Binding of galactose and lactose to ricin. Equilibrium studies.

The interaction of ricin, one of the two lectins of Ricinus sanguineus, with its specific ligands galactose and lactose (4-O-beta-D-galactopyranosyl-D-glucopyranose) has been studied by means of equilibrium dialysis, analytical ultracentrifugation and fluorescence polarization. In the studied concentration range, only one molecule of galactose is bound per molecule of ricin with an association constant, Ka = 6900 m-1 at 4 degrees C. Scatchard plots of equilibrium dialysis data show that two molecules of lactose bind to one molecule of ricin, without modification of molecular weight of the lectin. Together with results of microcalorimetric experiments and agglutination of erythrocytes by ricin, equilibrium dialysis data indicate that the lectin contains two distinct saccharide binding sites. Regardless of the existence of extended sites, it is not possible to select between the two models: (a) two independent sites (Ka1 = 35 000 M-1, Ka2 = 2800 M-1 at 4 degrees C) or (b) two identical sites with negative cooperativity.

Purification of 125I-labelled lysine-vasopressin by affinity chromatography on sepharose-bound neurophysins.

Purification of 125I-labelled lysine-vasopressin has been achieved by affinity chromatography on a Sepharose 4 B conjugate of porcine neurophysins. This affinity absorbent did not retain halogenated hormone, while native lysine-vasopressin was bound on the solid support. The specific activity of purified iodinated lysine-vasopressin was 1700-1800 Ci/g, corresponding to one iodine atom per mole. By comparison with an unpurified tracer, a five times increase in the minimum detectable dose was obtained in the vasopressin radioimmunoassay.

Nature of the interaction between Ricinus communis agglutinin and blood cells.

Binding of Ricinus communis agglutinin (RCA 120) to carbohydrate receptors of human lymphocytes and erythrocytes is enthalpically driven. As in the case of simple saccharides, the delta S contribution is always unfavorable to the interaction. This result is different from that observed for other lectins and might indicate that hydrophobic interactions do not play a dominant role in binding of RCA 120 to cell surfaces.

Subunit structure of human alpha 2-macroglobulin (alpha 2-MG) with respect to its interaction with trypsin.

SDS-polyacrylamide gel electrophoresis of a recently prepared alpha 2-macroglobulin solution showed only the polypeptide chains of 190,000 molecular weight. Reduction-alkylation of this preparation followed by gel-filtration on a Sephadex G-200 column in 5.2 M guanidine hydrochloride was unable to separate a fraction of 83,000 molecular weight as previously described. Nevertheless, after incubation of a mixture alpha 2-macroglobulin-trypsin during 45 minutes at 37 degrees C, approximately 60 per cent of the preparation were converted in a component with 83,000 molecular weight as detected in SDS polyacrylamide gel. That component was isolated on Sephadex G-200 in guanidine hydrochloride and corresponds to the subunit, fraction II. According to the results of the present work together with those of previous studies, it can be assumed that alpha 2-MG is a 780,000 molecular weight protein (19S) formed of two half-molecules of equal weight (11-12S). The half-molecule contains two polypeptide chains of 180,000-190,000 molecular weight, each of them having, in its middle, a specific region particularly susceptible to attack by proteases.

[Interaction between ricin hemagglutinin and its ligands, galactose and lactose. Microcalorimetry and equilibrium dialysis]. / Interaction entre l'hémagglutinine de ricin et ses ligands, galactose et lactose. Etude par microcalorimétrie et dialyse à l'équilibre.

The interaction of Ricinus communis hemagglutinin with galactose and lactose has been studied by means of microcalorimetry, equilibrium dialysis and analytical ultracentrifugation. A first class of beta-galactoside-binding sites involves two similar and independent sites of which affinity constants are 2600 M-1 for galactose and 26700 M-1 for lactose at 25 degrees C. The binding of one galactose or one lactose molecule leads to enthalpy changes of--12.3 Kcal and--11 Kcal, respectively. Considering the negative entropy changes of the association, and as for ricin, the binding of galactosides with hemagglutinin is driven by favorable enthalpic contributions. In presence of high lactose concentrations, a second endothermic step of the calorimetric titration curve was observed. This result and the biphasic nature of Scatchard plots of equilibrium dialysis suggest the existence of a second class of binding sites on the lectin molecule. As for ricin, the interaction between these secondary sites and lactose would be entropically driven.

Use of quantitative image microfluorometry to follow fluorescent ricin internalization in single living cells.

We used microspectrofluorometry and videomicrofluorometry to follow the binding and internalization of fluorescein-labeled toxic lectin ricin in living Zajdela hepatoma cells. Microspectrofluorometry showed that when ricin was specifically labeled on its B-chain with one molecule of fluorescein (ABF), its fluorescence spectrum did not alter during its binding to the cell surface and subsequent internalization. This enabled us to use image analysis to follow cell internalization of labeled ricin. Accordingly, we measured the appropriate fluorescent cell parameters, comprising total fluorescence intensity, cell surface area, mean fluorescence intensity and its standard error, and used the measurements for mono- and biparametric studies of cell fluorescence distribution. The results showed that (a) ricin binds two different subpopulations of Zajdela hepatoma cells, (b) Zajdela hepatoma cells internalize ricin rapidly and after a relatively stable period of 1-2 hr, internalization starts again at 4 hr, and (c) the distribution of intracellular fluorescence is heterogeneous and ABF accumulates in certain cellular localizations. Our results demonstrate that quantitative microfluorometry is an effective and interesting approach for real-time studies of macromolecule internalization in living cells.

Effect of physical environment on the conformation of ricin. Influence of low pH.

The molecular properties of ricin (the toxic lectin from Ricinus communis seeds, RCA II or RCA 60) were evaluated by analytical ultracentrifugation, viscosimetry, c.d., fluorescence and equilibrium dialysis. Measurements of sedimentation (S0(20,W) = 4.60 S) and viscosity (eta = 2.96 X 10(-2) dl/g) indicated that, at neutral pH, the ricin molecule is very compact. Various transitions were explored, and a pH-triggered change in the ricin conformation was observed between pH 7 and 4. In this range, the sedimentation coefficient, far-u.v. c.d. and fluorescence altered simultaneously without unfolding. Below pH 7 the change in the ricin conformation was accompanied by a decrease in the affinity of ricin for galactosides, and at pH 4.0 by an alteration in its binding capacity. These effects of low pH are discussed in relation to the physical conditions encountered by ricin molecules during their entry into living cells.

Interaction of rice (Oryza sativa) lectin with N-acetylglucosaminides. Fluorescence studies.

The interaction of lectin isolated from rice (Oryza sativa) embryos with N-acetylglucosaminides was studied by equilibrium dialysis and fluorescence. Equilibrium dialysis with 4-methylumbelliferyl-(GlcNac)2 showed that rice lectin (Mr 38000) contains four equivalent saccharide-binding sites. Addition of the N-acetylglucosaminides GlcNac, (GlcNac)2 and (GlcNac)3 enhanced the intrinsic fluorescence of rice lectin and this was accompanied by a 10nm blue-shift of its maximum fluorescence with (GlcNac)2 and (GlcNac)3. These changes in intensity allowed determination of the association constants, which increased with the number of saccharide units: at 20 degrees C, Ka = (1.3 +/- 0.1) X 10(3), (5.1 +/- 0.4) X 10(4) and (2.6 +/- 0.1) X 10(5) M-1 for GlcNac, (GlcNac)2 and (GlcNac)3 respectively. The binding enthalpy, delta H0, for the three glucosaminides were very low and ranged from -12.1 to -20.6 kJ X mol-1. The results are compared with those obtained with wheat-germ agglutinin, another GlcNac-specific gramineaous lectin.

Separation of ricin A- and B-chains after dithiothreitol reduction.

After complete cleavage of ricin interchain disulfide bridge by 0.05 M dithiothreitol in nondenaturing conditions at 37 degrees C during 1 h 30 min, A- and B-chains were separated on a lactosaminyl-aminoethyl Biogel P-150 column at 4 degrees C, in the presence of 0.01 M dithiothreitol and 0.5 M MgCl2. A- and B-chains have been characterized by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and immunology. Their specific activities have been tested by protein synthesis inhibition in a cell-free assay (rabbit reticulocyte lysate) and on whole cells (Zajdela hepatoma cells) and by hemagglutination. From these tests, the apparent cross contamination of the chains was about 0.1%.

Stability and subunit structure of human alpha2-macroglobulin.

The molecular weights of alpha2-macroglobulin and its non-covalent subunits have been determined by equilibrium centrifugation. The secondary structure of the native and the thermally denatured molecules has been analyzed by circular dichroic measurements. In contrast to most proteins the thermally denatured form contains a slightly more highly organized polypeptide chain than the native form. The relaxation time of the native protein, as determined by fluorescence polarization measurements, indicates that alpha2-macroglobulin is composed of domains smaller than that of the two subunits. The transitions in acid, alkali, and at high temperatures have been explored in order to establish the pH and thermal range of stability of alpha-macroglobin.

Cryoinsolubility of peanut agglutinin. Effect of saccharides and neutral salts.

Peanut agglutinin (PNA) has been shown to be insoluble at low temperatures. This cryoinsolubility has been studied by means of absorption spectroscopy, fluorescence, circular dichroism, and analytical ultracentrifugation. It was found to be dependent on pH, temperature, and protein concentration. No effects on dimer-tetramer equilibrium could be determined nor any conformational changes provoked by exposure of the PNA preparation to low temperatures. The dimer half-molecule apparently does not precipitate. The cryoinsolubility was partially reversible and totally inhibited in the presence of galactosides, the specific ligands of PNA. Their efficacy as inhibitors of cryoinsolubility was related to their affinity for the lectin. The effects of neutral salts and particularly inhibition of the insolubility by strongly chaotropic salts indicate that charge-charge interactions are of little importance and that hydrogen bonds and/or van der Waals interactions are most probably responsible for the formation of the cryoprecipitate.

Interactions of ricin with sinusoidal endothelial rat liver cells. Different involvement of two distinct carbohydrate-specific mechanisms in surface binding and internalization.

We have investigated the interactions of the plant toxin ricin with sinusoidal endothelial rat liver cells (EC). In these cells, ricin can be bound and internalized via either cell surface galactosyl residues or mannose receptors. Binding and uptake via galactosyl residues and mannose receptors was studied in the presence of mannan (1 mg/ml) and lactose (50 mM) respectively. Whereas most of the ricin binding was accounted for by cell surface galactosyl residues, uptake of ricin via mannose receptors was much more efficient than uptake via galactosyl residues. Internalized ricin is subject to extensive retroendocytosis (recycling to the cell surface from an early endocytic compartment). Retroendocytosis occurs after internalization of ricin via either pathway and to a much greater extent than for other glycoproteins taken up via mannose receptors of the EC. Hyperosmolarity (150 mM-sucrose), which is known to inhibit endocytosis from coated pits, strongly inhibited ricin uptake via mannose receptors, but had less effect on uptake via galactosyl residues. This suggests that only part of the galactose-specific uptake takes place from coated pits. Protein synthesis in EC was very sensitive to ricin [concn. causing half-maximal inhibition (IC50) = 1.3 x 10(-13) M]. Mannan was slightly more effective than lactose in protecting the EC protein synthesis from ricin toxicity.

Structure and stability of Ricinus communis haemagglutinin.

The molecular properties of the haemagglutinin of Ricinus communis (RCA I or RCA 120) were evaluated by analytical ultracentrifugation, light-scattering, c.d. and fluorescence. The native molecule had a fairly expanded structure (f/f0 = 1.43) and dissociated into two subunits of equal size in 6 M-guanidinium chloride. This native structure was stable in alkali (up to pH 11) and resistant to thermal denaturation at neutrality. A pH-triggered change in the haemagglutinin conformation was observed and characterized by analytical ultracentrifugation, c.d. and fluorescence between pH 7 and 4.5, the range in which its affinity for galactosides decreased [Yamasaki, Absar & Funatsu (1985) Biochim, Biophys. Acta 828, 155-161]. These results are discussed in relation to those reported in the literature for other lectins and more especially ricin, for which a pH-dependent conformation transition has been observed in the same range of low pH.

4-Methylumbelliferyl-glycosides as fluorescence probes of sugar-binding sites on lectin molecules: spectral properties and dependence of fluorescence on polarity and viscosity.

The spectral properties of 4-methylumbelliferyl-glycosides (MeUmb-glycosides) were investigated in order to assess their usefulness as probes of the microenvironment of sugar binding sites on lectin molecules. It was shown that the abnormally high values for fluorescence polarization of free MeUmb-glycosides (from 0.07 to 0.251) were due neither to their molecular size nor to the blockade of their movement, but to the short lifetimes (less than 0.55 ns) of the excited state of these compounds. Working essentially with two MeUmb-monosaccharides and one MeUmb-disaccharide (MeUmb-alpha-D-galactopyranoside, MeUmb-beta-D-galactopyranoside, and MeUmb-2-acetamido-2-deoxy-3-O-(beta-D-galactopyranosyl)-beta-D- galactopyranoside) which were solubilized in various solvents, it was demonstrated that solvent polarity and viscosity definitely affected the fluorescence intensity of MeUmb-glycosides. A low-polarity medium reduced this intensity, and high viscosity enhanced it. The implications of these findings are discussed in relation to the variations in the fluorescence intensity of MeUmb-glycosides when these compounds were bound to lectins.

Rat angiotensinogen and des(angiotensin I)angiotensinogen: purification, characterization, and partial sequencing.

Rat angiotensinogen was completely purified by a six-step procedure including (1) ammonium sulfate precipitation, (2) affinity chromatography on Affi-gel blue, (3) chromatography on DEAE-Sephacel, (4) chromatography on hydroxylapatite, (5) chromatography on Ultrogel AcA 54, and (6) isoelectric focusing. Two peaks of pure angiotensinogen were obtained, distinguishable by their isoelectric points (4.55 and 4.75). Both contained 23 microgram of angiotensin I/mg of protein. Sodium dodecyl sulfate--polyacrylamide gel electrophoresis of the peak with pI = 4.55 revealed two protein bands (respectively Mr 57000 and 59000) and a single protein band (Mr 57000) for the peak with pI = 4.75. The molecular weight of the latter homogeneous form, as determined by sedimentation equilibrium, was 55000. Only one immunoprecipitin line was observed when antiserum reacted with the heterogeneous angiotensinogen in Ouchterlony gels. The first 17 amino acids of the N-terminal region of the angiotensinogen with pI = 4.75 are reported. The amino acids in positions 10 and 11 which correspond to the renin cleavage site are leucyl-leucyl. The des(angiotensin I)angiotensinogen obtained after hydrolysis of angiotensinogen with pure mouse submaxillary gland renin was found to consist of a single protein band with an Mr of 56000 as revealed by sodium dodecyl sulfate--polyacrylamide gel electrophoresis. Only one N-terminal residue (leucyl) was obtained for this des(angiotensin I)angiotensinogen. These findings establish that renin only cleaves angiotensinogen at a single site.