A new silver sulfadiazine water soluble gel

Silver sulfadiazine is the most commonly used topical antibacterial agent for the treatment of burn wounds. It has many clinical advantages, including a broad spectrum of antimicrobial activity, low toxicity, and minimal pain on application. The current formulation of silver sulfadiazine contains a lipid soluble carrier, polypropylene glycol, that has certain disadvantages, including pseudo-eschar formation and the need for twice daily application. The purpose of this investigation was to describe a new formulation of silver sulfadiazine in a water soluble gel, poloxamer 188. The antibacterial activity of this new gel has been compared to that of the commercially available silver sulfadiazine cream by in vitro and in vivo testing. The results of the in vitro antibacterial testing of these two different agents demonstrated the superiority of the new gel formulation. In experimental wounds, the antibacterial activity of the gel and the commercially available silver sulfadiazine cream were not significantly different when applied once a day. The antibacterial activity of the gel when applied once a day was comparable to that encountered by twice daily applications of the silver sulfadiazine cream by experimental wounds. The major advantage of this gel was its ease of application and removal that is attributed to its water solubility.     

Characterization of the independent and combined effects of two inhibitors on oxidative drug metabolism in rat liver microsomes

To evaluate how two inhibitors influence oxidative drug metabolism, this study investigated the inhibitory effects of mexiletine with cimetidine and mexiletine with lidocaine, both individually and in combination, on the oxidative metabolism of two probe substrates, aminopyrine and aniline in rat liver microsomes. Mexiletine was a competitive inhibitor of aminopyrine N-demethylation, whereas cimetidine was a mixed type of inhibitor (Ki = 2.00 +/- 0.04 and 0.20 +/- 0.02 mM, respectively). For aniline hydroxylation, mexiletine exhibited a mixed type of inhibition, whereas lidocaine was a noncompetitive inhibitor (Ki = 0.60 +/- 0.07 and 8.50 +/- 0.12 mM, respectively). The combined inhibition of either mexiletine with cimetidine or mexiletine with lidocaine on aminopyrine and aniline metabolism was close to the fully additive effects of the individual compounds when their individual concentrations were below a 2-fold Ki concentration, regardless of the apparent kinetic inhibition type. The combined inhibition was less than fully additive when the individual concentrations were twice the Ki or above. These results demonstrate that, when two inhibitors of oxidative drug metabolism are combined, both the Ki values and the concentrations of inhibitors play important roles in determining the extent of additive inhibition of enzyme activity.     

Alkaline phosphatase (EC 3.1.3.1) in serum is inhibited by physiological concentrations of inorganic phosphate

Natural and artificial manipulation of tissue nonspecific alkaline phosphatase activity indicates that pyrophosphate, phosphoethanolamine, and pyridoxal 5'-phosphate are among the natural substrates for this enzyme. Although inorganic phosphate has been recognized as a competitive inhibitor of this enzyme for many years, the influence of phosphate on alkaline phosphatase activity in serum under physiological conditions has not been previously reported. We examined the kinetics of tissue nonspecific alkaline phosphatase from bovine kidney and sera from 49 patients with a wide range of endogenous phosphate concentrations using pyridoxine 5'-phosphate as a substrate at pH 7.4. For the bovine kidney enzyme, the Km was 0.42 +/- 0.04 micromol/L, and the Ki for phosphate was 2.4 +/- 0.2 micromol/L. Analysis of the kinetics using pyridoxine 5'-phosphate in undiluted serum from 10 subjects with phosphorus ranging from 0.5-2.1 mmol/L and alkaline phosphatase activity ranging from 41-165 nmol/min x mL gave estimates for the Km of 56 +/- 11 micromol/L and for the Ki of 540 +/- 82 micromol/L for phosphate. This indicates that under physiological conditions alkaline phosphatase activity toward pyridoxine 5'-phosphate is reduced approximately 50% by the normal phosphate concentration and that it will increase or decrease significantly in response to changes in phosphate concentration within the ranges observed clinically.     

Ornithine decarboxylase from Leishmania mexicana promastigotes: interaction with pyridoxal 5'-phosphate and alpha-difluoromethylornithine

The catalytic properties of ornithine decarboxylase (ODC) from Leishmania mexicana as well as the interaction with its cofactor pyridoxal 5'-phosphate (PLP) and the irreversible inhibitor alpha-difluoromethylornithine (DFMO) have been studied using partially purified preparations of the enzyme obtained from parasite promastigotes. Leishmania extracts prepared in the presence of saturating concentrations of PLP yielded an enzyme considerably more resistant to heat inactivation and with a three-fold higher activity than the ODC obtained without the addition of cofactor. The complete removal of PLP by treatment with hydroxylamine yielded the apoenzyme which shows an absolute requirement for PLP to recover its enzymatic activity. The Km values for L-ornithine and PLP were 0.7 mM and 25 microM, respectively, while Ki for DFMO was 0.2 mM. The restoration of ODC activity from apoenzyme and cofactor seems to involve time and temperature-dependent activation processes. L. mexicana ODC has an apparent molecular mass of 240 +/- 20 kDa.     

Amino acid sequence of piguamerin, an antistasin-type protease inhibitor from the blood sucking leech Hirudo nipponia

Dimethylamine:5-hydroxybenzimidazolylcobamide methyltransferase (DMA-MT) was purified from cells of Methanosarcina barkeri Fusaro grown on trimethylamine. In the presence of methylcobalamine:coenzyme M methyltransferase isoenzyme II [MT2(II)] the enzyme quite specifically catalyzed the stoichiometric conversion of dimethylamine (apparent Km = 0.45 mM) and 2-mercaptoethane-sulfonate (coenzyme M) to monomethylamine and methyl-coenzyme M. Monomethylamine was a competitive inhibitor of the reaction (Ki = 4.5 mM). The apparent molecular mass of DMA-MT was 100 kDa and the enzyme was found to be a dimer, composed of identical 50-kDa subunits. A corrinoid content of 0.9 +/- 0.1 mol B12/mol holoenzyme was calculated from HPLC analysis. The as-isolated methyltransferase was inactive, but it could be reductively reactivated. Activation required the presence of methyltransferase-activating protein, ATP and dimethylamine. Incubation with these compounds resulted in the methylation of the corrinoid prosthetic group.     

Anandamide hydrolysis by human cells in culture and brain

Anandamide (arachidonylethanolamide; AnNH) has important neuromodulatory and immunomodulatory activities. This lipid is rapidly taken up and hydrolyzed to arachidonate and ethanolamine in many organisms. As yet, AnNH inactivation has not been studied in humans. Here, a human brain fatty-acid amide hydrolase (FAAH) has been characterized as a single protein of 67 kDa with a pI of 7.6, showing apparent Km and Vmax values for AnNH of 2.0 +/- 0.2 microM and 800 +/- 75 pmol.min-1.mg of protein-1, respectively. The optimum pH and temperature for AnNH hydrolysis were 9.0 and 37 degreesC, respectively, and the activation energy of the reaction was 43.5 +/- 4.5 kJ.mol-1. Hydro(pero)xides derived from AnNH or its linoleoyl analogues by lipoxygenase action were competitive inhibitors of human brain FAAH, with apparent Ki values in the low micromolar range. One of these compounds, linoleoylethanolamide is the first natural inhibitor (Ki = 9.0 +/- 0.9 microM) of FAAH as yet discovered. An FAAH activity sharing several biochemical properties with the human brain enzyme was demonstrated in human neuroblastoma CHP100 and lymphoma U937 cells. Both cell lines have a high affinity transporter for AnNH, which had apparent Km and Vmax values for AnNH of 0.20 +/- 0.02 microM and 30 +/- 3 pmol.min-1.mg of protein-1 (CHP100 cells) and 0.13 +/- 0.01 microM and 140 +/- 15 pmol.min-1.mg of protein-1 (U937 cells), respectively. The AnNH carrier of both cell lines was activated up to 170% of the control by nitric oxide.     

Purification, crystallization and properties of triosephosphate isomerase from human skeletal muscle

1. Triosephosphate isomerase (D-glyceraldehyde-3-phosphate ketoisomerase, EC 5.3.1.1) from human skeletal muscle was purified to homogeneity and crystallized. The crystalline enzyme preparation was resolved on polyacrylamide-gel electrophoresis into three isoenzymes. 2. The molecular weight of the enzyme estimated by gel filtration method was found to be 57,400 +/- 3000. Molecular weight determination under dissociation conditions indicated a dimeric subunit structure of the enzyme. 3. The apparent Km for D-glyceraldehyde-3-phosphate as substrate is 0.34 mM, and for dihydroxyacetone phosphate, 0.61 mM. Vmax of the reaction is, respectively, 7200 and 660 units/mg protein at 25 degrees C and pH 7.5. 4. Molecular and kinetic properties of triosephosphate isomerase from human skeletal muscle are very similar to those of rabbit muscle enzyme.     

In vitro antimicrobial activity of a new antiseptic central venous catheter

A central venous catheter coated with a new antiseptic combination, silver chloride (AgCl) and benzalkonium chloride (BKC) in a polymer matrix, was developed. The antimicrobial efficacy and the ability to prevent surface colonization, after elution in both serum and saline, were evaluated and compared to catheters coated with silver sulfadiazine/chlorhexidine. The results of in vitro assays demonstrated that the AgCl-BKC coated catheters had a broad spectrum of activity against bacteria and C. albicans and prolonged antimicrobial activity for extraction periods of up to 30 days. These data suggest that AgCl-BKC coated catheters may provide another solution for reduction of catheter-related infections.     

Interactions between transport of triiodothyronine and tryptophan in JAR cells

We studied the effect of a number of amino acids on uptake of L-triiodothyronine (T3) in the human choriocarcinoma cell line, JAR. Tryptophan inhibited saturable T3 uptake by about 57% without any significant effect on the non-saturable uptake. Michaelis constant (Km) for T3 uptake was 1.06 +/- 0.15 microM (n = 15) with the corresponding maximum velocity (Vmax) of 24.2 +/- 3.1 pmol/min/mg cellular protein. For tryptophan uptake the Km was 1.31 +/- 0.26 microM (n = 7) and Vmax was 166.4 +/- 35.7 pmol/min/mg protein. The kinetic parameters for both uptake processes were similar to those reported in normal placenta. Uptake of T3 was inhibited by tryptophan but not phenylalanine, but tryptophan uptake was inhibited both by T3 and phenylalanine. Inhibition of T3 uptake by tryptophan was dose dependent, with an inhibition constant (Ki) of 2.9 +/- 0.5 mM. Similarly, tryptophan uptake was inhibited by T3 and phenylalanine in a dose dependent way with Ki values of 4.9 +/- 0.5 microM and 15.6 +/- 4.8 microM respectively. Km for T3 uptake was significantly increased to 1.86 +/- 0.42 microM (n = 4) in the presence of 3 mM unlabelled tryptophan and, similarly, Km for tryptophan uptake was significantly increased to 9.91 +/- 2.57 microM (n = 3) in the presence of 5 microM unlabelled T3. Efflux of T3 was progressively inhibited by increasing concentrations of both ligands, i.e. was saturable. We conclude that there is mutual competitive inhibition between uptake systems for T3 and tryptophan in JAR cells, but the kinetic parameters of cross-inhibition of uptake by the substrates suggest that the carriers are distinct. T3 may be transported in JAR cells by at least two transport systems with differing substrate specificities. We also demonstrated the presence of a saturable membrane carrier mediating the efflux of T3 from the cells which was subject to trans-inhibition by T3 and tryptophan.     

Identification and characterization of high and low affinity transport systems for reduced glutathione in liver cell canalicular membranes

Driving forces and substrate specificity for transport of reduced glutathione (GSH) across rat liver cell canalicular membrane were examined in vesicles isolated from this plasma membrane domain. In contrast to previous studies indicating a single saturable component of canalicular GSH transport, the present results demonstrate the presence of both high and low affinity components with apparent Km values of 0.24 +/- 0.04 and 17.4 +/- 2.1 mM and Vmax values of 0.09 +/- 0.01 and 2.3 +/- 0.3 nmol.mg-1.20 s-1, respectively. The Km values in two previously published reports are discordant, 0.33 versus 16 mM, but are comparable with the two transport components identified in the present study. To further characterize these GSH transport mechanisms, [3H]GSH uptake by canalicular vesicles was measured at concentrations of 50 microM, where transport is expected to occur largely on the high affinity component, and at 5 mM, where the low affinity system should predominate. Neither component of GSH transport was affected by ATP or a Na+ gradient, but both were stimulated by a valinomycin-induced membrane potential, indicating electrogenic transport pathways. The high affinity component was cis-inhibited by glutathione S-conjugates (1 mM), other gamma-glutamyl compounds (5 mM), and 4,4'-diisothiocyanatostilbene-2,2'-disulfonic acid (0.1 mM), whereas these agents had no effect on the low affinity component at similar inhibitor concentrations. Sulfobromophthalein (BSP, 0.1 mM) inhibited both GSH transport components. However, neither component was affected by taurocholate (0.5 mM) or L-glutamate (10 mM). The inhibition by S-butylglutathione, the GSH analogue ophthalmic acid, and by BSP was competitive in nature, although BSP also produced a slight decrease in Vmax, suggesting a mixed type of inhibition. Ophthalmic acid and some glutathione S-conjugates were also able to trans-stimulate high affinity GSH uptake. These results indicate the presence of at least two ATP-independent, electrogenic glutathione transport mechanisms on the canalicular membrane; the high affinity component may function to deliver some glutathione S-conjugates, gamma-glutamyl compounds, and other anions into bile, whereas the low affinity system probably functions as a high capacity transporter capable of delivering large amounts of GSH into bile.     

Mechanism-based inactivation of serine transhydroxymethylases by D-fluoroalanine and related amino acids

Serine transhydroxymethylase, from lamb or rabbit liver, is known to catalyze slow transamination of D-alanine, but not of L-amino acids, in a tetrahydrofolate-independent reaction. Both enzymes will process the D-isomer of beta-fluoroalanine for alpha, beta-elimination of HF to yield an aminoacrylate-pyridoxal-P-enzyme intermediate. This intermediate partitions between harmless hydrolysis to pyruvate, NH4+, and active enzyme-pyridoxal-P (catalytic turnover) and suicidal enzyme alkylation by covalent modification with an average partition ratio of 40-60 turnovers/inactivation event/monomer unit of this tetrameric enzyme. Enzyme inactivation occurs with stoichiometric incorporation of radioactive label from D-[1,2-14C]fluoroalanine. Titration of enzymic cysteinyl --SH groups with 5,5'-dithiobis(2-nitrobenzoate) indicates loss of 1 --SH group on inactivation. Acid hydrolysis of radioactive-inactive enzyme confirms cysteine residue modification. Treatment of inactive enzyme with 6 M urea, then KBH4, followed by acid hydrolysis yields two radioactive compounds, lanthionine and S-carboxyhydroxyethylcysteine, in about equal amounts. The addition of tetrahydrofolate stimulates both pyruvate production and inactivation to equal extents with about a 200-fold rate acceleration at 0.5 mM tetrahydrofolate to turnover numbers of approximately 120 min-1. The Km for D-fluoroalanine is high, 10-60 mM, and this low substrate affinity suggests D-fluoroalanine will not be a useful in vivo agent for selective inactivation of liver cell serine transhydroxymethylases.     

Properties of electrogenic Pi transport by a human renal brush border Na+/Pi transporter

Inorganic phosphate (Pi) induced an inward current (IP) in Xenopus oocytes expressing the human renal Na+/Pi cotransporter NaPi-3. At 100mM Na+, Pi-transport was independent of the holding potential and resulted in an apparent Km of 0.08 mM; lowering the Na+ concentration to 50 mM resulted in an increase of the apparent Km to 0.22 mM at -50 mV and to 0.31 mM at -90 mV. In contrast, the apparent Km for Na+ was not significantly influenced by the holding potential. A decrease of the pH from 7.8 to 6.8 resulted in a decrease of IP at 50 mM Na+, but not at 150 mM Na+. Arsenate induced inward currents through NaPi-3 and decreased the apparent Km in measurements of IP. Phosphonoformic acid itself induced no currents, but inhibited Pi-induced currents with an apparent Ki of 3.6 mM. In summary, NaPi-3 displays characteristic Na+/Pi cotransporter properties with relevant interactions with arsenate (transport substrate) and phosphonoformic acid (inhibitor). Monovalent and divalent Pi both appear to be transported by NaPi-3.     

A silicone-coated nylon dressing reduces healing time in burned paediatric patients in comparison with standard sulfadiazine treatment: a prospective randomized trial

Mepitel is a new grid like silicone coated nylon dressing containing no additional biological compounds. We describe a prospective randomized pilot study comparing Mepitel to the standard silver sulfadiazine cream (Flamazine) dressing for the topical treatment of paediatric burns. Seventy-six children presenting within 24 h of injury with a non previously treated burn were randomly assigned to Mepitel treatment (group M) or Flamazine treatment (group F). Age, sex, surface area of burn and causal agent were noted at admission. The depth of the burn, cumulative number of dressings, presence or absence of a complete epithelial cover, infection, bleeding and allergy were noted at each dressing change. There were 41 children in group M and 35 children in group F. Five children were subsequently withdrawn from each group because they required skin grafting. Analysis of the above mentioned criteria showed no statistical difference between the two groups except for the healing time (group M: 7.58+/-3.12, group F: 11.26+/-6.02, p < 0.01) and the number of dressings (group M: 3.64+/-1.5, group F: 5.13+/-2.9, p < 0.05). Mepitel has proved to be an easy-to-remove dressing, adhering only to intact skin. The faster healing time found in the Mepitel group may be related to a direct effect of silicone on epithelial growth or to a decrease in surface-cell damage compared to the silver sulfadiazine group. This attractive product will be further assessed on a larger scale trial to confirm our observations.     

L-serine and L-threonine dehydratase from Clostridium propionicum. Two enzymes with different prosthetic groups

L-Serine dehydratase from the Gram-positive bacterium Peptostreptococcus asaccharolyticus is novel in the group of enzymes deaminating 2-hydroxyamino acids in that it is an iron-sulfur protein and lacks pyridoxal phosphate [Grabowski, R. and Buckel, W. (1991) Eur. J. Biochem. 199, 89-94]. It was proposed that this type of L-serine dehydratase is widespread among bacteria but has escaped intensive characterization due to its oxygen lability. Here, we present evidence that another Gram-positive bacterium, Clostridium propionicum, contains both an iron-sulfur-dependent L-serine dehydratase and a pyridoxal-phosphate-dependent L-threonine dehydratase. These findings support the notion that two independent mechanisms exist for the deamination of 2-hydroxyamino acids. L-Threonine dehydratase was purified 400-fold to apparent homogeneity and revealed as being a tetramer of identical subunits (m = 39 kDa). The purified enzyme exhibited a specific activity of 5 mu kat/mg protein and a Km for L-threonine of 7.7 mM. L-Serine (Km = 380 mM) was also deaminated, the V/Km ratio, however, being 118-fold lower than the one for L-threonine. L-Threonine dehydratase was inactivated by borohydride, hydroxylamine and phenylhydrazine, all known inactivators of pyridoxal-phosphate-containing enzymes. Incubation with NaB3H4 specifically labelled the enzyme. Activity of the phenylhydrazine-inactivated enzyme could be restored by pyridoxal phosphate. L-Serine dehydratase was also purified 400-fold, but its extreme instability did not permit purification to homogeneity. The enzyme was specific for L-serine (Km = 5 mM) and was inhibited by L-cysteine (Ki = 0.5 mM) and D-serine (Ki = 8 mM). Activity was insensitive towards borohydride, hydroxylamine and phenylhydrazine but was rapidly lost upon exposure to air. Fe2+ specifically reactivated the enzyme. L-Serine dehydratase was composed of two different subunits (alpha, m = 30 kDa; beta, m = 26 kDa), their apparent molecular masses being similar to the ones of the two subunits of the iron-sulfur-dependent enzyme from P. asaccharolyticus. Moreover, the N-terminal sequences of the small subunits from these two organisms were found to be 47% identical. In addition, 38% identity with the N-terminus of one of the two L-serine dehydratases of Escherichia coli was detected.     

Complete inactivation of Escherichia coli uridine phosphorylase by modification of Asp5 with Woodward's reagent K

Woodward's reagent K (WRK) completely inactivated Escherichia coli uridine phosphorylase by reversible binding in the active site (Ki = 0.07 mM) with subsequent modification of a carboxyl (k2 = 1.2 min-1). Neither substrate alone protected uridine phosphorylase from inactivation. The presence of phosphate did not affect the Ki and k2 values. The addition of uracil or uridine led to a significant increase of both Ki (to 2.5 or 2.1 mM, respectively) and k2 (to 6.1 or 4.8 min-1, respectively) values. Thus, WRK could react in accordance with slow (high affinity) and fast (low affinity) mechanisms. Combined addition of phosphate and uracil completely protected uridine phosphorylase. Tryptic digestion yielded a single modified peptide (Ser4-Asp(WRK)-Val-Phe-His-Leu-Gly-Leu-Thr-Lys13). Treatment of the modified enzyme with hydroxylamine led to removal of the bulky WRK residue and replacement of the Asp5 carboxyl by a hydroxamic group. The enzyme thus obtained recovered about 10% of initial specific activity, whereas its substrate binding ability changed only moderately; the Km values for phosphate and uridine were changed from 5.1 and 0.19 mM (or 7.3 and 0.14 mM according to Leer et al. (Leer, J.C., Hammer-Jespersen, K., and M. Schwartz (1977) Eur. J. Biochem. 75, 217-224)) to 22.6 and 0.12 mM, respectively. The hydroxamic enzyme had higher thermostability than the native enzyme. The results obtained demonstrated the importance of the carboxyl at position 5. The loss of activity after selective group replacement is due to impaired stabilization of the transition state rather than to a decline in substrate affinity or change of the active site structure.     

Inhibitory actions of emodin on arylamine N-acetyltransferase activity in strains of Helicobacter pylori from peptic ulcer patients

Arylamine N-acetyltransferase (NAT) activities with p-aminobenzoic acid and 2-aminofluorene were determined in Helicobacter pylori, a gram-negative rod bacteria collected from peptic ulcer patients. The NAT activity was determined using a acetyl CoA recycling assay and HPLC. Cytosols or suspensions of H. pylori with and without selected concentrations of emodin co-treatment showed different percentages of 2-aminofluorene and p-aminobenzoic acid acetylation. The data indicate that there were decreased NAT activity associated with increased emodin in H. pylori cytosols. As 400 microns of emodin can obviously inhibit NAT activity both in vitro and in vivo (inhibition rate 90% and 93% for 2-aminofluorene and p-aminobenzoic acid in vitro, and 90% and 92%, respectively, for both substrate in vivo). For in vitro examination, the apparent values of Km and Vmax were 3.12 +/- 0.38 mM and 15.20 +/- 3.16 nmol/min/mg protein for 2-aminofluorene, and 0.56 +/- 0.12 mM and 0.74 +/- 0.09 nmol/min mg protein for p-aminobenzoic acid. However, when emodin was added to the reaction mixtures, the values of apparent Km and Vmax were 2.40 +/- 0.32 mM and 10.62 +/- 0.04 nmol/min/mg protein for 2-aminofluorene, and 0.23 +/- 0.02 mM and 0.62 +/- 0.08 nmol/min/mg protein for p-aminobenzoic acid. For in vivo examination, the apparent Km and Vmax were 0.82 +/- 0.18 mM and 0.92 +/- 0.21 nmol/min/10 x 10(10) colony forming units (CFU) for 2-aminofluorene, and 0.78 +/- 0.14 mM and 0.52 +/- 0.06 nmol/min/ 10 x 10(10) (CFU) for p-aminobenzoic acid. However, when emodin was added to the reaction mixtures, the values of apparent Km and Vmax were 0.50 +/- 0.08 mM and 0.62 +/- 0.22 nmol/min/ 10 x 10(10) (CFU) for 2-aminofluorene, and 0.52 +/- 0.21 mM and 0.26 +/- 0.04 nmol/min/ 10 x 10(10) (CFU) for p-aminobenzoic acid. This report is the first finding of emodin inhibition of arylamine N-acetyltransferase activity in a strain of H. pylori.     

Effect of extracellular pH on the myo-inositol transporter SMIT expressed in Xenopus oocytes

The myo-inositol transporter SMIT is expressed particularly at high extracellular osmolarity and serves to accumulate the osmolyte myo-inositol. Transport of myo-inositol is coupled to the cotransport of Na+ and is electrogenic. In Xenopus oocytes injected with mRNA encoding SMIT but not in water-injected oocytes, myo-inositol creates an inward current that is dependent on the ambient Na+ concentration. The present study has been performed to elucidate the pH dependence of myo-inositol-induced currents. Therefore, Xenopus oocytes were injected with mRNA encoding SMIT and two-electrode voltage-clamp studies were performed. The myo-inositol-induced currents in oocytes expressing SMIT were found to have a sigmoidal dependence on the ambient pH between pH 5.5 and 8.5 with an apparent Ki of 0.21+/-001 microM H+ and a Hill coefficient of 1.80+/-0.16. Kinetic analysis of the myo-inositol-induced currents at pH 8.0 and -90 mV holding potential revealed a Hill coefficient of 0.93+/-0.07 and an apparent Km for myo-inositol of 0.031+/-0.003 mM as well as a Hill coefficient of 1. 64+/-0.24 and an apparent Km of 38.8+/-4.1 mM for Na+. A decrease of the Na+ concentra-tion from 150 mM to 50 mM significantly altered the maximal observed current and increased the apparent Km for myo-inositol. Acidification to pH 6.5 significantly increased the apparent Km for myo-inositol and for Na+ to 0.057+/-0.005 mM and 73. 9+/-4.8 mM, respectively. The Hill coefficients for myo-inositol and Na+ were not affected and remained close to 1 for myo-inositol and 2 for Na+. In summary, acidification impedes SMIT-mediated myo-inositol transport at least partially by decreasing the affinity of the carrier for Na+. The impaired Na+ binding subsequently decreases binding and transport of myo-inositol.     

p-aminohippurate uptake by syncytial microvillous membrane vesicles of human term placenta

The mechanism of uptake of p-aminohippurate (PAH) by syncytial microvillous membrane vesicles of human term placenta was investigated. Initial PAH uptake and efflux were increased in the presence of a pH-gradient and a Cl(-)-gradient, respectively. Forced negative and positive membrane potentials did not influence the uptake, which indicated that the transport is not electrogenic. The pH-dependent increase is probably the result of a higher rate of diffusion due to a lower degree of dissociation of PAH. Because several organic anions failed to transstimulate PAH uptake and FCCP did not decrease the uptake in the presence of an inwardly directed H(+)-gradient, ruling out a PAH/OH- antiport, an anion exchange system does not appear to be present in these membranes. Since electrogenicity and anion exchange seem not to be involved in the Cl(-)-dependent increase, an allosteric effect of Cl- on the transporter might be possible. Various organic anions were able to inhibit pH-stimulated PAH uptake significantly. Kinetic analysis of the probenecid sensitive part of uptake provided further evidence for mediated transport of PAH (Km = 7.4 +/- 2.6 mM and Vmax = 2.0 +/- 0.4 nmol/mg/15 s). Non-inhibitable diffusion accounted for the main part of total transport. Concentration dependent inhibition of PAH transport by probenecid showed a Ki of 2.5 +/- 0.9 mM. It is concluded that human placental syncytial microvillous membrane vesicles possess a low affinity transport mechanism for PAH with low specificity. The importance of this system, for placental excretion of anionic drugs, will depend on the intrasyncytial concentration of these drugs, caused by the transport across the basal membrane.     

The effects of temperature and some inhibitors on the carnitine exchange system of heart mitochondria

1. 11-Trimethylamino-undecanoyl-L-carnitine (Ki = 56 muM) and 6-trimethylaminohexanoyl-L-carnitine (Ki = 1.3 mM) are competitive inhibitors of the ox heart mitochondrial carnitine exchange system. The latter is itself a poor substrate, with a V about 0.7% of that for carnitine. These compounds have been used as 'stop inhibitors' in a study of the effect of temperature on the carnitine exchange. 2. The carnitineout in equilibrium carnitinein exchange has a temperature coefficient (Q10) of 8.5 and an activation energy of 176 kJ/mol which is constant from 0 degrees - 18 degrees C. The Km for L-carnitine (5.3 mM) is also constant over this temperature range. 3. The slow efflux (leak) of mitochondrial L-[14C]carnitine in the absence of external substrate has a similar temperature dependence and is also inhibited by 11-trimethylamino-undecanoyl-L-carnitine. It is probably mediated by the same system as the very much faster exchange with external carnitine.     

Substituted glycals as probes of glycosidase mechanisms

D-Glucal and a series of substituted derivatives have been tested as substrates, inhibitors and inactivators of the Agrobacterium faecalis beta-glucosidase in order to probe structure/function relationships in this enzyme. D-Glucal is shown to be a substrate (kcat = 2.3 min-1, Km = 0.85 mM) undergoing hydration with stereospecific protonation from the alpha-face to yield 2-deoxy-beta-D-glucose. 1-Methyl-D-glucal surprisingly serves as only a poor substrate (kcat = 0.056 min-1, Km = 57 mM), also undergoing protonation from the alpha-face. 2-Fluoro-D-glucal, however is completely inert, as a result of inductive destabilisation of the oxocarbenium ion-like transition state for protonation, and functions only as a relatively weak (Ki = 24 mM) inhibitor. Similar behaviour was seen with almond beta-glucosidase and yeast alpha-glucosidase and for the interaction of 2-fluoro-D-galactal with Escherichia coli beta-galactosidase. A series of of alpha, beta-unsaturated glucal derivatives was also synthesised and tested as potential substrates, inhibitors or inactivators of A. faecalis beta-glucosidase. Of these only 1-nitro-D-glucal functioned as a time dependent, irreversible inactivator (ki = 0.011 min-1, Ki = 5.5 mM), presumably acting as a Michael acceptor. Electrospray mass spectrometric analysis revealed multiple labeling of the enzyme by this inactivator, lessening its usefulness as an affinity label. Less reactive Michael acceptor glycals which might have been more specific (1-cyano-, 2-cyano-, 1-carboxylic acid, 1-carboxylic acid methyl ester) unfortunately did not function as inactivators or substrates, only as relatively weak reversible inhibitors (Ki = 3-96 mM).