Elucidation of the mechanism of interaction of sheep spleen galectin-1 with splenocytes and its role in cell-matrix adhesion.

The binding of a 14 kDa beta-galactoside animal lectin to splenocytes has been studied in detail. The binding data show that there are two classes of binding sites on the cells for the lectin: a high-affinity site with a K(a) ranging from 1.1 x 10(6) to 5.1 x 10(5) M (-1) and a low affinity binding site with a K(a) ranging from 7.7 x 10(4) to 3.4 x 10(4) M (-1). The number of receptors per cell for the high- and low-affinity sites is 9 +/- 3 x 10(6) and 2.5 +/- 0.5 x 10(6), respectively. The temperature dependence of the K value yielded the thermodynamic parameters. The energetics of this interaction shows that, although this interaction is essentially enthalpically driven (DeltaH - 21 kJ lambdamol(-1)) for the high-affinity sites, there is a very favorable entropy contribution to the free energy of this interaction (-TDeltaS - 17.5 Jmol(-1)), suggesting that hydrophobic interaction may also be playing a role in this interaction. Lactose brought about a 20% inhibition of this interaction, whereas the glycoprotein asialofetuin brought about a 75% inhibition, suggesting that complex carbohydrate structures are involved in the binding of galectin-1 to splenocytes. Galectin-1 also mediated the binding and adhesion of splenocytes to the extracellular matrix glycoprotein laminin, suggesting a role for it in cell-matrix interactions.

Comparative studies on kinetics of inhibition of protein synthesis in intact cells by ricin and a conjugate of ricin B-chain with momordin.

Ribosome inactivating proteins from plants have been widely used for the preparation of immunotoxins and hormonotoxins, which have potential application in the therapy of diseases such as cancer. However, these hybrid toxins have been found to be less cytotoxic than native ribosome inactivating proteins. Therefore, it is important to understand the factors that control the intrinsic toxicity of RIPs and the hybrid toxins prepared using them. Here, a hybrid toxin has been prepared by coupling ricin B-chain to momordin and the cytotoxicity of this hybrid toxin has been compared to that observed in case of native ricin. In the two cell types used here, thymocytes and macrophages, the conjugate was found to be about 40 fold less toxic than native ricin. Kinetics of inhibition of protein synthesis showed that prior to onset of inhibition the conjugate exhibits a longer lag phase than native ricin. The rates of inhibition of protein synthesis by the conjugate were also found to be slower than ricin. Analysis of the results suggests that in addition to cell surface binding, the B-chain of ricin facilitates another step in the transmembrane translocation of ricin A-chain to the cytosol.

Calorimetric studies on the stability of the ribosome-inactivating protein abrin II: effects of pH and ligand binding.

The effects of pH and ligand binding on the stability of abrin II, a heterodimeric ribosome-inactivating protein, and its subunits have been studied using high-sensitivity differential scanning calorimetry. At pH7.2, the calorimetric scan consists of two transitions, which correspond to the B-subunit [transition temperature (Tm) 319.2K] and the A-subunit (Tm 324.6K) of abrin II, as also confirmed by studies on the isolated A-subunit. The calorimetric enthalpy of the isolated A-subunit of abrin II is similar to that of the higher-temperature transition. However, its Tm is 2.4K lower than that of the higher-temperature peak of intact abrin II. This indicates that there is some interaction between the two subunits. Abrin II displays increased stability as the pH is decreased to 4.5. Lactose increases the Tm values as well as the enthalpies of both transitions. This effect is more pronounced at pH7.2 than at pH4.5. This suggests that ligand binding stabilizes the native conformation of abrin II. Analysis of the B-subunit transition temperature as a function of lactose concentration suggests that two lactose molecules bind to one molecule of abrin II at pH7.2. The presence of two binding sites for lactose on the abrin II molecule is also indicated by isothermal titration calorimetry. Plotting DeltaHm (the molar transition enthalpy at Tm) against Tm yielded values for DeltaCp (change in excess heat capacity) of 27+/-2 kJ.mol-1.K-1 for the B-subunit and 20+/-1 kJ.mol-1.K-1 for the A-subunit. These values have been used to calculate the thermal stability of abrin II and to surmise the mechanism of its transmembrane translocation.

The effect of pH on the unfolding pathway and stability of ribosome-inactivating protein abrin-II.

The effect of pH on the unfolding pathway and the stability of the toxic protein abrin-II have been studied by increasing denaturant concentrations of guanidine hydrochloride and by monitoring the change in 8,1-anilino naphthalene sulfonic acid (ANS) fluorescence upon binding to the hydrophobic sites of the protein. Intrinsic protein fluorescence, far and near UV-circular dichroism (CD) spectroscopy and ANS binding studies reveal that the unfolding of abrin-II occurs through two intermediates at pH 7.2 and one intermediate at pH 4.5. At pH 7.2, the two subunits A and B of abrin-II unfold sequentially. The native protein is more stable at pH 4.5 than at pH 7.2. However, the stability of the abrin-II A-subunit is not affected by a change in pH. These observations may assist in an understanding of the physiologically relevant transmembrane translocation of the toxin.

Evolution of tetrameric lectin Ricinus communis agglutinin from two variant groups of ricin toxin dimers.

Seeds of Ricinus communis contain two types of lectins; the toxin ricin (approximately 60 kDa) and R. communis agglutinin (approximately 120 kDa). The toxin is a heterodimer composed of a toxic A subunit and a lectin B subunit, while R. communis agglutinin is a tetramer, constituted of two ricin-like dimers held together by non-covalent forces. The lactamyl Sepharose affinity-purified ricin consists of two major groups of variants designated ricin II and III [Hegde, R. & Podder, S. K. (1992) Eur. J. Biochem. 204, 155-164]. The purified A subunits of all the variants of ricins and R. communis agglutinin show heterogeneity in the molecular mass as shown for ricin before [Fulton, J. R., Blakey, C. D., Knowles, P. P., Uhr, J. W., Thorpe, P. E. & Vitetta, E. S. (1986) J. Biol. Chem. 261, 5314-5319]. Since the isoelectric points of the R. communis agglutinin variants fall between the isoelectric points of ricin II and III, we investigated the possibility that this lectin is made up of ricin II and III. The isoelectric points of the purified B subunits of R. communis agglutinin matched well with those of ricin II and III on urea-polyacrylamide isoelectric focussing gel. Further, two-dimensional electrophoretic analysis of the ricin constituants of R. communis agglutinin in the presence of 9 M urea, showed two protein bands, differing by nearly pH 2 in their isoelectric points, the more alkaline one corresponding to that of ricin III analyzed under the same conditions, while the other, although a higher molecular mass variant, corresponding well with ricin II in its isoelectric point. Based on these results and those obtained from adenine binding to A chains of both ricin and R. communis agglutinin, we provide a plausible evolutionary relationship between R. communis agglutinin and two groups of ricin variants; ricin II and III. The model predicts that one half of R. communis agglutinin is derived from ricin I and II, and the other half from ricin III. The results presented, contrary to the existing notion, unequivocally show that the two halves of R. communis agglutinin are not identical protein units, but differ both in surface charge and molecular mass.

Evaluation of the stoichiometry and energetics of carbohydrate binding to Ricinus communis agglutinin: a calorimetric study.

High-sensitivity isothermal titration calorimetry has been used to investigate the thermodynamics of binding of Ricinus communis agglutinin to galactose, lactose and their derivatives in the temperature range 280.5-298 K. The present study unequivocally establishes the carbohydrate-binding stoichiometry of the tetrameric agglutinin from castor bean as two, i.e. the (As-sB)2-type tetramer of the agglutinin has two equivalent sites that are non-interacting and independent. The site binding constants range from 2.2x10(3) M-1 at 282 K for galactose to 4.84x10(4) M-1 at 281 K for N-acetyl-lactosamine. The binding enthalpies range from -21.9 kJ. mol-1 at 293 K for 4-methylumbelliferyl-beta-galactoside to -50.2 kJ. mol-1 at 292.9 K for thiodigalactoside. The observation of limited entropy-enthalpy compensation for binding of the sugars to the lectin indicates that reorganization of water molecules plays an important role in binding. As the slope of the compensation plot is greater than unity, the reactions are largely enthalpically driven. These studies show that the stronger binding of N-acetyl-lactosamine than lactose is due to a favourable interaction between the acetamido group of the reducing-end N-acetylglucosamine of the former and the corresponding loci in the agglutinin molecule. Preferential binding of methyl-beta-galactoside over methyl-alpha-galactoside also indicates the apolar nature of the interaction with the methyl group of the former sugar.

A- and B-subunit variant distribution in the holoprotein variants of protein toxin abrin: variants of abrins I and III have constant toxic A subunits and variant lectin B subunits.

The cytotoxic lectin abrin shows more than 30 variant forms (R. Hegde, T. K. Maiti, and S. K. Podder, 1991, Anal. Biochem. 194, 101-109). The lectin B subunit as cause for variance in abrins I and III was detected by a combination of one- and two-dimensional electrophoresis and Western blotting. Intriguingly, in abrin I but not in abrin III, association of a single A subunit with the variant B subunits shifts the holoprotein pI toward the alkaline side indicating that the subunit association involves neutralization of few negative charges. The B-subunit variants of abrins I and III overlap in their pI, and the A-subunit association gives the holoproteins a distinctness on isoelectric focusing gel. The results were also confirmed by analyzing the pH titration curves. These differences in the subunit association pattern between abrins I and III are in corroboration with the previously observed differences in the kinetics of protein synthesis inactivation and accessibility of the disulfide bridge to reducing agents in the presence or absence of putative receptor (R. Hegde, A. Karande, and S. K. Podder, 1993 Eur. J. Biochem. 215, 411-419). Further, the genetic origin of variance was confirmed by peptide mapping of the individual subunit variants. Considering a theoretical value of 0.1 to 0.2 pI/charge, a 15-17 charge difference could be predicted between the variants of two extreme pIs. The fact that the A subunits are not shared between the groups was taken to interpret that the protein synthesized as prepro form is processed posttranslationally and the processing takes place only after the disulfide bond formation between A and B subunits. The N-terminal 16 amino acids of A subunits of abrins I and III showed 26% dissimilarity. The A subunits of abrins I and III did not react with concanavalin A, indicating that the heterogeneity in the molecular weight is because of differential processing but not because of glycosylation.

Unusual structural stability and ligand induced alterations in oligomerization of a galectin.

L-14, a 14-kDa S-type lectin shows the jelly roll tertiary structural fold akin to legume lectins yet, unlike them, it does not dissociate on thermal unfolding. In the absence of ligand L-14 displays denaturation transitions corresponding to tetrameric and octameric entities. The presence of complementary ligand reduces the association of L-14, which is in stark contrast with legume lectins where no alterations in quaternary structures are brought about by saccharides. From the magnitude of the increase in denaturation temperature induced by disaccharides the binding constants calculated from differential scanning calorimetry are comparable with those extrapolated from titration calorimetry indicating that L-14 interacts with ligands essentially in the folded state.

Basis for dosing time-dependent changes in the ocular and systemic absorption of topically applied timolol.

The objective of the present study was to determine the basis for dosing time-dependent changes in the ocular and systemic absorption of topically applied timolol in pigmented rabbits. The gamma scintigraphic technique was used to monitor the changes in precorneal solution retention following instillation. Changes in timolol concentration in the plasma over 120 min and in various anterior segment eye tissues at 30 min following the topical instillation of 25 microliters of 0.65% timolol maleate solutions at various dosing times were monitored using reversed phase HPLC. Corneal and conjunctival permeability at various dosing times was evaluated in the modified Ussing chamber. The results indicated that precorneal solution drainage was slowest at noon. Suppressing tear production by anesthesia led to an increase in ocular timolol absorption at 6 a.m. but not at other dosing times, in spite of the lowest corneal permeability then. There was no statistically significant dosing time influence on systemic timolol absorption following nasal or conjunctival dosing. In conclusion, possible diurnal changes in precorneal solution clearance may be the main factor underlying the diurnal changes in ocular as well as systemic timolol absorption in rabbits. In addition, diurnal changes in corneal permeability may also contribute to diurnal changes in ocular timolol absorption.

Energetics of carbohydrate binding by a 14 kDa S-type mammalian lectin.

The thermodynamics of the binding of derivatives of galactose and lactose to a 14 kDa beta-galactoside-binding lectin (L-14) from sheep spleen has been studied in 10 nM phosphate/150 mM NaCl/10 mM beta-mercaptoethanol buffer, pH 7.4, and in the temperature range 285-300 K using titration calorimetry. The single-site binding constants of various sugars for the lectin were in the following order: N-acetyl-lactosamine thiodigalactoside > 4-methylumbelliferyl lactoside > lactose > 4-methylumbelliferyl alpha-D-galactoside > methyl-alpha-galactose > methyl-beta-galactose. Reactions were essentially enthalpically driven with the binding enthalpies ranging from -53.8 kJ/mol for thiodigalactoside at 301 K to -2.2 kJ/mol for galactose at 300 K, indicating that hydrogen-bonding and van der Waals interactions provide the major stabilization for these reactions. However, the binding of 4-methylumbelliferyl-alpha-D-galactose displays relatively favourable entropic contributions, indicating the existence of a non-polar site adjacent to the galactose-binding subsite. From the increments in the enthalpies for the binding of lactose, N-acetyl-lactosamine and thiodigalactoside relative to methyl-beta-galactose, the contribution of glucose binding in the subsite adjacent to that for galactose shows that glucose makes a major contribution to the stability of L-14 disaccharide complexes. Observation of enthalpy-entropy compensation for the recognition of saccharides such as lactose by L-14 and the absence of it for monosaccharides such as galactose, together with the lack of appreciable changes in the heat capacity (delta Cp), indicate that reorganization of water plays an important role in these reactions.

Ricin-membrane interaction: membrane penetration depth by fluorescence quenching and resonance energy transfer.

The entry of the plant toxin ricin and its A- and B-subunits in model membranes in the presence as well as absence of monosialoganglioside (GM1) has been studied. Dioleoylphosphatidylcholine and 5-, 10-, and 12-doxyl- or 9,10-dibromophosphatidylcholines serve as quenchers of intrinsic tryptophan fluorescence of the proteins. The parallax method of Chattopadhyay and London [(1987) Biochemistry 26, 39-45] has been employed to measure the average membrane penetration depth of tryptophans of ricin and its B-chain and the actual depth of the sole Trp 211 in the A-chain. The results indicate that both of the chains as well as intact ricin penetrate the membrane deeply and the C-terminal end of the A-chain is well inside the bilayer, especially at pH 4.5. An extrinsic probe N-(iodoacetyl)-N'-(5-sulfo-1-naphthyl)ethylenediamine (I-AEDANS) has been attached to Cys 259 of the A-chain, and the kinetics of penetration has been followed by monitoring the increase in AEDANS fluorescence at 480 nm. The insertion follows first-order kinetics, and the rate constant is higher at a lower pH. The energy transfer distance analysis between Trp 211 and AEDANS points out that the conformation of the A-chain changes as it inserts into the membrane. CD studies indicate that the helicity of the proteins increases after penetration, which implies that some of the unordered structure in the native protein is converted to the ordered form during this process. Hydrophobic forces seem to be responsible for stabilizing a particular protein conformation inside the membrane.

The variants of the protein toxins abrin and ricin. A useful guide to understanding the processing events in the toxin transport.

Kinetic data on inhibition of protein synthesis in thymocyte by three abrins and ricin have been obtained. The intrinsic efficiencies of A chains of four toxins to inactivate ribosomes, as analyzed by ki-versus-concentration plots were abrin II, III > ricin > abrin I. The lag times were 90, 66, 75 and 105 min at a 0.0744 nM concentration of each of abrin I, II, III and ricin, respectively. To account for the observed differences in the dose-dependent lag time, functional and structural variables of toxins such as binding efficiency of B chains to receptors and low-pH-induced structural alterations have been analyzed. The association constants obtained by stopped flow studies showed that abrin-I (4.13 x 10(5) M-1 s-1) association with putative receptor (4-methylumbelliferyl-alpha-D-galactoside) is nearly two times more often than abrin III (2.6 x 10(5) M-1 s-1) at 20 degrees C. Equilibrium binding constants of abrin I and II to thymocyte at 37 degrees C were 2.26 x 10(7) M-1 and 2.8 x 107 M-1 respectively. pH-induced structural alterations as studied by a parallel enhancement in 8-anilino-L-naphthalene sulfonate fluorescence revealed a high degree of qualitative similarity. These results taken with a nearly identical concentration-independent lag time (minimum lag of 41-42 min) indicated that the binding efficiencies and internalization efficiencies of these toxins are the same and that the observed difference in the dose-dependent lag time is causally related to the proposed processing event. The rates of reduction of inter-subunit disulfide bond, an obligatory step in the intoxication process, have been measured and compared under a variety of conditions. Intersubunit disulfide reduction of abrin I is fourfold faster than that of abrin II at pH 7.2. The rate of disulfide reduction in abrin I could be decreased 11-fold by adding lactose, compared to that without lactose. The observed differences in the efficiencies of A chains, the dose-dependent lag period, the modulating effect of lactose on the rates of disulfide reduction and similarity in binding properties make the variants a valuable tool to probe the processing events in toxin transport in detail.

Segmental differences in drug permeability, esterase activity and ketone reductase activity in the albino rabbit intestine.

Possible segmental differences in drug permeability as well as esterase and ketone reductase activities in the albino rabbit intestine were investigated. Beta adrenergic antagonists and timolol prodrugs spanning four orders of magnitude in distribution coefficient were used as model drugs. Drug penetration was evaluated in Ussing chambers using isolated segments of the duodenum, jejunum, ileum, ascending colon, descending colon, and rectum. Esterase and ketone reductase activities were determined in homogenates of the above segments using timolol ester prodrugs and levobunolol as substrates, respectively. The results indicate that the hydrophilic beta adrenergic antagonists atenolol and sotalol and moderately lipophilic metoprolol penetrated all intestinal segments equally well, whereas moderately lipophilic timolol and lipophilic propranolol, levobunolol and betaxolol were better absorbed from the large than from the small intestinal segments. Changes in lipophilicity exerted a more pronounced effect on the penetration of beta adrenergic antagonists in the large than the small intestinal segments. A similar pattern existed for timolol prodrugs. In addition to segmental differences in drug permeability, segmental differences in esterase and ketone reductase activities also existed. The level of esterase and ketone reductase activities in the small intestinal segments was, on average, 12 times and 5 times higher, respectively, than in the large intestinal segments. The implication of the above findings is that segmental differences in drug permeability and metabolism must be considered in the design of oral drug delivery systems.

Lectin binding to complex carbohydrate at the interface: a study by resonance energy transfer.

The binding affinity of the oligosaccharide moiety of a neutral glycosphingolipid, asialoGM1, towards Ricinus communis agglutinin (RCAI) was determined for the first time by fluorescence resonance energy transfer (RET). The asialoGM1 was incorporated into a phospholipid (DMPC) vesicle doped with dansylated DPPE and then titrated with an increasing amount of the galactose specific RCAI. The efficiency of RET was determined by a saturable increase in the quenching of 'donor' fluorescence, i.e. the 'trp' residue of RCAI, due to the energy transfer from the 'acceptor' dansyl group on the surface of the vesicle. The apparent binding constant was found to be in the range of 10(5)-10(6) M-1 at 27 degrees C.

Complex carbohydrate-lectin interaction at the interface: a model for cellular adhesion. II. Reactivity of both the oligosaccharide chain and sugar-binding domain of a glycoprotein lectin.

We describe studies of a new model cell adhesion system involving liposomes bearing lectins and the glycosphingolipid, asialomonosialoganglioside (asialoGM1). The model provides a simple analysis of experimental data to elucidate the mechanism of heterophilic cell-cell adhesion mediated by multiple protein-carbohydrate interactions. Phospholipid vesicles bearing the fatty acid conjugate of a glycoprotein lectin from Ricinus communis (RCAI vesicle) are shown to react with vesicles bearing the fatty acid conjugate of Concanavalin A (Con A) and asialoGM1 (Con A vesicle). The kinetics of aggregation and monosaccharide-induced disaggregation of the two types of vesicles were followed by monitoring the time-dependent change in turbidity. Depending on the surface density of the asialoGM1, 40-60% of the resulting precipitin complex was dissociable only in the presence of both RCAI-specific galactose and Con A-specific alpha-methyl-D-mannoside. Results indicate simultaneous participation of both the saccharide-binding domain and carbohydrate sequence of RCAI, a model cell adhesion molecule, to stabilize the encounter complex by two types of interactions. These findings support the possibility of stable cell-cell adhesion in vivo occurring via interactions between cell adhesion molecules on apposing cell-surface membranes.

Improving the safety of topically applied timolol in the pigmented rabbit through manipulation of formulation composition.

The objective of this study was to compare the efficiency of various formulations in maximizing the ratio of ocular to systemic absorption of topically applied timolol in the pigmented rabbit. Formulations of various pHs, tonicities, and concentrations of benzalkonium chloride, EDTA, poly(vinyl alcohol), poly(vinylpyrrolidone), hydroxypropylcellulose, and Na hyaluronate were tested. Ocular absorption was determined by monitoring the timolol concentration in various anterior segment tissues 30 min after instillation of timolol solution, while systemic absorption was determined by monitoring the time course of timolol concentration over 120 min. Timolol was assayed by reversed-phase HPLC. Lowering the solution tonicity to 80 mosmol kg-1 and incorporating polymers into the formulation were the only approaches that promised to improve the safety of topically applied timolol, since they afforded the desired increase in ocular absorption and reduction in systemic absorption. Lowering the solution pH to 6.4 and increasing the tonicity of the solution to 600 mosmol kg-1 reduced systemic absorption but caused either no change or a decrease in ocular absorption. Raising the solution pH to 8.4 and incorporating 0.025% benzalkonium chloride and 0.5% EDTA into the formulation led to an undesirable increase in systemic absorption although ocular absorption was also increased. In the final analysis, the net effect of formulation changes on the ratio of ocular to systemic absorption depended on the interplay of changes in solution drainage; permeability of the cornea, conjunctiva, and nasal mucosa; and fraction of drug in the preferentially absorbed form.

Studies on the variants of the protein toxins ricin and abrin.

This study elucidates some structural and biological features of galactose-binding variants of the cytotoxic proteins ricin and abrin. An isolation procedure is reported for ricin variants from Ricinus communis seeds by using lactamyl-Sepharose affinity matrix, similar to that reported previously for variants of abrin from Abrus precatorius seeds [Hegde, R., Maiti, T. K. & Podder, S. K. (1991) Anal. Biochem. 194, 101-109]. Ricin variants, subfractionated on carboxymethyl-Sepharose CL-6B ion-exchange chromatography, were characterized further by SDS/PAGE, IEF and a binding assay. Based on the immunological cross-reactivity of antibody raised against a single variant of each of ricin and abrin, it was established that all the variants of the corresponding type are immunologically indistinguishable. Analysis of protein titration curves on an immobilized pH gradient indicated that variants of abrin I differ from other abrin variants, mainly in their acidic groups and that variance in ricin is a cause of charge substitution. Detection of subunit variants of proteins by two-dimensional gel electrophoresis showed that there are twice as many subunit variants as there are variants of holoproteins, suggesting that each variant has a set of subunit variants, which, although homologous, are not identical to the subunits of any other variant with respect to pI. Seeds obtained from polymorphic species of R. communis showed no difference in the profile of toxin variants, as analyzed by isoelectric focussing. Toxin variants obtained from red and white varieties of A. precatorius, however, showed some difference in the number of variants as well as in their relative intensities. Furthermore, variants analyzed from several single seeds of A. precatorius red type revealed a controlled distribution of lectin variants in three specific groups, indicating an involvement of at least three genes in the production of Abrus lectins. The complete absence or presence of variants in each group suggested a post-translational differential proteolytic processing, a secondary event in the production of abrin variants.

Pharmacokinetic basis for nonadditivity of intraocular pressure lowering in timolol combinations.

The authors determined whether the ocular absorption of topically applied timolol in the pigmented rabbit was affected significantly by coadministration with either pilocarpine or epinephrine in the same drop to explain the nonadditivity in intraocular pressure lowering (IOP) seen clinically. They instilled 25 microliters of 0.65% timolol maleate solution (equivalent to 0.5% timolol), both in the presence and absence of 2.6% pilocarpine nitrate or 1% epinephrine bitartrate, into pigmented rabbit eyes. The time course of timolol concentration in the conjunctiva, anterior sclera, corneal epithelium, corneal stroma, aqueous humor, iris-ciliary body, and lens was monitored for 360 min by using reversed-phase high-performance liquid chromatography. The area under the timolol concentration-time curve in all but one of the anterior segment tissues was reduced by 20-50% (mean, 40%) when timolol was coadministered with pilocarpine and by 20-70% (mean, 42%) when timolol was coadministered with epinephrine. Such an effect was not a result of alterations in corneal permeability or aqueous humor turnover rate, nor was it related to the extent of systemic absorption caused by pilocarpine and epinephrine. Rather, the reduction in ocular timolol absorption may have been caused by the accelerated washout of timolol by tears stimulated by the coadministered drugs and, to a lesser extent, by the loss of timolol through binding to the increased amount of tear proteins induced by the coadministered drugs. Thus, the nonadditivity in IOP lowering from timolol-pilocarpine and timolol-epinephrine combinations is probably caused by changes in precorneal timolol clearance.

Formulation influence on conjunctival penetration of four beta blockers in the pigmented rabbit: a comparison with corneal penetration.

The objective of this study was to compare the influence of pH, tonicity, benzalkonium chloride, and EDTA on the conjunctival and corneal penetration of four beta blockers--atenolol, timolol, levobunolol, and betaxolol. Drug penetration was evaluated using the isolated pigmented rabbit conjunctiva and cornea in the modified Ussing chamber. The conjunctiva was more permeable than the cornea to all four beta blockers. Formulation changes caused larger changes in corneal than in conjunctival drug penetration, especially for the hydrophilic beta blockers, atenolol and timolol. Raising the solution pH to 8.4 caused the largest increase in corneal penetration for all drugs except atenolol. This increase was greater than that obtained by removing the corneal epithelium. The same formulation also increased conjunctival drug penetration, although to a lesser extent. In the case of timolol, the formulation changes evaluated brought about similar changes in its ocular and systemic absorption with good in vitro-in vivo correlations. The above findings indicate that in making formulation changes to maximize corneal drug penetration, it is necessary to evaluate possible changes in conjunctival drug penetration, hence systemic absorption. Moreover, because the conjunctiva plays an active role in the noncorneal route of ocular drug absorption, the relative contribution of the noncorneal to the corneal routes to ocular drug absorption may also be altered by formulation changes.

Purification and characterization of three toxins and two agglutinins from Abrus precatorius seed by using lactamyl-Sepharose affinity chromatography.

Three toxins, abrin-I, -II, and -III, and two agglutinins, APA-I and -II, were purified from the seeds of Abrus precatorius by lactamyl-Sepharose affinity chromatography followed by gel filtration and DEAE-Sephacel column chromatography. Abrin-I did not bind on DEAE-Sephacel column chromatography and the bound abrin-II, abrin-III, APA-I, and APA-II were eluted with a sodium acetate gradient. The identity of each protein was established by sodium dodecylsulfate-polyacrylamide gel electrophoresis and isoelectric focusing. The relative molecular weights are abrin-I, 64,000; abrin-II and abrin-III, 63,000 each: APA-I, 130,000; and APA-II, 128,000. Isoelectric focusing revealed microheterogeneity due to the presence of isoforms in each protein. Toxicity and binding studies further confirmed the differences among the lectins. The time course of inhibition of protein synthesis in thymocytes by the toxins showed lag times of 78, 61, and 72 min with Ki's of 0.55, 0.99, and 0.74 ms-1 at a 0.63 nM concentration of each of abrin-I, -II, and -III, respectively. A Scatchard plot obtained from the equilibrium measurement for the lectins binding to lactamyl-Sepharose beads showed nonlinearity, indicating a cooperative mode of binding which was not observed for APA-I binding to Sepharose 4B beads. Further, by the criterion of the isoelectric focusing profile, it was shown that the least toxic abrin-I and the highly toxic abrin-II isolated by lactamyl-Sepharose chromatography were not retained on a low-affinity Sepharose 4B matrix, which signifies the necessity of using a high-affinity matrix for the purification of the lectins.