study of plasma protein adsorption on these polymers has revealed that bovine serum albumin as well as bovine serum fibrinogen adsorbs to a lesser extent as the hydrophilicity of the polymers increases. Platelet deposition and fibrin formation, examined using platelet-rich plasma, have been found to take place less significantly on PVA as well as sodium acrylate- and acrylamide-grafted polyethylene than on nongrafted and acrylic acid-grafted polyethylene. Ex vivo experiments with canine whole blood have shown that formation of thrombus on PVA is less than on siliconized glass but increases upon heat treatment which reduces the hydrophilicity. When PVA tubes of about 1 mm diameter are anastomosed to the carotid artery of rat, the patency rate is found to depend strongly on the anastomotic technique. From the results on the zeta potential and the experiments in vitro and ex vivo it can be concluded that the material having a surface from which solvated, neutral chains are extended into the outer aqueous phase may exhibit excellent resistance to thrombus formation.

Preferential Hydration of Bovine Serum Albumin in Polyhydric Alcohol-Water Mixtures

Abstract: The preferential solvent interaction with bovine serum albumin in aqueous solution of polyhydric alcohols (ethylene glycol, glycerol, xylitol, sorbitol, mannitol, and inositol) was investigated by a densimetric method with the application of multicomponent theory. This proteins was preferentially hydrated in all solvent systems examined: the extent depended on the number and the steric configuration of the hydroxyl groups of alcohols. The absolute interactions of these alcohols with the protein were estimated by assuming that the amount of hydration of protein at every solvent composition used is identical with that in pure water. The preferential hydration of the protein in 30% aqueous solutions of glycerol and sorbitol was found to decrease as the temperature was increased, indicating that the increase in chemical potential of protein on transferring it from water to both aqueous solvents is generated by a large positive enthalpy change, sufficient to compensate for the positive entropy change in the transfer process. On the basis of these results, and mechanism of stabilization of protein structure by these alcohols was discussed from the viewpoint of the solvation of protein.

The role of vesicles in the transport of ferritin through frog endothelium.

Abstract: 1. The transport of ferritin molecules by endothelial cell vesicles has been quantitatively investigated by electron microscopy. Single mesenteric capillaries of pithed frogs were perfused with solutions containing 6.7 g ferritin 100 ml.-1 for known periods before fixation in situ with osmium tetroxide. 2. Two series of experiments were carried out: in the first series the perfusate contained bovine serum albumin (1.0 g 100 ml.-1); in the second series the perfusate contained no protein other than the ferritin. To assess the molecular radius of ferritin in solution, the free diffusion coefficient of ferritin was measured in the presence and absence of albumin. 3. The free diffusion coefficient of ferritin in saline solution (110 m-mole 1.-1) was found to be 0.35 X 10(-6) cm2 sec-1 at 21 degrees C and was not affected by the presence of bovine serum albumin. This indicates that there is no significant binding of albumin to ferritin in solution and yields a value for the Stokes-Einstein radius of ferritin of 6.1 nm. 4. In all perfusion experiments the percentage of luminal vesicles containing ferritin exceeded the percentage of labelled cytoplasmic vesicles, which in turn exceeded the percentage of labelled abluminal vesicles. 5. Labelling of all vesicle populations was seen after perfusions lasting less than 1 sec. At this time luminal vesicles were more heavily labelled in the absence of albumin. 6. The labelling of luminal vesicles increased with lengthening perfusion times up to 30-40 sec, after which steady levels of labelling were achieved. The rate of rise in luminal labelling and the steady-state levels reached were both greater in the absence of albumin. By contrast cytoplasmic labelling increased above its initial value only after perfusions of longer than 10 sec. 7. In the steady state, labelled cytoplasmic vesicles contained, on average, fewer ferritin molecules than labelled luminal vesicles. This finding is inconsistent with translocation of labelled luminal vesicles across the cell. 8. It is suggested that the early constant labelling of cytoplasmic and abluminal vesicles is consistent with the existence of vesicular channels. Later cytoplasmic labelling may result from the transient fusion of cytoplasmic vesicles with labelled luminal vesicles for periods long enough to allow mixing of vesicular contents. Albumin may affect vesicular transport by its interaction with the endothelial glycocalyx.

Interaction of Pasteurella haemolytica with bovine neutrophils: identification and partial characterization of a cytotoxin.

Abstract: Timed cultures of Pasteurella haemolytica 12296 strain in RPMI 1640 medium (with L-glutamine, pH 7.4) were used to determine the correlation between cytotoxin production and the age of the culture. Cytotoxic activity was measured by a 51Cr-release assay and trypan blue exclusion test with bovine neutrophils as target cells. Results demonstrated that optimal cytotoxin production occurred during the logarithmic phase (peaked at 6 hours) and decreased during the stationary phase of bacterial growth. The cytotoxin was concentrated by sequential ultrafiltration on Diaflo XM 50, XM 100, and XM 300 membranes. The cytotoxin was retained on an XM 300 membrane. These studies indicated that the molecular weight of cytotoxic substance was 300,000 or more. The cytotoxin was heat labile, oxygen stable, and susceptible to extremes of pH and killed bovine neutrophils and mononuclear leukocytes. It was not hemolytic to bovine or ovine RBC. The cytotoxic activity was inactivated by trypsin and did not contain any detectable endotoxin. Bovine fetal serum and serum collected before immunization from neonatal calves did not neutralize the cytotoxic effects of toxin on neutrophils. However, adult bovine serum from 6 cows and an antiserum (against the cytotoxin) neutralized the cytotoxin, as revealed by both the 51Cr-release assay and the trypan blue exclusion test. This was confirmed by transmission electron microscopy. These results indicated that the cytotoxin may be antigenic in cattle. The significance and implications of these findings to bovine pasteurellosis are discussed.

Covalent Binding of Methotrexate to Immunoglobulins and the Effect of Antibody-linked Drug on Tumor Growth in Vivo

Abstract: In an attempt to design cancer chemotherapeutic agents of improved specificity by linking them to antibodies against tumor-associated antigens, we studied the binding of methotrexate (MTX) to immunoglobulins by three different methods, initially using a model system that consisted of bovine serum albumin and rabbit anti-bovine serum albumin immunoglobulin G (IgG). One method used coupling via water-soluble 1-ethyl-3-(3′-dimethylaminopropyl)carbodiimide (ECDI), whereas two others used reactive intermediate derivatives of MTX, i.e. , an active ester formed by reaction with N -hydroxysuccinimide or a mixed anhydride formed by reaction with acetic anhydride. The ECDI and active ester methods yielded covalent conjugates as established by: ( a ) lack of binding of MTX to IgG in the absence of ECDI or when MTX was added without prior activation; and ( b ) the presence of a constant amount of MTX in the IgG fraction after gel filtration. The active ester method was the most effective. Ten mol of MTX per mol of IgG could be incorporated with retention of 90% of the anti-bovine serum albumin activity of an equimolar amount of unconjugated IgG and with recovery of 70% of the original protein. In the ECDI procedure, there was 50% loss of protein, and the recovered protein retained only 50% antibody activity at an incorporation level of 8 mol of MTX per mol of IgG. Conjugates containing 12 mol of drug per mol of IgG lost all antibody activity. MTX conjugated by both the active ester and ECDI methods partially retained the ability to inhibit dihydrofolate reductase. The mixed anhydride procedure failed to incorporate more than 2 to 3 mol of MTX per mol of IgG even when there was a 200-fold molar excess of anhydride in the reaction mixture. The conjugate retained full antibody activity, but neither it nor the product resulting from the reaction of MTX with acetic anhydride inhibited dihydrofolate reductase. The demonstrated superiority of the active ester method in producing active conjugates prompted us to use this technique for linking MTX to a rabbit IgG antibody against the mouse EL4 lymphoma. Conjugates containing 13 mol of MTX per mol of IgG retained their reactivity with EL4 cells on membrane immunofluorescence assay. When tumor-inoculated mice (104 EL4 cells/animal) were given injections of this conjugate on Days 1, 4, and 7 after inoculation (MTX dose, 4 mg/kg/injection), they survived significantly longer than did mice treated with equivalent amounts of drug alone, antitumor IgG alone, drug followed by antibody, or drug linked to non-tumor-specific IgG. Eight of 11 mice treated with this MTX-anti-EL4 IgG conjugate survived tumor free for an observation period of 90 days, whereas control mice died at 17.3 ± 2.47 (S.D.) days after tumor inoculation.

UltrasOnic determination of the nonlinearity parameter B/A for biological media

Abstract: The nonlinearity parameter B/A has been determined for solutions of bovine serum albumin, of dextrans, of sucrose, of hemoglobin extracted from blood, and for fresh whole blood. The value of B/A for solutions of bovine serum albumin has been observed to increase nearly linearly with concentration. Further, B/A appears to exhibit no dependence upon the molecular weight of dextrose and dextrans over the molecular weight range from 102 to 106.

Development of gastrointestinal mucosal barrier. II. The effect of natural versus artificial feeding on intestinal permeability to macromolecules.

Abstract: We have recently reported that the intestinal transport of intact macromolecules into the circulation decreases with age presumably due to maturation of mucosal barrier factors. To extend this observation and determine the effect of natural versus artificial feeding on maturation of intestinal mucosal "barrier function" we conducted experiments which assessed both macromolecular transport and epithelial cell morphology. To study barrier function, we gavage fed a physiologic quantity (100 mg) of bovine serum albumin (BSA) to weight-matched breast- and bottle-fed infant rabbits at 1 and 2 wk of age and quantitated intestinal macromolecular transport by measuring circulating plasma concentrations of the intact antigen 4 hr later. a significant decrease (P less than 0.02) in immunoreactive bovine serum albumin (I-BSA) concentration was noted in breast-fed (6.12 +/- 0.77 micrograms I-BSA per ml plasma) compared with bottle-fed (9.19 +/- 0.93 micrograms I-BSA per ml plasma) animals at one wk. However, at 2 wk, no difference could be demonstrated between the two groups. Furthermore, small intestinal morphology evaluated by light and electron microscopy was similar in both groups each age. To determine if the lower plasma I-BSA noted at one wk in naturally fed animals was related to the presence of anti-BSA antibodies in breast milk and/or in plasma of the pups, breast milk and plasma from the breast-fed animals was evaluated by counterimmunoelectrophoresis and hemagglutination. No anti-BSA antibodies were detected. Moreover, plasma from breast- and artificially fed rabbits not gavage fed BSA contained no I-BSA. These data suggest that intestinal transport of antigens in the immediate neonatal period is decreased earlier in breast- as compared to bottle-fed animals. Therefore, we suggest that breast milk may exert a protective function to control the transport of potentially antigenic molecules into the systemic circulation of newborn animals by either facilitating the early maturation of intestinal barrier function or by providing passive barrier factors until the newborn's natural barrier can develop.

Peroxidase-Mediated Formation of Reactive Metabolites of Acetaminophen

Abstract: Acetaminophen (49-hydroxyacetanilide) is metabolized by horseradish peroxidase to a reactive metabolite or metabolites that become covalently (irreversibly) bound to either mouse liver microsomal protein or bovine serum albumin. The time-dependent reaction requires the presence of both the enzyme and hydrogen peroxide. Although the binding is almost completely inhibited by either catalase (0.2 mg/ml) or ascorbic acid (1.5 mM), it is unaffected by superoxide dismutase (20 µg/ml). Glutathione also inhibits the binding (∼50% at a concentration of 0.1 mM) with formation of the same glutathione conjugate that is produced from acetaminophen and glutathione in the presence of mouse liver microsomal oxygenases. An acetaminophen radical is generated by horseradish peroxidase in the presence of hydrogen peroxide as determined by electron paramagnetic resonance spectroscopy. The radical rapidly disappears in the presence of microsomal protein, bovine serum albumin, or glutathione, and is quenched by ascorbic acid with the concomitant formation of the ascorbate radical. The acetaminophen radical can be photolytically generated and spin-trapped with 5,5-dimethyl-1-pyrroline-1-oxide. The hyperfine splitting constants AHβ (18.7 G) and AN (15.2 G) strongly suggest that the radical is primarily centered on a carbon atom. These results indicate that a reactive metabolite of acetaminophen is formed in a peroxidase-mediated reaction with properties similar to that which is produced in microsomal incubations, and that an acetaminophen radical is formed under the same peroxidative conditions.

Radioimmunoassay of the leukotrienes of slow reacting substance of anaphylaxis

Abstract: A rabbit immunized with a conjugate of leukotriene D4 (LTD4) and bovine albumin via the icosanoid carboxyl produced antibodies with comparable affinities for leukotrienes C4, D4, and E4 (LTC4, LTD4, and LTE4) and their 11-trans stereoisomers The antibodies bound 3H-labeled 11-trans-LTC4 and 11-trans-LTC4 with the same average association constant (Ka) of 28 x 10(9) M-1 at 37 degrees C and were present at a concentration of 032 microgram/ml of the immune rabbit plasma When 95 microliter of anti-LTD4 and 108 pmol of 11-trans-[3H]LTC4 (40 Ci/mmol) were incubated in a volume of 300 microliter with LTC4, LTD4, LTE4, or their 11-trans stereoisomers, 50% inhibition of 11-trans-[3H]LTC4 binding was achieved at levels varying between 03 and 07 ng As assessed with synthetic analogs of the natural leukotrienes, the antibodies recognized neither those changes within the 6-sulfidopeptide unit of LTD4 produced by deamination or modest peptide lengthening nor the specific stereochemistry of the delta 14-cis double bond However, the antibodies did recognize the triene lipid domain and the position and spatial orientation of the glutathione or cysteinylglycine function Binding of 11-trans-[3H]LTC4 by anti-LTD4 was not inhibited by glutathione, cystinylbisglycine, arachidonic acid, or 5-hydroxy-6,8,11,14-icosatetraenoic acid, and leukotriene B4 (LTB4) was about 1/1000th as active as LTC4, LTD4, or LTE4 Mouse lymphoma (WEHI-5) and rat basophil leukemia (RBL-1) cells, when stimulated with calcium ionophore A23187, each produced immunoreactive leukotrienes; and LTC4, LTD4, and LTE4 from RBL-1 cells were individually quantitated by radioimmunoassay after resolution by high-performance liquid chromatography

Acyl exchange between oleoyl-CoA and phosphatidylcholine in microsomes of developing soya bean cotyledons and its role in fatty acid desaturation

Abstract: Microsomes of developing soya bean cotyledons transfer oleate from oleoyl-CoA to phosphatidylcholine (PC) by two different mechanisms: one in which oleate transfer is accompanied by the release of free CoA and another which results in the exchange of oleate from oleoyl-CoA for unsaturated 18-carbon fatty acids of PC. The acyl exchange can be demonstrated only when bovine serum albumin is present in the incubation medium. ATP-dependent acyl-CoA synthetase is not involved in the exchange process, which apparently does not require any cofactors. In light of this exchange process, the oleate desaturase system was reinvestigated in order to determine what the actual substrate for this system is. Upon incubation of microsomes with high concentrations of [14C] oleoyl-CoA, bovine serum albumin and NADH, it could be conclusively demonstrated that most oleic acid is desaturated while part of the PC molecule. The amounts of [14C] linoleoyl-CoA formed could be explained entirely by the acyl exchange. The physiological significance of the acyl exchange system is discussed. A new method for separation of acyl-CoA from other lipids and free CoA using reversed phase column chromatography also is described.