Inhibition of (Na(+)/K(+))-ATPase by Cibacron Blue 3G-A and its analogues.

A specific feature of anthraquinone dyes (AD) is to mimic the adenine nucleotides ATP, ADP, NAD and NADH, enabling them to act as ligands in interaction with nucleotide-binding sites of several enzymes and receptors. In the present study, the interactions and/or inhibitory effects of eight AD, including Cibacron Blue 3G-A (Reactive Blue 2), Procion Blue MX-R (Reactive Blue 4) and Remazol Brilliant Blue R (Reactive Blue 19) on the activity of (Na(+)/K(+))-ATPase were investigated. The AD used in this paper could be divided into two groups: i) AD1-AD4 that do not contain the triazine moiety; ii) AD5-AD8 that contain the triazine moiety. Interaction affinity between the respective dye and (Na+/K+)-ATPase was characterized by means of enzyme kinetics. All AD, excluding AD1 and AD2 (which were practically ineffective) exerted effective competitive inhibition to the (Na(+)/K(+))-ATPase activity. Present study is devoted to elucidation of relationship between the inhibitory efficacy of AD against (Na(+)/K(+))-ATPase activity, their acid-basic properties and their three dimensional structure. From the results obtained, the following conclusions could be driven: 1. Similarities in the mutual position of positively and negatively charged parts of ATP and AD are responsible for their interaction with ATP-binding site of (Na(+)/K(+))-ATPase. This may be documented by fact that mutual position of 1-aminogroup of anthraquinone and -SO3(-) group of benzenesulphonate part of respective AD plays crucial role for inhibition of this enzyme. Distances of these two groups on all effective AD were found to be similar as the distance of the 6-aminogroup of adenine and the second phosphate group on ATP molecule. This similarity could be responsible for biomimetic recognition of AD in ATP-binding loci of (Na(+)/K(+))-ATPase. 2. The affinity of AD to ATP binding site of (Na(+)/K(+))-ATPase increases with increasing values of molar refractivity, i. e., with increasing molecular volume and polarizability.

Prolongation of pentoxifylline aliphatic side chain positively affects the reversal of P-glycoprotein-mediated multidrug resistance in L1210/VCR line cells.

We reported previously that derivatives of pentoxifylline (PTX) reverse multidrug resistance (MDR) in P-glycoprotein (P-gp) positive L1210/VCR cells. Based on the results of a recent study using 25 N-alkylated methylxanthines with carbohydrate side-chains of various lengths, we formulated the following design criteria for a methylxanthine molecule to effectively reverse P-gp mediated MDR: i) a massive substituent at the N1 position is crucial for MDR reversal potency; ii) elongation of the substituents at the N3 and N7 positions (from methyl to propyl) increases the efficacy of a xanthine to reverse MDR; iii) elongation of the substituent at the C8 position (from H to propyl) decreases the efficacy of a xanthine to reverse MDR. Based on these criteria, we synthesized and tested for potency to reverse MDR a new PTX derivative, 1-(10-undecylenyl)-3-heptyl-7-methyl xanthine (PTX-UHM), with prolonged substituents at the N1 and N3 positions. The derivative was obtained by alkylation of 3-heptyl-7-methyl xanthine with 1-methylsulfonyloxy-10-undecylenyl. NMR and IR structural analyses proved the identity of the product. Cytotoxicity study showed that PTX-UHM is only slightly more toxic to L1210/VCR cells than PTX. We found that both PTX-UHM and PTX were able to reverse vincristine resistance of L1210/VCR cells, yet PTX-UHM was significantly more efficient in the reversal than PTX.

Carbonyl group of aliphatic side chain of pentoxifylline does not play role for P-glycoprotein antagonizing effect of pentoxifylline.

Previously we have found that pentoxifylline (PTX), but not caffeine, theophylline, or 1-methyl-3-isobutylxanthine, affects sensitivity of L1210/VCR cells, a line with multidrug resistance mediated by P-glycoprotein (P-gp) to vincristine (VCR) and doxorubicine. Comparison of chemical structure of PTX with other above xanthines has revealed only one marked difference. PTX contains extended aliphatic chain containing reactive electrophilic carbonyl group in the position N1. The investigation of possibility that this group is crucial for PTX-induced MDR reversal represents the aim of the current paper. To prove this hypothesis, we used the new synthesized PTX derivative in which the carbonyl group is modified by a substance containing amino-group and the product of reaction is the respective Schiff base (SB). Successful reaction was observed when PTX reacted with 3,5-diaminobenzenesulfonyl acid (DABS). The product of reaction of DABS with carbonyl group of aliphatic part of PTX was proved using NMR and IR spectroscopy. We found that the resulting PTX derivative PTX-SB revealed higher cytotoxicity on both sensitive L1210 and multidrug resistant L1210/VCR cells than PTX. Moreover, PTX-SB exerts more pronounced MDR reversal effect on L1210/VCR cells than PTX. These results indicate that electrophilic carbonyl group on aliphatic chain located in position N1 of PTX is not essential for MDR reversal effects of PTX.

Cytotoxic activity of several unrelated drugs on L1210 mouse leukemic cell sublines with P-glycoprotein (PGP) mediated multidrug resistance (MDR) phenotype. A QSAR study.

L1210/VCR-1 and L1210/VCR-2 cell lines are multidrug resistant (MDR) sublines obtained by adaptation of mouse leukemic cell line L1210 to vincristine and, the development of MDR in these cell lines has been found to be associated with an overexpression of P-glycoprotein (PGP). In the present work we studied the relationship between the structure of 15 cytotoxic active substances (drugs) and their cytotoxicities on L1210/VCR-1 and L1210/VCR-2 resistant cell lines. The resistance of these MDR cells to the respective drugs was expressed as the ratio of IC50 values obtained for resistant and sensitive cells. These values of resistance were correlated with the following physico-chemical constants of the test substances: binding energy, Ebind; total energy of the molecule, Esum; aromaticity, Kpi; molecular weight, Mw; acidobasic constant, pKa; partition coefficient in water/octanol two phase system, log(p). It has been found that according to the cytotoxic effects the tested drugs may be divided into three groups: (i) drugs with higher cytotoxicity to the resistant cell lines as to sensitive cells (collateral hypersensitivity); (ii) drugs exhibiting approximately similar effects on sensitive and resistant cell lines; (iii) drugs with weaker cytotoxicity to resistant cells than to sensitive cells. No direct correlations with any physico-chemical constant described above could be established for cell resistance to the drug studied. However, resistance values could be fitted by multiple exponential regression with all described physico-chemical constants implied as six independent variables. The latter procedure made us to conclude that the ability of a drug to be a substrate for PGP is connected with its fulfilling the following criteria: (i) flexible structure of its molecule; (ii) molecular weight lower than approximately 1,300 g/mol; (iii) nonprotonized character at pH 7.0.

Examination of bioaffinity immobilization by precipitation of mannan and mannan-containing enzymes with legume lectins.

The interaction of four lectins from crops of the legume family with Saccharomyces cerevisiae alpha-mannan, and also with two glycoenzymes containing mainly alpha-mannan moieties, has been studied. The interaction was characterized by a quantitative precipitation assay. The results of precipitation differ with respect to both quality (the point of maximum precipitation) and of the quantity (the amount of aggregated lectin and saccharide). The lectin concanavalin A [Con A, from jack bean (Canavalia ensiformis)] was observed to form more extensive precipitates with Saccharomyces cerevisiae mannan and glycoenzymes than did lectins from Lens culinaris (lentil) and Pisum sativum (garden pea), while in the case of Vicia faba (broad or fava bean) no interaction was found with either the examined mannans or with glycosylated enzymes. The complete precipitation of invertase and glucoamylase with Con A (enzymes and also Con A; up to 100%) was achieved at a Con A glycoenzyme molar ratio of 20.2 and 2.3 respectively, whereby about 85% of precipitated and also of initial activities of glycoenzymes were determined in the aggregates. More valuable results were achieved by the technique of enzyme immobilization called 'multiple bioaffinity layering' which is based on the stepwise biospecific adsorption of the glycosylated enzymes and Con A on a matrix precoupled with Con A. A 3-fold repetition of the layering procedure afforded up to a 10-fold increase in catalytic activity of the immobilized invertase, in contrast with a 2.1-fold increase in catalytic activity of the immobilized glucoamylase.

Prevention of processes coupled with free radical formation prevents also the development of calcium-resistance in the diabetic heart.

Recently it was shown that besides their negative role in pathogenesis of diabetes, reactive oxygen species (ROS) and particularly the products of non-enzymatic glycation of proteins (NEGP) may also participate in some processes of adaptation of the myocardium to diabetes, such as in the mechanism of development of calcium resistance of the heart. Our study revealed that the hearts of rats with experimentally induced diabetes (single dose of streptozotocin, 45 mg/kg i.v., 6 U/kg insulin daily) develop considerable resistance against calcium overload (induced by means of Ca-paradox). On the day 63 after the beginning of experiment, when the diabetic cardiomyopathy became fully developed but the hearts were still not failing, their calcium resistance was increased to 83.33%. Our results provide evidence that, when applied in a special regimen, resorcylidene aminoguanidine (RAG, 4 mg/kg) prevented both, the formation of fructosamine (a source of ROS generation), and also that of the advanced Maillard products, in the heart sarcolemma of diabetic rats. The effect of RAG was accompanied by a decrease in calcium resistance in the group of rats with chronic diabetes (63 days) from 83.3 to 46.7%. It is concluded that NEGP and ROS formation are inevitably needed for development of calcium resistance in the diabetic hearts.

Interaction of lactate dehydrogenase with anthraquinone dyes: characterization of ligands for dye-ligand chromatography.

Anthraquinone dyes (ADs), originally developed for the textile industry, are useful nucleotide-specific ligands for the purification of proteins by affinity techniques. Their specific feature is to mimic the adenine nucleotides ATP, ADP, NAD, NADH, which enables them to interact with the nucleotide-binding sites of enzymes such as dehydrogenases, kinases and ATPases. In the present study, the interactions and/or inhibitory effects of seven ADs, including Cibacron Blue F3G-A, Remazol Brilliant Blue R, on the activity of lactate dehydrogenase (LDH) were investigated. The ADs used in this paper could be divided into two groups: (i) AD1-AD3 which do not contain a triazine moiety; (ii) AD4-AD7 which contain the triazine moiety. Enzyme kinetics and zonal affinity chromatography were used for the characterization of the interaction affinity between the dye and LDH. Enzyme kinetic measurements were carried out at three different pH values: 6.5, 7.5 and 8.5. The relationship between physical and chemical properties of ADs (e.g., acid-basic properties, three dimensional structure of the respective dyes) and their interaction efficiency with LDH was studied. LDH activity was inhibited by all ADs, excluding AD1 (precursor of the blue dyes) and inhibition was always competitive. Similarity in the mutual position of the acidic and basic groups in NADH and the respective AD molecule was found to be a crucial factor for influencing the inhibitory action of the substance. The existence of ADs in the protonated form should be considered as another factor, important for the ADs inhibitory action on this enzyme.

Amplification of flow-microcalorimetry signal by means of multiple bioaffinity layering of lectin and glycoenzyme.

This paper demonstrates a positive influence of a special, stepwise technique of enzyme immobilization based on the biospecific adsorption of the glycoenzyme invertase on immobilized concanavalin A (Con A), subsequent adsorption of the free Con A on the immobilized invertase:Con A support and repeated adsorption of invertase on the support. A 3-fold repetition of the same procedure designed preliminarily as bioaffinity layering afforded up to a 10-fold increase in catalytic activity of the immobilized invertase. Reactive hydrogels based on bead cellulose and bead poly(glycidyl methacrylate) were used as immobilization supports for the preparation of these highly active preparations. The enhancement in catalytic activity of immobilized invertase preparations was demonstrated thermometrically, by flow microcalorimetry. Further attractive aspects for utilizing the signal amplification of biosensors with immobilized enzymes are discussed.

Competitive inhibition of (Na/K)-ATPase by furylethylenes with respect to potassium ions.

The effects of newly synthetized derivatives of furylethylene: i) 1-(5-nitro-2-furyl)-2-phenylsulfonyl-2-furylcarbonyl ethylene (FE1), ii) 1-(5-phenylsulfonyl-2-furyl)-2-phenylsulfonyl-2-furylcarb onyl ethylene (FE2), iii) 1-(5-phenylsulfonyl-2-furyl)-2-phenylsulfonyl-2-tienocarb onyl ethylene (FE3), on the reaction kinetics of the dog kidney (Na/K)-ATPase were tested. Besides the conjugated triene moiety of the furylethylene skeleton, the groups responsible for the reaction with nucleophilic groups, the formyl group that connects the second furyl ring in FE1 and FE2 and the formyl group that connects the thienyl ring to the furylethylene moiety in FE3. Among the furylethylenes tested, only FE1 was found to react effectively with beta-mercaptoethanol (beta ME) and glycine (GLY) as model substances containing nucleophilic groups, and also exhibit an inhibitory interaction with the (Na/K)-ATPase. A suppression of the reactivity of the formyl group due to the replacement of the furyl ring with the more aromatic thienyl ring in FE3 did not induce any significant change in the reactivity of the compound with the model substances or with (Na/K)-ATPase. On the other hand, replacement of the NO2 group on the furylethylene moiety (in FE1) by the less electron-attracting phenylsulfonyl group (in FE2 and FE3) yielded a considerable suppression of the inhibitory effect on (Na/K)-ATPase. Moreover, in comparison to FE1, FE2 and FE3 were found to react less potently with the model nucleophilic substances. The results indicated that the conjugated triene moiety on the furylethylene part of the molecule of FE1 may be made responsible for the inhibitory interaction with the nucleophilic aminoacid residue on the (Na/K)-ATPase molecule. FE1 interfered competitively with the (Na/K)-ATPase activation by increasing amounts of potassium. This was manifested by a significant increase in the apparent K0.5App value and a decrease in the apparent cooperativity constant, nApp, for potassium ions, but had no influence on the apparent VmaxApp value for potassium. With respect to the activation of the enzyme with sodium ions and ATP, only FE1 decreased the VmaxApp values while having no considerable influence on the other kinetic variables. It was concluded that FE1 inhibits the (Na/K)-ATPase by selective interaction with some essential nucleophilic (probably SH and/or NH2) aminoacid residues located in, or closed to the potassium binding site of the enzyme molecule.

New approaches for verification of kinetic parameters of immobilized concanavalin A: invertase preparations investigated by flow microcalorimetry.

In our preceding article, we demonstrated a procedure based upon enzymic flow microcalorimetry using an enzyme thermistor (ET) to characterize the microkinetic properties of an immobilized enzyme (IME) and its further application in the screening of IMEs. To consider the ET method (single ET unit, ET system 1) as standard, it was necessary to show that the estimated relative kinetic parameter (DeltaT(max)) calorimetrically corresponds with the absolute value for the reaction rate within the whole measurement range. This article presents three experimental verification procedures. Two procedures are based on adaptation of the flow-through ET column to a mini-differential-reactor (DR) system with substrate recirculation and post-ET-column methods for determination of the concentration change of the product (spectrophotometrically in ET system 2) or the substrate (calorimetrically in ET system 3) with the IME-catalyzed enzymic hydrolysis. The third procedure is an independently operating DR system which spectrophotometrically estimates the concentration change of the product. The results obtained exhibited good correlation (r = 0.921) between the relative kinetic parameter DeltaT(max), as determined calorimetrically by ET system 1, and the absolute value for the reaction rate (r(max)) as determined by ET systems 2 and 3. These data proved that, within the whole range of experimental conditions applied in this study, the parameter DeltaT(max) instead of the true reaction rate could be employed for the IME screening. Moreover, the generality of the detection principle and the standardized configuration of the ET favor ET systems 2 and 3 for normal screening of IMEs and as miniaturized DR systems allowing dual measurements of kinetic parameters.

Inhibition of (Na/K)-ATPase by electrophilic substances: functional implications.

The effect of electrophilic substances: p-bromophenylisothiocyanate (PBITC); fluoresceinisothiocyanate (FITC); [4-isothiocyanatophenyl-(6-thioureidohexyl)-carbamoylmethyl] -ATP (ATPITC); 2,4,6-trinitrobezenesulfonic acid (TNBS); 1-(5-nitro-2-furyl)-2-phenylsulfonyl-2-furylcarbonyl ethylene (FE1); 1-(5-phenylsulfonyl-2-furyl)-2-phenylsulfonyl-2-furylcarb onyl ethylene (FE2) and 1-(5-phenylsulfonyl-2-furyl)-2-phenylsulfonyl-2-tienocarb onyl ethylene (FE3) on the sarcolemmal (Na/K)-ATPase isolated from guinea-pig hearts was studied. FITC and PBITC were found to inhibit competitively the activation of (Na/K)-ATPase by ATP. Being for the enzyme inhibitor and substrate at the same time ATPITC does not offered clear kinetic behavior. However, the activation of (Na/K)-ATPase by sodium and potassium ions was inhibited non-competitively by all three isothiocyanates. These data indicated that isothiocyanates may interact predominantly in the ATP-binding site of the enzyme molecule. In contrary to isothiocyanates TNBS and FE1 (FE2 and FE3 were ineffective) inhibited the activation of (Na/K)-ATPase by ATP non-competitively i.e., their interaction in the ATP-binding site seemed to be improbable. Nevertheless, TNBS and FE1 both manifested affinities to that moiety of (Na/K)-ATPase molecule which is binding potassium. More specific was the effect of FE1 that showed clearly competitive inhibition of potassium-stimulation of the enzyme activity. FE1 exerted also an ouabain-like effect on the mechanical activity of isolated perfused guinea-pig heart. This result indicates that FE1 seems to exert a selective inhibition of the (Na/K)-ATPase not only in vitro but also in integrated cardiac tissue.

Affinity chromatography of invertase on concanavalin A-bead cellulose matrix: the case of an extraordinary strong binding glycoenzyme.

This work presents confirmation of the biospecific character of the interaction of Concanavalin A (Con A) immobilized on bead cellulose with invertase. In spite of the extraordinary strong binding of invertase to this Con A conjugate (KD = 5 x 10(-9) M), conditions have been found to use Con A-bead cellulose as an affinity chromatography medium. The effective factor in the release of the bound invertase by the counter ligand (alpha-methyl-D-mannopyranoside) is the time of incubation. This phenomenon was demonstrated in both batch and flow-through experiments. A concentration of 1.5 mg Con A per ml of gel was found to be suitable with regard to the maximal invertase/Con A binding ratio and the optimal invertase recovery (94%). As a result of the strong biospecific interaction the purification of invertase was very effective (above ten-fold). Verification by FPLC and PAGE of the product purity revealed only one significant protein band.

Inhibition of (Na/K)-ATPase by NFE induces an increase in mechanical activity of perfused guinea-pig heart.

The effect of 1-(5-nitro-2-furyl)-2-(phenylsulfonyl)-2-(furylcarbonyl)- ethylene (NFE) on stimulation of (Na/K)-ATPase by sodium and potassium ions was tested in isolated, partially purified sarcolemmal preparation from guinea-pig hearts. NFE inhibited competitively the stimulation of the enzyme by increasing concentrations of potassium. This inhibition was characterized by a significant (p < 0.001) increase of the K0.5 value, a considerable decrease of the Hill's cooperativity constant n as well as by an insignificant diminution of the Vmax value. Contrary to the effect on stimulation by potassium, NFE inhibited non-competitively the stimulation of the ATPase by sodium ions with a significant (p < 0.001) depression of Vmax but without any considerable effect on the K0.5 and n values. These results indicated that NFE may interact with the molecule of (Na/K)-ATPase in a locus close to or identical with the potassium binding site of the enzyme, i.e., in a similar mode as it was well documented for ouabain. This possibility was strongly supported by the finding that NFE administered at the concentration of 0.1 mumol/l in the perfusion medium increased significantly (p < 0.01) the mechanical activity of isolated perfused guinea-pig heart (Langendorff preparation). Nevertheless, it also caused some adverse effects such as a slight increase in coronary flow resistance and in heart rate as well as in the left ventricular end-diastolic pressure.(ABSTRACT TRUNCATED AT 250 WORDS)

Screening of concanavalin A-bead cellulose conjugates using an enzyme thermistor with immobilized invertase as the reporter catalyst.

Screening and design of immobilized biocatalysts (IMBs) is a time-consuming process. An ideal process should be universal, fast, convenient, precise, and reproducible. Many of these requirements are met by enzymic flow microcalorimeters, also known as enzyme thermistors (ETs) or thermal assay probes (TAPs). Adaptation of ETs to real measurements of reaction rates requires coupling of the mathematical description of the reaction-diffusion phenomena in the ET column with heat balance and, subsequently, experimental verification of the mathematical model. This article presents such a process developed as an adaptation of ETs for the characterization of the microkinetic properties of IMBs and their further application for screening of IMBs. The IMBs characterized were the preparations of invertase, biospecificaly adsorbed on concanavalin A conjugated to activated bead cellulose. (c) 1994 John Wiley & Sons, Inc.

Overcoming of vincristine resistance in L1210/VCR cells by several corticosteroids. Collateral sensitivity of resistant cells.

Five corticosteroids were tested to determine whether they are able to overcome the multidrug resistance of vincristine resistant mouse leukemia cells L1210/VCR. The most effective in reversing multidrug resistance were cortisone and dexamethasone, less effective as reversing agents were 11-deoxycorticosterone, 1-dehydrocortisone and hydrocortisone. By testing of collateral sensitivity of vincristine resistant cell line to these corticosteroids it was found that only 11-deoxycorticosterone and dexamethasone were toxic to multidrug resistant cells at doses much lower than required for toxicity to the drug-sensitive L1210/S cells. Using thin layer chromatography the polarity of tested corticosteroids was estimated, and good correlation was found between polarity of corticosteroids and their increased toxicity to vincristine resistant cells.