G. Entlicher

Studies on phytohemagglutinins III. Isolation and characterization of hemagglutinins from the pea (Pisum sativum L.)

Abstract A mixture of two closely related phytohemagglutinins have been isolated from crude water extracts of the garden pea seeds ( Pisum sativum L. var. Pyram) by (NH 4 ) 2 SO 4 precipitation of the active protein fraction, chromatography on hydroxylapatite and DEAE-cellulose as well as by specific adsorption on Sephadex. The two phytohemagglutinins were separated by further chromatography on DEAE-cellulose. Both are homogenous proteins exhibiting a single band by electrophoresis on starch and polyacrylamide gels as well as on cellulose-acetate strips at different pH values. Their electrophoretic mobility is similar to that of human IgG globulin, their isoelectric point lies at pH 7.7–7.9. They both reveal a single symmetrical peak on ultra-centrifugation and a s 20. w of 3.5 S. The molecular weights of 54 000 and 53 000 have been found for phytohemagglutinin 1 and phytohemagglutinin II, respectively, by the sedimentation equilibrium method. Both phytohemagglutinins show the same degree of nonspecific hemagglutinating activity against all types of human red blood cells of the ABO system. They have perceptible activity up to a dilution of 2.5 μg/ml. The amino acid compositions of both pea phytohemagglutinins are very similar. They contain high amounts of aspartic acid and threonine, but no cystine and methionine. The DNP method of N-terminal amino acid analysis of phytohemagglutinin H and the mixture of both phytohemagglutinins shows the presence of equal amounts of valine and threonine.

Studies on phytohemagglutinins. IV. Isolation and characterization of a hemagglutinin from the lentil, Lens esculenta, Moench

Abstract The presence of two nonspecific phytohemagglutinins was shown in seeds of the lentil, Lens esculenta , Moench. One of them was isolated by water extraction, followed by ammonium sulfate fractionation and specific adsorption on Sephadex. Hemagglutinin exhibited a single symmetrical peak on ultracentrifugation and a single band on polyacrylamide and starch gel electrophoreses. It has a sedimentation coefficient s 20, w 3.78 S, as calculated from the sedimentation velocity data. A molecular weight of 42 000 was estimated by gel filtration at pH 7.4. In acid medium, dissociation into smaller subunits of uniform molecular weight occurs; the dissociation can be reversed under alkaline conditions. The lentil hemagglutinin contains high amounts of aspartic acid and threonine, about 1.5% of neutral sugar and no sulfur-containing amino acids. The presence of about 1 gatom of Mn per 66 500 g of agglutinin and 1 gatom of Ca per 11 000 g of agglutinin was shown. The minimal hemagglutinating dose of the purified preparation is 4.5 μg/ml. The hemagglutinating activity of the purified lentil agglutinin can be increased by some bivalent cations, especially by Mn 2+ .

Studies on phytohemagglutinins: VII. Effect of Mn2+ and Ca2+ on hemagglutination and polysaccharide precipitation by phytohemagglutinin of Pisumsativum L

Abstract The “demetallization” procedure used to remove Mn and Ca from concanavalin A has been applied to pea phytohemagglutinin and was shown to decrease the Ca content but to leave the Mn content unaffected. “Demetallization” completely abolished the precipitation of mannan by the hemagglutinin and decreased hemagglutination to 25% of its original activity. Addition of Mn2+ and Ca2+ enhanced both the hemagglutinating and precipitating activities. Mn2+ was more active in promoting hemagglutination while Ca2+ showed a more pronounced stimulation of mannan precipitation. EDTA inhibited both hemagglutination and precipitation of mannan. Mannan precipitation by the phytohemagglutinin was more sensitive to the effect of EDTA than hemagglutination.

Studies on phytohemagglutinins. VIII. Isoelectric point and multiplicity of purified concanavalin A

Abstract Isoelectric focusing of purified concanavalin A reveals a heterogeneity of this phytohemagglutinin. The main components show pI (isoelectric point) values in the pH range of 4.5–5.5 and about equal hemagglutinating activity against untreated rabbit erythrocytes. A simultaneous determination of pI by electrophoretic mobility on cellulose acetate strips gives a pI value of 5.1. These pI values are in disagreement with values reported recently and support the original observations of earlier workers.

Studies on phytohemagglutinins: XXII. Isolation and characterization of a lymphocyte receptor for concanavalin A

Abstract A receptor glycopeptide for concanavalin A was isolated from calf thymocytes by a method originally devised for the isolation of a lectin receptor from human erythrocytes (Kubanek, J., Entlicher, G.; and Kocourek, J. [1973] Biochim, Biophys. Acta 304, 93–102). The method consisted of pronase digestion of the lipid-depleted thymocyte membrane material followed by ethanol fractionation, separation on Sephadex and preparative paper electrophoresis. The isolated glycopeptide contains 10.4% of neutral sugar. The molar ratios of the sugar components mannose, galactose, glucosamine, glucose, fucose and sialic acid are 3 : 2 : 2 : 1 : 1 : 1. The minimum molecular weight calculated from the sugar composition is about 12 000. Concanavalin A receptor activity of the glycopeptide was demonstrated in three different ways: (i) Reduction of the 125 I-labeled concanavalin A binding to thymocytes. (ii) Prevention of concanavalin A induced agglutination of calf thymocytes. (iii) Inhibition of concanavalin A stimulated DNA synthesis in calf and rabbit thymocytes and rabbit lymph node lymphocytes cultivated in vitro. The isolated glycopeptide seems to be involved in the interaction of lymphocytes with concanavalin A and the subsequent stimulation.

Studies on phytohemagglutinins: IX. Metal content and activity of the hemagglutinin from the lentil (Lens esculenta moench)

Abstract “Native” phytohemagglutinin of the lentil containing 0.13% and 0.37% Mn 2+ and Ca 2+ respectively, takes up Mn 2+ and Ca 2+ in solution, up to a content of 0.21% and 0.58% respectively, under a simultaneous 4- to 8-fold increase in hemagglutinating activity and about a 2-fold increase in the mannan precipitating activity. The increased metal content as well as the activity are maintained after prolonged dialysis against distilled water and lyophylization. The “demetallization” procedure does not affect the Ca 2+ content. However, it brings about nearly complete loss of Mn 2+ and results in a 94% decrease of hemagglutinating activity that cannot be reversed by “reactivation” with Mn 2+ and Ca 2+ . Mn 2+ and Ca 2+ enhance both the hemagglutinating and polysaccharide precipitating activities: Mn 2+ is more active in promoting hemagglutination, Ca 2+ shows a more pronounced stimulation of the mannan precipitation. EDTA strongly inhibits mannan precipitation and, to a smaller degree, erythrocyte agglutination.

Studies on phytohemagglutinins: XI. Importance of tryptophan residues for the activity of pea phytohemagglutinin☆

Abstract Methylene blue-sensitized photooxidation of pea phytohemagglutinin at pH 8.2 leads to stepwise inactivation of this protein. Initial destruction of 1 tryptophan, 3 histidine, 3 arginine, and 4 tyrosine residues is accompanied by a 50% inactivation. Further activity decrease (from 50 to 1% of the original activity) follows after destruction of 1 tyrosine and 2 tryptophan residues. 36% of the original amount of the protein-bound Mn is released during photooxidative inactivation. Photooxidatively inactivated protein shows a sedimentation coefficient practically identical to the native substance. The conformation of the phytohemagglutinin molecule as estimated on the basis of circular dichroism spectra remains unchanged after photooxidative inactivation. Like concanavalin A, the pea phytohemagglutinin consists essentially of a mixture of β-form and random coil. The possible presence of tryptophan in the binding site of the pea phytohemagglutinin is discussed.

Studies on phytohemagglutinins XVI. Subunit structure of the pea isophytohemagglutinins

Abstract The existence of subunits in the molecules of the two pea isophytohemagglutinins was demonstrated by sodium dodecylsulfate polyacrylamide gel electrophoresis, gel filtration and ultracentrifugation in concentrated guanidine · HCl solutions. Molecules of both pea isophytohemagglutinins (mol. wt 55 000) were found to be composed of two heavy (mol. wt 18 000) and two light (mol. wt 10 500) subunits. Subunits are bound together in the intact isophytohemagglutinin molecules apparently by non-covalent bonds. As far as molecular weight and N- and C-terminal amino acids are concerned the heavy and the light subunits of both isophytohemagglutinins are identical. The light subunit contains valine as the N-terminal amino acid and serine as the C-terminal. The C-terminal sequence Ser-Val-Leu …… has been found. Threonine is the N-terminal amino acid of the heavy subunit polypeptide chain. Unlike the native isophytohemagglutinins both the subunits are devoid of manganese and exhibit no hemagglutinating activity.

Possible mechanism of nitric oxide production from NG-hydroxy-l-arginine or hydroxylamine by superoxide ion

It has been speculated the N(G)-hydroxy-L-arginine (OH-L-Arg), which is an intermediate in NO production from L-arginine, may be converted to NO by superoxide ion. However, there is still no direct evidence for this conversion. In the present study this was investigated using superoxide ion generated either in acellular or cellular systems. It was found that OH-L-Arg and hydroxylamine were converted to nitrite and nitrate apparently via NO by superoxide ion in aqueous solution. Arginine remained unaffected. These changes were observed during reaction of chemical substances as well as in a biological system (zymosan-activated macrophages in culture). Superoxide dismutase prevented this transformation. OH-L-Arg was also spontaneously hydrolysed to hydroxylamine and L-citrulline, however this occurred at pH > 9 only. Activated microsomes (containing different isoforms of cytochrome P450) were unable to replace NO-synthase in its ability to produce OH-L-Arg from L-arginine. These data support the hypothesis that a pathway alterative to the well-known synthesis of NO by NO-synthase via OH-L-Arg exists. This pathway may involve the production of OH-L-Arg by NO-synthase and decomposition of OH-L-Arg to NO by the action of superoxide ion. Alternatively, hydrolysis of OH-L-Arg to hydroxylamine may occur followed by its oxidation to NO, again by superoxide ion.

Studies on phytohemagglutinins: XXIV. Isoelectric point and hybridization of the pea (Pisum sativum L.) isophytohemagglutinins

The isoelectric point of the two pea isophytohemagglutinins varies from pH 5.7 to pH 8.4 depending on the composition of the buffer used. Isoelectric focusing reveals three main molecular species with pI at pH 5.90, 6.35 and 7.00. Molecular species with pI at pH 5.9 and 7.0 correspond to the two pea isophytohemagglutinins which can be obtained by DEAE-cellulose chromatography (Entlicher, G., Kostír, J.V. and Kocurek, J. (1970) Biochim. Biophys. Acta 221, 272-281). A molecular species with pI at pH 6.35 is formed from the two pea isophytohemagglutinins by hybridization. According to the hybridization pattern and subunit composition of the pea isophytohemagglutinins the subunit composition AABB, AACC and AABC can be proposed for the three molecular species with respect to ionic properties of the subunits.