Zinc uptake by isolated rat enterocytes: effect of low molecular weight ligands

The luminal phase of zinc intestinal absorption has not been well characterized. This study was intended to elucidate the possible role of low molecular weight (LMW) ligands in zinc intestinal transport in an isolated rat enterocyte system. Under these in vitro conditions, zinc uptake by the isolated enterocytes was rapid, leveling off within 1 min. Kinetic analysis revealed that both a mediated and diffusion component were involved in zinc uptake in the absence of LMW ligands by the cells. For the mediated component of zinc transport, the Kt and Vmax were 64.1 microM and 13.9 nmol/20 sec/mg protein, respectively. Zinc uptake was not affected by the addition of metabolic inhibitors. In the presence of histidine or cysteine (2:1 ligand:zinc molar ratio), zinc uptake was greatly reduced and occurred solely via mediated transport. Zinc uptake was also significantly decreased upon the addition of EDTA to the assay media. Other amino acids tested had no effect on zinc uptake by the cells. Albumin markedly reduced zinc uptake by the cells. Histidine and other potential LMW ligands were unable to facilitate albumin-inhibited zinc uptake. The results of this study suggest that the intestinal absorption of zinc may not be effected in the form of chelates with LMW ligands. Amino acids such as histidine and cysteine significantly reduce the uptake of the metal by isolated rat enterocytes, making questionable their putative role as necessary vehicles in the luminal phase of zinc absorption.     

Intestinal absorption of copper: effect of sodium

The mechanisms of copper (Cu) absorption from the small intestinal lumen are poorly understood. In this study we investigated the role of sodium (Na) during the removal of Cu from the lumen of jejunal and ileal segments, using an in situ perfusion procedure in the anesthetized rat. Intestinal absorption of Cu from a 31 microM solution was highest in the presence of an isotonic concentration of NaCl, as compared to solutions containing either glycerol (GRL) or N-methyl-D-glucamine (NMG) as osmotic agents. In the jejunum, mean +/- SEM Cu absorption rates in the presence of the following solutes were: with NaCl, 57.5 +/- 10.5 pmole/min X cm; with GRL, 13.3 +/- 14.7 (P less than 0.05); with NMG, 18.4 +/- 10.1 (P less than 0.05). In the ileum, copper absorption in the presence of NaCl was 64.4 +/- 9.6; with GRL, 24.3 +/- 10.1 (P less than 0.01); with NMG, 15.8 +/- 3.7 (P less than 0.001). Kinetic analysis of the carrier-mediated component of Cu absorption in rat jejunum yielded a Vmax = 47.5 pmole/min X cm and an apparent Kt = 21 microM. The diffusion coefficient was calculated to be 1.4 X 10(-5) cm2/sec. The absorption of Cu was independent of net water absorption, which was highest in the presence of GRL and abolished and reversed into secretion by NMG. The data obtained are indicative of a significant role of Na in the small intestinal transport of Cu, in vivo, although not directly related to unidirectional water fluxes. The cation specificity of Na in this process remains to be elucidated, although the results support earlier studies which postulated that mediated transport may constitute a major component of Cu absorption in the mammalian small intestine.     

Inhibition of intestinal copper absorption by divalent cations and low-molecular-weight ligands in the rat

l-histidine (His) has been shown to enhance the inhibitory effect of zinc on intestinal copper absorption. This study was aimed at examining whether this effect of His was also extended to the interactions of other divalent cations: ferrous iron, tin, and cobalt, using an in vivo perfusion system in rats. Copper absorption and intestinal content of this element significantly decreased in the presence of 2 mM His and ferrous iron. Iron accumulation was greater when His was present than when omitted. A fivefold excess of tin inhibited copper absorption only when His was present. Citrate, at the same concentration as His, had no effect on copper absorption, but hepatic copper levels were increased, as compared to the absence of either His or citrate. Addition of 0.5 or 1.0 mM cobaltous salt plus His resulted in a sharp decrease in copper intestinal absorption, with an increase in intestinal tissue retention. These results confirm earlier findings with zinc and His, and suggest that a general phenomenon, either accelerating the removal of copper from the intestinal lumen or increasing, the retention of this element by the intestinal tissue, is a common feature of the interaction between cations of similar electronic configuration to copper and a high-affinity ligand, such as His.     

Copper-sodium linkage during intestinal absorption: inhibition by amiloride.

The possible association between copper and sodium small intestinal absorption in the rat was investigated in the presence or absence of the electrolyte transport inhibitors amiloride, acetazolamide, and furosemide, at pharmacologic concentrations, using an in situ perfusion procedure. Amiloride (1 mM) produced a significant decrease in copper, net water, and sodium absorption, in solutions with sodium. Copper tissue retention was not altered, but was much higher in the absence of sodium. Acetazolamide and furosemide (1 mM), in separate experiments, had no effect on copper removal from the lumen, but generally reduced sodium and water transport. The presence or absence of sodium in the perfusate influenced rates of copper uptake. These data are compatible with a more effective passage of copper across the enterocyte basolateral membrane in the presence of sodium than in its absence.     

The effects of severe zinc deficiency on intestinal amino acid losses in the rat

To determine whether intestinal amino acid losses might occur during zinc deficiency, labeled aminoisobutyric acid was given parenterally to zinc deficient rats and to appropriate zinc-sufficient controls. After 24 hours, the aminoisobutyric acid loss into the intestinal lumen was measured by in situ perfusion of isolated intestinal segments under conditions of either net water absorption or water secretion. Net amino acid losses were larger in the jejunum of the zinc deficient rats and losses were exacerbated during net water secretion in the jejunum and colon segments. The contribution of amino acid losses to fecal nitrogen, particularly during osmotic diarrhea, may be important in the growth retardation of zinc deficiency. Further, these alterations may indicate defective enterocyte transport functions during severe deficiency.     

Inhibition of copper absorption by zinc

Copper and zinc interact at the intestinal mucosal level, affecting copper absorption. Amino acids, such as histidine, may affect the absorption of these two elements by chelating these cations. The two mechanisms could have additive potential. This possibility was investigated using a duodenal-jejunal single-pass perfusion procedure in anesthetized rats. Copper absorption and tissue retention from solutions containing 0.1 mM copper were determined in the presence of either no zinc or equimolar zinc, or at a zinc/copper ratio of 10/1, either without histidine or with histidine at a 10/1 or 20/1 ratio to copper. Copper removal from the intestinal lumen was decreased by zinc, and further reduced by increasing concentrations of histidine. There was a greater accumulation of copper in the small intestine, reaching a maximum with a 10-fold excess of histidine. With zinc at a 10/1 ratio to copper, the addition of a 10- or 20-fold molar excess of histidine further decreased the net uptake of copper from the perfusate while greater copper accumulation in the tissue occurred. Histidine thus enhances the inhibitory effects of zinc on copper absorp|tion, suggesting the application of convergent mechanisms for diminishing copper uptake. This could be relevant for the treatment of Wilson’s disease.     

Adaptation of electrolytes and fluid transport in rat small and large intestine after distal small bowel resection

Na+, Cl- and water transport were studied in jejunum, caecum and colon after either 50% or 80% of small bowel resection (SBR). Four weeks after surgery, dry and wet weights, net absorption in vivo of sodium, chloride and water were determined. There was a significant intestinal growth after 50% or 80% SBR except for the colon which only showed increased tissue mass after 80% SBR. Net transport was stimulated both, per organ and per unit mass. In the small intestine and caecum both organ growth and changes in cell function appear to be involved in the adaptive response, regardless the extent of the small intestine resected. In the colon, compensatory growth appear to contribute to the adaptive response only after 80% SBR, whilst the transport function of the colonocytes seems to be stimulated after both types of SBR.     

Low-molecular-weight zinc-binding ligand: a regulatory modulator for intestinal zinc transport

1. In the last two decades, vast numbers of studies on the zinc nutriture of animals and man have been made. However, the biochemical and physiological events in controlling zinc nutrition are still poorly understood. This report concerns the progress made toward understanding the intestinal zinc absorption and secretion mechanisms. 2. Evidence is accumulating that zinc absorption is a facilitated diffusion while zinc secretion is an active transport. 3. It is known that a low molecular weight zinc-binding ligand (LMW-ZBL) is a key regulator of intestinal zinc absorption, possibly a carrier molecule across the intestinal mucosal cells. Some high molecular weight zinc-binding ligands also appear to be involved in regulating intestinal zinc transport. 4. The identity of the LMW-ZBL is a matter of controversy and its specific role in regulating intestinal zinc transport is not well defined. 5. According to the available literature, no systemic investigations have been made to elucidate the intestinal zinc transport mechanisms, and much more information is needed to fully understand them.     

Physiological response of Pseudomonas putida S12 subjected to reduced water activity

Abstract The effect of osmotic stress, given as decreased water activity (a_w), on growth and the accumulation of potassium and the compatible solute betaine by Pseudomonas putida S12 was investigated. Reduced a_w was imposed by addition of sodium chloride, sucrose, glycerol or polyethylene glycol to the growth medium. Accumulation of potassium and betaine was established when sodium chloride and sucrose were used to cause osmotic stress. No accumulation of these solutes was found in the presence of glycerol. Addition of polyethylene glycol to the medium strongly decreased the growth rate in comparison with the other osmolytes tested at the corresponding a_w. Although polyethylene glycol did decrease the a_w, neither potassium nor betaine was accumulated by the cells.     

Altered intestinal chloride transport in cystic fibrosis

Sodium ion and chloride transport was studied in vitro in small intestinal and colonic tissue from patients with cystic fibrosis (CF) and from non-CF control subjects matched as to age and sex. Normal histological appearance and substantial response to mucosal glucose (5 mM, ileum) or mucosal amiloride (10(-5) M, colon) indicated normal tissue viability in both control and CF tissues. Electroneutral NaCl absorption was demonstrated in the small intestine of control subjects and CF patients. Small intestinal and colonic tissues of control subjects responded to four secretagogues (theophylline, 5 mM; prostaglandin E2, 10(-6) M; calcium ionophore (A23187), 10(-5) M; bethanechol, 5 x 10(-5) M), with electrogenic chloride secretion. The tissues of CF patients, however, did not respond to any of the test secretagogues. These studies demonstrate that an abnormality in chloride transport is present in the small intestinal and colonic epithelia of CF patients. Unlike airway epithelia, which secrete chloride in response to Ca ionophore, the intestinal epithelia of CF patients do not respond to either cAMP- or Ca-mediated secretagogues. This abnormality in intestinal electrolyte transport may play a role in the pathogenesis of meconium impactions in CF patients.     

UV spectroscopic studies of human erythrocyte superoxide dismutase

Using UV absorption spectroscopy, first derivative spectroscopy, and UV difference spectroscopy, the active site of human superoxide dismutase is probed. First derivative spectra (dA/d lambda versus lambda) show the HESOD spectrum to be a composite of Phe and Trp absorbance. The 278 and 288 nm Trp absorbance peaks are sensitive to solvent polarity. A 5-10% decrease in these peaks accompanies copper removal from the active site indicating greater solvent access to Trp in the apoenzyme than the holoenzyme. A Trp UV difference peak at 305-310 nm documents the presence or absence of copper at the active site, and documents also the movement of a nonbridging copper-binding His (His 46 or 120) when HESOD is inhibited by azide or when the copper moiety is reduced. Trp absorbances indicate that neither cyanide nor KCl inhibition affects the Cu(II)-His bonds. Phe UV absorbance is increased by the presence of copper at the active site and increased further by the addition of cyanide or azide. Neither Trp nor Phe responds to the presence of zinc in the active site. A molecular graphics program, FRODO, shows Trp and the four Phe residues lying in an approximate ring around the active site of HESOD and thus excellently placed to report on active site perturbations.     

Liposome-loaded phenylalanine or tryptophan as sickling inhibitor: a possible therapy for sickle cell disease

Phenylalanine or tryptophan entrapped in small unilamellar liposomes was used to transport Phe or Trp across the red blood cell membrane. The incorporation of Phe or Trp into RBCs via liposomes markedly inhibited and reversed the in vitro sickling of deoxy Hb S. Furthermore, normal and SS RBCs loaded with Phe or Trp did not exhibit significant change in osmotic fragility, mechanical fragility, autohemolysis, and glycolysis when compared to untreated RBCs. In addition, the oxygen affinity measured as the P50 and concentrations of 2,3-DPG and ATP were not affected by the incorporation of Phe or Trp into AA or SS RBCs. These results demonstrate that this liposomal transport system which transferred Phe and Trp into intact RBCs did not have any adverse effect on RBC metabolism and function, and may have therapeutic implications in the treatment of sickle cell disease.     

Metabolism of chloride in halophilic prokaryotes

While much understanding has been achieved on the intracellular sodium and potassium concentrations of halophilic and halotolerant microorganisms and on their regulation, we know little on the metabolism of anions. Archaea of the family Halobacteriaceae contain molar concentrations of chloride, which is pumped into the cells by cotransport with sodium ions and/or using the light-driven primary chloride pump halorhodopsin. Most halophilic and halotolerant representatives of the bacterial domain contain low intracellular ion concentrations, with organic osmotic solutes providing osmotic balance. However, some species show a specific requirement for chloride. In Halobacillus halophilus certain functions, such as growth, endospore germination, motility and flagellar synthesis, and glycine betaine transport are chloride dependent. In this organism the expression of a large number of proteins is chloride regulated. Other moderately halophilic Bacteria such as Halomonas elongata do not show a specific demand for chloride. A very high requirement for chloride was demonstrated in two groups of Bacteria that accumulate inorganic salts intracellularly rather than using organic osmotic solutes: the anaerobic Halanaerobiales and the aerobic extremely halophilic Salinibacter ruber. It is thus becoming increasingly clear that chloride has specific functions in haloadaptation in different groups of halophilic microorganisms.     

Detection of open and closed conformations of tryptophan synthase by 15N-heteronuclear single-quantum coherence nuclear magnetic resonance of bound 1-15N-L-tryptophan

1-15N-L-Tryptophan (1-15N-L-Trp) was synthesized from 15N-aniline by a Sandmeyer reaction, followed by cyclization to isatin, reduction to indole with LiAlH4, and condensation of the 15N-indole with L-serine, catalyzed by tryptophan synthase. 1-15N-L-Trp was complexed with wild-type tryptophan synthase and beta-subunit mutants, betaK87T, betaD305A, and betaE109D, in the absence or presence of the allosteric ligands sodium chloride and disodium alpha-glycerophosphate. The enzyme complexes were observed by 15N-heteronuclear single-quantum coherence nuclear magnetic resonance (15N-HSQC NMR) spectroscopy for the presence of 1-15N-L-Trp bound to the beta-active site. No 15N-HSQC signal was detected for 1-15N-L-Trp in 10 mm triethanolamine hydrochloride buffer at pH 8. 1-15N-L-Trp in the presence of wild-type tryptophan synthase in the absence or presence of 50 mm sodium chloride showed a cross peak at 10.25 ppm on the 1H axis and 129 ppm on the 15N axis as a result of reduced solvent exchange for the bound 1-15N-L-Trp, consistent with formation of a closed conformation of the active site. The addition of disodium alpha-glycerophosphate produced a signal twice as intense, suggesting that the equilibrium favors the closed conformation. 15N-HSQC NMR spectra of betaK87T and betaE109D mutant Trp synthase with 1-15N-L-Trp showed a similar cross peak either in the presence or absence of disodium alpha-glycerophosphate, indicating the preference for a closed conformation for these mutant proteins. In contrast, the betaD305A Trp synthase mutant only showed a 15N-HSQC signal in the presence of disodium alpha-glycerophosphate. Thus, this mutant Trp synthase favored an open conformation in the absence of disodium alpha-glycerophosphate but was able to form a closed conformation in the presence of disodium alpha-glycerophosphate. Our results demonstrate that the 15N-HSQC NMR spectra of 1-15N-L-Trp bound to Trp synthase can be used to determine the conformational state of mutant forms in solution rapidly. In contrast, UV-visible spectra of wild-type and mutant Trp synthase in the presence of L-Trp with NaCl and/or disodium alpha-glycerophosphate are more difficult to interpret in terms of altered conformational equilibria.     

Distribution,pharmacological characterization and function of the 18 kDa translocator protein in rat small intestine

Background information. The TSPO (18 kDa translocator protein) is a mitochondrial transmembrane protein involved in cholesterol transport in organs that synthesize steroids and bile salts. Different natural and synthetic high‐affinity TSPO ligands have been characterized through their ability to stimulate cholesterol transport, but also to stimulate other physiological functions including cell proliferation, apoptosis and calcium‐dependent transepithelial ion secretion. Here, we investigate the localization and functions of TSPO in the small intestine. Results. TSPO was present in enterocyte mitochondria but not in rat intestinal goblet cells. Enterocyte cytoplasm also contained the endogenous TSPO ligand, polypeptide DBI (diazepam‐binding inhibitor). Whereas intestinal TSPO had high affinity for the synthetic ligand PK 11195, the pharmacological profile of TSPO in the duodenum was distinct from the jejunum and ileum. Specifically, benzodiazepine Ro5‐4864 and protoporphyrin IX showed 5–13‐fold lower affinity for duodenal TSPO. The mRNA and protein ratios of TSPO to other mitochondrial membrane proteins VDAC (voltage‐dependent anion channel) and ANT (adenine nucleotide transporter) were significantly different. PK 11195 stimulated calcium‐dependent chloride secretion in the duodenum and calcium‐dependent chloride absorption in the ileum, but did not affect jejunum ion transport. Conclusions. The functional differences in subpopulations of TSPO in different regions of the intestine could be related to structural organization of mitochondrial protein complexes that mediate the ability of TSPO to modulate either chloride secretion or absorption in the duodenum and ileum respectively.     

Factors Affecting Autocatalysis of Botulinum A Neurotoxin Light Chain*

The light chain of botulinum neurotoxin serotype A undergoes autocatalytic fragmentation into two major peptides during purification and storage (Ahmed S. A. et al. 2001, J. Protein Chem. 20:221–231) by both intermolecular and intramolecular mechanisms (Ahmed S. A. etal. 2003, Biochemistry 42:12539–12549). In this study, we investigated the effects of buffers and salts on this autocatalytic reaction in the presence and absence of zinc chloride. In the presence of zinc chloride, the fragmentation reaction was enhanced in each of acetate, MES, HEPES and phosphate buffers with maximum occurring in acetate when compared to those in the absence of zinc chloride. Adding sodium chloride in phosphate buffer in the presence of zinc chloride increased the extent of proteolysis. Irrespective of the presence of zinc chloride, adding sodium chloride or potassium chloride in phosphate buffer elicited an additional proteolytic reaction. Higher concentrations of sodium phosphate buffer enhanced the autocatalytic reaction in the absence of zinc chloride. In contrast, in the presence of zinc chloride, higher concentrations of sodium phosphate decreased the autocatalytic reaction. Optimum pH of autocatalysis was not affected significantly by the absence or presence of zinc chloride. Like zinc chloride, other chlorides of divalent metals, such as magnesium, cobalt, iron and calcium also enhanced the autocatalytic reaction. Polyols such as ethylene glycol protected the light chain from fragmentation. Exposure of light chain to UV radiation led to enhanced fragmentation. In order to avoid fragmentation, the protein should be stored frozen in a low concentration buffer of neutral or higher pH devoid of any metal. Our results provide a choice of buffers and salts for isolation, purification and storage of intact botulinum neurotoxin serotype A light chain.     

Water and NaCl absorption by the intestine of the tilapiaSarotherodon mossambicus adapted to fresh water or seawater and the possible role of prolactin and cortisol

Summary Rates of intestinal water, sodium and chloride absorption in tilapia, adapted to fresh water (FW) and seawater (SW), were measured in vitro, using noneverted sacs made from the anterior, middle and posterior intestinal regions. The anterior intestine from SW fish showed considerably less water, sodium and chloride absorption compared with that seen in FW fish. The middle intestine showed either minimal absorption or some secretion in both FW and SW. In the posterior intestine, water absorption was only limitedly affected by SW-adaptation, but sodium and chloride absorption rates were significantly lower in SW fish. Reductions in water absorption were already evident in the anterior intestine 24 h after transfer to 1/3 SW but reached lower levels 3 to 5 days following transfer to 100% SW. Thus, the anterior intestine of tilapia responds to increased environmental salinity by decreasing uptake of ions, whereas the posterior intestine maintains similar water absorption in both FW and SW, although ion absorption is lower in SW.Prolactin administration to SW fish augmented sodium and water absorption in the anterior intestine but had no effect on chloride absorption. In contrast, cortisol administration to FW fish decreased absorption of sodium, chloride and water to levels usually seen in SW fish. The observed effects of these hormones in tilapia intestinal absorption may be confined to the specialized anterior intestinal region in this species; hormonal effects on the rest of the intestine were not examined.     

Inhibition of intestinal uptake of amino acids by unconjugated bile salt.

The unconjugated bile salt, sodium deoxycholate, at a concentration of 0.5 mM was shown to inhibit the intestinal uptake of the amino acids L-glycine, L-leucine, L-proline, L-lysine and L-tyrosine in rats in vitro. This effect was acutely reversible except for the basis amino acid L-lysine and is therefore not simply due to tissue damage. These results, and the recent finding that sodium deoxycholate inhibits intestinal absorption of amino acids in vivo, suggest that impaired intestinal amino acid transport may contribute to hypoproteinaemia in patients with bacterial overgrowth in the upper small intestine in whom deoxycholate is present in the small intestinal lumen in excessive concentrations.     

The role of zinc in the methylation of the coenzyme M thiol group in methanol:coenzyme M methyltransferase from Methanosarcina barkeri.

Methanol:coenzyme M methyltransferase from methanogenic archaea is a cobalamin-dependent enzyme composed of three different subunits: MtaA, MtaB and MtaC. MtaA is a zinc protein that catalyzes the methylation of coenzyme M (HS-CoM) with methylcob(III)alamin. We report zinc XAFS (X-ray absorption fine structure) results indicating that, in the absence of coenzyme M, zinc is probably coordinated by a single sulfur ligand and three oxygen or nitrogen ligands. In the presence of coenzyme M, one (N/O)-ligand was replaced by sulfur, most likely due to ligation of the thiol group of coenzyme M. Mutations in His237 or Cys239, which are proposed to be involved in ligating zinc, resulted in an over 90% loss in enzyme activity and in distinct changes in the zinc ligands. In the His237-->Ala and Cys239-->Ala mutants, coenzyme M also seemed to bind efficiently by ligation to zinc indicating that some aspects of the zinc ligand environment are surprisingly uncritical for coenzyme M binding.     

Yeast alpha mating factor structure-activity relationship derived from genetically selected peptide agonists and antagonists of Ste2p.

alpha-Factor, a 13-amino-acid pheromone secreted by haploid alpha cells of Saccharomyces cerevisiae, binds to Ste2p, a seven-transmembrane, G-protein-coupled receptor present on haploid alpha cells, to activate a signal transduction pathway required for conjugation and mating. To determine the structural requirements for alpha-factor activity, we developed a genetic screen to identify from random and semirandom libraries novel peptides that function as agonists or antagonists of Ste2p. The selection scheme was based on autocrine strains constructed to secrete random peptides and respond by growth to those that were either agonists or antagonists of Ste2p. Analysis of a number of peptides obtained by this selection procedure indicates that Trp1, Trp3, Pro8, and Gly9 are important for agonist activity specifically. His2, Leu4, Leu6, Pro10, a hydrophobic residue 12, and an aromatic residue 13 are important for both agonist and antagonist activity. Our results also show that activation of Ste2p can be achieved with novel, unanticipated combinations of amino acids. Finally, the results suggest the utility of this selection scheme for identifying novel ligands for mammalian G-protein-coupled receptors heterologously expressed in S. cerevisiae.