Entamoeba histolytica: an ecto-phosphatase activity regulated by oxidation-reduction reactions

In this work, an ecto-phosphatase activity of Entamoeba histolytica was characterized using intact cells. This activity presented the following biochemical characteristics: (i) it hydrolyzes p-NPP with V(max) of 8.00+/-0.22 nmol p-NP x h(-1) x 10(-5) cells and K(m) of 2.68+/-0.25 mM; (ii) it is inhibited by acid phosphatase inhibitors, such as sodium molybdate (K(i)=1.70+/-0.24 microM) and sodium fluoride (K(i)=0.25+/-0.02 mM); (iii) it also showed high sensitivity to phosphotyrosine phosphatase inhibitors, such as sodium orthovanadate (K(i)=1.07+/-0.14 microM), bpV-PHEN (K(i)=0.38+/-0.02 microM) and mpV-PIC (K(i)=0.39+/-0.04 microM). Zn(2+), an oxidizing agent, decreased the enzymatic activity in 50%. DTT and GSH, two reducing agents, enhanced the activity twofold. The non-invasive E. histolytica and free-living E. moshkovskii were less efficient in hydrolyzing p-NPP than the pathogenic E. histolytica suggesting that this enzyme could represent a virulence marker for this cell.     

Bis(sulfonamide) isosters of mycophenolic adenine dinucleotide analogues: inhibition of inosine monophosphate dehydrogenase

Synthesis of novel inhibitors of human IMP dehydrogenase is described. These inhibitors are isosteric methylenebis(sulfonamide) analogues 5-8 of earlier reported mycophenolic adenine methylenebis(phosphonate)s 1-3. The parent bis(phosphonate) 1 and its bis(sulfonamide) analogue 5 showed similar sub-micromolar inhibitory activity against IMPDH2 (K(i) approximately 0.2 microM). However, the bis(sulfonamide) analogues 6 and 8 substituted at the position 2 of adenine were approximately 3- to 10-fold less potent inhibitors of IMPDH2 (K(i)=0.3-0.4 microM) than the corresponding parent bis(phosphonate)s 2 and 3 (K(i)=0.04-0.11 microM), respectively.     

Synthesis and glycosidase inhibitory activities of 2-(aminoalkyl)pyrrolidine-3,4-diol derivatives

Several 2-(aminomethyl)-and 2-(2-aminoethyl)-pyrrolidine-3,4-diol derivatives have been assayed for their inhibitory activities towards glycosidases. Good inhibitors of alpha-mannosidases must have the (2R,3R,4S) configuration and possess 2-(benzylamino)methyl substituents. Stereomers with the (2S,3R,4S) configuration are also competitive inhibitors of alpha-mannosidases, but less potent as they share the configuration of C(1), C(2), C(3) of beta-D-mannosides rather than that of alpha-D-mannosides. Interestingly, (2S,3R,4S)-2-[2-[(4-phenyl)phenylamino]ethyl]pyrrolidine-3,4-diol (12g) inhibits several enzymes, for instance alpha-L-fucosidase from bovine epididymis (K(i)=6.5microM, competitive), alpha-galactosidase from bovine liver (K(i)=5microM, mixed) and alpha-mannosidase from jack bean (K(i)=102microM, mixed). Diamines such as (2R,3S,4R)-2-[2-(phenylamino) or 2-(benzylamino)ethyl]pyrrolidine-3,4-diol (ent-12a, ent-12b) inhibit beta-glucosidase from almonds (K(i)=13-40microM, competitive).     

Malathion detoxification by human hepatic carboxylesterases and its inhibition by isomalathion and other pesticides

The organophosphorothioate (OPT) pesticide malathion (MAL) in mammals is readily hydrolyzed by mammalian carboxylesterases (CE). The reaction competes with the CYP-catalyzed formation of malaoxon (MOX), the toxic metabolite. Alterations or individual variations in CE activity may result in increased MOX formation, enhancing MAL toxicity. We have characterized the human hepatic CE activity in a panel of 18 human liver microsomes as well as the inhibitory effect of IsoMAL, a major impurity of MAL commercial formulations, parathion (PAR), chlorpyrifos (CPF), and chlorpyrifos-oxon (CPFO). CE activity showed a low level of variation among individuals (4-fold). The reaction consists of two different phases, differing in their affinity for mal (k(m1)=0.25-0.69 microm; K(m2)=10.3-26.8 microM). The relatively low K(m1) values confirmed that human CE efficiently detoxify MAL. IsoMAL was shown to be a potent noncompetitive inhibitor of MAL detoxification (K(i)=0.6 microM), with a higher inhibitory potency than CPF and PAR (K(i)=7.5 microM and 50 microM, respectively). These two latter compounds very likely act as mixed inhibitors. CPFO showed the highest inhibitory potency toward CE-mediated detoxification, being characterized by a K(i)=22 nM. The present results provide useful information for a better understanding of possible interactions between different OPTs and for assessing the potential cumulative risk for exposure to OPT mixtures.     

Intermediate analogue inhibitors of mandelate racemase: N-Hydroxyformanilide and cupferron

Mandelate racemase (MR) catalyzes the 1,1-proton transfer that interconverts the enantiomers of mandelate. The transition state/intermediate analogues N-hydroxyformanilide (K(i)=2.79+/-0.19 microM) and cupferron (K(i)=2.67+/-0.09 microM) are identified as potent competitive inhibitors of MR. The pH-pK(i) profile indicates that MR can bind either the protonated or deprotonated forms of N-hydroxyformanilide, with a 10-fold greater affinity for the latter form.     

Human CYP1A1 variants lead to differential eicosapentaenoic acid metabolite patterns

To answer the question whether the most common allelic variants of human CYP1A1, namely CYP1A1.1 (wild type), CYP1A1.2 (Ile462Val), and CYP1A1.4 (Thr461Asn), differ in their catalytic activity towards eicosapentaenoic acid (EPA), in vitro enzymatic assays were performed in reconstituted CYP1A1 systems. All CYP1A1 variants catalyzed EPA epoxygenation and hydroxylation to 17(R),18(S)-epoxyeicosatetraenoic acid (17(R),18(S)-EETeTr) and 19-OH-EPA, yet with varying catalytic efficiency and distinct regiospecificity. CYP1A1.1 and CYP1A1.4 formed 17(R),18(S)-EETeTr as main product (K(m)=53 and 50 microM; V(max)=0.60 and 0.50 pmol/min/pmol; V(max)/K(m)=0.11 and 0.10 microM(-1)min(-1), respectively), followed by 19-OH-EPA (K(m)=76 and 93 microM; V(max)=0.37 and 0.37 pmol/min/pmol; V(max)/K(m)=0.005 and 0.004 microM(-1)min(-1), respectively). The variant CYP1A1.2 produced almost equal amounts of both metabolites, but its catalytic efficiency for hydroxylation was five times higher (K(m)=66 microM; V(max)=1.7 pmol/min/pmol; V(max)/K(m)=0.026 microM(-1)min(-1)) and that for epoxygenation was twice higher (K(m)=66 microM; V(max)=1.5 pmol/min/pmol; V(max)/K(m)=0.023 microM(-1)min(-1)) than those of the wild-type enzyme. Thus, the Ile462Val polymorphism in human CYP1A1 affects EPA metabolism and may contribute to interindividual variance in the local production of physiologically active fatty acid metabolites in the cardiovascular system and other extrahepatic tissues, where CYP1A1 is expressed or induced by polycyclic aromatic hydrocarbons and other xenobiotics.     

Metabolic differences between attached and free-living marine bacteria: inadequacy of liquid cultures for describing in situ bacterial activity

Marinobacter sp. strain CAB was cultivated with or without porous glass beads as solid support. Two substrates were used: the hydrophilic sodium lactate and a hydrophobic C(18)-isoprenoid ketone (6,10,14-trimethylpentadecan-2-one (TMP)). The substrate adsorption onto the beads was measured. Bacterial adhesion was determined by a direct count technique and amounted to 70% of total cells. In the immobilised cell cultures (ICC), generation times were 1.5 and 1.8 times shorter than in the planktonic cultures (FCC) with sodium lactate and with TMP, respectively. In ICC, the growth yields were lower (15.3(FCC) x 10(9) and 0.8(ICC) x 10(9) bacteria mg(-1) of sodium lactate; 50(FCC) x 10(9) and 35(ICC) x 10(9) bacteria mg(-1) of TMP). The mineralisation of substrates was estimated after mass spectrometric determination of the CO2 production rates of both free and immobilised cell cultures. The results indicated a higher specific CO2 production rate in the ICC with sodium lactate (3.1(FCC)+/-0.2 and 3.5(ICC)+/-0.3 nmol CO2 mg(-1) protein min(-1)) but not in the ICC with TMP (1.9(FCC)+/-0.7 and 0.5(ICC)+/-0.3 nmol CO2 mg(-1) protein min(-1)). The affinities for the two substrates were lower in the presence of the solid support (K(m,ICC)=18.2+/-0.2 microM and 37.1+/-2.0 microM, for sodium lactate and TMP, respectively) than without support (K(m,FCC)=8.5+/-1.5 microM and 8.4+/-1.2 microM, for sodium lactate and TMP, respectively). Moreover, the presence of a solid support showed a lower inhibition by the TMP (K(i,FCC)=3.8+/-1.0 microM and K(i,ICC)=12.2+/-2.5 microM) which may explain why the immobilised cell cultures degraded hydrophobic TMP more efficiently than the planktonic cultures.     

Novel 5-substituted 1-pyrazolol analogues of ibotenic acid: synthesis and pharmacology at glutamate receptors

5-Substituted 1-pyrazolol analogues of ibotenic acid have been synthesized and pharmacologically characterized on ionotropic and metabotropic glutamate receptors (iGluRs and mGluRs). The syntheses involved introduction of bromide, alkyls, phenyl and arylalkyls in the 5-position of 1-benzyloxypyrazole leading to 5-substituted (RS)-2-amino-(1-hydroxy-4-pyrazolyl)acetic acids (5a-l). The pharmacological activities of the synthesized analogues ranged from the 5-cyclopropylmethyl analogue (5f) with weak but selective affinity for NMDA receptors (IC(50)=35 microM), over the 5-n-propyl analogue (5c), which was a selective mGluR2 agonist (EC(50)=72 microM), to the 5-cyclohexylmethyl analogue (5g), which was a selective mGluR2 antagonist (K(i)=32 microM), and the 5-phenylethyl analogue (5j), which was a weak but apparently selective mGluR1 antagonist (K(i)=230 microM). This series of compounds afforded GluR ligands with a broad spectrum of pharmacological profiles, and showing potential for development of new compounds with subtype-selective activities at various GluRs.     

Synthetic FP-prostaglandin-induced contraction of rat uterus smooth muscle in vitro

Numerous synthetic FP-class prostaglandin (PG) analogs stimulated the contraction of isolated non-pregnant female rat uterus in a concentration-dependent manner with the following agonist potencies: bimatoprost acid (17-phenyl-trinor PGF(2alpha); EC(50)=0.68+/-0.06 nM)=cloprostenol (EC(50)=0.73+/-0.01 nM)>travoprost acid (EC(50)=1.3+/-0.07 nM)>latanoprost acid (EC(50)=2.7+/-0.08 nM)>PGF(2alpha) (EC(50)=52+/-11 nM)>unoprostone (UF-021; EC(50)=310+/-101 nM)>S-1033 (EC(50)=610+/-4 nM)>bimatoprost (EC(50)=1130+/-173 nM). The FP-receptor antagonist, AL-8810, antagonized the contractile effects of PGF(2alpha) (K(i)=2.9+/-0.2 microM), travoprost acid (K(i)=0.6+/-0.1 microM) and bimatoprost (K(i)=0.2+/-0.02 microM). Agonist and antagonist potencies for rat uterus contraction by these PGs compared well with their potencies for inducing/blocking functional responses in other systems (r=0.83-0.94) except with bovine iris sphincter (r=0.2; p<0.7). In conclusion, the rat uterus contains functionally active FP-receptors whose activation by a variety of free acid and an amide forms of synthetic PGs leads to the contraction of this tissue and which can be pharmacologically blocked by an FP-receptor antagonist, AL-8810.     

Aporphine metho salts as neuronal nicotinic acetylcholine receptor blockers

(S)-Aporphine metho salts with the 1,2,9,10 oxygenation pattern displaced radioligands from recombinant human alpha7 and alpha4beta2 neuronal nicotinic acetylcholine receptors (nAChR) at low micromolar concentrations. The affinity of the nonphenolic glaucine methiodide (4) (vs [(3)H]cytisine) was the lowest at alpha4beta2 nAChR (K(i)=10 microM), and predicentrine methiodide (2) and xanthoplanine iodide (3), with free hydroxyl groups at C-2 or C-9, respectively, had the highest affinity at these receptors (K(i) approximately 1 microM), while the affinity of the diphenolic boldine methiodide (1) was intermediate between these values. At homomeric alpha7 nAChR, xanthoplanine had the highest affinity (K(i)=10 microM) vs [(125)I]alpha-bungarotoxin while the other three compounds displaced the radioligand with K(i) values between 15 and 21 microM. At 100 microM, all four compounds inhibited the responses of these receptors to EC(50) concentrations of ACh. The effects of xanthoplanine iodide (3) were studied in more detail. Xanthoplanine fully inhibited the EC(50) ACh responses of both alpha7 and alpha4beta2 nACh receptors with estimated IC(50) values of 9+/-3 microM (alpha7) and 5+/-0.8 microM (alpha4beta2).     

Modulation by ammonium ions of gill microsomal (Na+,K+)-ATPase in the swimming crab Callinectes danae: a possible mechanism for regulation of ammonia excretion

The modulation by Na(+), K(+), NH(4)(+) and ATP of the (Na(+),K(+))-ATPase in a microsomal fraction from Callinectes danae gills was analyzed. ATP was hydrolyzed at high-affinity binding sites at a maximal rate of V=35.4+/-2.1 Umg(-1) and K(0.5)=54.0+/-3.6 nM, obeying cooperative kinetics (n(H)=3.6). At low-affinity sites, the enzyme hydrolyzed ATP obeying Michaelis-Menten kinetics with K(M)=55.0+/-3.0 microM and V=271.5+/-17.2 Umg(-1). This is the first demonstration of a crustacean (Na(+),K(+))-ATPase with two ATP hydrolyzing sites. Stimulation by sodium (K(0.5)=5.80+/-0.30 mM), magnesium (K(0.5)=0.48+/-0.02 mM) and potassium ions (K(0.5)=1.61+/-0.06 mM) exhibited site-site interactions, while that by ammonium ions obeyed Michaelis-Menten kinetics (K(M)=4.61+/-0.27 mM). Ouabain (K(I)=147.2+/-7.microM) and orthovanadate (K(I)=11.2+/-0.6 microM) completely inhibited ATPase activity, indicating the absence of contaminating ATPase and/or neutral phosphatase activities. Ammonium and potassium ions synergistically stimulated the enzyme, increasing specific activities up to 90%, suggesting that these ions bind to different sites on the molecule. The presence of each ion modulates enzyme stimulation by the other. The modulation of (Na(+),K(+))-ATPase activity by ammonium ions, and the excretion of NH(4)(+) in benthic crabs are discussed.     

A fluorimetric method for the determination of pepsin activity

An intramolecularly quenched fluorogenic peptide containing o-aminobenzoyl (Abz) and ethylenediamine 2,4-dinitrophenyl (Eddnp) groups at amino- and carboxyl-terminal amino acid residues, Abz-Lys-Pro-Ile-Glu-Phe-Phe-Arg-Leu-Eddnp, was hydrolyzed by purified human pepsin, gastricsin, and gastric juice uniquely at the Phe-Phe bond. Kinetic parameters determined for purified pepsin were K(m)=0.68+/-0.11 microM; k(cat)=6.3+/-0.16s(-1); k(cat)/K(m)=9.26s(-1) microM(-1); Gastricsin showed K(m)=2.69+/-0.18 microM; k(cat)=0.03+/-0.005s(-1); k(cat)/K(m)=0.011s(-1) microM(-1). Gastric juice (21 samples) from subjects without gastric disorders at endoscopy examination showed activities varying from 0.0008 to 9.72 micromolml(-1)min(-1). Pepstatin A inhibition of gastric juice enzymatic activity was complete at 3.4x10(-5)M (final concentration) inhibitor. In the proposed method the presence of a unique scissile bond in the synthetic substrate provides a direct ratio between enzymatic activity and amount of substrate hydrolyzed, and a unique step reaction facilitates the use of this assay for the determination of the activity of aspartic proteinases in biological fluids and during enzyme purification procedures.     

Convenient synthesis and evaluation of enzyme inhibitory activity of several N-alkyl-, N-phenylalkyl,and cyclic isourea derivatives of 5a-carba-alpha-DL-fucopyranosylamine

Convenient synthesis and chemical modification of the potent alpha-L-fucosidase inhibitor, 5a-carba-alpha-DL-fucopyranosylamine (1), are described. Among seven N-substituted and three cyclic isourea derivatives newly prepared, the N-octyl derivative was found to be the strongest inhibitor of alpha-L-fucosidase (bovine kidney) more potent (K(i)=0.016 microM) than deoxyfuconojirimycin (K(i)=0.031 microM) with p-nitrophenyl-alpha-L-fucopyranoside as the substrate.     

Dual acting antioxidant A1 adenosine receptor agonists

Herein we report the synthesis and biological evaluation of some potent and selective A(1) adenosine receptor agonists, which incorporate a functionalised linker attached to an antioxidant moiety. N(6)-(2,2,5,5-Tetramethylpyrrolidin-1-yloxyl-3-ylmethyl)adenosine (VCP28, 2e) proved to be an agonist with high affinity (K(i)=50nM) and good selectivity (A(3)/A(1) > or = 400) for the A(1) adenosine receptor. N(6)-[4-[2-[1,1,3,3-Tetramethylisoindolin-2-yloxyl-5-amido]ethyl]phenyl]adenosine (VCP102, 5a) has higher binding affinity (K(i)=7 nM), but lower selectivity (A(3)/A(1)= approximately 3). All compounds bind weakly (K(i)>1 microM) to A(2A) and A(2B) receptors. The combination of A(1) agonist activity and antioxidant activity has the potential to produce cardioprotective effects.     

Isolation and characterization of mitochondrial F(1)-ATPase from crayfish (Orconectes virilis) gills

A soluble F(1)-ATPase was isolated from the mitochondria of crayfish (Orconectes virilis) gill tissue. The maximal mitochondrial disruption rate (95%) was obtained by sonicating for 4 min at pH 8.6. A 15-fold purification was estimated. The properties for both soluble and membrane-bound enzyme were studied. Both enzyme forms were stable at 4 to -70 degrees C when kept in 20% glycerol. Soluble F(1)-ATPase was more stable at room temperature than membrane-bound enzyme. It displayed a narrower pH profile (pK(1) =6.58, pK(2)=7.68) and more acid pH optimum (7.13) than membrane-bound enzyme (pK(1)=6.42, pK(2)=8.55, optimum pH 7.49). The anion-stimulated activities were in the order HCO(3)(-)>SO(4)(2-)>Cl(-). The apparent K(a) values for soluble enzyme were 11.4, 11.2, and 10.9 mM, respectively, but the K(a) of HCO(3)(-) for membrane-bound enzyme (14.9 mM) was higher than for soluble enzyme. Oligomycin and DCCD inhibited membrane-bound F(1)-ATPase with I(50) of 18.6 ng/ml and 2.2 microM, respectively, but were ineffective in inhibiting soluble enzyme. Both enzyme forms shared identical sensitivity to DIDS (I(50)=12.5 microM) and vanadate (I(50)=9.0 mM). Soluble ATPase was significantly more sensitive to pCMB (I(50)=0.15 microM) and NO(3)(-) (I(50)=28.6 mM) than membrane-bound enzyme (I(50)=1.04 microM pCMB and 81.5 mM NO(3)(-)). In addition, soluble F(1)-ATPase was slightly more sensitive to azide (I(50)=91.8 microM) and NBD-Cl (I(50)=9.18 microM) than membrane-bound enzyme (I(50)=111.6 microM azide and 12.88 microM NBD-Cl). These data suggest a conformational change transmission between F(0) and F(1) sectors and slight conformational differences between soluble F(1) and membrane-bound F(1). In addition, an unmodified F(0) stabilizes F(1) and decreases F(1) sensitivities to inhibitors and modulators.     

In vitro metabolism of zolmitriptan in rat cytochromes induced with beta-naphthoflavone and the interaction between six drugs and zolmitriptan

Zolmitriptan is a novel and highly selective 5-HT(1B/1D) receptor agonist used as an acute oral treatment for migraine. There are few reports regarding the in vitro metabolism of zolmitriptan. Previous studies indicated zolmitriptan was metabolized via CYP1A2 in human hepatic microsomes. In order to study the enzyme kinetics and drug interaction, the metabolism of zolmitriptan and possible drug-drug interactions were investigated in rat hepatic microsomes induced with different inducers. An active metabolite, N-demethylzolmitriptan, was detected and another minor, inactive metabolite that was reported in human hepatic microsomes was not detected in this study. The enzyme kinetics for the formation of N-demethylzolmitriptan from zolmitriptan in rat liver microsomes pretreated with BNF were 96+/-22 microM (K(m)), 11+/-3 pmol min(-1)mg protein(-1) (V(max)), and 0.12+/-0.02 microl min(-1)mg protein(-1) (CL(int)). Fluvoxamine and diphenytriazol inhibited zolmitriptan N-demethylase activity catalyzed by CYP1A2 (K(i)=3.8+/-0.3 and 3.2+/-0.1 microM, respectively). Diazepam and propranolol elicited a slight inhibitory effect on the metabolism of zolmitriptan (K(i)=70+/-11 and 90+/-18 microM, respectively). Cimetidine and moclobemide produced no significant effect on the metabolism of zolmitriptan. Fluvoxamine yielded a k(inactivation) value of 0.16 min(-1), and K(i) of 57 microM. The results suggest that rat hepatic microsomes are a reasonable model to study the metabolism of zolmitriptan, although there is a difference in the amount of minor, inactive metabolites between human hepatic microsomes and rat liver microsomes. The results of the inhibition experiments provided information for the interactions between zolmitriptan and drugs co-administrated in clinic, and it is helpful to explain the drug-drug interactions of clinical relevance on enzyme level. This study aso demonstrated that fluvoxamine may be a mechanism-based inactivator of CYP1A2.     

A simple two step procedure for purification of the catalytic domain of chicken tryptophan hydroxylase 1 in a form suitable for crystallization

Tryptophan hydroxylase (TPH) [EC 1.14.16.4] catalyzes the conversion of tryptophan to 5-hydroxytryptophan, which is the first and rate-determining step in the biosynthesis of the neurotransmitter serotonin. We have expressed the catalytic domain of chicken (Gallus gallus) TPH isoform 1 in Escherichia coli in high yield. The enzyme was highly purified using only one anion exchange and one gel filtration, with a yield of 11 mg/L culture and a specific activity of 0.60 micromol/min/mg. The K(m) values were determined to K(m, tryptophan)=7.7+/-0.7 microM, K(m, BH4)=324+/-10 microM and K(m, O2)=39+/-2 microM. Substrate inhibition by tryptophan was observed at concentrations above 15 microM. Furthermore, the purified enzyme has been crystallized without 7,8-dihydro-L-biopterin and a data set to 3A resolution has been collected.     

Kinetic properties of human placental glucose-6-phosphate dehydrogenase

The kinetic properties of placental glucose-6-phosphate dehydrogenase were studied, since this enzyme is expected to be an important component of the placental protection system. In this capacity it is also very important for the health of the fetus. The placental enzyme obeyed "Rapid Equilibrium Ordered Bi Bi" sequential kinetics with K(m) values of 40+/-8 microM for glucose-6-phosphate and 20+/-10 microM for NADP. Glucose-6-phosphate, 2-deoxyglucose-6-phosphate and galactose-6-phosphate were used with catalytic efficiencies (k(cat)/K(m)) of 7.4 x 10(6), 4.89 x 10(4) and 1.57 x 10(4) M(-1).s(-1), respectively. The K(m)app values for galactose-6-phosphate and for 2-deoxyglucose-6-phosphate were 10+/-2 and 0.87+/-0.06 mM. With galactose-6-phosphate as substrate, the same K(m) value for NADP as glucose-6-phosphate was obtained and it was independent of galactose-6-phosphate concentration. On the other hand, when 2-deoxyglucose-6-phosphate used as substrate, the K(m) for NADP decreased from 30+/-6 to 10+/-2 microM as the substrate concentration was increased from 0.3 to 1.5 mM. Deamino-NADP, but not NAD, was a coenzyme for placental glucose-6-phosphate dehydrogenase. The catalytic efficiencies of NADP and deamino-NADP (glucose-6-phosphate as substrate) were 1.48 x 10(7) and 4.80 x 10(6) M(-1)s(-1), respectively. With both coenzymes, a hyperbolic saturation and an inhibition above 300 microM coenzyme concentration, was observed. Human placental glucose-6-phosphate dehydrogenase was inhibited competitively by 2,3-diphosphoglycerate (K(i)=15+/-3 mM) and NADPH (K(i)=17.1+/-3.2 microM). The small dissociation constant for the G6PD:NADPH complex pointed to tight enzyme:NADPH binding and the important role of NADPH in the regulation of the pentose phosphate pathway.     

Protein tyrosine kinase-dependent modulation of voltage-dependent potassium channels by genistein in rat cardiac ventricular myocytes

Effects of the isoflavone protein tyrosine kinase (PTK) inhibitor genistein on voltage-dependent K(+) currents, i.e., transient outward K(+) current (I(to)), sustained K(+) current (I(ss)), and inward rectifier K(+) current (I(K1)) were studied in rat cardiac ventricular myocytes. It was found that I(to) was reversibly inhibited by genistein in a concentration-dependent manner (IC(50)=28.1 microM), while I(ss) was suppressed by genistein with IC(50) of 18.5 microM. In addition, I(K1) (at -50 mV) was significantly decreased by 36.3+/-4.4% with 25 microM genistein. The inhibition of I(to), I(ss), and I(K1) by genistein was significantly reversed by the application of the protein tyrosine phosphatase inhibitor sodium orthovanadate (1 mM). However, I(to), I(ss), and I(K1) were not affected by the non-isoflavone PTK inhibitor tyrphostin A23 (100 microM) and PP2 (1 microM). These results indicate that activation of I(to), I(ss), and I(K1) channels is modulated by genistein-sensitive PTKs in rat ventricular myocytes.     

A detailed kinetic study of Mox-1, a plasmid-encoded class C beta-lactamase

Surveys of beta-lactamases in different parts of the world show an important increase in class C beta-lactamases, thus the study of these enzymes is becoming an important issue. We created an overproduction system for Mox-1, a plasmid class C beta-lactamase, by cloning the gene encoding this enzyme, and placing it under the control of a T7 promoter, using vector pET 28a. The enzyme, purified by ion exchange chromatography, was used to obtain the molecular mass (38246), the N-terminal sequence (GEASPVDPLRPVV), and pI (8.9), and to perform a detailed kinetic study. Cephalotin was used as reporter substrate in the case of poor substrates. The kinetic study showed that benzylpenicillin, cephalotin, cefcapene and moxalactam were good substrates for Mox-1 (k(cat)/K(m) values >2.5 x 10(6) M(-1) s(-1)). On the other hand, ceftazidime and cefepime were poor substrates for this enzyme (K(m) values >200 microM). Clavulanic acid had no inhibitory effect on Mox-1 (K(m)=30.2 mM), however aztreonam behaved as an inhibitor of Mox-1 (K(i)=2.85 microM).