[Interaction of the Laburnum anagyroides lectin with fucoantigens].

We studied interaction of the lectin from the bark of Golden Rain shrub (Laburnum anagyroides, LABA) with a number of basic fucose-containing carbohydrate antigens by changes in its tryptophan fluorescence. The strongest LABA binding was observed for the trisaccharide H of type 6 [alpha-L-Fucp-(1-2)-beta-D-Galp-(1-4)-D-Glc, Ka= 4.2 x 10(3) M(-1)]. The following antigens were bound with a weaker affinity: H-disaccharide alpha-L-Fucp-(1-2)-D-Gal, a glucoanalogue of tetrasaccharide Ley alpha-L-Fucp-(1-2)-beta-D-Galp-(1-4)-[alpha-L-Fucp-(1-3)]-D-Glc, and 6-fucosyl-N-acetylglucosamine, a fragment of core of the N-glycans family (Ka 1.1-1.7 x 10(3) M(-1)). The lowest binding was observed for L-fucose (Ka = 2.7 x 10(2) M-1) and trisaccharide Lea, (3-Galp-(1-3)-[a-L-Fucp-(1-4)]-GlcNAc (Ka = 6.4 x 10(2) M(-1)). The Lea, Lea, and Lex pentasaccharides and Leb hexasaccharide were not bound to LABA.

[Interaction of perch fucolectin with Lewis antigens]. / Vzaimodeistvie fukolektina okunia s l'iuisovymi antigenami.

Interaction of fucolectin of perch Perca fluviatilis (PFL) with a set of Lewis antigens was studied by monitoring changes in its tryptophan fluorescence. PFL bound Le(c) (H type 1)-pentasaccharide (Ka = 6.6 x 10(3) M(-1)) and H type 6-trisaccharide (Ka = 2.5 x 10(3) M(-1)); essentially weaker, with Le(b)-hexasaccharide (Ka = = 4.0 x 10(2) M(-1)); and failed to interact with Le(a)-, Le(x)-, and Le(d)-containing oligosaccharides. PFL belongs to a new type of the fucolectins recognizing H-disaccharide Fuc alpha1-2Gal within various antigens, including H type 1/2 and Le(b).

Interaction of smooth muscle caldesmon with calmodulin mutants.

The interaction of avian smooth muscle caldesmon with calmodulin (CaM) was investigated by studying the ability of selected mutant calmodulins to induce fluorescence changes in caldesmon. Different types of CaM mutants were used including point charge mutants, cluster mutations, and mutations which alter the calcium binding of CaM. The caldesmon binding properties were only slightly affected by E84K-CaM or by the double mutation E84Q/E120Q-CaM. Affinity of calmodulin to caldesmon was decreased 2-4 times by point mutation G33V-CaM, double mutation E84K/E120K-CaM, deletion of residues 82-84, and by cluster mutations DEE118-120-->KKK or EEE82-84-->KKK. Mutations of the first (E31A-CaM) and the second (E67A-CaM) calcium binding sites reduced the affinity of calmodulin to caldesmon by at least 5-fold; in addition these calmodulin mutants exhibited smaller changes in the fluorescence spectra of caldesmon. Simultaneous mutation of the two negatively charged clusters of calmodulin EEE82-84-->KKK and DEE118-120-->KKK resulted in a more than 15-fold decrease in the affinity of calmodulin for caldesmon. The data indicate that charged and uncharged amino acids in both halves of CaM play an important role in the binding of calmodulin to caldesmon, and that Ca2+ binding must be maintained in the amino-terminal sites for maximal interaction with caldesmon.

[Structure-functional properties of conjugates of proteins with polyalkylene oxides: study by a fluorescence method]. / Strukturno-funktsional'nye svoistva kon''iugatov belkov s polialkilenoksidami: issledovanie metodom fluorestsentsii.

A fluorescent study of some structural and functional properties of conjugates of a number of proteins (bovine serum albumin, pyruvate kinase, alpha-chymotrypsin, and the two toxic proteins of plant origin--ricin and viscumin) with polyalkylene oxides (polyethylene glycol and pluronic) has been carried out. Analysis of the intrinsic protein fluorescence showed that the structure and stability of various protein conjugates to denaturing agents change only slightly: the conformational mobility of tryptophan residues accessible to the solvent decreases, whereas that of tryptophan residues localized in the protein regions of low polarity remains unchanged. Besides, the conjugates display a higher thermal stability in comparison with their native proteins. The fluorescence of 1-anilinonaphthalene-8-sulfonic acid and water insoluble 2',3',4',5'-tetrabenzoylriboflavin bound to the native and modified proteins indicated that modification of the proteins with polyalkylene oxides decreased the polarity and increased the viscosity of the microenvironment. Hence, this modification makes it possible to change some functional characteristics of the protein without causing any significant changes in its structure.

[Interaction of the toxic plant protein--ricin, with model membranes. A fluorescence method of study]. / Vzaimodeistvie rastitel'nogo toksichnogo belka--ritsina s model'nymi membranami. Issledovanie metodom fluorestsentsii.

The fluorescence method has been used to investigate ricin and its isolated subunits interaction with some model membranes. Three liposome types were used as a model of biological membrane: 1) liposomes constructed from lecithin and cholesterol (9:1, M:M) 2) from ganglioside receptors GM1 and 3) from the mixture of GM1, lecithin and cholesterol (1:9:1). Interaction of the protein with liposome evokes changes in the parameters of both intrinsic protein fluorescence and fluorescence of the covalently bound dansyl. Binding constants were calculated from a decrease of the intrinsic fluorescence intensity as well as from the changes in the dansyl rotation anisotropy. Measurements were carried out at neutral and acidic pH. There was good correlation of the results obtained by different methods. It was shown that association constants were different for intact ricin and its subunits. The constants also depend on liposome composition and pH of the solution. The present study has demonstrated that interaction of ricin with liposome is accounted for not only by receptor centers but also by other hydrophobic regions of ricin that are inaccessible in the native toxin and may represent the region of the subunits interaction.

[Elongation factor EF-Ts interacts with the aminoacyl-tRNA.EF-Tu.GTP complex]. / Faktor élongatsii EF-Ts vzaimodeistvuet s kompleksom aminoatsil-tRNK.EF-Tu.GTP.

The fluorescence polarization technique has been used to study the interaction of the EF-Ts dansyl derivative with EF-Tu after nucleotide exchange and binding of the aminoacyl-tRNA to EF-Tu.GTP. It is shown that the ternary complex formation results in the increase of EF-Ts affinity to EF-Tu and EF-Ts remains bound to EF-Tu up to the GTP hydrolysis stage on the ribosome.

[Features of the structure of catalytic subunits of toxins, inhibiting protein synthesis. I. The effect of pH and interaction with the B-chain of ricin]. / Osobennosti struktury kataliticheskikh sub''edinits toksinov, ingibiruiushchikh belkovyi sintez. I. Vliianie pH, vzaimodeistvie s B-tsep'iu ritsina.

A comparative study of gelonin and A-chains of ricin, mistletoe lectin I and diphtheria toxin was undertaken. The effect of pH was studied on: a) the conformation of the proteins under study using intrinsic fluorescence; b) interaction of these proteins with ricin B-chain using gel-filtration. Structural stability of the proteins was assessed according to denaturing action of guanidine hydrochloride and temperature, and localization of tryptophan residues was determined using fluorescence quenching by I-, Cs+ and acrylamide. All investigated proteins were shown to undergo the conformational changes when a environment became acidic. In comparison with an intact protein--gelonin, the A-chains of ricin, a mistletoe lectin and a diphtheria toxin are less stable. At pH less than 5.0 tryptophan residues became more accessible to quencher and a positive charge of the surrounding area increases (in the case of gelonin it is negatively charged). No reliable interaction of a ricin B-chain with both gelonin and A-chain of diphtheria toxin was observed. The interaction of a ricin B-chain with a A-chain of mistletoe lectin I is weaker than that with ricin A-chain and is practically pH-independent.

Immunotoxin with mistletoe lectin I A-chain and ricin A-chain directed against CD5 antigen of human T-lymphocytes; comparison of efficiency and specificity.

Monoclonal anti-CD5 antibody was coupled to the enzymatically active subunit of plant toxin [either mistletoe lectin I (ML) or ricin]. The obtained conjugates proved to be selectively toxic to CD5-bearing target cells. The immunotoxin prepared from ML A-chain (MLA) was as toxic as native ML and approximately 80-fold more active than the corresponding conjugate with ricin A-chain (RTA). The comparative studies of the structural properties of isolated MLA and RTA were carried out using intrinsic fluorescence spectroscopy. The results showed similar properties for both proteins. No antigenic cross-reactivity against both toxins was detected when using polyclonal antibodies. The results suggest that MLA-antibody conjugates may be potential candidates for therapeutical use.

[Recombination of micellar complexes of apolipoprotein A1--phosphatidylcholine in the presence of complex components]. / Rekombinatsiia mitselliarnykh kompleksov apolipoprotein A1--fosfatidilkholin v prisutstvii komponentov kompleksa.

Interaction of micellar complexes apolipoprotein A1--phosphatidyl choline (apoA1--DMPC and apoA1--EPC) with complex components: apoA1 (dansyl-A1) and phosphatydil cholines (DMPC, EPC and spin labelled PC) was studied in the absence of lipoproteins and plasma components. Recombination of the complexes (changes in complex sizes and stoichiometry) was shown to occur in the presence of the complex components. Interaction of lipid-free apoA1 with the complex is a fast process; incorporation of PC molecule takes place more slowly. This recombination is considered to be a kinetikally complicated process, the rate of recombination depending on PC exchange and interconversion.

[Ricin structure: the study by the fluorescence quenching method]. / Struktura ritsina: issledovanie metodom tusheniia fluorestsentsii.

To elucidate the details of pH-induced conformational transformation of ricin [I] in the region surrounding tryptophan residues, we studied parameters of fluorescence of the native toxin and its isolated A- and B-subunits at pH 4.0, 5.0 and 7.4. The studies were carried out using resolution of fluorescence spectra according to different degree of tryptophan accessibility to ionic (iodide) and non-ionic organic (acrylamide) quenchers. Application of the new method allowed to reveal three classes of tryptophan residues differing in their accessibility to quenchers alpha-residues are accessible neither to ions nor to organic molecules; beta-residues are accessible only to organic molecules; while surface gamma-residues are accessible to both types of quenchers. The fluorescence spectra were assessed for each class of tryptophan residues. The major part of them was shown to be localized in apolar rigid microenvironment. Fluorescence of ricin and especially of its isolated subunits proved to be strongly dependent on the pH value. At pH less than 5 the structure of B-chain loosens, this process being reflected by an increase in accessibility of tryptophan residues to quenchers. In acidic solution at least one out of seven tryptophan residues in the ricin molecule undergoes conformational transformation. Positive charge prevails in the regions surrounding quencher-accessible tryptophan residues. Binding of lactose leads to a slight compactization of the toxin structure that causes, in its turn, short-wave shifts of the fluorescence spectra and reduction of Stern-Volmer constants for intraglobular tryptophan residues.

[Relative location of anthroyl derivatives of stearic acid in low density lipoproteins]. / Otnositel'noe raspolozhenie antroil'nykh proizvodnykh stearinovoi kisloty v lipoproteidakh nizkoi plotnosti.

Effective quenching constants (K'sv) of 2-, 7- and 12-(9-anthroyloxy)stearic acid (n-AS) fluorescence in LDL were determined. Spin probes I(m, n) (n = 3, 7, 10, 14) and I- anions were used as quenchers. Quenching of 2-AS and 12-AS fluorescence by I(m, n) was the more effective, the deeper spin probe nitroxyl fragment was located (the greater n was); maximal K'sv value corresponded to I(1,14). By contrast, for 7-AS the quenching by I(12,3) was the most effective. 2-AS and 12-AS spectra maxima and fluorescence polarization were similar. We concluded that the 2-AS chromophore was located deeper in LDL phospholipid monolayer than chromophore of 7-AS (as was the case for 12-AS).

[Physico-chemical properties of low density lipoproteins isolated in an equilibrium density gradient]. / Fiziko-khimicheskie svoistva podfraktsii lipoproteidov nizkoi plotnosti, poluchennykh v ravnovesnom gradiente plotnosti.

The tryptophanyls of total low density lipoproteins (LDL) (1.006-1.063 g/ml) from coronary heart disease (CHD) patients and subjects without CHD signs had different accessibility to fluorescence quenchers (I-and acrylamide). LDL were separated into subfractions in equilibrium density gradient. The coefficient of extinction , quantum yield and other spectral characteristics of LDL intrinsic fluorescence, rotational mobility of maleimide spin labels and fatty acid spin probe were different in LDL subfractions from healthy subjects. LDL subfractions with hydrated density 1.045-1.05 g/ml bound to B,E-receptors of cultured fibroblasts more effectively than did subfractions with density 1.01-1.03 g/ml. Structural differences of apo-B in the particles with different lipid to protein ratio are supposed.

[Formation of micellar complexes of apolipoprotein A-I with dimyristoylphosphatidylcholine]. / Obrazovanie mitselliarnykh kompleksov apolipoproteina A-I s dimiristoilfosfatidilkholinom.

Structural changes of apolipoprotein AI from human plasma high density lipoproteins in 2-chloroethanol solutions were studied using spin label and fluorescence techniques. Reversible changes in spectral parameters occur in 2-chloroethanol concentration range 0-40% and are affected by dimyristoylphosphatidylcholine, of 2-chloroethanol concentration does not exceed 30%. Dialysis experiments demonstrated the absence of binding of monomer phosphatidylcholine with apolipoprotein AI. It thus follows that formation of complexes of apolipoprotein AI with dimyristoylphosphatidylcholine is caused by lipid micella aggregation.

Caldesmon-calmodulin interaction. Study by the method of protein intrinsic tryptophan fluorescence.

Addition of calmodulin to caldesmon causes a concentration-dependent shortwave shift and an increase of fluorescence intensity of caldesmon tryptophan residues. The existence of a protein complex is confirmed by the increase of the caldesmon sedimentation coefficient s0(20,w) from 3.0 S to 4.5 S in the presence of calmodulin. These findings allow application of the method of protein intrinsic tryptophan fluorescence for quantitative study of unmodified caldesmon and calmodulin in solution. The affinity of the caldesmon-calmodulin interaction (Kass = 1.8 x 10(6) M-1, in 0.1 M-KCl at 25 degrees C) and Ca2+ requirement (half-maximum binding at 0.8 microM-Ca2+) determined by means of the fluorescence technique are in agreement with previously reported values, thus confirming the validity of the method. The same approach was further used to provide information about the nature of interactions stabilizing the caldesmon-calmodulin complex. Association of the proteins and dissociation of the complex were studied in different physicochemical conditions, including variation of pH, temperature and ionic strength and the addition of quenchers, denaturants and anticalmodulin drugs. The results obtained suggest that caldesmon tryptophan residues, together with charged groups, are involved in calmodulin binding. Hydrophobic, electrostatic and hydrogen interactions contribute to the stability of the protein complex, making it insensitive to variations of physicochemical conditions within physiological limits.

[The structure of toxic protein, the mistletoe lectin, at different pH: the study using intrinsic fluorescence method]. / Struktura toksichnogo belka-lektina iz omely-pri razlichnykh pH: issledovanie metodom sobstvennoi fluorestsentsii.

The effects of pH on the conformation of mistletoe lectin I and its isolated A- and B-subunits has been investigated by using the methods of intrinsic fluorescence. By the denaturating action of guanidine hydrochloride and the influence of the quenchers (I-, Cs+, acrylamide) the structural stability of the native protein and its isolated subunits was estimated. Treatment of the protein with the denaturant and quenchers revealed its different structure at pH 7.0 and 4.0. At pH 4.0 tryptophan residues become more accessible to quenchers, positive charge of the surrounding area increases and the protein becomes more stable to the action of denaturant. The structure of the isolated A- and B-chains of mistletoe lectin I differs considerably from that of the whole protein: a) its stability to the action of guanidine hydrochloride is lower; b) it depends on the ionic strength of the solvent; c) it is characterized by increased accessibility of tryptophan residues to quenchers (for B-chain). Differences between the conformations of the isolated chains at pH 7.0 and 4.0 are marked more strongly; moreover, at pH 4.5 the B-chain undergoes structural transition. The possible relationship between structural peculiarities of mistletoe lectin I and the mechanism of its transmembrane transfer is discussed.