The raz1 mutant of Arabidopsis thaliana lacks the activity of a high-affinity amino acid transporter

The raz1 mutant of Arabidopsis thaliana (L.) Heynh. has been selected as resistant to the toxic proline analogue, azetidine-2-carboxylic acid (2AZ). Seedlings of the mutant tolerated fivefold higher concentrations of 2AZ (ED₅₀ = 0.25 mM) than the wild-type seedlings (ED⁵⁰ = 0.05 mM). The mutant gene was found to be semi-dominant and the corresponding RAZ1 locus was mapped on chromosome 5 at 69.6±1.8 cM. The resistance to 2AZ could be fully and exclusively accounted for by the lower uptake rate of the proline analogue in the mutant. The influx of L-proline in roots of wild-type seedlings could be dissected into two components: (i) a component with a high affinity and a low capacity for l-proline (K _m≈20 gmM, V _max≈60 nmol·(g FW)^-1·h^-1) and also a high affinity for L-2AZ (K _i≈40 μM) and (ii) a low-affinity, high-capacity component (K _m≈5 mM: V _max = 1300 nmol·(g FW)^-1·h^-1). Clearly, the raz1 mutation affects the activity of a high-affinity transporter, because the high-affinity uptake of proline in the mutant was at least fivefold lower than in the wild-type, whereas the low-affinity uptake was unchanged.     

Analysis of the <Emphasis Type="Italic">xynB5</Emphasis> gene encoding a multifunctional GH3-BglX β-glucosidase-β-xylosidase-α-arabinosidase member in <Emphasis Type="Italic">Caulobacter crescentus</Emphasis>

The Caulobacter crescentus (NA1000) xynB5 gene (CCNA_03149) encodes a predicted β-glucosidase-β-xylosidase enzyme that was amplified by polymerase chain reaction; the product was cloned into the blunt ends of the pJet1.2 plasmid. Analysis of the protein sequence indicated the presence of conserved glycosyl hydrolase 3 (GH3), β-glucosidase-related glycosidase (BglX) and fibronectin type III-like domains. After verifying its identity by DNA sequencing, the xynB5 gene was linked to an amino-terminal His-tag using the pTrcHisA vector. A recombinant protein (95 kDa) was successfully overexpressed from the xynB5 gene in E. coli Top 10 and purified using pre-packed nickel-Sepharose columns. The purified protein (BglX-V-Ara) demonstrated multifunctional activities in the presence of different substrates for β-glucosidase (pNPG: p-nitrophenyl-β-D-glucoside) β-xylosidase (pNPX: p-nitrophenyl-β-D-xyloside) and α-arabinosidase (pNPA: p-nitrophenyl-α-L-arabinosidase). BglX-V-Ara presented an optimal pH of 6 for all substrates and optimal temperature of 50 °C for β-glucosidase and α-l-arabinosidase and 60 °C for β-xylosidase. BglX-V-Ara predominantly presented β-glucosidase activity, with the highest affinity for its substrate and catalytic efficiency (K_m 0.24 ± 0.0005 mM, V_max 0.041 ± 0.002 µmol min^?1 mg^?1 and K_cat/K_m 0.27 mM^?1 s^?1), followed by β-xylosidase (K_m 0.64 ± 0.032 mM, V_max 0.055 ± 0.002 µmol min^?1 mg^?1 and K_cat/K_m 0.14 mM^?1s^?1) and finally α-l-arabinosidase (K_m 1.45 ± 0.05 mM, V_max 0.091 ± 0.0004 µmol min^?1 mg^?1 and K_cat/K_m 0.1 mM^?1 s^?1). To date, this is the first report to demonstrate the characterization of a GH3-BglX family member in C. crescentus that may have applications in biotechnological processes (i.e., the simultaneous saccharification process) because the multifunctional enzyme could play an important role in bacterial hemicellulose degradation.     

Evaluation of role of the peptide transport system in absorption of dipeptides in the rat small intestine in chronic experiments <Emphasis Type="Italic">in vivo</Emphasis>

Hydrolysis and absorption of glycylglycine and glycyl-L-leucine as well as absorption of glycine and leucine were studied in chronic experiments on rats with their isolated small intestine loop. Values of the “true” kinetic constants (with taking into account effect of the preepithelial layer) were determined to be as follows: (1) K _t = 46.7 ± 4.0 and 2.15 ± 0.59 mM, J _max = 0.74 ± 0.15 and 0.16 ± 0.03 μmol min^?1 cm^?1 (for transport of free glycine and leucine, respectively); (2) K _t = 4.4 ± 0.6 and 4.8 ± 0.9 mM, J _max = 0.24 ± 0.02 and 0.23 ± 0.02 μmol min^?1 cm^?1 (for transport of glycylglycine and glycyl-L-leucine, respectively); (3) K _M = 5.4 ± 1.0 and 38.2 ± 4.4 mM, V _max = 0.09 ± 0.02 and 0.24 ± 0.07 μmol min^?1 cm^?1 (for membrane hydrolysis of these dipeptides, respectively). According to our calculations, in the wide range of the initial glycylglycine concentrations (2.5–40 mM) a part of the peptide component in its total absorption accounts for 0.77–0.80. In the case of glycyl-L-leucine a part of the peptide component in the total glycine absorption decreases from 0.89 to 0.84, while in the total leucine absorption—from 0.86 to 0.71, the initial dipeptide concentration rising from 5 to 40 mM. The obtained results show that the peptide component prevails in absorption of the studied dipeptides in the rat small intestine, but its role is much lesser than what many authors believe. In the case of glycyl-L-leucine, the peptide component can achieve saturation in the range of high substrate concentrations, its part decreasing essentially to become compared with absorption of free amino acids formed as a result of the dipeptide membrane hydrolysis.     

Fasting and refeeding modulate neutral amino acid transport activity in the basolateral membrane of the rat exocrine pancreatic epithelium: fasting-induced insulin insensitivity

The effects of fasting and refeeding on amino acid transport in the perfused rat exocrine pancreas were investigated using a rapid dual tracer dilution technique. Unidirectional amino acid influx (15 s) was quantified (relative to the extracellular tracer d-mannitol) over a wide range of perfusate concentrations in pancreata isolated frm fed and 24 h, 48 h, and 72 h fasted and 72 h fasted and refed (24 h) animals. In fed animals transport of phenylalamine (1–24 mM) and l-serine (1–50 mM) was saturable and weighted non-linear regression analyses of the overall transport indicated an apparent K_t=10±3mM and V_max=7.0±1.0 μmol/min per g (n = 7) for phenylalanine and K_t=16±3 mM and V_max=20.6±2.1 μmol/min per g (n = 5) for serine. Fasting animals for 24 h or 48 h did not change the kinetics of either phenylalanine or serine transport. After a 72 h fast the rate of phenylalanine transport (V_max=15.9±2.9 μmol/min per g, (n = 5) was enhanced whereas the transport affinity (K_t=11±3 mM) remained unaltered. l-Serine transport was essentially unaltered. When 72 h fasted animals were refed for 24 h the V_max for the phenylalanine transport was reduced to values observed in fed animals. In parallel experiments refeeding had no significant effect on serine transport. Perfusion of pancreata isolated from 72 h fasted animals with bovine insulin (1 mU/ml or 1 μU/ml) did not stimulate either phenylalanine or serine transport. The fasting-induced stimulation of transport may provide a mechanism by which the extracellular supply of essential amino acids as phenylalanine is increased to meet the demands of continued proteolytic and lipolytic enzyme synthesis.     

Catalytic properties of <Emphasis Type="Italic">Rhodotorula aurantiaca</Emphasis> KM-1 phenylalanine ammonia-lyase

L-Phenylalanine ammonia-lyase (PAL, EC 4.3.1.5) of the Rhodotorula aurantiaca strain KM-1 deaminates L-phenylalanine according to the Michaelis-Menten kinetics with K _M 1.75 ± 0.44 mM and V _max 3.01 ± 0.43 units/mg. The enzyme is competitively inhibited by D-phenylalanine with K _i 3.38 ± 0.32 mM. The Michaelis-Menten kinetics was analyzed, the inhibition type (competitive, noncompetitive, and mixed) was identified, and corresponding kinetic parameters were calculated using the computer programs written in Gauss 4.0. PAL was most stable at pH 6.55 and lacked approximately 50% of its activity after incubation at 57°C for 15 min. The yield of L-phenylalanine increased in the presence of mercaptoethanol, sodium ethylenediaminetetraacetate (EDTA), and ascorbic acid. The effects of EDTA and ascorbic acid were additive.     

Trypanosoma lewisi: alterations in membrane function in the rat.

DEAE-cellulose-purified Trypanosoma lewisi from 4-day (dividing trypanosomes) and 7-day (non-dividing trypanosomes) infections in rats were compared for initial uptake of glucose, leucine, and potassium. Glucose entered the parasitic cells by mediated (saturable) processes, whereas leucine and K⁺ entered by mediated processes and diffusion. Glucose entry was significantly elevated in 4-day cells (V_max 4.00 ± 1.02 nmoles/ 1 × 10⁸ cells/min) with respect to 7-day cells (V_max 1.83 ± 0.62 nmoles 1 × 10⁸ cells/min). Likewise, the affinity of the glucose carrier was significantly greater in 4-day cells (K_m = 0.30 ± 0.02 mM) than in 7-day cells (K_m = 0.59 ± 0.11 mM). When leucine and K⁺ transport were compared in 4- and 7-day populations, significant elevations in the rate of entry (V_max) of both substrates were observed for 4-day cells; K_m values for leucine and K⁺ were not altered by the stage of infection. For leucine, the V_max and K_m for 4-day cells were 2.40 ± 0.50 nmoles/1 × 10⁸ cells/30 sec and 78 ± 7 μM, respectively; corresponding values in 7-day cells were 1.06 ± 0.02 nmoles/1 × 10⁸ cells/30 sec and 66 ± 11 μM. For K⁺, the V_max and K_m for 4-day cells were 15.97 ± 0.38 nmoles/1 × 10⁸ cells/min and 1.2 mM, respectively; corresponding values in 7-day cells were 4.76 ± 1.82 nmoles/1 × 10⁸ cells/min and 1.05 mM. The observed increase in the rate of K⁺ entry into 4-day cells was attributable to enhanced influx; no significant difference in the rate of K⁺ efflux was noted when 4- and 7-day cells were compared ($\text{t}{}_{\mathrm{<mtext>1</mtext><mtext>2</mtext>}}$ of K⁺ leak for 4- and 7-day cells were 68.1 ± 9.3 and 67.9 ± 15.2 min, respectively). Potassium influx was ouabain insensitive. Membrane function in 7-day cells was not uniformly inhibited. No significant difference in the activity of the membrane-bound enzyme, 5′-nucleotidase, was observed when 4- and 7-day cells were compared.     

Catalytic properties of aminoacylase of strain <Emphasis Type="Italic">Rhodococcus armeniensis</Emphasis> AM6.1

Studies of substrate specificity revealed that the D-aminoacylase of Rhodococcus armeniensis AM6.1 strain exhibits absolute stereospecificity to the D-stereoisomers of N-acetyl-amino acids. The enzyme is the most active reacted with N-acetyl-D-methionine, as well as with aromatic and hydrophobic N-acetylamino acids and interacts weakly with the basic substrates. It is practically not reacted with acidic and hydrophilic N-acetyl-amino acids. Michaelis constants (K_m) and maximum reaction velocities (V_max) were calculated, using linear regression analysis, for the following substrates: N-acetyl-D-methionine, N-acetyl-D-alanine, N-acetyl-D-phenylalanine, N-acetyl-D-tyrosine, N-acetyl-D-valine, N-acetyl-D-oxyvaline, N-acetyl- D-leucine. Substrate inhibition of D-aminoacylase was displayed with N-acetyl-D-leucine (K_s = 35.5 ± 28.3 mM) and N-acetyl-DL-tyrosine (K_s = 15.8 ± 4.5 mM). Competitive inhibition of the enzyme with product–acetic acid (K_i = 104.7 ± 21.7 mM, K_m = 2.5 ± 0.5 mM, V_max = 25.1 ± 1.5 U/mg) was observed.     

Kinetic Studies on the Inhibition of GABA-T by γ-Vinyl GABA and Taurine

γ-Aminobutyric acid transaminase (GABA-T, EC 2.6.1.19) is a pyridoxal phosphate (PLP) dependent enzyme that catalyzes the degradation of γ-aminobutyric acid. The kinetics of this reaction are studied in vitro, both in the absence, and in the presence of two inhibitors: γ-vinyl GABA (4-aminohex-5-enoic acid), and a natural product, taurine (ethylamine-2-sulfonic acid). A kinetic model that describes the transamination process is proposed. GABA-T from Pseudomonas fluorescens is inhibited by γ-vinyl GABA and taurine at concentrations of 51.0 and 78.5?mM. Both inhibitors show competitive inhibition behavior when GABA is the substrate and the inhibition constant (K_i) values for γ-vinyl GABA and taurine were found to be 26±3?mM and 68±7?mM respectively. The transamination process of α-ketoglutarate was not affected by the presence of γ-vinyl GABA, whereas, taurine was a noncompetitive inhibitor of GABA-T when α-ketoglutarate was the substrate. The inhibition dissociation constant (K_ii) for this system was found to be 96±10?mM. The Michaelis-Menten constant (K_m) in the absence of inhibition, was found to be 0.79±0.11?mM, and 0.47±0.10?mM for GABA and α-ketoglutarate respectively.     

Ligand-induced conformations of rat brain hexokinase: studies with hexoses, hexose 6-phosphates, and nucleoside triphosphates leading to development of a new model for the enzyme

Various ligands of rat brain hexokinase (ATP:d-hexose 6-phosphotransferase, EC 2.7.1.1) have been found to protect the enzyme against either (or both) chymotryptic digestion or inactivation by glutaraldehyde. Using this protective effect, the K_d for various enzyme-ligand complexes has been estimated: hexokinase-Glc, K_d = 0.24 ± 0.03mM (chymotryptic digestion), K_d = 0.26 ± 0.07mM (glutaraldehyde inactivation); hexokinase-Glc-6- P, K_d = 0.041 ± 0.005m M (glutaraldehyde inactivation); hexokinase-ATP, K_d = 1.01 ± 0.28mM (chymotryptic digestion); hexokinase-ATP-Mg ²⁺, K_d = 0.07-0.08mM (chymotryptic digestion). Other nucleoside triphosphates (UTP, ITP, GTP, and CTP) were much less effective than ATP at protecting against chymotrypsin. Various hexoses were tested for their ability to protect against glutaraldehyde. Only ?good” substrates (mannose, 2-deoxyglucose) protected; nonsubstrates (galactose, arabinose) and N-acetylglucosamine, a competitive inhibitor of Glc binding, were not effective. Various hexose 6-phosphates were tested for their ability to protect against glutaraldehyde inactivation. Glc-6-P was much more effective than were mannose-6-P, galactose-6-P, or fructose-6-P. It was observed that ?good” substrates (Glc, mannose) increased the effectiveness of Glc-6-P at solubilizing the mitochondrial form of the enzyme; galactose and N-acetylglucosamine had no effect on solubilization by Glc-6-P. These results are taken as an indication of enhanced Glc-6-P binding in the presence of Glc, as previously reported by Ellison et al. (J. Biol. Chem., 250, 1864–1871, 1975). Along with previous studies on ligand-induced conformations and kinetics of this enzyme, these results form the basis for a new model for brain hexokinase. This model specifically takes into account the ligand-induced conformations at various points in the catalytic cycle and specifically accounts for the ability of various hexoses to serve as substrates and hexose 6-phosphates to serve as inhibitors in terms of their ability to induce specific conformations of the enzyme. The properties of the various conformations involved in the model are designated by a four-letter code which facilitates comparison and discussion.     

Effect of zinc and calcium ions on the rat kidney membrane-bound form of dipeptidyl peptidase IV

Dipeptidyl peptidase IV (DPP-IV) is an ectopeptidase with many roles, and a target of therapies for different pathologies. Zinc and calcium produce mixed inhibition of porcine DPP-IV activity. To investigate whether these results may be generalized to mammalian DPP-IV orthologues, we purified the intact membrane-bound form from rat kidney. Rat DPP-IV hydrolysed Gly-Pro-p-nitroanilide with an average V_max of 0.86±0.01 μmol min^–1mL^–1 and K_M of 76±6 μM. The enzyme was inhibited by the DPP-IV family inhibitor l-threo-Ile-thiazolidide (K_i=64.0±0.53 nM), competitively inhibited by bacitracin (K_i=0.16±0.01 mM) and bestatin (K_i=0.23±0.02 mM), and irreversibly inhibited by TLCK (IC₅₀ value of 1.20±0.11 mM). The enzyme was also inhibited by divalent ions like Zn²⁺ and Ca²⁺, for which a mixed inhibition mechanism was observed (K_i values of the competitive component: 0.15±0.01 mM and 50.0±1.05 mM, respectively). According to bioinformatic tools, Ca²⁺ ions preferentially bound to the β-propeller domain of the rat and human enzymes, while Zn²⁺ ions to the α-β hydrolase domain; the binding sites were essentially the same that were previously reported for the porcine DPP-IV. These data suggest that the cationic susceptibility of mammalian DPP-IV orthologues involves conserved mechanisms.     

Modulation of alfa-adrenoceptor activity by beta-adrenoceptor agonists and antagonists in rat brain cortex membranes

The influence of β-adrenoceptor activation and inhibition by isoprenaline and propranolol on the specific binding of nonselective α₁- and α₂-adrenoceptor antagonists [³H]prazosin and [³H]RX821002 in rat cerebral cortex subcellular membrane fractions was studied. It was established that for the α₁- and α₂-adrenoceptors the ligand–receptor interaction corresponds to the model of one affinity pool of receptors and binding of two ligand molecules by one dimer receptor. The parameters of [³H]prazosin binding to α₁-adrenoceptors were: K _d = 1.85 ± 0.16 nM, B _max = 31.14 ± 0.35 fmol/mg protein, n = 2. The parameters of [³H]RX821002 binding to α₂-adrenoceptors were: K _d = 1.57 ± 0.27 nM, B _max = 7.2 ± 1.6 fmol/mg protein, n = 2. When β-adrenoceptors were activated by isoprenaline, the binding of radiolabelled ligands with α₁- and α₂-adrenoceptors occurred according to the same model. The affinity to [³H]prazosin and the concentration of active α₁-adrenoceptors increased by 27% (K _d = 1.36 ± 0.03 nM) and 84% (B _max = 57.37 ± 0.28 fmol/mg protein), respectively. The affinity of α₂-adrenoceptors to [³H]RX821002 decreased by 56% (K _d = 3.55 ± 0.02 nM), and the concentration of active receptors increased by 69% (B _max = 12.24 ± 0.06 fmol/mg protein). Propranolol alters the binding character of both ligands. For [³H]prazosin and [³H]RX821002, two pools of receptors were detected with the following parameters: K _d1 = 1.13 ± 0.09, K _d2 = 6.07 ± 1.06 nM, B _m1 = 11.36 ± 1.77, Bm2 = 51.09 ± 0.41 fmol/mg protein, n = 2 and K _d1 = 0.61 ± 0.02, K _d2 = 3.41 ± 0.13 nM, B _m1 = 1.88 ± 0.028, B _m2 = 9.27 ± 0.08 fmol/mg protein, n = 2, respectively. The concentration of active receptors (B _max) increased twofold for both ligands. It was suggested that α₁- and α₂-adrenoceptors in rat cerebral cortex subcellular membrane fractions exist as dimers. A modulating influence of isoprenaline and propranolol on the specific binding of the antagonists to α₁- and α₂- adrenoceptors was revealed, which was manifested in the activating effect on the [³H]prazosin binding parameters, in the inhibitory effect on the [³H]RX821002 binding parameters, and in a change of the general character of binding for both ligands.     

ATP and ADP hydrolysis in cell membranes from rat myometrium

Extracellular nucleotides affect female reproductive functions, fertilization, and pregnancy. The aim of this study was to investigate biochemical characteristics of ATP and ADP hydrolysis and identify E-NTPDases in myometrial cell membranes from Wistar albino rats. The apparent K _m values were 506.4?±?62.1 and 638.8?±?31.3?μM, with a calculated V _max (app) of 3,973.0?±?279.5 and 2,853.9?±?79.8?nmol/min/mg for ATP and ADP, respectively. The enzyme activity described here has common properties characteristic for NTPDases: divalent cation dependence; alkaline pH optimum for both substrates, insensitivity to some of classical ATPase inhibitors (ouabain, oligomycine, theophylline, levamisole) and significant inhibition by suramine and high concentration of sodium azides (5?mM). According to similar apparent K_m values for both substrates, the ATP/ADP hydrolysis ratio, and Chevillard competition plot, NTPDase1 is dominant ATP/ADP hydrolyzing enzyme in myometrial cell membranes. RT-PCR analysis revealed expression of three members of ectonucleoside triphosphate diphosphohydrolase family (NTPDase 1, 2, and 8) in rat uterus. These findings may further elucidate the role of NTPDases and ATP in reproductive physiology.     

Identification and characterization of a 20β-HSDH from the anaerobic gut bacterium Butyricicoccus desmolans ATCC 43058

Members of the gastrointestinal microbiota are known to convert glucocorticoids to androstanes, which are subsequently converted to potent androgens by other members of the gut microbiota or host tissues. Butyricicoccus desmolans and Clostridium cadaveris have previously been reported for steroid-17,20-desmolase and 20β-hydroxysteroid dehydrogenase (HSDH) activities that are responsible for androstane formation from cortisol; however, the genes encoding these enzymes have yet to be reported. In this work, we identified and located a gene encoding 20β-HSDH in both B. desmolans and C. cadaveris. The 20β-HSDH of B. desmolans was heterologously overexpressed and purified from Escherichia coli. The enzyme was determined to be a homotetramer with subunit molecular mass of 33.8 ± 3.7 kDa. The r20β-HSDH displayed pH optimum in the reductive direction at pH 9.0 and in the oxidative direction at pH 7.0–7.5 with (20β-dihydro)cortisol and NAD(H) as substrates. Cortisol is the preferred substrate with K_m, 0.80 ± 0.06 μM; V_max, 30.36 ± 1.97 μmol·min⁻¹; K_cat, 607 ± 39 μmol·μM⁻¹·min⁻¹; K_cat/K_m, 760 ± 7.67. Phylogenetic analysis of the 20β-HSDH from B. desmolans suggested that the 20β-HSDH is found in several Bifidobacterium spp., one of which was shown to express 20β-HSDH activity. Notably, we also identified a novel steroid-17,20-desmolase-elaborating bacterium, Propionimicrobium lymphophilum, a normal inhabitant of the urinary tract.     

Methylenebisphosphonate and triphosphate derivatives of the mevalonate pathway are substrates of yeast UTP:glucose-1-phosphate uridylyltransferase

UTP:glucose-1-phospate uridylyltransferase (EC 2.7.7.9) from Saccharomyces cerevisiae transfers the uridylyl moiety of UDP-glucose onto methylenebisphosphonate (pCH₂p) yielding uridine 5′-(β,γ-methylenetriphosphate) (UppCH₂p). The following bisphosphonates were not acceptors of UMP: alendronate, pamidronate, clodronate and etidronate. UDP-glucose serves as uridylyl donor to triphosphate derivatives of the mevalonate pathway: farnesyl (far-PPP), geranyl (ger-PPP) and isopentenyl (iso-PPP), with formation of farnesyl-tetraphosphouridine (far-ppppU); geranyl-tetraphosphouridine (ger-ppppU) and isopentenyl-tetraphosphouridine (iso-ppppU). The K_m (mM) and V_max (mU/mg protein) values determined for these substrates were: 0.32 ± 0.07 and 4.9 ± 0.6; 0.21 ± 0.06 and 5.7 ± 0.8; 0.51 ± 0.14 and 2.0 ± 0.2, respectively. The K_m and V_max values for methylenebisphosphonate were 1.1 ± 0.2 mM and 4055 ± 96 mU/mg protein, respectively.     

Transport of hemicellulose monomers in the xylose-fermenting yeastCandida shehatae

Summary Cells ofCandida shehatae repressed by growth in glucose- or D-xylose-medium produced a facilitated diffusion system that transported glucose (K _s±2 mM,V _max±2.3 mmoles g⁻¹ h⁻¹),d-xylose (K _s±125 mM,V _max±22.5 mmoles g⁻¹ h⁻¹) and D-mannose, but neither D-galactose norl-arabinose. Cells derepressed by starvation formed several sugar-proton symports. One proton symport accumulated 3-0-methylglucose about 400-fold and transported glucose (K _s±0.12 mM,V _max ± 3.2 mmoles g⁻¹ h⁻¹) andd-mannose, a second proton symport transportedd-xylose (K _s± 1.0 mM,V _max 1.4 mmoles g⁻¹ h⁻¹) andd-galactose, whilel-arabinose apparently used a third proton symport. The stoicheiometry was one proton for each molecule of glucose or D-xylose transported. Substrates of one sugar proton symport inhibited non-competitively the transport of substrates of the other symports. Starvation, while inducing the sugar-proton symports, silenced the facilitated diffusion system with respect to glucose transport but not with respect to the transport of D-xylose, facilitated diffusion functioning simultaneously with thed-xylose-proton symport.     

Investigation of the effects of some sulfonamide derivatives on the activities of glucose-6-phosphate dehydrogenase, 6-phospho gluconate dehydrogenase and glutathione reductase from human erythrocytes

In this study, the in vitro effects of some sulfonamide derivatives, which are carbonic anhydrase inhibitors, on the enzymes activities of glucose-6-phosphate dehydrogenase, 6-phospho gluconate dehydrogenase and glutathione reductase were investigated. For this purpose, these three enzymes were purified from human erythrocytes. Purification procedure composed of four steps; preparation of the hemolysate, ammonium sulfate precipitation, 2′,5′-ADP Sepharose 4B affinity chromatography, and gel filtration chromatography on Sephadex G-200. 5-(3α-Hydroxy-5-β-cholanamido)-1,3,4-thiadiazole-2-sulfonamide (1), 5-(3α,12α-Dihydroxy-5-β-cholanamido)-1,3,4-thiadiazole-2-sulfonamide (2), 5-(3α,7α,12α-Trihydroxy-5-β-cholanamido)-1,3,4-thiadiazole-2-sulfonamide (3), 5-(3α,Acetoxy-5-β-cholanamido)-1,3,4-thiadiazole-2-sulfonamide (4), 5-(3α,7α,12α-Triacetoxy-5-β-cholanamido)-1,3,4-thiadiazole-2-sulfonamide (5), 5-(3,7,12-Trioxo-5-β-cholanamido)-1,3,4-thiadiazole-2-sulfonamide (6), acetazolamide, and dorzolamide were tested in this experiment. Compounds 3, 5, and dorzolamide showed inhibitory effects on the activity of 6-phosphogluconate dehydrogenase, and I₅₀ values and K_i constants were calculated as 0.0601 mM, 0.00253 mM, and 1.41 mM and 0.0878 ± 0.0274 mM, 0.0042 ± 0.0009 mM, and 3.1446 ± 0.2081 mM, respectively. Glutathione reductase was also inhibited by 1 and 2. I₅₀ values and K_i constants were 0.0471 mM and 0.0723 ± 0.0388 mM for 1 and 0.0045 mM and 0.0061 ± 0.0014 mM, for 2. If these sulfonamide derivatives are proposed as drugs, some of which are being used in glaucoma treatment such as acetazolamide and dorzolamide, these results should be taken into consideration concerning via these enzymes.     

Heterologous expression and characterization of a proxidomal ascorbate peroxidase from <Emphasis Type="Italic">Populus tomentosa</Emphasis>

The present study reported for the first time, cloning, expression and characteristics of a Proxidomal APX gene (PpAPX) from Populus tomentosa. The PpAPX gene encodes a protein of 287 amino acid residues with a calculated molecular mass of 31.58 kDa. The over-expressed recombinant PpAPX protein showed high activity towards the substrates ascorbate aicd (ASA) and H₂O₂. At fixed ASA concentrations, the K _m and V _max values were 0.12 ± 0.01 mM and 23.4 ± 4.2 mmol/min mg for H₂O₂. And at fixed H₂O₂ concentrations, the K _m and V _max values were 0.53 ± 0.04 mM and 20.0 ± 2.3 mmol/min mg for ASA.     

In Vitro Functional Characterisation of Cytochrome P450 (CYP) 2C19 Allelic Variants <Emphasis Type="Italic">CYP2C19*23</Emphasis> and <Emphasis Type="Italic">CYP2C19*24</Emphasis>

Cytochrome P450 (CYP) 2C19 is essential for the metabolism of clinically used drugs including omeprazole, proguanil, and S-mephenytoin. This hepatic enzyme exhibits genetic polymorphism with inter-individual variability in catalytic activity. This study aimed to characterise the functional consequences of CYP2C19*23 (271 G>C, 991 A>G) and CYP2C19*24 (991 A>G, 1004 G>A) in vitro. Mutations in CYP2C19 cDNA were introduced by site-directed mutagenesis, and the CYP2C19 wild type (WT) as well as variants proteins were subsequently expressed using Escherichia coli cells. Catalytic activities of CYP2C19 WT and those of variants were determined by high performance liquid chromatography-based essay employing S-mephenytoin and omeprazole as probe substrates. Results showed that the level of S-mephenytoin 4′-hydroxylation activity of CYP2C19*23 (V _max 111.5 ± 16.0 pmol/min/mg, K _m 158.3 ± 88.0 μM) protein relative to CYP2C19 WT (V _max 101.6 + 12.4 pmol/min/mg, K _m 123.0 ± 19.2 μM) protein had no significant difference. In contrast, the K _m of CYP2C19*24 (270.1 ± 57.2 μM) increased significantly as compared to CYP2C19 WT (123.0 ± 19.2 μM) and V _max of CYP2C19*24 (23.6 ± 2.6 pmol/min/mg) protein was significantly lower than that of the WT protein (101.6 ± 12.4 pmol/min/mg). In vitro intrinsic clearance (CL_int = V _max/K _m) for CYP2C19*23 protein was 85.4 % of that of CYP2C19 WT protein. The corresponding CL_int value for CYP2C19*24 protein reduced to 11.0 % of that of WT protein. These findings suggested that catalytic activity of CYP2C19 was not affected by the corresponding amino acid substitutions in CYP2C19*23 protein; and the reverse was true for CYP2C19*24 protein. When omeprazole was employed as the substrate, K _m of CYP2C19*23 (1911 ± 244.73 μM) was at least 100 times higher than that of CYP2C19 WT (18.37 ± 1.64 μM) and V _max of CYP2C19*23 (3.87 ± 0.74 pmol/min/mg) dropped to 13.4 % of the CYP2C19 WT (28.84 ± 0.61 pmol/min/mg) level. Derived from V _max/K _m, the CL_int value of CYP2C19 WT was 785 folds of CYP2C19*23. K _m and V _max values could not be determined for CYP2C19*24 due to its low catalytic activity towards omeprazole 5′-hydroxylation. Therefore, both CYP2C19*23 and CYP2C19*24 showed marked reduced activities of metabolising omeprazole to 5-hydroxyomeprazole. Hence, carriers of CYP2C19*23 and CYP2C19*24 allele are potentially poor metabolisers of CYP2C19-mediated substrates.     

Inhibition of dopamine β-hydroxylase by bidentate chelating agents

1-2H-Phthalazine hydrazone (hydralazine; HYD), 2-1H-pyridinone hydrazone (2-hydrazinopyridine; HP), 2-quinoline-car☐ylic acid (QCA), 1-isoquinolinecar☐ylic acid (IQCA), 2,2′-bi-1H-imidazole (2,2′-biimidazole; BI), and 1H-imidazole-4-acetic acid (imidazole-4-acetic acid; IAA) directly and reversibly inhibit homogeneous soluble bovine dopamine β-hydroxylase (3,4-dihydroxyphenethylamine, ascorbate:oxygen oxidoreductase (β-hydroxylating), EC 1.14.17.1). HYD, QCA and IAA show competitive allosteric inhibition of dopamine β-hydroxylase with respect to ascorbate (K_is = 5.7(±0.9) μM, 0.14(±0.03) mM, 0.80(±0.20) mM; n_H= 1.4(±0.1), 1.8(±0.4), 2.8(±0.6), respectively). HYD and IAA show slope and intercept mixed-type allosteric inhibition of dopamine β-hydroxylase with respect to tyramine. QCA shows allosteric uncompetitive inhibition of dopamine β-hydroxylase with respect to tyramine. HP, BI and IQCA all show linear competitive inhibition (K_is = 1.9(±0.3) μM, 21(±6) μM, and 0.9(±0.3) μM, respectively) with respect to ascorbate. HP and BI show linear mixed-type while IQCA shows linear uncompetitive inhibition of dopamine β-hydroxylase with respect to tyramine. In the presence of HP, HYD or IAA intersecting double-reciprocal plots of the initial velocity as a function of tyramine concentration at differing fixed levels of ascorbate are observed. These findings are consistent with a uni-uni-ping-pong-ter-bi kinetic mechanism for dopamine β-hydroxylase that involves a ternary enzyme-ascorbate-tyramine-oxygen complex. The results for HYD, QCA and IAA are the first examples of allosteric inhibitor interactions with dopamine β-hydroxylase.     

Identity of β-alanine-oxo-glutarate aminotransferase and L-β-aminoisobutyrate aminotransferase in rat liver

L-β-Aminoisobutyrate served as an amino donor for purified β-alanine-oxo-glutarate aminotransferase from rat liver when 2-oxoglutarate was employed as an amino acceptor, but he D-isomer did not. L-β-Aminoisobutyrate acted as a competitive inhibitor with respect to β-alanine and had a K_i of approximately 2.6 mM, which is the same value as the K_m of 2.7 mM. When the crude extract was applied to a DEAE-Sepharose CL-6B column, L-β-aminoisobutyrate aminotransferase and β-alanine-oxo-glutarate aminotransferase activities were found in the same fractions with a single peak. Antiserum to rat liver β-alanine-oxo-glutarate aminotransferase inhibited L-β-aminoisobutyrate aminotransferase activity in rat liver in the same way as β-alanine-oxo-glutarate aminotransferase activity.