Inhibition of human plasma cholinesterase by malachite green and related triarylmethane dyes: mechanistic implications

The inhibitory effects of the cationic triarylmethane (TAM+) dyes, pararosaniline (PR+), malachite green (MG+), and methyl green (MeG+) on human plasma cholinesterase (BChE) were studied at 25 degrees C in 100 mM Mops, pH 8.0, with butyrylthiocholine as substrate. PR+ and MG+ caused linear mixed inhibition of enzyme activity. The respective inhibitory parameters were K(i) = 1.9 +/- 0.23 microM, alpha = 13 +/- 48, beta = 0 and K(i) = 0.28 +/- 0.037 microM, alpha = 23 +/- 7.4, beta = 0. MeG+ acted as a competitive inhibitor with K(i) = 0.12 +/- 0.017 microM (alpha, infinity, beta, not applicable). The K(i) values were within the same range reported for a number of ChE inhibitors including propidium ion, donepezil, and the phenothiazines, suggesting that TAM+s are active site ligands. On the other hand, the alpha values failed to correlate with values previously reported for a number of ChE inhibitors. It appears that mixed inhibition is the combined result of more than one type of binding and S-I interference. The impact of ligands at the choline-specific and peripheral anionic sites (or, possibly, accessory structural domains) on BChE activity needs to be studied in further detail.     

Effect of angiotensin II infusion in rats on Na,K-ATPase activity in renal cortical microsomal preparations

Direct dose-dependent effects of angiotensin II on renal tubular sodium reabsorption have been demonstrated. Alterations in tubular sodium reabsorption may occur via modulation of renal Na,K-ATPase activity. Thus, these experiments were undertaken to ascertain whether angiotensin II could influence renal cortical Na,K-ATPase activity. Angiotensin II, 495 ng/microliters/h, or vehicle (controls) was infused for 24 h via miniosmotic pumps 48 h after rats were adrenalectomized and implanted with osmotic pumps containing 12.5 micrograms/microliters corticosterone (Treatment I) or both corticosterone and 0.2 microgram/microliter aldosterone (Treatment II), and in rats receiving 3% NaCl in their food (sodium loaded, Treatment III). Rats receiving Treatments I and III received saline to drink. Renal cortical microsomal membranes were prepared, and the effects of angiotensin II infusion on the K1/2 and Vmax for Na, K, and ATP determined. Angiotensin II infusions were associated with (i) a decrease (P less than 0.001) in the K1/2 for Na activation of Na,K-ATPase from 14 +/- 3 to 6 +/- 1 (n = 4 experiments), 16 +/- 1 to 12 +/- 1 (n = 5), and 12 +/- 3 to 7 +/- 1 (n = 5) mM (means +/- SE) for treatments I, II, and III, respectively; (ii) no changes in the K1/2 for K activation or the Km for ATP; (iii) no changes in the Vmax for Na, K, or ATP; and (iv) no change in Mg-ATPase activity. We conclude that angiotensin II infusion is associated with a decrease in the K1/2 of renal cortical Na,K-ATPase activity for sodium. This action of angiotensin II on the enzyme activity may contribute to the regulation of tubular sodium transport.     

The inhibitory effect of some new synthesized xanthates on mushroom tyrosinase activities

Three iso-alkyldithiocarbonates (xanthates), as sodium salts, C3H7OCS2Na (I), C4H9OCS2Na (II) and C5H11OCS2Na (III), were synthesized, by the reaction between CS2 with the corresponding iso-alcohol in the presence of NaOH, and examined for inhibition of both cresolase and catecholase activities of mushroom tyrosinase (MT) from a commercial source of Agricus bisporus. 4-[(4-methylbenzo)azo]-1,2-benzendiol (MeBACat) and 4-[(4-methylphenyl)azo]-phenol (MePAPh) were used as synthetic substrates for the enzyme for the catecholase and cresolase reactions, respectively. Lineweaver-Burk plots showed different patterns of mixed and competitive inhibition for the three xanthates and also for cresolase and catecholase activities of MT. For cresolase activity, I and II showed a mixed inhibition pattern but III showed a competitive inhibition pattern. For catecholase activity, I showed mixed inhibition but II and III showed competitive inhibition. These new synthesized compounds are potent inhibitors of MT with K(i) values of 9.8, 7.2 and 6.1 microM for cresolase inhibitory activity, and also 12.9, 21.8 and 42.2 microM for catecholase inhibitory activity for I, II and III, respectively. They showed a greater inhibitory potency towards the cresolase activity of MT. Both substrate and inhibitor can be bound to the enzyme with negative cooperativity between the binding sites (alpha > 1) and this negative cooperativity increases with increasing length of the aliphatic tail in these compounds in both cresolase and catecholase activities. The cresolase inhibition is related to the chelating of the copper ions at the active site by a negative head group (S-) of the anion xanthate, which leads to similar values of K(i) for all three xanthates. Different K(i) values for catecholase inhibition are related to different interactions of the aliphatic chains of I, II and III with hydrophobic pockets in the active site of the enzyme.     

Transferrins, the mechanism of iron release by ovotransferrin.

Iron release from ovotransferrin in acidic media (3 < pH < 6) occurs in at least six kinetic steps. The first is a very fast (相似文献   

Characterization of an NADH-linked cupric reductase activity from the Escherichia coli respiratory chain.

Previous results from our laboratory have shown that NADH-supported electron flow through the Escherichia coli respiratory chain promotes the reduction of cupric ions to Cu(I), which mediates damage of the respiratory system by hydroperoxides. The aim of this work was to characterize the NADH-linked cupric reductase activity from the E. coli respiratory chain. We have used E. coli strains that either overexpress or are deficient in the NADH dehydrogenase-2 (NDH-2) to demonstrate that this membrane-bound protein catalyzes the electron transfer from NADH to Cu(II), but not to Fe(III). We also show that purified NDH-2 exhibits NADH-supported Cu(II) reductase activity in the presence of either FAD or quinone, but is unable to reduce Fe(III). The K(m) values for free Cu(II) were 32 +/- 5 pM in the presence of saturating duroquinone and 22 +/- 2 pM in the presence of saturating FAD. The K(m) values for NADH were 6.9 +/- 1.5 microM and 6.1 +/- 0.7 microM in the presence of duroquinone and FAD, respectively. The quinone-dependent Cu(II) reduction occurred through both O(*-)(2)-mediated and O(*-)(2)-independent pathways, as evidenced by the partial inhibitory effect (30-50%) of superoxide dismutase, by the reaction stoichiometry, and by the enzyme turnover numbers for NADH and Cu(II). The cupric reductase activity of NDH-2 was dependent on thiol groups which were accessible to p-chloromercuribenzoate at low, but not at high, ionic strength of the medium, a fact apparently connected to a conformational change of the protein. To our knowledge, this is the first protein with cupric reductase activity to be isolated and characterized in its biochemical properties.     

Inhibition of choline oxidase by quinoid dyes

Choline oxidase catalyzes the oxidation of choline to glycine-betaine, with betaine-aldehyde as intermediate and molecular oxygen as primary electron acceptor. This study reports on the inhibitory effects of triarylmethanes (cationic malachite green; neutral leukomalachite green), phenoxazines (cationic, meldola blue and nile blue; neutral nile red) and a structurally-related phenothiazine (methylene blue) on choline oxidase, assayed at 25 degrees C in 50 mM MOPS buffer, pH 7, using choline as substrate. Methylene B acted as a competitive inhibitor with K(i) = 74 +/- 7.2 microM, pointing to the choline-binding site of the enzyme as a target site. Nile B caused noncompetitive inhibition of enzyme activity with K(i) = 20 +/- 4.5 microM. In contrast to methylene B and nile B, malachite G and meldola B caused complex, nonlinear inhibition of choline oxidase, with estimated K(i) values in the micromolar range. The difference in kinetic pattern was ascribed to the differential ability of the dyes to interact (and interfere) with the flavin cofactor, generating different perturbations in the steady-state balance of the catalytic process.     

Purine Nucleoside Phosphorylase Activity in Rat Cerebrospinal Fluid

The sequential hydrolysis of purines is present in rat CSF and generates nucleosides as inosine and guanosine that are usual substrates for purine nucleoside phosphorylase (PNP). PNP catalyzes phosphorolysis of the purine nucleosides and deoxynucleosides releasing purine bases. Here we investigated the presence of PNP in CSF of rats using: i) a specific chromophoric analogue of nucleosides, 2-amino-6-mercapto-7-methylpurine ribonucleoside (MESG), and ii) an inhibitor of PNP activity, immucillin-H. Additionally, we performed a preliminary kinetic characterization (K(M): Henry-Michaelis-Menten constant; V: maximal velocity) for MESG and inorganic phosphate (Pi). The values of K(M) and V for MESG (n = 3, mean+/-SD) were 142.5+/-29.5 microM and 0.0102+/-0.0006 U mg(-1), respectively. For Pi (n=3, mean+/-SD), the K(M) values and V were 186.8+/-43.7 microM and 0.0104+/-0.0016 U mg(-1), respectively. The results indicated that PNP is present in rat CSF and provided a preliminary kinetic characterization.     

The inhibitory effects of flavonoids and antiestrogens on the Glut1 glucose transporter in human erythrocytes

Flavonoids and isoflavonoids are potent inhibitors of glucose efflux in human erythrocytes. Net changes of sugars inside the cells were measured by right angle light scattering. The inhibitory potency of hydroxylated flavonoids depends on the pH of the medium. The apparent affinity is maximal at low pH where the molecule is in the undissociated form. The following K(i)-values at pH 6.5 in microM have been obtained: phloretin 0.37+/-0.03, myricetin 0.76+/-0.42, quercetin 0.93+/-0.28, kaempferol 1.33+/-0.17, isoliquiritigenin 1.96, genistein 3.92+/-0.62, naringenin 8.88+/-1.88, 7-hydroxyflavone 17.58+/-3.15 and daidzein 18.62+/-2.85. Flavonoids carrying hydroxyl groups are weak acids and are deprotonated at high pH-values. From spectral changes pK-values between 6.80 (naringenin) and 7.73 (myricetin) have been calculated. No such pK-value could be obtained from quercetin which was rather unstable at alkaline pH. Flavone itself without a hydroxyl group does not demonstrate any absorbance changes at different pH-values and no significant change in inhibition of glucose transport with pH (K(i)-value around 35 microM). In this respect it is similar to the antiestrogens diethylstilbestrol, tamoxifen and cyclofenil with K(i)-values for glucose efflux inhibition of 2.61+/-0.30, 6.75+/-2.03 and 3.97+/-0.54 microM. Except for phloretin, the flavonoids investigated have planar structures. The inhibitory activity in glucose efflux of planar flavonoids increases exponentially with the number of hydroxyl groups in the molecule.     

Modulation of cyclin-dependent kinase 4 by binding of magnesium (II) and manganese (II)

All kinases require an essential divalent metal for their activity. In this study, we investigated the metal dependence of cyclin-dependent kinase 4 (CDK4). With Mg(2+) as the essential metal and MgATP being the variable substrate, the maximum velocity, V, was not affected by changes in metal concentration, whereas V/K was perturbed, indicating that the metal effects were mainly derived from a change in the K(m) for MgATP. Analysis of the metal dependence of initial rates according to a simple metal binding model indicated the presence on enzyme of one activating metal-binding site with a dissociation constant, K(d(a)), of 5 +/-1 mM, and three inhibitory metal-binding sites with an averaged dissociation constant, K(d(i)), of 12+/-1 mM and that the binding of metal to the activating and inhibitory sites appeared to be ordered with binding of metal to the activating site first. Substitution of Mn(2+) for Mg(2+) yielded similar metal dependence kinetics with a value of 1.0+/-0.1 and 4.7+/-0.1 for K(d(a)) and K(d(i)), respectively. The inhibition constants for the inhibition of CDK4 by MgADP and a small molecule inhibitor were also perturbed by Mg(2+). K(d(a)) values estimated from the metal variation of the inhibition of CDK4 by MgADP (6+/-3 mM) and a small molecule inhibitor (3+/-1 mM), were in good agreement with the K(d(a)) value (5+/-1 mM) obtained from the metal variation of the initial rate of CDK4. By using the van't Hoff plot, the temperature dependence of K(d(a)) and K(d(i)) yielded an enthalpy of -6.0 +/- 1.1 kcal/mol for binding of Mg(2+) to the activating site and -3.2 +/- 0.6 kcal/mol for Mg(2+) binding to the inhibitory sites. The values of associated entropy were also negative, indicating that these metal binding reactions were entirely enthalpy-driven. These data were consistent with metal binding to multiple sites on CDK4 that perturbs the enzyme structure, modulates the enzyme activity, and alters the affinities of inhibitor for the metal-bound enzyme species. However, the affinities of small molecule inhibitors for CDK4 were not affected by the change of metal from Mg(2+) to Mn(2+), suggesting that the structures of enzyme-Mg(2+) and enzyme-Mn(2+) were similar.     

Novel amidino-substituted benzimidazoles: synthesis of compounds and inhibition of dipeptidyl peptidase III

Dipeptidyl peptidase III (DPP III), also known as enkephalinase B, is a zinc-hydrolase with an indicated role in the mammalian pain modulatory system. In order to find a potent antagonist of this enzyme, we synthesized and screened the effect of a small set of benzimidazole derivatives on its activity. To improve the inhibitory potential, a cyclobutane ring was introduced as rigid conformation support to the diamidino substituted dibenzimidazoles. Two such compounds (1' and 4') from the group of cyclobutane derivatives containing amidino-substituted benzimidazole moieties, obtained by photochemical cyclization in water and by respecting rules of the "green chemistry" approach, were found to be strong DPP III inhibitors, with IC(50) value below 5 microM. Compound 1' displayed time-dependent inhibition towards human DPP III, characterized by the second-order rate constant of 6924+/-549 M(-1)min(-1) (K(i)=0.20 microM). The peptide substrate valorphin protected the enzyme from inactivation by 1'.     

Oleyl sulfate reveals allosteric inhibition of soybean lipoxygenase-1 and human 15-lipoxygenase

Inhibition of lipoxygenase (LO) is currently an important goal of biomedical research due to its critical role in asthma, atherosclerosis, and cancer regulation. Steady-state kinetic data indicate that oleic acid (OA) is a simple competitive inhibitor for soybean lipoxygenase; however, kinetic isotope effect (KIE) data suggest a more complicated inhibitory mechanism. To investigate the inhibitory effects of fatty acids on lipoxygenase more thoroughly, we have synthesized a novel inhibitor to lipoxygenase, (Z)-9-octadecenyl sulfate (oleyl sulfate, OS), which imparts kinetic properties that are inconsistent with simple competitive inhibition for both SLO-1 and 15-HLO. The KIE exhibits a hyperbolic rise with addition of OS, indicating the formation of a catalytically active ternary complex with K(D) values of 0.6 +/- 0.2 and 0.4 +/- 0.05 microM for SLO-1 and 15-HLO, respectively. The steady-state kinetics show that SLO-1 proceeds through a hyperbolic mixed-type inhibition pathway, where OS binding (K(i) = 0.7 +/- 0.3 microM) causes an approximate 4-fold increase in the K(m)(app) (alpha = 4.6 +/- 0.5) and a decrease in the k(cat) by approximately 15% (beta = 0.85 +/- 0.1). 15-HLO also exhibits a hyperbolic saturation of k(cat)/K(m) consistent with the observed rise in its KIE. Taken together, these findings indicate the presence of an allosteric site in both SLO-1 and 15-HLO and suggest broad implications regarding the inhibition of LO and the treatment of LO-related diseases.     

Inhibition of testicular 17 alpha-hydroxylase and 17,20-lyase but not 3 beta-hydroxysteroid dehydrogenase-isomerase or 17 beta-hydroxysteroid oxidoreductase by ketoconazole and other imidazole drugs

Ketoconazole, an orally active antifungal drug, is known to inhibit testicular androgen production both in vitro and in vivo. The aim of the present study was to examine the effect of ketoconazole and 13 other imidazole drugs on rat testicular microsomal 17 alpha-hydroxylase, 17,20-lyase, 3 beta-hydroxysteroid dehydrogenase-isomerase (3 beta-HSD-I) and 17 beta-hydroxysteroid oxidoreductase (17 beta-HSOR). The order of decreasing inhibitory effect (determined from Ki values) on 17 alpha-hydroxylase (substrate [3H]progesterone; Km = 89 +/- 0.65 nmol/l; SEM, n = 8) was bifonazole (Ki = 86 +/- 3.3 nmol/l; SEM, n = 4) greater than ketoconazole (160 +/- 4.92) greater than clotrimazole (170 +/- 5.81) greater than miconazole (599 +/- 7.22) greater than econazole (688 +/- 6.98) greater than tioconazole (901 +/- 1.71) greater than isoconazole (1090 +/- 6.96) and on 17,20-lyase (substrate, [3H]17 alpha-hydroxyprogesterone; Km = 250 +/- 0.75 nmol/l; SEM, n = 8) was bifonazole (56.5 +/- 3.4) greater than clotrimazole (81.5 +/- 3.1) greater than ketoconazole (84 +/- 3.5) greater than miconazole (243 +/- 6.3) greater than econazole (325 +/- 5.1) greater than tioconazole (505 +/- 5.2) greater than isoconazole (610 +/- 6.34). However, these imidazole drugs did not inhibit the 3 beta-HSD-I or 17 beta-HSOR activities. A common structural feature of the imidazole drugs having an inhibitory effect was the presence of one or more aromatic rings on the imidazole side chain. In contrast, the imidazole drugs having the imidazole ring fused to a benezene ring, i.e. benzimidazoles (astemizole, mebendazole, thiabendazole) and those having an aliphatic side chain on the N-1 of the imidazole ring (carbimazole, metronidazole, nimorazole, tinidazole) did not inhibit 17 alpha-hydroxylase, 3 beta-HSD-I or 17 beta-HSOR enzyme activities. However some did inhibit 17,20-lyase activity but only at high concentrations. The results of the present study suggest that some imidazole drugs may be useful in clinical situations requiring the suppression of androgen production, for example in the treatment of hormone-dependent prostatic cancer.     

A Streptomyces collinus thiolase with novel acetyl-CoA:acyl carrier protein transacylase activity

Acetyl-CoA:acyl carrier protein (ACP) transacylase (ACT) activity has been demonstrated for the 3-ketoacyl-ACP synthase III (KASIII) which initiates fatty acid biosynthesis in the type II dissociable fatty acid synthases of plants and bacteria. Several lines of evidence have indicated the possibility of ACT activity being associated with proteins other than KASIII. Using a crude extract of Streptomyces collinus, we have resolved from KASIII an additional protein with ACT activity and subsequently purified it 85-fold in five chromatographic steps. The 45 kDa protein was shown by gel filtration to have a molecular mass of 185 +/- 35 kDa, consistent with a homotetrameric structure for the native enzyme. The corresponding gene (fadA) was cloned and sequenced and shown to encode a protein with amino acid sequence homology to type II thiolases. The fadA was expressed in Escherichia coli, and the resulting recombinant FadA enzyme purified by metal chelate chromatography was shown to have both ACT and thiolase activities. Kinetic studies revealed that in an ACT assay FadA had a substrate specificity for a two-carbon acetyl-CoA substrate (K(m) 8.7 +/- 1.4 microM) but was able to use ACPs from both type II fatty acid and polyketide synthases (Streptomyces glaucescens FabC ACP, K(m) 10.7 +/- 1.4 microM; E. coli FabC ACP, K(m) 8.8 +/- 2 microM; FrenN ACP, K(m) 44 +/- 12 microM). In the thiolase assay kinetic analyses revealed similar K(m) values for binding of substrates acetoacetyl-CoA (K(m) 9.8 +/- 0.8 microM) and CoA (K(m) 10.9 +/- 1.8 microM). A Cys92Ser mutant of FadA possessed virtually unchanged K(m) values for acetoacetyl-CoA and CoA but had a greater than 99% decrease in k(cat) for the thiolase activity. No detectable ACT activity was observed for the Cys92Ser mutant, demonstrating that both activities are associated with FadA and likely involve formation of the same covalent acetyl-S-Cys enzyme intermediate. An ACT activity with ACP has not previously been observed for thiolases and in the case of the S. collinus FadA is significantly lower (k(cat) 3 min(-1)) than the thiolase activity of FadA (k(cat) 2170 min(-1)). The ACT activity of FadA is comparable to the KAS activity and significantly higher than the ACT activity, reported for a streptomycete KASIII.     

Cyclic and linear peptides derived from alpha-amylase inhibitory protein tendamistat

Tendamistat is a strong inhibitory protein of porcine pancreatic alpha-amylase (PPA) with a K(i) value of 0.2 nM. To develop potent alpha-amylase inhibitors, we synthesized six odd-length cyclic peptides (5-15 residues) and four even-length cyclic peptides (10 and 12 residues) having the inhibitory sequence of tendamistat. Their PPA inhibitory activities were evaluated, and, among them, the 11-residue cyclic peptide Ten(15-23) (K(i) = 0.27 microM) exhibited the strongest inhibitory activity (K(i) = 0.27-1.41 microM). To examine the effect of cyclic structure on PPA inhibition, ten linear peptides corresponding to the cyclic peptides were also synthesized, and their PPA inhibitory activities were evaluated (K(i) = 0.28-1.00 microM). Interestingly, the 11-residue linear peptide Ten(15-23) exhibited almost the same inhibitory activity (K(i) = 0.28 microM) as that of cyclic Ten(15-23). The results of a circular dichroism study indicated that stabilization of the beta-hairpin structure occurred only for cyclic Ten(15-23). Also, the results of proteolytic digestion experiments of the cyclic and linear Ten(15-23) peptides by trypsin and chymotrypsin suggested no differences in protease resistance between the cyclic and linear structures. Therefore, we demonstrated that both cyclic and linear peptides containing the inhibitory sequence of tendamistat exhibit potent PPA inhibitory activity.     

The effect of extracellular potassium on the intracellular potassium ion activity and transmembrane potentials of beating canine cardiac Purkinje fibers

We used open tip microelectrodes containing a K+-sensitive liquid ion exchanger to determine directly the intracellular K+ activity in beating canine cardiac Purkinje fibers. For preparations superfused with Tyrode's solution in which the K+ concentration was 4.0 mM, intracellular K+ activity (ak) was 130.0+/-2.3 mM (mean+/-SE) at 37 degrees C. The calculated K+ equilibrium potential (EK) was -100.6+/- 0.5 mV. Maximum diastolic potential (ED) and resting transmembrane potential (EM) were measured with conventional microelectrodes filled with 3 M KCl and were -90.6+/-0.3 and -84.4+/-0.4 mV, respectively. When [K+]o was decreased to 2.0 mM or increased to 6.0, 10.0, and 16.0 mM, ak remained the same. At [K+]o=2.0, ED was -97.3+/-0.4 and Em - 86.0+/-0.7 mV; at [K+]o=16.0, ED fell to -53.8+/-0.4 mV and Em to the same value. Over this range of values for [K+]o, EK changed from - 119.0+/-0.3 to -63.6+/-0.2 mV. These values for EK are consistent with those previously estimated indirectly by other techniques.     

Electroporative fast pore-flickering of the annexin V-lipid surface complex, a novel gating concept for ion transport

In contact with lipid bilayers and Ca2+-ions, the intracellular protein human annexin V (wild-type), Mr = 35,800, forms two types of cation-selective channels for the transport of Ca2+-, K+-, Na+- and Mg2+-ions, depending on the protein concentration [AN]. Type (I) channel events are large and predominant at high values [AN] > or = K = 5 nM at 296 K. At 50 mM Ca2+, symmetrical on both membrane sides, AN added at the cis side, the conductance is gCa(I) = 22 +/- 2 pS and at symmetrical 0.1 M K+-conditions: gK(I) = 32 +/- 3 pS, associated with two mean open-times tau1(I) = 0.68 +/- 0.2 ms and tau2(I) = 31 +/- 2 ms. Monoclonal anti-AN antibodies added to the trans-side first increase the mean open-times and then abolish the channel activity, suggesting that type (I) channels refer to a membrane spanning protein complex, probably a trimer T, which at [AN] > K changes its membrane organization to a higher oligomer, probably to the side-by-side double-trimer T2. The smaller type (II) channel events are predominant at low [AN] < or = K and refer to the (electroporative) adsorption complex of the monomer. The conductances g(i)(II) for symmetrical concentrations depend non-linearly on the voltage Um = Uext + U(AN), where U(AN) = 0.02 +/- 0.002 V is the electrostatic contribution of the Ca2+-AN complex and Uext the externally applied voltage. There is only one mean open-time tau(o)(II) which is voltage-dependent according to a functional of b x Um2 where b = 113.9 +/- 15 V(-2), yielding an activation Gibbs free energy of Ga = RT x b x Um2. The conformational flicker probability f(i)(II) in g(i)(II) = g(i)0(II) x gamma(i) x f(i)(II) is non-linearly voltage-dependent according to a functional of a x Um2. The Nernst term gamma(i) refers to asymmetrical ion concentrations. From a = 50 V(-2), independent of the ion type, we obtain f(i)0(II) = 0.03 +/- 0.002 and the conductances for the fully open-channel states: gCa0(II) = 69 +/- 3 pS (0.05 M Ca2+) and gK0(II) = 131 +/- 5 pS (1.2 M K+). From the electroporation term a = pi[r(p)2]epsilon0(epsilon(w) - epsilon(m))/(2 kTd) we determine the mean pore radius of the complex in its fully open state as r(p)= 0.86 +/- 0.05 nm. The adsorbed annexin V (Ca2+) monomer appears to electrostatically facilitate the electric pore formation at the contact interface between the protein and the lipid phase. The complex rapidly flickers and thus limits the ion transport in a voltage-dependent manner.     

Effects of magnesium and ATP on pre-steady-state phosphorylation kinetics of the Na+,K(+)-ATPase.

The aim of the present work was to elucidate the role played by ATP and Mg2+ ions in the early steps of the Na+,K(+)-ATPase cycle. The approach was to follow pre-steady-state phosphorylation kinetics in Na(+)-containing K(+)-free solutions under variable ATP and MgCl2 concentrations. The experiments were performed with a rapid mixing apparatus at 20 +/- 2 degrees C. The concentrations of free and complexes species of Mg2+ and ATP were calculated on the basis of a dissociation constant of 0.091 +/- 0.004 mM, estimated with Arsenazo III under identical conditions. A simplified scheme were ATP binds to the ENa enzyme, which is phosphorylated to MgEPNa and consequently dephosphorylated returning to the ENa form, was used. In the absence of ADP and phosphate four rate constants are relevant: k1 and k-1, the on and off rate constants for ATP binding; k2, the transphosphorylation rate constant and k3, the constant that governs the dephosphorylation rate. The values obtained were: k1 = 0.025 +/- 0.003 microM-1 ms-1 for both free ATP and ATPMg; k-1 = 0.038 +/- 0.004 ms-1 for free ATP and 0.009 +/- 0.002 ms-1 for ATPMg; k2 = 0.199 +/- 0.005 ms-1; k3 = 0.0019 +/- 0.0002 ms-1. The model that seems best to explain the data is one where (i) the role of true substrate can be played equally well by free ATP or ATPMg, and (ii) free Mg2+, an essential activator, acts by binding to a specific Mg2+ site on the enzyme molecule.     

Mechanisms of inhibition of phenylalanine ammonia-lyase by phenol inhibitors and phenol/glycine synergistic inhibitors

Phenylalanine ammonia-lyase (PAL) catalyzes the beta-elimination of ammonia from L-phenylalanine to trans-cinnamic acid. A study of inhibition of PAL by phenol, ortho-cresol, and meta-cresol gave mixed inhibition; para-cresol is not an inhibitor. The calculated values of K(i) and alphaK(i) are phenol, K(i)=2.1+/-0.5 mM and alphaK(i)=3.45+/-0.95 mM; ortho-cresol, K(i)=0.8+/-0.2 mM and alphaK(i)=3.4+/-0.2 mM; meta-cresol, K(i)=2.85+/-0.15 mM and alphaK(i)=18.5+/-1.5 mM. The synergistic inhibition of the same inhibitors with glycine showed a lack of inhibition with the para-cresol/glycine pair, while mixed inhibition was observed with the ortho-cresol/glycine pair (K(i)=0.038+/-0.008 mM, alphaK(i)=0.13+/-0.04 mM) and phenol/glycine pair (K(i)=0.014+/-0.003 mM, alphaK(i)=0.058+/-0.01 M). The meta-cresol/glycine pair gave competitive inhibition (K(i)=0.36+/-0.076 mM). The strong synergistic inhibition observed implies that the inhibitors bind at the active site: in fact, the inhibitors used imitate the structure of the substrate. The order of synergistic inhibition is the same for the sites related to K(i) and alphaK(i). These results are in agreement with the inhibitors entering two active sites located in two different subunits.     

Sequestration of biogenic amines by alginic and fulvic acids

The interaction of natural (alginic and fulvic acids) and synthetic (polyacrylic acid 2.0 kDa) polyelectrolytes with some protonated polyamines [diamines: ethylendiamine, 1,4-diaminobutane (or putrescine), 1,5-diaminopentane (or cadaverine); triamines: N-(3-aminopropyl)-1,4-diaminobutane (or spermidine), diethylenetriamine; tetramine: N,N'-bis(3-aminopropyl)-1,4-diaminobutane (or spermine); pentamine: tetraethylene-pentamine; hexamine: pentaethylenehexamine] was studied at T=25 degrees C by potentiometry and calorimetry. Measurements were performed without supporting electrolyte, in order to avoid interference, and results were reported at I=0 mol L(-)(1). For all the systems, the formation of (am)L(2)H(i) species was found (am=amine; L=polyelectrolyte; i=1...4, depending on the amine considered). The stability of polyanion-polyammonium cation complexes is always significant, and for high-charged polycations, we observe a stability comparable to that of strong metal complexes. For example, by considering the formation reaction (am)H(i)+2L=(am)L(2)H(i) we found log K(i)=6.0, 6.5 and 10.8 for i=1, 2 and 3, respectively, in the system alginate-spermidine. Low and positive formation DeltaH(degrees) values indicate that the main contribution to the stability is entropic in nature. The sequestering ability of polyelectrolytes toward amines was modelled by a sigmoid Boltzman type equation. Some empirical relationships between stability, charges and DeltaG(degrees) and TDeltaS(degrees) are reported. Mean values per salt bridge of formation thermodynamic parameters (DeltaX(degrees) (n)) are DeltaG(degrees) (n)=-5.8+/-0.4, DeltaH degrees (n)=0.7+/-0.5 and TDeltaS(degrees) (n)=6.5+/-0.5 kJmol(-)(1) for all the systems studied in this work.     

Type III phosphodiesterase plays a necessary role in the growth-promoting actions of insulin, insulin-like growth factor-I, and Ha p21ras in Xenopus laevis oocytes.

Three phosphodiesterase (PDE) type III inhibitors were tested and found to inhibit Xenopus oocyte maturation induced by insulin with apparent IC50 values of 2.2 +/- 0.2 microM Cl-930, 25 +/- 3 microM imazodan (Cl-914), and 786 +/- 237 microM piroximone (MDL 19,205). The same rank order of potencies was observed for inhibition of insulin-like growth factor-I (IGF-I)-induced oocyte maturation, with IC50 values of 5.5 +/- 0.9 microM Cl-930, 54 +/- 4 microM imazodan, and 1190 +/- 395 microM piroximone. Oocyte maturation induced by microinjection of Ha p21ras was also inhibited by pretreatment of oocytes with Cl-930 or imazodan, with IC50 values of 4.3 +/- 1.2 and 59 +/- 4 microM, respectively. Progesterone-induced maturation was not affected by PDE III inhibitor action; and, neither type IV PDE inhibitors (Ro 20, 1724 or rolipram) nor dipyridamole (a type V PDE inhibitor) inhibited cell division induced by IGF-I or microinjected Ha p21ras. In addition, while insulin-stimulated oocyte PDE activity measured in vivo after microinjection of 200 microM [3H] cAMP was inhibited by nonselective and type III-specific drugs (with IC50 values of 4.2 +/- 1.8 microM Cl-930 and 26 +/- 6 microM imazodan), type IV and type V inhibitors did not inhibit hormone-stimulated enzyme activity. This pharmacological evidence demonstrates a necessary role for PDE III in insulin-, IGF-I-, and p21ras-induced meiotic cell division in Xenopus laevis oocytes.