Covalently cross-linked monovalent, divalent, and tetravalent derivatives of concanavalin A.

Dimeric succinyl-concanavalin A was cross-linked with ethylenediamine using 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide a condensing agent. Thus, a divalent dimer and a tetravalent tetramer composed mostly of covalently cross-linked subunits bearing altered net charges were obtained. Photoaffinity labeling of the cross-linked dimer with p-azidophenyl alpha-D-mannopyranoside resulted in a specific label for its saccharide-binding site and yielded a nonvalent dimer and a monovalent dimer (showing no subunit exchange). However, hemagglutination and glycogen precipitation data suggested that the labeled binding site is shielded but not destroyed by the label and can still bind weakly an external saccharide ligand possibly due to unsteadiness of the shielding label. Although nonvalent and monovalent derivatives were mitogenic as well as divalent and tetravalent derivatives for mouse splenic lymphocytes, binding and stimulation experiments indicated that their stimulating efficiencies after binding to the cells were far lower than those of the multivalent counterparts. Their activities were inhibited by methyl alpha-D-mannopyranoside, suggesting that the weak activities of nonvalent and monovalent derivatives were due to the labeled sites entirely and partly, respectively. We suggest that the triggering of lymphocyte mitogenesis by concanavalin A may depend on cross-linkage of cell surface receptors.     

Metabolic stimulation of polymorphonuclear leucocytes: Effects of tetravalent and divalent concanavalin A

Summary Polymorphonuclear leucocytes (PMNL) undergo a marked activation of their oxidative metabolism upon interaction with surface-reactive soluble stimuli as well as with phagocytosable objects. To get some insight into the mechanism of this stimulation, we have compared the stimulatory activity of the tetravalent lectin concanavalin A (Con A) with that of the divalent derivative succinyl-Con A (S-Con A). Both lectins bind to the PMNL surface to the same extent. S-Con A, however, is much less efficient in stimulating the PMNL metabolism. When S-Con A-treated PMNL are further reacted with antiserum to Con A, a potentiation of the metabolic stimulation is observed. Normal serum or addition to PMNL of antiserum to Con A in the absence of lectin has no effect. Furthermore, if S-Con A is displaced from its receptors on the cell membrane with -methyl mannopyranoside, the addition of antiserum fails to cause a respiratory stimulation. These results suggest that the initial triggering of the metabolic stimulation of PMNL is in part accomplished through cross-linkage of membrane constituents.     

Effect of divalent metal ions on the digestibility of concanavalin A by endopeptidases.

Demetallized concanavalin A is degraded rapidly at pH 7.0 and 8.2 by alpha-chymotrypsin, thermolysin or trypsin, yielding peptide fragments devoid of ability to bind to Sephadex G-75. Addition of Ni2+ and of Ca2+ confers on concanavalin A high resistance towards proteolytic attack so that even after long periods of exposure to the enzymes, almost all of the saccharide-binding capacity is preserved. Ni2+ alone protects strongly at pH 7.0 but not at pH 8.2. Apparently, both the transition metal ion and Ca2+ play an important role in stabilizing the native conformation of the protein molecule. Digestion of demetallized concanavalin A with alpha-chymotrypsin or thermolysin readily yields small peptide fragments (Mr less than 10 000), while trypsin yields as the major product(s) larger peptide(s) (Mr approximately 20 000) of appreciable resistance to further fragmentation.     

Effect of monovalent and divalent ions upon the activity of nisin againstMicrococcus flavus

Effect of monovalent and divalent ions on the activity of nisin againstMicrococcus flavus has been studied and Mg²⁺ ions have been found to reverse the effect of nisin. The protection by Mg²⁺ ions has been found to be due to complex formation with the antibiotic.     

Immunocytochemical localization of procollagen and fibronectin in human fibroblasts: effects of the monovalent ionophore, monensin

The monovalent ionophore monensin inhibits the secretion of both procollagen and fibronectin from human fibroblasts in culture. The distribution of these proteins in control and inhibited (5 x 10(-7) M monensin) cells has been studied by immunofluorescence microscopy. In control cells, both antigens are present throughout the cytoplasm and in specific deposits in a region adjacent to the nucleus, which we identify as a Golgi zone by electron microscopy. Treatment of cells with monensin causes intracellular accumulation of procollagen and fibronectin, initially in the juxta-nuclear region and also subsequently in peripheral regions. Electron microscope studies reveal that in such cells the juxta-nuclear Golgi zone becomes filled with a new population of smooth-membraned vacuoles and that normal Golgi complexes are not found. Immunocytochemically detected procollagen and fibronectin are localized in the region of these vacuoles, whereas more peripheral deposits correspond to the dilated cisternae of rough endoplasmic reticulum, which are also caused by monensin. Procollagen and fibronectin are often codistributed in these peripheral deposits. Accumulation of exportable proteins in Golgi-related vacuoles is consistent with previous analyses of the monensin effect. The subsequent development of dilated rough endoplasmic reticulum also containing accumulated proteins may indicate that there is an additional blockade at the exit from the endoplasmic reticulum, or that the synthesized proteins exceed the capacity of the Golgi compartment and that their accumulation extends into the endoplasmic reticulum.     

Synthesis and processing of arylsulfatase A in human skin fibroblasts

Biosynthesis of arylsulfatase A in normal and mutant human fibroblasts was studied by growing cells in the presence of L-[4,5-3H] leucine or [2-3H] mannose, isolation of labelled arylsulfatase A by immune precipitation and visualization of electrophoretically separated polypeptide by fluorography. Arylsulfatase A was synthesized as a precursor with a mean apparent molecular mass of 62 kDa. Intracellularly the precursor was converted into a 60.5 kDa polypeptide within a chase period of 1 to 7 days. The 60.5 kDa product in polyacrylamide corresponded to one of two polypeptides present in arylsulfatase A isolated from human placenta. In fibroblasts from a patient with metachromatic leukodystrophy no immune precipitable polypeptides of arylsulfatase A were detected. In normal fibroblasts less than 10% of the precursor of arylsulfatase A was secreted into the medium, whereas in mucolipidosis II fibroblasts and in control fibroblasts grown in the presence of NH4Cl up to 90% of the precursor of arylsulfatase A, appeared in the medium and remained there without change in the apparent molecular mass for at least 7 days. Arylsulfatase A polypeptides appear to contain two carbohydrate side chains. In about 90% of the polypeptides both side chains are cleaved by endo-beta-N-acetylglucosaminidase H, whereas in the remaining chains one of the two oligosaccharides is not cleaved.     

Effect of concanavalin A and succinyl-concanavalin A on nucleoside and sugar uptake in mouse embryo fibroblasts

We have examined the effect of the tetrameric and dimeric form of Con A at a dose of 50μg ml^?1 on nucleoside and glucose uptake using synchronized mouse embryo fibroblasts undergoing S phase. We have found that thymidine and uridine uptake were depressed by Con A but not significantly by succinyl-Con A. The inhibition was gradual, as it required a suitable time of incubation to become fully manifest and it was of non-competitive type. By contrast the uptake of 2-deoxy glucose was inhibited promptly and to a similar extent by Con A regardless of molecular structure. Kinetic analysis of the modalities of the sugar uptake process indicated an inhibition of competitive type.     

Synthesis of collagen, collagenase and collagenase inhibitors by cloned human gingival fibroblasts and the effect of concanavalin A

Individual clones of human gingival fibroblasts that differ in morphology and growth characteristics have been found to synthesize collagen (types I, III and V), collagenase and collagenase inhibitors, and to be capable of degrading native collagen mats. Although collagenase activity was normally low, synthesis of the enzyme could be stimulated ten-fold by Concanavalin A. These results demonstrate that individual fibroblasts have the ability to both synthesize and degrade collagen.     

Binding of concanavalin A by normal, herpes virus-transformed, and trypsin-treated hamster embryo fibroblasts

Using fluorescein isothiocyanate-labeled concanavalin A (ConA), it was shown that normal hamster embryo fibroblast cells appear to bind less ConA than do herpes virus-transformed cells. However, the binding capacity of normal HEF cells could be increased following trypsin treatment.     

Effect of adenosine on concanavalin A agglutination of human erythrocytes

We have attempted to correlate the functional activity of protein 3 with its activity as a receptor for concanavalin A. The concanavalin A agglutination of human erythrocytes is enhanced by adenosine. It varies with time of storage of the blood and is dependent on the concentration of adenosine in the medium. Adenine and/or inosine, which increase cellular ATP, do not substitute for adenosine in enhancing agglutination, and adenosine enhances agglutination of fresh erythrocytes with normal levels of ATP. Thus, it appears that cellular ATP levels are not directly involved in modulation of concanavalin A agglutination by adenosine. Trypsin, which hydrolyzes most of the exposed proteins of the cell surface but does not alter protein 3, enhances concanavalin A agglutination without altering the relative response of the cell to adenosine.Glucose, as well as the glucose transport inhibitors maltose and cellobiose, inhibits agglutination. High concentrations of adenosine reverse the inhibition by glucose and enhance agglutination in the presence of maltose and cellobiose.Treatment of erythrocytes with 4,4′-diisothiocyanostilbene-2,2-disulfonic acid disodium salt, which selectively inhibits the anion transport function of protein 3, substantially inhibits adenosine-supported concanavalin A agglutination.Treatment of erythrocytes with iodoacetate under conditions in which it selectively reacts with glyceraldehyde-3-phosphate dehydrogenase inhibits agglutination. Adenosine protects this dehydrogenase in erythrocytes from inactivation by iodoacetate, over the same concentration range in which it enhances agglutination.     

Negative surface charge near sodium channels of nerve: divalent ions, monovalent ions, and pH.

Evidence is given for a high density of negative surface charge near the sodium channel of myelinated nerve fibres. The voltage dependence of peak sodium permeability is measured in a voltage clamp. The object is to measure voltage shifts in sodium activation as the following external variables are varied: divalent cation concentration and type, monovalent concentration, and pH. With equimolar substitution of divalent ions the order of effectiveness for giving a positive shift is: Ba equals Sr less than Mg less than Ca less than Co approximately equal to Mn less than Ni less than Zn. A tenfold increase of concentration of any of these ions gives a shift of +20 to +25 mV. At low pH, the shift with a tenfold increase in Ca-2+ is much less than at normal pH, and conversely for high pH. Soulutions with no added divalent ions give a shift of minus 18 mV relative to 2 mM Ca-2+. Removal of 7/8 of the cations from the calcium-free solution gives a further shift of minue 35 mV. All shifts are explained quantitatively by assuming that changes in an external surface potential set up by fixed charges near the sodium channel produce the shifts. The model involves a diffuse double layer of counterions at the nerve surface and some binding of H+ions and divalent ions to the fixed charges. Three types of surface groups are postulated: (1) an acid pKa equals 2.88 charge density minus 0.9 nm- minus 2; (i) an acid pKa equals 4.58, charge density minus 0.58 nm- minus 2; (3) a base pKa equals 6.28, charge density +0.33 nm- minus 2. The two acid groups also bind Ca-2+ ions with a dissociation constant K equals 28 M. Reasonable agreement can also be obtained with a lower net surface charge density and stronger binding of divalent ions and H+ ions.     

Effect of hyaluronan on the elastase-type activity of human skin fibroblasts.

The influence of hyaluronan (HA) on the expression of human skin fibroblast elastase-type protease (HSFEp) (Homsy et al, 1988) was studied. At confluency of HSF cultures, hyaluronan increased the level of HSFEp in a time and dose-dependent fashion, Optimal effect was observed after 48 h of culture and at 2 mg/ml HA concentration; the stimulatory, effect of HA could be suppressed by 1 μM cycloheximide. The enhancement of enzyme biosynthesis by HA was dependent on cell proliferation but quasi invariant with HSF passage number (from 7-21).     

Differential effects of monovalent, divalent and trivalent metal ions on rat brain hexokinase

The effects of monovalent (Li+, Cs+) divalent (Cu2+, Ca2+, Sr2+, Ba2+, Zn2+, Cd2+, Hg2+, Pb2+, Mn2+, Fe2+, Co2+, Ni2+) and trivalent (Cr3+, Fe3+, Al3+) metals ions on hexokinase activity in rat brain cytosol were compared at 500 microM. The rank order of their potency as inhibitors of brain hexokinase was: Cr3+ (IC50 = 1.3 microM) greater than Hg2+ = Al3+ greater than Cu2+ greater than Pb2+ (IC50 = 80 microM) greater than Fe3+ (IC50 = 250 microM) greater than Cd2+ (IC50 = 540 microM) greater than Zn2+ (IC50 = 560 microM). However, at 500 microM Co2+ slightly stimulated brain hexokinase whereas the other metal ions were without effect. That inhibition of brain glucose metabolism may be an important mechanism in the neurotoxicity of metals is suggested.