Purification and immunohistochemical localization of the auxin binding site i protein from maize coleoptiles

An auxin binding protein fraction prepared by means of affinity chromatography on 2-OH-3,5-diiodobenzoic acid-Sepharose and gel filtration was used as antigen. The obtained rabbit antisera contained antibodies against the auxin, binding protein (ABP) and several contaminating proteins (nonABP). The nonABP could be separated on an appropriate affinity matrix omitting the TIBA analogue. After their immobilization on Sepharose antibodies directed towards contaminating, the proteins were isolated and immobilized, too. This IgGanti nonABP-Sepharose retains almost all contaminating proteins present in the specific eluates of the auxin affinity matrix. In a final affinity chromatography step on IgG-Sepharose a highly purified ABP could be eluted. This ABP was immobilized on Sepharose for the separation of monospecific antibodies against ABP (IgGanti abp). Using these antibodies the ABP could be localized within the outer epidermal cells of the coleoptile by immunofluorescence microscopy. From the inhibition of auxin induced elongation of coleoptile tissue by IgGanti abp it is concluded that the ABP is localized at the plasmalemma of the epidermal cells and that the ABP is involved in auxin action as a true hormone receptor. Presented at the International Symposium “Plant Growth Regulators” held on June 18–22, 1984 at Liblice, Czechoslovakia.     

Assessing the role of tryptophan residues in the binding site

Instead of looking at the interfacial area as a measure of the extent of a protein--protein recognition site, a new procedure has been developed to identify the importance of a specific residue, namely tryptophan, in the binding process. Trp residues which contribute more towards the free energy of binding have their accessible surface area reduced, on complex formation, for both the main-chain and side-chain atoms, whereas for the less important residues the reduction is restricted only to the aromatic ring of the side chain. The two categories of residues are also distinguished by the presence or absence of hydrogen bonds involving the Trp residue in the complex. A comparison of the observed change in the accessible surface area with the value calculated using an analytical expression provides another way of characterizing the Trp residues critical for binding and this has been used to identify such residues involved in binding non-proteinaceous molecules in protein structures.     

Riboflavin binding protein contains a type II copper binding site

Riboflavin binding protein, purified from egg white, binds copper(II) under dialysis conditions in an approximately 1:1 molar ratio. Results further indicate a small, but not negligible, amount of copper is present in the protein as purified from egg white. Electron paramagnetic resonance indicates a single type II copper site present in the protein. These results suggest the possibility of a previously unknown function of riboflavin binding protein in the storage or transport of copper.     

The binding of auxin to the Arabidopsis auxin influx transporter AUX1

The cellular import of the hormone auxin is a fundamental requirement for the generation of auxin gradients that control a multitude of plant developmental processes. The AUX/LAX family of auxin importers, exemplified by AUX1 from Arabidopsis (Arabidopsis thaliana), has been shown to mediate auxin import when expressed heterologously. The quantitative nature of the interaction between AUX1 and its transport substrate indole-3-acetic acid (IAA) is incompletely understood, and we sought to address this in the present investigation. We expressed AUX1 to high levels in a baculovirus expression system and prepared membrane fragments from baculovirus-infected insect cells. These membranes proved suitable for determination of the binding of IAA to AUX1 and enabled us to determine a K(d) of 2.6 mum, comparable with estimates for the K(m) for IAA transport. The efficacy of a number of auxin analogues and auxin transport inhibitors to displace IAA binding from AUX1 has also been determined and can be rationalized in terms of their physiological effects. Determination of the parameters describing the initial interaction between a plant transporter and its hormone ligand provides novel quantitative data for modeling auxin fluxes.     

Reconstitution of the photosystem II Ca2+ binding site

The roles of Ca(2+) in H(2)O oxidation may be as a site of substrate binding, and as a structural component of the photosystem II O(2)-evolving complex. One indication of this dual role of the metal is revealed by probing the Mn cluster in the Ca(2+) depleted O(2) evolving complex that retains extrinsic 23- and 17-kDa polypeptides with reductants (NH(2)OH and hydroquinone) [Biochemistry 41 (2002) 958]. Calcium appears to bind to photosystem II at a site where it could bind substrate H(2)O. Equilibration of Ca(2+) with this binding site is facilitated by increased ionic strength, and incubation of Ca(2+) reconstitution mixtures at 22 degrees C accelerates equilibration of Ca(2+) with the site. The Ca(2+) reconstituted enzyme system regains properties of unperturbed photosystem II: Sensitivity to NH(2)OH inhibition is decreased, and Cl(-) binding with increased affinity can be detected. The ability of ionic strength and temperature to facilitate rebinding of Ca(2+) to the intact O(2) evolving complex suggests that the structural environment of the oxidizing side of photosystem II may be flexible, rather than rigid.     

Induction of a high affinity binding site for auxin in Avena root membrane

A membrane preparation from Avena sativa root has been found to contain only one low-affinity IAA-binding site having a K_d value of 8.4 ×     

Patch-clamp analysis establishes a role for an auxin binding protein in the auxin stimulation of plasma membrane current in Zea mays protoplasts

The electrical response of Zea mays protoplasts to different auxins and to antibodies raised against an ER-located auxin binding protein from maize (Zm-ERabp1), was investigated using the patch-clamp technique (whole-cell configuration). Following a lag-phase of 30–40 seconds, indole-3-acetic acid and 1-naphthylacetic acid induced an outwardly directed current of positive charge in a concentration-dependent manner. This current was further increased by the fungal toxin fusicoccin (FC). The current was observed only in the presence of Mg²⁺-ATP in the patch-pipette and was abolished after addition of erythrosin B, an inhibitor of H⁺-ATPase, to the protoplasts indicating that the plasma membrane H⁺-ATPase is activated by auxins and fusicoccin. Addition of antibodies directed against Zm-ERabp1 abolished the current induced by auxins, without affecting the response of protoplasts to fusicoccin. Antibodies directed against a peptide representing part of the putative auxin binding domain of Zm-ERabp1 showed auxin agonist activity, stimulating an outwardly directed membrane current in the absence of auxin. These results suggest that (i) Zm-ERabp1 or antigenically related proteins represent a site for auxin perception through which the plasma membrane H⁺-ATPase is activated, and (ii) that the activation of the H⁺-ATPase by such proteins is initiated from outside the plasma membrane.     

Functional characterization of the secondary actin binding site of myosin II

The role of the interaction between actin and the secondary actin binding site of myosin (segment 565-579 of rabbit skeletal muscle myosin, referred to as loop 3 in this work) has been studied with proteolytically generated smooth and skeletal muscle myosin subfragment 1 and recombinant Dictyostelium discoideum myosin II motor domain constructs. Carbodiimide-induced cross-linking between filamentous actin and myosin loop 3 took place only with the motor domain of skeletal muscle myosin and not with those of smooth muscle or D. discoideum myosin II. Chimeric constructs of the D. discoideum myosin motor domain containing loop 3 of either human skeletal muscle or nonmuscle myosin were generated. Significant actin cross-linking to the loop 3 region was obtained only with the skeletal muscle chimera both in the rigor and in the weak binding states, i.e., in the absence and in the presence of ATP analogues. Thrombin degradation of the cross-linked products was used to confirm the cross-linking site of myosin loop 3 within the actin segment 1-28. The skeletal muscle and nonmuscle myosin chimera showed a 4-6-fold increase in their actin dissociation constant, due to a significant increase in the rate for actin dissociation (k(-)(A)) with no significant change in the rate for actin binding (k(+A)). The actin-activated ATPase activity was not affected by the substitutions in the chimeric constructs. These results suggest that actin interaction with the secondary actin binding site of myosin is specific for the loop 3 sequence of striated muscle myosin isoforms but is apparently not essential either for the formation of a high affinity actin-myosin interface or for the modulation of actomyosin ATPase activity.     

The donor side of Photosystem II as the copper-inhibitory binding site

We have measured, under Cu (II) toxicity conditions, the oxygen-evolving capacity of spinach PS II particles in the Hill reactions H₂OSiMo (in the presence and absence of DCMU) and H₂OPPBQ, as well as the fluorescence induction curve of Tris-washed spinach PS II particles. Cu (II) inhibits both Hill reactions and, in the first case, the DCMU-insensitive H₂O SiMo activity. In addition, the variable fluorescence is lowered by Cu (II). We have interpreted our results in terms of a donor side inhibition close to the reaction center. The same polarographic and fluorescence measurements carried out at different pHs indicate that Cu (II) could bind to amino acid residues that can be protonated and deprotonated. In order to reverse the Cu (II) inhibition by a posterior EDTA treatment, in experiments of preincubation of PS II particles with Cu (II) in light we have demonstrated that light is essential for the damage due to Cu (II) and that this furthermore is irreversible.Abbreviations DCMU 3-(3,4-dichlorophenyl)-1, 1-dimethyl urea - DCIP 2,6-dichlorophenolindophenol - DPC 1,5-diphenilcarbazide - F_o initial non-variable fluorescence - F_I intermediate fluorescence yield - F_m maximum fluorescence yield - F_v variable fluorescence yield - Mes 2,-(N-morpholino)ethanosulfonic acid - OEC oxygen-evolving complex - P680 Primary electron donor chlorophyll - Pheo pheophytin - PPBQ phenyl-p-benzo-quinone - PS II Photosystem II - SiMo Silicomolybdate - Q_B secondary quinone acceptor - Q_A primary quinone aceptor - Tris N-tris(hydroxymethyl)amino ethane - Tyr_z electron carrier functioning between P680 and the Mn cluster This article is dedicated to Prof. Dr. Harmut Lichtenthaler on the occasion of his 60th birthday.     

Effects of inorganic solutes on the binding of auxin

The binding of α-naphthaleneacetic acid (¹⁴C-NAA) to pelletable particulates from corn (Zea mays) coleoptiles was found to be influenced by inorganic solutes. La³⁺, Ca²⁺, and Mg²⁺ increased the binding whereas monovalent cations did not. The concentrations of CaCl₂ which increased auxin binding were similar to those which inhibited coleoptile elongation in the presence of auxin. These results are interpreted as suggesting that the alteration of hormonal effectiveness by some inorganic solutes involves alterations in the attachment of the hormone to binding sites in the cell.     

Insights into the role of the metal binding site in methionine-R-sulfoxide reductases B

Methionine sulfoxide reductases B (MsrBs) catalyze the reduction of methionine-R-sulfoxide via a three-step chemical mechanism including a reductase step, formation of an intradisulfide bond followed by a thioredoxin recycling process. Fifty percent of the MsrBs, including the Escherichia coli enzyme, possess a metal binding site composed of two CXXC motifs of unknown function. It is located on the opposite side of the active site. The overexpressed E. coli MsrB tightly binds one atom of zinc/iron. Substitution of the cysteines of E. coli MsrB results in complete loss of bound metal and reductase activity, and leads to a low-structured conformation of the protein as shown by CD, fluorescence, and DSC experiments. Introduction of the two CXXC motifs in Neisseria meningitidis MsrB domain leads to a MsrB that tightly binds one atom of zinc/iron, shows a strongly increased thermal stability and displays a reductase activity similar to that of the wild-type but lacking thioredoxin recycling activity. These results demonstrate the stabilizing effect of the metal and the existence of a preformed metal binding site in the nonbound metal MsrB. The data also indicate that metal binding to N. meningitidis MsrB induces subtle structural modifications, which prevent formation of a competent binary complex between oxidized MsrB and reduced thioredoxin but not between reduced MsrB and substrate. The fact that the E. coli and the N. meningitidis MsrBs exhibit a similar thermal stability suggests the existence of other structural factors in the nonbound metal MsrBs that compensate the metal bound stabilizing effect.     

The action of specific inhibitors of auxin transport on uptake of auxin and binding of N-1-naphthylphthalamic acid to a membrane site in maize coleoptiles

Using both 1-mm segments of corn (Zea mays L.) coleoptiles and a preparation of membranes isolated from the same source, we have compared the effectiveness of several inhibitors of geotropism and polar transport in stimulating uptake of auxin (indole-3-acetic acid, IAA) into the tissue and in competing with N-1-naphthylphthalamic acid (NPA) for a membrane-bound site. Low concentrations of 2,3,5-triiodobenzoic acid (TIBA), NPA, 2-chloro-9-hydroxyfluorene-9-carboxylic acid (morphactin), and fluorescein, eosin, and mercurochrome all stimulated net uptake of [³H]IAA by corn coleoptile tissues while higher concentrations reduced the uptake of both [³H]IAA and another lipophilic weak acid, [¹⁴C]benzoic acid. Since low concentrations of fluorescein and its derivatives competed for the same membrane-bound site in vitro as did morphactin and NPA, the basis for both the specific stimulation of auxin accumulation and the inhibition of polar auxin transport by all these compounds may be their ability to interfere with the carrier-mediated efflux of auxin anions from cells. At higher concentrations, the decrease in accumulation of weak acids was nonspecific and thus may be the result of acidification of the cytoplasm and a general decrease in the driving force for uptake of the weak acids. Triiodobenzoic acid was an exception. Low concentration of TIBA (0.1–1 M) were much less effective than NPA in competing for the NPA receptor in vitro, but little different from NPA in ability to stimulate auxin uptake. One possibility is that TIBA, a substance which is polarly transported, may compete with auxin for the polar transport site while NPA, morphactin, and the fluorescein derivatives may render this site inactive.Abbreviations C1-NPA 2,3,4,5-tetrachloro-N-1-naphthylphthalamic acid - IAA indole-3-acetic acid - -NAA -naphthaleneacetic acid - -NAA -naphthalenacetic acid - NPA N-1-naphthylphthalamic acid - TIBA 2,3,5-triiodobenzoic acid     

Reassessing the role of YUCCAs in auxin biosynthesis

It is remarkable that although auxin was the first growth-promoting plant hormone to be discovered, and although more researchers work on this hormone than on any other, we cannot be definitive about the pathways of auxin synthesis in plants. In 2001, there appeared to be a dramatic development in this field, with the announcement of a new gene,¹ and a new intermediate, purportedly from the tryptamine pathway for converting tryptophan to the main endogenous auxin, indole-3-acetic acid (IAA). Recently, however, we presented evidence challenging the original and subsequent identifications of the intermediate concerned.²Key words: auxin synthesis, YUCCA, tryptamine, N-hydroxytryptamineThe new gene was termed YUC, and the putative intermediate is N-hydroxytryptamine. It was claimed that the YUC protein, a flavin-containing monooxygenase, converts tryptamine (formed from tryptophan by decarboxylation) to N-hydroxytryptamine, which is converted via other intermediates to IAA. When the YUC gene was expressed in E. coli and the resulting protein incubated with tryptamine, a weak TLC spot resulted, which produced a mass spectrum said to match that expected from N-hydroxytryptamine.¹ However, the authors did not report mass spectral data from authentic N-hydroxytryptamine, and their suggested fragmentation pattern breaks a fundamental rule of mass spectrometry (the even-electron rule).² Nevertheless, N-hydroxytryptamine has been added to virtually all IAA synthesis schemes published since 2001.³In 2010, LeClere et al. expressed a maize YUC gene in E. coli,⁴ and again claimed that the resulting protein converted tryptamine to N-hydroxytryptamine. This time, the mass spectrum was of better quality, but we have shown that it does not match that of authentic N-hydroxytryptamine, synthesised in our laboratory.^2,5 We have demonstrated by electrospray tandem mass spectrometry that the protonated molecule of N-hydroxytryptamine (m/z 177) fragments to give an abundant ion at m/z 144. This was the crucial piece of evidence that the product obtained by LeClere et al. was not N-hydroxytryptamine, since their compound gave an abundant ion at m/z 160, and no ion at m/z 144.⁴The m/z 144 ion is formed by loss of NH₂OH (hydroxylamine), as shown by accurate mass determinations (Fig. 1). In other words, it is the alkyl-amine bond that is broken; this is also the case for tryptamine and serotonin. In the latter case, an m/z 160 ion results through loss of ammonia, because the hydroxyl group on the indole ring (at position 5) is retained in the fragment. The compound obtained by LeClere et al. when protonated, also had a mass of 177, consistent with a hydroxylated tryptamine, and the abundant m/z 160 ion indicates that this fragment, as in serotonin, retains the hydroxyl group.⁴ However, we believe that the LeClere et al. product is not serotonin, because of dissimilar behavior on thin layer chromatography. Apart from the probability that it is a hydroxylated tryptamine, the identity of the LeClere et al. product is not known.Open in a separate windowFigure 1Fragmentation of (A) N-hydroxytryptamine, (B) tryptamine and (C) 5-hydroxytryptamine (serotonin), as determined by MS/MS analysis.² The m/z ratios of the fragments produced are indicated. The loss of neutral hydroxylamine (A) or ammonia (B and C) involves heterolytic cleavage and/or hydrogen atom rearrangement, and consequent retention of the positive charge on the remaining indole-containing fragment.¹⁴It is interesting to contrast the previous “identifications” of N-hydroxytryptamine1,⁴ with the identification of gibberellins, during the period when most of the gibberellins were identified (1970–1990). There were rigorous criteria for the identification of these compounds, imposed by a triumvirate of “Gibberellin Godfathers”: Jake MacMillan, Nobatuka Takahashi and the late Bernard Phinney, and more latterly by Caporegimes such as Peter Hedden and Yuji Kamiya. Three of the present authors (James B. Reid, Noel W. Davies and John J. Ross) experienced at first hand the rigour with which these criteria were applied.Essentially, any identification of an endogenous gibberellin was viewed with suspicion unless a synthesized form of that compound (a standard, confirmed by NMR) was available for comparison. For a firm identification, the retention time on GC should be identical between the standard and the putative compound, on the same GC instrument. Next, the electron ionization fragmentation patterns of the compound of interest and the standard should match, again on the one GC-MS system. It was not considered adequate to compare a spectrum of the compound of interest with published spectra from another laboratory. Often a spectrum from a plant extract might contain extra ions, contributed by “interfering” compounds and this was sometimes acceptable. However, the absence of ions that should be present was usually sufficient to render the identification unconvincing. Electrospray mass spectra are intrinsically much poorer in information than electron ionisation spectra since most or all of the signal is concentrated in the protonated molecule, and tandem mass spectrometry (MS/MS) is required to create diagnostic fragment ions. The MS/MS spectrum of another hydroxylated tryptamine that we have examined is dominated by a strong m/z 160 ion, and discrimination between hydroxylated tryptamines on the basis of MS alone could be problematic. N-Hydroxytryptamine is the exception in this regard, and it can be easily distinguished.Another technique that has been used extensively in gibberellin research, and in early auxin research as well, is “feeding” labelled compounds and determining the fate of the label concerned (often deuterium or ¹³C). This technique contributed strongly to the identification of most of the candidate auxin pathways.⁶ Its power should not be underestimated, and yet in the auxin field, it was under-utilised during much of the later 1990s and the 2000s. We have used this technique to demonstrate that tryptamine is not converted to N-hydroxytryptamine in pea roots or seeds.^2,5 In fact, to our knowledge, N-hydroxytryptamine has not yet been identified in any plant species.N-Hydroxytryptamine has been the main link between the YUC genes and the tryptamine pathway, and this link is now called into question. In fact, there are some indications that YUCCAs might not be concerned with IAA synthesis at all. The floozy mutant of petunia has a strong phenotype but normal levels of IAA.⁷ The yuc1 yuc2 yuc4 yuc6 quadruple mutant of Arabidopsis also exhibits a whole-plant phenotype but again its IAA content is not reduced compared with WT plants.⁸ Indeed, as yet there is not a single instance where knocking out YUC function has been shown to significantly reduce IAA content. We should note also that while overexpression of YUC genes does lead to elevated IAA content, the increase is small (up to about 2-fold^1,9) compared with the increases recorded for other IAA over-producing mutants; for example, sur1 (also known as rty) and sur2, which can contain 5 to 20 times more IAA than the WT.¹⁰-12Therefore, it is possible that YUC catalyses a reaction or reactions in another pathway leading to another compound that is required for normal plant development; hence the phenotypic consequences of loss-of-function yuc mutants. This compound might be another auxin or auxinlike compound, which might explain why elevating auxin content genetically sometimes rescues yuc phenotypes.¹³ The suggestion that YUC controls the synthesis of another compound was made as early as 2002,⁷ but has attracted little attention from auxin biologists. There seems little doubt that YUCCAs play essential roles in plant development, as evidenced by the phenotypes of knockout mutants, even though it is sometimes necessary to construct multiple mutants to observe strong phenotypes.¹³ Furthermore, YUC genes are found in a wide range species, and we recently extended the list to include pea.² However, the almost universal placement of N-hydroxytryptamine in auxin synthesis schemes since 2001 is now called into question by the recently published evidence.²     

Reassessing the role of N-hydroxytryptamine in auxin biosynthesis

The tryptamine pathway is one of five proposed pathways for the biosynthesis of indole-3-acetic acid (IAA), the primary auxin in plants. The enzymes AtYUC1 (Arabidopsis thaliana), FZY (Solanum lycopersicum), and ZmYUC (Zea mays) are reported to catalyze the conversion of tryptamine to N-hydroxytryptamine, putatively a rate-limiting step of the tryptamine pathway for IAA biosynthesis. This conclusion was based on in vitro assays followed by mass spectrometry or HPLC analyses. However, there are major inconsistencies between the mass spectra reported for the reaction products. Here, we present mass spectral data for authentic N-hydroxytryptamine, 5-hydroxytryptamine (serotonin), and tryptamine to demonstrate that at least some of the published mass spectral data for the YUC in vitro product are not consistent with N-hydroxytryptamine. We also show that tryptamine is not metabolized to IAA in pea (Pisum sativum) seeds, even though a PsYUC-like gene is strongly expressed in these organs. Combining these findings, we propose that at present there is insufficient evidence to consider N-hydroxytryptamine an intermediate for IAA biosynthesis.     

Modelling a binuclear metal binding site in the photosynthetic reaction centre II

Based on the experimental data and homologous sites in Protein Data Bank (PDB) a model for metal binding sites in D1/D2 heterodimer has been proposed. On searching for tetranuclear and binuclear Mn binding sites in the PDB, a suitable sequence homology in thermolysin and D1 could be observed. From the homology and site-directed mutagenesis data, a model for binuclear Mn-Ca or Mn-Mn has been built and it is extended to a tetranuclear Mn centre.     

Specificity of Mg2+ binding at the Group II intron branch site

Metal ions play a crucial role in the conformation and splicing activity of Group II introns. Results from 2-aminopurine fluorescence and solution NMR studies suggest that metal ion binding within the branch site region of native D6 of the Group II intron is specific for alkaline earth metal ions and involves inner sphere coordination. Although Mg(2+) and Ca(2+) still bind to a mutant stem loop sequence from which the internal loop had been deleted, ion binding to the mutant RNA results in decreased, rather than increased, exposure of the branch site residue to solvent. These data further support the role of the internal loop in defining branch site conformation of the Group II intron. The specific bound Mg(2+) may play a bivalent role: facilitates the extrahelical conformation of the branch site and has the potential to act as a Lewis acid during splicing.     

The high-affinity binding site for manganese on the oxidizing side of Photosystem II

Electron donation to Photosystem II (PS II) by diphenylcarbazide (DPC) is interrupted by the presence of endogenous Mn in PS II particles. Removal of this Mn by Tris treatment greatly stimulates the electron transport with DPC as donor. Binding of low concentration of exogenous Mn(II) to Tris-treated PS II particles inhibits DPC photooxidation competitively with DPC. This phenomenon was used to locate a highly specific Mn(II) binding site on the oxidizing side of Photosystem II with dissociation constant about 0.15 μM. The binding of Mn(II) is electrostatic in nature. Its affinity depends not only on the ionic strength, but also on the anion species of the salt in the medium. The effectiveness in decreasing the affinity follows the order F⁻ > SO²⁻₄ > CH₃COO⁻ > CI⁻ > Br⁻ > NO₃⁻. This observation is interpreted as follows: smaller ions, like F⁻, CH₃COO⁻, and larger ions, like SO²⁻₄, have inhibitory effects on Mn(II) binding, whereas ions with optimal size, like Cl⁻, Br⁻ and NO₃⁻, can stabilize the binding, resembling the anion requirement for reactivation of Cl⁻-depleted chloroplasts. We suggest that the binding site for Mn(II) we observed is the site for the endogenous Mn in the O₂-evolving complex of PS II. This site remains after Tris treatment, which removes all the endogenous Mn as well as the three extrinsic proteins, indicating that it is on the intrinsic component(s) of PS II reaction centers. Furthermore, the Cl⁻ requirement for O₂ evolution may be attributed, at least partly to its stabilizing effect on Mn binding.     

Reevaluation of ANS binding to human and bovine serum albumins: key role of equilibrium microdialysis in ligand - receptor binding characterization

In this work we return to the problem of the determination of ligand-receptor binding stoichiometry and binding constants. In many cases the ligand is a fluorescent dye which has low fluorescence quantum yield in free state but forms highly fluorescent complex with target receptor. That is why many researchers use dye fluorescence for determination of its binding parameters with receptor, but they leave out of account that fluorescence intensity is proportional to the part of the light absorbed by the solution rather than to the concentration of bound dye. We showed how ligand-receptor binding parameters can be determined by spectrophotometry of the solutions prepared by equilibrium microdialysis. We determined the binding parameters of ANS - human serum albumin (HSA) and ANS - bovine serum albumin (BSA) interaction, absorption spectra, concentration and molar extinction coefficient, as well as fluorescence quantum yield of the bound dye. It was found that HSA and BSA have two binding modes with significantly different affinity to ANS. Correct determination of the binding parameters of ligand-receptor interaction is important for fundamental investigations and practical aspects of molecule medicine and pharmaceutics. The data obtained for albumins are important in connection with their role as drugs transporters.     

Studies on the role of the S4 substrate binding site of HIV proteinases

Kinetic analysis of the hydrolysis of the peptide H-Val-Ser-Gln-Asn-Tyr*Pro-Ile-Val-Gln-NH2 and its analogs obtained by varying the length and introducing substitutions at the P4 site was carried out with both HIV-1 and HIV-2 proteinases. Deletion of the terminal Val and Gln had only moderate effect on the substrate hydrolysis, while the deletion of the P4. Ser as well as P'3 Val greatly reduced the substrate hydrolysis. This is predicted to be due to the loss of interactions between main chains of the enzyme and the substrate. Substitution of the P4 Ser by amino acids having high frequency of occurrence in beta turns resulted in good substrates, while large amino acids were unfavorable in this position. The two proteinases acted similarly, except for substrates having Thr, Val and Leu substitutions, which were better accommodated in the HIV-2 substrate binding pocket.