Mercurial perturbation of brush border membrane permeability in rabbit ileum

The sulfhydryl reagents Hg++ and p-chloromercuribenzene sulfonate (PCMBS) at millimolar concentrations reduced the mucosal entry of sugars and amino acids to 80-90% of control levels within several minutes. Based on 50% levels of inhibition, Hg++ proved to be 20 and 10 times as potent as PCMBS in blocking sugar and amino acid transport, respectively; both systems were equally sensitive to Hg++. Concomitant measurements of 203Hg-PCMBS demonstrated a progressive tissue uptake, which, unlike inhibition, did not saturate with increasing times of exposure, thus suggesting appreciable epithelial entry with prolonged exposures (less than 30 min at 1 mM). At similar dose levels, no significant change in mucosal Na+ entry was detected. Inhibition was not reversed by 30-min washes in cholinesalt solutions; however, 10-min exposures to dithiothreitol [10 mM] reversed Hg++ and PCMBS inhibition by 40 and 100%, respectively. Alanine and galactose influx kinetics measured at concentrations of 0-100 mM exhibited a linear or diffusional entry component in addition to the usual saturable component for both control and Hg++-treated ileum. The presence of a diffusional term in the flux equation resulted in two sets of parameters giving nearly equal fits to these measurements. It was shown that this ambiguity could be resolved by determining the change in diffusional entry with Hg++ treatment. A 20-min exposure to 0.5 mM Hg++ caused an increase from 0.050 and 0.045 to 0.064 and 0.070 cm/hr in the coefficient of diffusional entry for alanine and galactose, respectively. On the basis of this increase, it is argued that Hg++ causes a decrease in Jmax and little change in Km for both transport mechanisms. This analysis has a general bearing on kinetic measurements of transport in which passive fluxes are comparable to those mediated by specific pathways. The alanine results are consistent with bimolecular reactions between mercurial and two membrane inhibitory sites, each producing approximately 40% reduction in membrane translocation rate. The estimated reaction rate constants were 5.0 and 0.4 mM min.     

Absorption of 5-methyltetrahydrofolate in rat jejunum with intact blood and lymphatic vessels

Transport results from in vitro studies may not be applicable to in vivo situations. In this study, we extended our previous in vitro observations regarding the intestinal transport of 5-methyltetrahydrofolate to in vivo studies in the unanesthetized rat and examined the effect of the unstirred water layer on the absorption process. We used a well defined intestinal perfusion technique. Absorption of 0.5 and 5 microM 5-methyltetrahydrofolate proceeded in a linear manner for 40 min of perfusion at 0.31 and 1.74 nmol/100 cm per min, respectively. Absorption of 0.5 microM 5-methyltetrahydrofolate increased with increasing perfusate flow-rate from 0.5 to 2 to 4 ml/min, indicating an unstirred water layer influence on the absorption rate. Absorption of the substrate was saturable with an apparent Kt of 5.7 microM and Vmax of 3.45 nmol/100 cm per min. Absorption was pH-dependent, and was inhibited by structural analogues. In contrast to the in vitro data, addition of glucose (20 mM) to the perfusate was unnecessary for in vivo absorption to proceed. Unconjugated cholic (5 mM) and deoxycholic (1 mM) acids and the organic anion rose bengal (0.1 mM) inhibited the absorption of 0.5 microM 5-methyltetrahydrofolate when added to the perfusate. Conclusions: the results of previous in vitro studies of 5-methyltetrahydrofolate intestinal transport are applicable to in vivo situations, except that luminal glucose was found to be unnecessary in the latter. The unstirred water layer modulated the absorption of 5-methyltetrahydrofolate, while unconjugated bile acids and rose bengal inhibited it.     

Inhibition of sugar transport across rat jejunum, in vivo, by cupric ions

Cupric ions inhibit galactose absorption by in vivo perfused rat jejunum. It takes some delay for the inhibitory action to display its maximal levels, and previous exposure of the mucosa to Cu markedly increases inhibition. Copper effects were only scarcely reversed by saline solution washing, more effectively by EDTA and more so by dithioerythritol, in no case reaching control values. Absorption of L-sorbose, or that of galactose in the presence of 0.5 mM phlorizin, are not modified by 0.5 mM cupric ions. Cu action may be understood as a selective impairment of the phlorizin-sensitive sugar transport system by binding of the metal to prevailing thiol chemical groups of proteins at the brush border, located at different depth within the thickness of the membrane.     

The impact of heavy metals on the activity of some enzymes along the barley root

Barley seedlings 48 h after the onset of germination on filter paper treated for 24 h by 1 mM cadmium (Cd), 3 mM nickel (Ni) or 0.5 mM mercury (Hg) showed similar approximately 45% root growth inhibition. Although root growth inhibition was similar, loss of cell viability evaluated, as Evans blue uptake was distinct among Cd, Ni and Hg treated roots. While Cd and Hg caused cell death along the whole barley root (0–8 mm), Ni induced significant loss of cell viability only in root cells 6–8 mm distance from the root tip. Our results suggest that different metabolic processes are activated in different parts of barley root in relation to distance from the root tip during heavy metal (HM) treatment. Some of them are characteristic for several HMs such as inhibition of ascorbic acid oxidase or glutathione-S-transferase stimulation, while others are specific for individual HMs, e.g. activation of acid phosphatase and lipoxygenase by Cd and Hg, or inhibition of ascorbate peroxidase by Ni and Hg treatment.     

Inhibition by cadmium of D-galactose and L-phenylalanine transport by rat intestine in vitro

The effect of cadmium (CdCl2) on galactose and phenylalanine uptake by rat everted intestinal rings has been studied. The rings were preincubated (15 min) and incubated (5 min) in the presence of Cd. Galactose uptake (from 0.5 mM to 10 mM) was inhibited by 0.5 mM Cd about 25%. Only the phlorizin-dependent galactose transport was affected by cadmium, being a non-competitive type inhibition. A 15 min washing with saline solution significantly reduced the cadmium induced inhibition, which was practically reversed by washing with 5 mM EDTA. The uptake of 0.5 mM phenylalanine was not affected by 0.5 mM Cd but it was depressed by 1 mM Cd. Such inhibition was exerted on the sodium-dependent phenylaline transport. Washing with 5 mM EDTA diminished only slightly the inhibition of the transport by cadmium. It is suggested that the inhibition of intestinal transport of galactose and phenylalanine by cadmium may be due to its reversible interaction with metal-binding ligands, possibly sulfhydryl groups, related to the luminal transport systems.     

Role of -SH groups in rat sugar intestinal transport in vivo.

The effect of p-chloromercuribenzoic acid (pCMB), either alone or in the presence of 5,5'-dithiobis-(2-nitrobenzoic acid) (DTNB), on the 1 mM galactose absorption by in vivo perfused rat intestine has been studied. At 0.25 mM concentration, pCMB inhibits galactose absorption in about 32% but it does not modify the absorption of this sugar when the transport is blocked by 0.5 mM phlorizin, or that of the non-transportable monosaccharide derivative 2-deoxy-D-glucose. This shows that only the active transport component of galactose absorption is inhibited. A 2 min preexposure period is required for the inhibition to appear. The inhibition was not reversed by washing with saline solution even when it contained 0.5 mM dithioerythritol, 10 mM cysteine or 5 and 10 mM EDTA. The simultaneous exposure to 0.25 pCMB and 0.25 mM DTNB inhibits the total galactose entry in about 50%, an effect higher than the one exerted by each reagent separately and close to the one obtained with 0.5 mM phlorizin. Our results, in vivo, confirm the importance of the thiol groups in the cotransport of Na+ and sugar. As DTNB is an SH-reagent of lesser liposolubility than pCMB, the existence of two populations of sulfhydryl groups related to sugar transport which differ in their location within the brushborder membrane and in accessibility from the intestinal lumen, is suggested.     

Mercuric chloride inhibition of vasopressin release from the isolated neurointermediate lobe of the rat pituitary

The effects of HgCl2 and ouabain on vasopressin release and Ca2+ uptake and distribution was examined in the neurointermediate lobe of the rat pituitary. HgCl2 (0.5 mM) inhibited vasopressin release by approx. 90% in both basal and potassium depolarized states. With 0.1 mM HgCl2 vasopressin release was inhibited by 50% in the depolarized state, but release was not effected in basal state. On the other hand, ouabain (0.5 mM) caused a 3-fold stimulation of vasopressin release in the depolarized state. Both HgCl2 (0.5 mM) and ouabain (0.5 mM) increased net 45Ca+2 uptake by about 80% in groups of neurointermediate lobes. Following 45Ca+2 uptake, HgCl2 (0.5 mM), which is absorbed by the neurointermediate lobe, produced an increase in cytosolic 45Ca+2 content and a decrease in mitochondrial 45Ca+2 content compared to control. In comparison, ouabain (0.5 mM), which does not penetrate the neurointermediate lobe, gave no change in cytosolic 45Ca+2, but an increase in mitochondrial 45Ca+2. These results suggest that HgCl2 inhibits vasopressin release from the neurointermediate lobe of the rat pituitary at a point distal to Ca+2 uptake by the gland.     

Effects of metal elements on acid phosphatase activity in cucumber (Cucumis sativus L.) seedlings

Acid phosphatases (E.C.3.1.3.2) are a group of enzymes widely distributed in nature, which nonspecifically catalyze the hydrolysis of a variety of phosphate esters in pH ranges from 4 to 6 and play a major role in the supply and metabolism of phosphate in plants. The objective of the present study was to investigate the in vitro effects of some metals on the activity of acid phosphatase in cucumber seedlings (Cucumis sativus L.) and to determine their kinetic parameters. The enzyme was assayed with Hg, Cd, Mn, Pb, Zn, K and Na at the 0.001–1 mM range using ATP, PPi and β-glycerol phosphate as substrates. Mn, Na and Cd did not significantly alter the enzyme activity. K caused a broad activation at low concentrations and an inhibition at high concentrations (10 mM) and lead caused no inhibition. Acid phosphatase was inhibited by Hg and Zn and the inhibition type and IC₅₀ values were determined for these metals. Hg presented a mixed inhibition type with PPi and ATP as substrates and uncompetitive inhibition with β-glycerol phosphate as substrate. Zn presented competitive inhibition for ATP as substrate, and a mixed inhibition type with PPi and β-glycerol phosphate as substrate. IC₅₀ values were 0.02, 0.3 and 0.15 mM for Hg, and 0.056, 0.035 and 0.24 mM for Zn with ATP, PPi and β-glycerol phosphate as substrates, respectively. Analysis of these results indicates that Zn is a more potent inhibitor of acid phosphatase from cucumbers than Hg.     

Inhibition and activation of bovine heart NAD-specific isocitrate dehydrogenase by ATP

In the absence of added calcium, inhibition of NAD-specific isocitrate dehydrogenase by ATP occurred without ADP (I0.5 = 1.8 mM) and with 0.2 mM ADP3- (I0.5 = 1.0 mM) at subsaturating substrate concentrations at pH 7.4. Inhibition by ATP was competitive with NAD+ in the presence and absence of ADP and was not reversed by magnesium citrate. No reversal of ATP inhibition by free Ca2+ was observed in the presence of ADP (0.2 mM). However, when ADP was absent, increasing Ca2+ first caused progressive reversal of ATP inhibition followed by activation by ATP. Without ADP, the S0.5 for calcium activation was 80-140 microM at ATP concentrations between 0.6 and 3.0 mM. The S0.5 for ATP activation, in the absence of ADP, was 1.1 and 2.1 microM when free Ca2+ was held constant at 0.1 and 1.0 mM, respectively. As in activation by ADP, ATP decreased the S0.5 for magnesium isocitrate without affecting V. However, in contrast to ADP, the activation by ATP occurred without lowering the Hill coefficient for the substrate. GDP activated the enzyme at relatively high concentrations of Ca2+ but not without added Ca2+.     

Nonparticipation of extracellular glutathione in renal transport of dibasic amino acids

Microinjections of L-[14C]arginine (2.9 mM) and L-[14C]ornithine (3.4 mM) were made into renal proximal tubules of rats in the presence of methionine sulfoximine (MSO) (10, 20 mM), ATP (10 mM), and MgCl2 (20 mM) together. Absorption of both labelled amino acids dropped, respectively, by 31.1 and 49.1% compared with control microinjections. The MSO alone or ATP plus MgCl2 had no effect. These data suggest that the inhibition by MSO plus ATP plus MgCl2 is not due to direct competition between MSO and dibasic amino acids but rather to suppression of the renewal of intracellular glutathione. Such an effect is discussed in comparison with cycloleucine inhibition of dibasic amino acid transport. Addition of exogenous glutathione to microinjectates die not reverse either type of inhibition. This study shows that while intracellular glutathione may affect amino acid transport, extracellular glutathione has no effect.     

Inhibition of bovine kidney alpha-ketoglutarate dehydrogenase complex by reduced nicotinamide adenine dinucleotide in the presence or absence of calcium ion and effect of adenosine 5'-diphosphate on reduced nicotinamide adenine dinucleotide inhibition

Micromolar Ca2+ markedly reduces NADH inhibition of bovine kidney alpha-ketoglutarate dehydrogenase complex [Lawlis, V. B., & Roche, T. E. (1980) Mol. Cell. Biochem. 32, 147-152]. Product inhibition patterns from initial velocity studies conducted at less than 10(-9) M or at 1.5 X 10(-5) M Ca2+ with NAD+, CoA, or alpha-ketoglutarate as the variable substrate showed that NADH was a noncompetitive inhibitor with respect to each of these substrates, except at high NAD+ concentrations, where reciprocal plots were nonlinear and the inhibition pattern for NADH vs. NAD+ changed from a noncompetitive to a competitive pattern. From slope and intercept replots, 2-fold to 12-fold higher inhibition constants were estimated for inhibition by NADH vs. the various substrates in the presence of 1.5 X 10(-5) M Ca2+ than for inhibition at less than 10(-9) M Ca2+. These inhibition patterns and the lack of an effect of Ca2+ on the inhibition of the dihydrolipoyl dehydrogenase component suggested that Ca2+-modulated NADH inhibition occurs at an allosteric site with competitive binding at the site by high levels of NAD+. Decarboxylation of alpha-keto[1-14C]glutarate by the resolved alpha-ketoglutarate dehydrogenase component was investigated in the presence of 5.0 mM glyoxylate which served as an efficient acceptor. NADH (0.2 mM) or 1.0 mM ATP inhibited the partial reaction whereas 15 muM Ca2+, 1.0 mM ADP, or 10 mM NAD+ stimulated the partial reaction and reduced NADH inhibition of this reaction. Thus these effectors alter the activity of the alpha-ketoglutarate dehydrogenase complex by binding at allosteric sites on the alpha-ketoglutarate dehydrogenase component. Inhibition by NADH over a wide range of NADH/NAD+ ratios was measured under conditions in which the level of alpha-ketoglutarate was adjusted to give matching control activities at less than 10(-9) M Ca2+ or 1.5 X 10(-5) M Ca2+ in either the presence or the absence of 1.6 mM ADP. These studies establish that both Ca2+ and ADP decreased NADH inhibition under conditions compensating for the effects of Ca2+ and ADP on S0.5 for alpha-ketoglutarate. ADP was particularly effective in reducing NADH inhibition; further studies are required to determine whether this occurs through binding of NADH and ADP at the same, overlapping, or interacting sites.     

Effect of DTNB on rat intestinal galactose transport in vivo

The effect of the non-penetrating reagent of -SH groups: acid 5,5'-dithiobis (2-nitrobenzoic), (DTNB), on 1 mM galactose absorption in rat intestine in vivo has been studied. DTNB inhibits sugar absorption in about 35%, which is due to an action on the mediated transport component, but without affecting the diffusional passive one. Consequently it does not modify galactose absorption in the presence of 0.5 mM phlorizin or that of the non-transportable sugar 2-deoxy-glucose. Galactose transport inhibition appears after a not longer than 5 min preexposure period and it remains constant at least up to 30 min. The inhibitory effect does not vary between 0.1 and 1 mM DTNB and it reverses completely with 0.5 mM dithioerythritol. Protection by excess of substrate has not been observed. Results show that DTNB affects sulfhydryl groups very probably located at the luminal side and related to the proteins of the cotransport system.     

A novel nonhemorragic protease from the African puff adder (Bitis arietans) venom.

A nonhemorrhagic proteinase B-20 from the venom of Bitis arietans has been purified to apparent electrophoretic homogeneity by chromatography on Sephadex G-100, Q-Sepharose, and CM-cellulose. It has a molecular weight of 20 k Da as determined by size exclusion chromatography on Sephadex G-100 and migrated as a single 20-k Da band on SDS polyacrylamide. It has an optimum pH of 6-8 and is inactive at pH 4.0. EDTA and 1,10-phenanthroline strongly inhibited the enzyme suggesting it is a metalloenzyme. Also it is inhibited by antipain but is unaffected by trasylol, antitrypsin, and pepsptatin. Colombin, an identified active component of Aristolochia albida used in the treatment of snake poisoning, did not inhibit the protease activity. It lost over 90% of its activity in the presence of 0.5 microM Hg(2+) but the inhibition was completely blocked in the presence of 10 microM mercaptoethanol implicating sulfhydryl groups in the catalytic entity of the protein. The activity was also inhibited competitively by glutathione and cysteine with inhibition binding constants K(i) of 240 and 40 microM, respectively. The enzyme is unaffected by several divalent cations but activated by 1 mM Fe(3+). It had a prolyl endopeptidase and thermolysin-like activity. The enzyme displayed a fast acting alpha-fibrinolytic and delayed gamma-fibrinolytic activity when tested on human fibrinogen. The relevance of these findings is discussed.     

Cyclic GMP efflux from liver slices

Cyclic GMP efflux from liver slices was studied when the intracellular cyclic GMP levels were elevated by incubation with N-methyl-N'-nitro-N-nitrosoguanidine (0.1 mM) and 3-isobutyl-1-methylxanthine (0.25 mM). A probenecid-sensitive and a probenecid-insensitive efflux system for cyclic GMP was found. It is suggested that the former system operates a carrier, which is saturable, the capacity of which is increased by alkylation with N-ethylmaleimide. The rate of net efflux of cyclic GMP increases 20-fold in the temperature interval 18-37 degrees C. Probenecid (0.5 mM) gives 50% inhibition of this system. The probenecid-insensitive cyclic GMP efflux component probably corresponds to passive diffusion. It appears that efflux may be an important complement to cyclic 3':5'-nucleotide phosphodiesterase activity in reducing intracellular cyclic GMP levels.     

Characterisation of the diol dehydratase pdu operon of Lactobacillus collinoides

Addition of copper (0.5-5 mM) or cadmium (1-5 mM) to the white rot fungus Pleurotus ostreatus cultivated in liquid nitrogen-limited medium for 12 days increased the activity of laccase. The addition of 2 mM Cd led to an 18.5-fold increase of activity, 1 mM Cu increased the activity eight-fold. When added earlier than 12 days, the activation of laccase was delayed (Cu) or decreased (Cd). Ag, Hg, Pb, Zn, and H(2)O(2) decreased laccase activity. To study the effect on native enzymes, purified laccase was incubated with Cd, Cu, and Hg. The addition of Hg decreased the activity of laccase immediately and reduced the temporal stability of the enzyme, while the addition of Cu (0.05-50 mM) increased both enzyme activity and stability. Laccase extracted at different stages of straw colonisation differed in its response to heavy metals.     

Neurospora fructose-1,6-diphosphate aldolase: inhibition by sodium pyruvate

$\text{Neurospora}$ fructose-1,6-diphosphate aldolase exhibited a hyperbolic substrate saturation curve which changed to sigmoidal in the presence of 0.5 mM sodium pyruvate. The S_0.5 value for fructose-1,6-diphosphate increased from 1.4 mM to 6.6 and 20 mM in the presence of 0.5 and 1.0 mM sodium pyruvate, respectively. The inhibition seems to be cooperative in nature and involves conformational changes. Potassium ions completely blocked the inhibition by sodium pyruvate.     

Purification and characterization of fructokinase from developing tomato (Lycopersicon esculentum Mill.) fruits

A procedure is described which allows the purification of fructokinase (EC 2.7.1.4) from young tomato fruit. The procedure yielded a 400-fold purification and two isoenzymes designated fructokinase I and II (FKI and FKII) were separated by anion-exchange chromatography. Using sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) the molecular mass was estimated to be 35 kDa. Gel filtration on Sepharose-12 indicated that for both fructokinases the functional form is a dimer. Two dimensional isoelectric focusing/SDS-PAGE combined with immunoblotting showed that FKI has two components with isoelectric points (pIs) of 6.42 and 6.55, while four components with pIs from 6.07 to 6.55 were detected for FKII. A mixture of both fructokinases showed that the components of FKI match the more alkaline components of FKII. The activity of both fructokinases increased with increasing pH to around 8.0 and equal activity was observed from 8.0 to 9.5. Both fructokinases were specific for fructose with K _m values for fructose of 0.131 and 0.201 mM for FKI and FKII, respectively. At high concentrations (> 0.5 mM), fructose was also a strong inhibitor with inhibition constants (K _i) of 1.82 and 1.39 mM for FKI and FKII, respectively. The preferred phosphate donor for both isoforms was ATP, and K _m values of 0.11 and 0.15 mM were observed for FKI and FKII. At low concentrations (0.05–0.2 mM), fructose exhibited noncompetitive inhibition with respect to ATP for both fructokinases. This inhibition pattern changed to uncompetitive when higher fructose concentrations (0.5–10 mM) were used. These data indicated that substrate addition is ordered, with ATP adding first. Inhibition by ADP was also affected by the fructose concentrations. At 0.5 mM fructose, FKI showed non-competitive inhibition by ADP with respect to ATP and this inhibition changed to uncompetitive when 3 mM fructose was used. The isoform FKII showed a competitive inhibition pattern for ADP at 0.5 mM fructose which also changed to uncompetitive when 3 mM fructose was used. The features of the regulation of both fructokinases suggest that this enzyme might have a relevant role in carbon metabolism during tomato fruit development.     

Active site histidine in pig liver aminolevulic acid dehydratase modified by diethylpyrocarbonate and protected by Zn2+ ions

1. The effect of diethylpyrocarbonate (DEP) (0.1-0.35 mM) on the purified pig liver amino-levulic acid dehydratase (ALA-D) containing 0.3 g-atoms Zn/subunit, under different pHs (6.0-7.5), temperature (0-18 degrees C) and time (0-60 min) was studied. 2. Three histidyl residues/subunit were modified by DEP (0.2 mM, pH 6.8), but activity was completely lost after the first one had reacted, indicating the presence of one histidine residue essential for ALA-D catalysis. Reactivation by treatment with hydroxylamine (0.7 mM, pH 7.0) confirmed that only histidine and no other nucleophile amino acids were directly involved in DEP inhibition. 3. Zn ions (0.5 mM) and the substrate ALA (5-10 mM) protected against DEP inactivation, protection was dependent on pH. 4. Sn, Se, Hg, Cd, Mn, Co and Pb (0.01-0.1 mM) did not significantly protect ALA-D against inactivation. 5. It is concluded that the substrate and Zn binding sites and the essential histidyl residues are in close proximity in the active center. It is proposed that in the catalytic synthesis of porphobilinogen from ALA, histidine groups have the specific role of transporting protons from the aqueous media to a hydrophobic active site.     

Inhibition of growth and respiration of Leishmania mexicana by the antitumor agent lonidamine

1. The antitumor drug lonidamine inhibited growth of promastigotes of Leishmania mexicana in axenic culture. 2. Fifty percent inhibition was attained at 0.42 mM, and was reflected mainly in an increase in lag time, with less effect on final cell yield. 3. The drug was leishmanistatic, since when a non-growing culture in the presence of 0.5 mM lonidamine was centrifuged and the cells resuspended in fresh medium, growth started and reached the control value. 4. Both coupled and FCCP-uncoupled respiration of intact promastigotes were inhibited by lonidamine; 50% inhibition was attained at 0.5 and 0.4 mM, respectively. 5. The results suggested that the mechanism of inhibition of growth of L. mexicana is, as proposed in the case of Trypanosoma cruzi epimastigotes and Trypanosoma brucei procyclic trypomastigotes, through inhibition of the energy metabolism.