The effect of various coupling methods on the adsorption of serum proteins by immobilized concanavalin A

Concanavalin A was coupled to Sepharose 6B after activation by cyanogen bromide, divinyl sulfone, or glutaraldehyde and its adsorption behavior toward human serum proteins was investigated. The capacity and selectivity of the lectin were influenced markedly by the method used for its immobilization. When coupled to agarose via CNBr, the resulting absorbent showed the highest capacity and the lowest selectivity relative to the other two derivatives. When coupled to agarose via divinyl sulfone, the lectin exhibited high selectivity but its adsorption capacity was significantly reduced. Coupling to agarose via glutaraldehyde gave an absorbent that behaved, in some respects, differently from the other two. The variability in the adsorption behavior of the immobilized concanavalin A is attributed in part to variations in the degree of multipoint attachment of the lectin or its subunits to the agarose matrix. The selectivity increases also with increasing sample load, irrespective of the coupling method used, apparently due to protein-protein displacement.     

The effect of superoxide dismutase on the radiosensitivity of bacterial spores at various oxygen concentrations

Summary Superoxide dismutases are considered to be essential protective agent against radiation injury. Experiments were carried out to investigate the effect of extracellulary added SOD on the radiosensitivity of Bacillus megaterium spores. 120 µg/ml SOD had no effect on the radiosensitivity of Bacillus megaterium spores at different oxygen concentrations.Relative enzyme activity obtained at various oxygen concentrations indicating the lack of oxygen effect in the radiation-induced inactivation of SOD.     

A novel early effect of concanavalin A on thymocytes.

Concanavalin A, added to freshly isolated rabbit thymocytes, markedly enhanced the extracellular appearance of non-immunoglobulin proteins. Time course studies revealed that the onset of enhancement occurred virtually without delay. The effect appeared to be restricted only to certain of the thymus-derived cells because thymocytes obtained from rabbits treated with hydrocortisone, as well as splenocytes derived from untreated rabbits essentially did not exhibit the enhancement. Stimulation by concanavalin A was specific in that pokeweed mitogen and lipopolysaccharide were without effect and also in that α-methyl-mannoside, but not galactose, abrogated the concanavalin A-mediated enhancement. Experiments with mouse thymocytes demonstrated that the cells which responded to concanavalin A were primarily cells that bear the θ-antigen on their surface (T-cells).     

The effect of lanthanum on the mechanical function of the isolated perfused rat heart at different extracellular calcium concentrations.

The effects of 50 microM lanthanum (La3+) on the contractile force, rate and coronary flow of rat hearts perfused with solutions containing 2.5, 5, 7.5 mM calcium (Ca2+) have been investigated. La3+ produced a rapid and marked decrease in contractile force within 1-3 min ("early La(3+)-effect"). The inhibition of contractility by La3+ was reduced progressively when the Ca2+ ion concentration in the perfusion fluid was raised from 2.5 to 7.5 mM. However, after 10-80 min of La3+ perfusion the contractile force was increased significantly ("late La(3+)-effect"). Elevation of Ca2+ during exposure to La3+ increased its effect. During the late La(3+)-effect, a marked decrease in heart rate and a significant increase in time to reach peak tension, time for half relaxation and twitch duration was observed. High concentrations of perfusate Ca2+ decreased the chronotropic response to La3+, in contrast, elevated Ca2+ potentiated La(3+)-induced increase in time to reach peak tension, time for half relaxation and twitch duration. La3+ produced a significant decrease in coronary flow. High Ca2+ augmented the decrease coronary flow. The findings indicate that La3+ may produce marked effects on myocardial function. High extracellular Ca2+ reduces the La(3+)-induced initial decrease in force of contraction, but potentiates the late increase in contractile force by La3+. Elevated external Ca2+ also increases the effects of La3+ on twitch parameters, heart rate and coronary flow.     

A decreased effect of organic phosphates on hemoglobin S at low concentrations.

Livers from fasted male rats were perfused with blood containing 30% carboxyhemoglobin. Chylomicron remnants (labelled with [³H] cholesterol and [¹⁴C] oleate), prepared in functionally hepatectomized rats, were added to the perfusate. Carboxyhemoglobin decreased hepatic uptake of remnant cholesterol and increased the amount of lipoprotein flushed out of the liver at the end of perfusion. Transfer of triacylglycerol fatty acids to phospholipid and formation of d>1.006 lipoproteins was diminished. Ketogenesis was increased and lipoprotein triacylglycerol secretion decreased. The data indicate an inhibition of hepatic remnant catabolism by carboxyhemoglobin and are discussed with reference to the possible role of smoking in atherosclerosis.     

Metabolism of palmitate in perfused rat liver. Effect of low and high ethanol concentrations at various concentrations of palmitate in the perfusion medium

1. The effect of ethanol on the metabolism of [1-(14)C]palmitate in rat liver was investigated in a single-pass perfusion system at concentrations of 10mm- or 80mm-ethanol and 0.2mm- or 1mm-palmitate. 2. After the perfusion the hepatic lipid was isolated in subcellular fractions. The two major fractions contained triacylglycerol from cytoplasmic lipid droplets and from endoplasmic reticulum plus Golgi apparatus respectively. 3. In experiments with 0.2mm-palmitate perfusion with 10mm- or 80mm-ethanol did not measurably increase the esterification, and the oxidation was markedly decreased and the fatty acid uptake was not affected. 4. Perfusion with ethanol, at 1mm-palmitate, increased the fatty acid uptake, increased esterification and decreased oxidation. The effects of 10mm- and 80mm-ethanol were similar. The incorporation of [1-(14)C]palmitate into triacylglycerol in cytoplasmic lipid droplets was not affected statistically significantly by ethanol. Ethanol increased the incorporation of [1-(14)C]palmitate into di- and tri-acylglycerol in the membranous fraction. Estimated chemically, the contents of di- and tri-acylglycerol were only slightly affected by ethanol. These results suggest that the effect of ethanol was to increase the turnover of fatty acids in triacylglycerol rather than to increase its accumulation. 5. The results indicate that an increased concentration of fatty acids is more important for the formation of acute fatty liver in fed rats than are the direct effects of ethanol on hepatic fatty acid metabolism.     

The effect of various iron hydroxide concentrations on the anaerobic fermentation of sulfate-containing model sewage

The addition of iron hydroxide and iron-reducing bacteria into a fermenter for anaerobic processing of sulfate-containing sewage was shown to decrease sulfate reduction and sulfide concentration, while increasing the total organic carbon (TOC) and methane production. The effect of iron (III) in sulfate-containing sewage depended on its dose, which can be expressed as molar ratio Fe(III)/SO4(2-). Sulfide concentration increased monotonically, reaching 91 mg/l and 45 mg/l after 15 days of processing at Fe(III)/SO4(2-) ratios of 0.06 and 0.5, respectively. However, soluble sulfide production was not observed at ratios equaling 1 and 2. At ratios of 0.06, 0.5, 1, and 2, the maximum rates of TOC removal were 0.75, 1.15, 1.39, and 1.55 g TOC/g of organic matter (OM) per 1 h. Methane production rates were 0.039, 0.047, 0.064, and 0.069 mg/g OM per 1 h, with the mean relative amounts of methane in the biogas being equal to 25, 41, 55, and 62%, respectively. These data can be applied to the development of new methods of anaerobic purification of sulfate-containing sewage.     

Modulation of cyclic AMP in purified rat mast cells. III. Studies on the effects of concanavalin A and anti-IgE on cyclic AMP concentrations during histamine release.

Changes in rat mast cell cyclic adenosine 3',5' monophosphate (cAMP) concentrations during stimulation of histamine release by concanavalin A (con A) and anti-IgE were studied. Con A caused an increase in cAMP with a mean peak level at 20 sec of 232% of control (range 164% to 365%). Con A-stimulated cells demonstrated falls toward control levels after 20 sec, but generally remained above control for at least 5 min. By 10 min cAMP had returned to control values. The con A effect on cAMP occurred in the absence of phosphatidyl serine but was markedly inhibited by 5 mM alpha-methyl-D-mannose. Anti-IgE induced a less marked increase in cAMP (157% of control, range 110% to 540% of control) which reached a peak at 20 sec. Two monospecific goat anti-rat myeloma IgE antisera induced similar changes in cAMP whereas normal goat IgG had no effect. These peak values were followed by a rapid decrease in cAMP. Within 2 min the cAMP content of anti-IgE stimulated cells had fallen to levels well below control and remained below control levels from 45 sec to over 15 min. Histamine release in both systems began after the peak cAMP levels, during the period of rapid destruction of cAMP.     

Sugar binding properties of various metal ion induced conformations in concanavalin A.

Concanavalin A is known to undergo a first-order conformational transition when metals are added to the demetallized protein at pH 5.6 (Brown, R.D., III, et al. (1977) Biochemistry 16, 3883--3896). The rate constants for this process, which wer have measured using a polarographic technique, are identical when zinc, cobalt, or manganese occupies S1 and calcium occupies S2. The reducible sugar, p-nitrophenyl alpha-D-mannopyranoside, binds only to the locked conformational structure which is formed upon the addition of metals. The affinity of the protein for sugars is dependent upon occupancy of S1 and S2 and quite sensitive to the identity of the metal in S2. The metals may be removed from the locked protein structure and the protein temporarily retains its ability to bind with sugars but with a considerably lower affinity. The locked form of concanavalin A is unstable at a pH near 2 and unfolds to the unlocked structure with a half-life of 25 min resulting in simultaneous loss of metal and sugar binding.     

Cooperative binding of concanavalin A to thymocytes at 4 degrees C and micro-redistribution of concanavalin A receptors.

The mode of binding of 125I-labelled concanavalin A and succinyl-concanavalin A to rat thymocytes at 4 degrees C was investigated. Simultaneously, the free binding sites of the cell-bound lectin molecules were quantified by horseradish peroxidase binding. Concanavalin A showed cooperative binding while succinyl-concanavalin A did not. The number of molecules of concanavalin A bound to the cell surface when it was saturated was twice the number of molecules of succinyl-concanavalin A. We interpret these results as showing that the binding of native concanavalin A to thymocytes at 4 degrees C brings about a cooperative modification of the membrane which leads to appearance of new receptors. Divalent succinyl-concanavalin A has no such effect. Horseradish peroxidase binding to cell-bound lectin was shown to be related to the immobilization of membrane receptors; the more they are immobilized, the more receptor-associated lectin can bind horseradish peroxidase. This allowed us to establish that post-binding events, which we called micro-redistribution, occurred at 4 degrees C when either concanavalin A or succinyl-concanavalin A binds to cells. A cooperative restriction of the micromobility of cell receptors is produced by increasing concentrations of concanavalin A. Succinyl-concanavalin A does not restrict cell receptor mobility at any concentration tested. The results are discussed in terms of cell stimulation and cell agglutination.     

The chromatographic heterogeneity of rat transferrin on immobilized concanavalin A and lentil lectin

A procedure was developed for the isolation of the microheterogeneous forms of rat transferrin consisting of anion-exchange and serial lectin affinity chromatographies. By deploying this technique, four to five different anionic species of the protein were detected in plasma. The two major components obtained, which encompassed 92-94% of the plasma transferrin, were further studied by sequential lectin chromatography. The larger of the two, representing 60-63% of plasma transferrin, was bound by concanavalin A - Sepharose, while the smaller one (30-32% of plasma transferrin) resolved into an unbound (25-27% of plasma transferrin) and a retarded (4-5% of plasma transferrin) fraction. The latter eluted from the column in a volume which was 1.9 times larger than that required for the passage of nonretarded transferrin. In accordance with their fucose contents, each of these three concanavalin A fractions resolved into a bound (20-29%) and an unbound (71-80%) subfraction by chromatography on lentil-Sepharose. It is concluded that there exist two kinds of glycan microheterogeneity in rat transferrin and that they are unrelated to each other. Consequently, at least six different forms of rat transferrin are available with respect to glycosylation. Epididymal fucosidase cleaved fucose from apotransferrin slowly and from the tryptic glycopeptide rapidly. Exploratory studies performed in vivo failed thus far to identify the significance of fucose in rat transferrin.     

The effect of endotoxemia on concanavalin A induced alterations in cytoplasmic free calcium in rat spleen cells as determined with Fluo-3.

The effects of acute (3 h) and chronic (30 h) in vivo infusions of Escherichia coli endotoxin on the Ca2+ homeostasis of rat spleen cells was investigated. Conditions were established for obtaining reliable estimates of [Ca2+]i in these cells using the newly-developed Ca2+ indicator Fluo-3. The resting [Ca2+]i of splenocytes and T lymphocyte-enriched preparations were 119 +/- 35 and 102 +/- 31 nM, respectively. Treatment of the cells with concanavalin A (Con A) resulted in a rapid increase in [Ca2+]i. The magnitude of the increase was positively correlated with the concentration of Con A, whereas the time required to reach the maximum [Ca2+]i was inversely related to the amount of Con A. The peak [Ca2+]i was attained more rapidly in splenocytes (i.e. less than or equal to 30 s) than in the T cell-enriched fraction (i.e. 1.5-2.0 min). Both the resting [Ca2+]i and the Con A-induced increase in [Ca2+]i were similar to values previously reported for other lymphocyte cell types using different Ca2+ indicators, thereby supporting the values obtained with Fluo-3. Infusions of saline or endotoxin prior to the isolation of the cells did not result in significant alterations of either resting [Ca2+]i or the cells' response to Con A. Since chronic infusions of endotoxin have previously been shown to cause a reduction in blastogenic responsiveness of splenocytes to Con A, these data suggest that the endotoxin-induced lesion occurs distal to the mobilization of intracellular Ca2+.     

Adaptation for growth at various saline concentrations by the archaebacterium Methanosarcina thermophila.

We report the ability of Methanosarcina thermophila TM-1 to adapt and grow in media containing NaCl concentrations of 0.005 to 1.2 M. When adapted to marine NaCl concentrations, this species ceased to produce the heteropolysaccharide outer layer typically formed by species of nonmarine origin. concomitant with this adaptation, M. thermophila ceased to grow as multicellular aggregates and existed solely in single-cell form. The sodium ion concentration was critical for the adaptation process, although magnesium ion appeared to contribute to the cell wall stability of single cells. The results suggest that these archaebacteria possess regulatory systems that enable them to adapt to environments with a wide range of saline concentrations.