Effects of natural (zinc,vitamin E) and synthetic (diludin) antioxidants on the intestinal permeability in chicks with vitamin A deficiency

The main concern of this work was to examine the relation between altered antioxidant status on the one hand and increase in L-tryptophan absorption in the small intestine in order to bring further information regarding to possible role of vitamin A and zinc to maintaining of intestinal epithelial barrier integrity, on the other hand. In control, only some ideal tight junctions at the tip of the villi were permeable to ZnC1(2), whereas in A-hypovitaminosis permeability increased significantly. Studies demonstrate that an increased L-tryptophan accumulation in the intestinal mucous may result from a free radical damage to the mucous surface with formation of "leaky" junctions in the ilea. The results suggest that the zinc plays a crucial role in stabilizing biomembranes.     

Effect of various methods of oxygenation in the isolated small intestine on the tryptophan and glycine accumulation

The impact of various oxygenation types of intestinal preparations (mucosal, serosal and bilateral) on the transepithelial transport of amino acids and peptide according to the character of amino acids, their stereoisometry, incubation time, intestinal gradient and other factors, was investigated in fowl. Against-the-gradient-transport of L-tryptophan and glycine was observed at various oxygenation types, and its intensity decreased in the following sequence: bilateral, mucosal, serosal oxygenation. At various oxygenation types, the accumulation of L-tryptophan occurs more efficiently than that of DL-tryptophan and D-tryptophan. Using an in vitro bilateral intestinal oxygenation model system, we have demonstrated the plasma membrane of enterocyte microvilli to be rapidly impaired after oxidative stress. Glycine, but not L-tryptophan, attenuates oxidative injury in brush border membrane and alterations in amino acid transport activity. Overall, our data indicates that in vitro serosal oxygenation of the duodenum markedly improves glycine absorption, possibly involving the basolateral transporters.     

Role of exchange transport in the absorption of L-tryptophan in the small intestine of chicks

1. In in vitro experiments with accumulating mucosal preparations (AMP) and everted intestinal sacs, as well as in in vivo experiments with isolated loops of the small intestine the stimulating effect of a number of amino acids on L-tryptophan uptake was investigated. 2. Under "switched off" active transport (anoxia, 2,4-DNF treatment, sodium ion replacement by lithium ions in the mucosal solution) an expressed stimulation of L-tryptophan transport was observed within the mucosa and across the wall of the small intestine in the presence of L-proline, glycine, L-alpha-alanine, L-histidine and L-lysine. 3. Preincubation of AMP in the solutions of glycine, L-alpha-alanine and L-lysine was characterized by a stimulation of L-tryptophan transport, and the increase of its concentration in tissue was accompanied by the exit of an equivalent amount of glycine from it. 4. These observations show the participation of exchange transport in the uptake of L-tryptophan in the small intestine of chicks. 5. The mechanism of exchange transport in chicks starts to function on the 25th day after hatching and its intensity depends on the character of amino acid-modifier participating in the process. 6. Maximum activity of the exchange transport of L-tryptophan is demonstrated in the middle ileum. 7. L-alpha-Alanine stimulates the absorption of L-tryptophan from the isolated intestinal loop proving the existence of an exchange transport mechanism in a living organism. 8. An increased intensity of exchange transport is observed when feeding chicks with diets deficient and enriched in tryptophan.     

Zinc tetra-4-(4'-carboxyphenoxy)phthalocyanine as a new site-specific marker for serum albumin

The interaction of zinc tetra-4-(4'-carboxyphenoxy)phthalocyanine with bovine serum albumin was studied via electron absorption spectroscopy and fluorescence spectroscopy. Being the associate in aqueous media, zinc tetra-4-(4'-carboxyphenoxy)phthalocyanine is converted to its monomeric form to be bound to albumin, with the monomeric form of the phthalocyanine being characterized by a fluorescence in the visible region of the spectrum. The displacement titration of albumin complexes with the known site-specific markers (such as warfarin, L-tryptophan, and hemin) and phthalocyanine was performed to show the location of zinc tetra-4-(4'-carboxyphenoxy)phthalocyanine within the IIA subdomain of albumin.     

Interactions of tryptophan synthase, tryptophanase, and pyridoxal phosphate with oxindolyl-L-alanine and 2,3-dihydro-L-tryptophan: support for an indolenine intermediate in tryptophan metabolism

We have examined the interaction of tryptophan synthase and tryptophanase with the tryptophan analogues oxindolyl-L-alanine and 2,3-dihydro-L-tryptophan. Since these analogues have tetrahedral geometry at carbon 3 of the heterocyclic ring, they are structurally similar to the indolenine tautomer of L-tryptophan, a proposed intermediate in reactions of L-tryptophan. Oxindolyl-L-alanine and 2,3-dihydro-L-tryptophan are potent competitive inhibitors of both tryptophan synthase and tryptophanase, with KI values (3-17 microM) 10-100-fold lower than the corresponding Km or KI values for L-tryptophan. Addition of oxindolyl-L-alanine or 2,3-dihydro-L-tryptophan to solutions of the alpha 2 beta 2 complex of tryptophan synthase results in new absorption bands at 480 or 494 nm, respectively, which are ascribed to a quinonoid or alpha-carbanion intermediate. Spectrophotometric titration data give half-saturation values of 5 and 25 microM, which are comparable to the KI values obtained in kinetic experiments. Our finding that both enzymes catalyze incorporation of tritium from 3H2O into oxindolyl-L-alanine is evidence that both enzymes form alpha-carbanion intermediates with oxindolyl-L-alanine. These results support the proposal that the indolenine tautomer of L-tryptophan is an intermediate in reactions catalyzed by both tryptophanase and tryptophan synthase. In addition, we have found that oxindolyl-L-alanine reacts irreversibly with free pyridoxal phosphate to form a covalent adduct.     

Ligand-mediated conformational changes in Trp repressor protein of Escherichia coli probed through limited proteolysis and the use of specific antibodies

Trp repressor of Escherichia coli K-12 is a dimeric protein (monomer size, 108 amino acids) that acquires high affinity for certain operator targets in double-stranded DNA upon interaction with L-tryptophan. High titer antiserum directed against E. coli Trp repressor protein, elicited in rabbits, was monospecific toward native or denatured Trp repressor. Using an enzyme-linked immunosorbent assay to measure antigen-antibody reaction, we found that the binding of L-tryptophan to Trp repressor was associated with a marked decrease in antibody reactivity that presumably accompanied a conformational change in this protein to a state with strong affinity for trp operator-bearing DNA. We analyzed the pattern of cleavage of Trp repressor by chymotrypsin and trypsin and the effect of L-tryptophan on such hydrolytic cleavages. Chymotrypsin cleaved Trp repressor mainly between residues 71 and 72. In the presence of L-tryptophan this cleavage was slowed. The first-order rate constants for chymotryptic digestion of Trp repressor were 7.6 X 10(-2) and 4.6 X 10(-2) min-1 in the absence and presence of L-tryptophan, respectively. Tryptic digestion was more complex. Initial cleavage of Trp repressor occurred with approximately equal facility between residues 69-70 or 84-85. Subsequent tryptic hydrolyses led eventually to a major core fragment containing the first 54 amino acids of Trp repressor plus four other fragments from the carboxyl-terminal half of the protein. In the presence of L-tryptophan, cleavage by trypsin between residues 54-55 and 84-85 was retarded, even when a previous hydrolytic event elsewhere in the protein had occurred. Tryptophan had essentially no effect on the tryptic hydrolysis of peptide bond 97-98, but accelerated cleavage at peptide bond 69-70. The first-order rate constants for the first tryptic cleavage of Trp receptor were 1.55 X 10(-1) and 1.33 X 10(-1) min-1 in the absence and presence of ligand, respectively. Our results are compatible with a structural model wherein certain amino acid side chains and peptide bonds of Trp repressor (specifically, those of residues 69-85) lie on or near the surface of the protein. This region of Trp repressor has been predicted to contain the operator recognition site. The susceptibility to proteolytic attack of at least four peptide bonds in this area changes when the protein interacts with L-tryptophan.     

Differences in the nature of the interaction of insulin and proinsulin with zinc

1. The reversible interaction of zinc with pig insulin and proinsulin has been studied at pH7 by equilibrium dialysis (ultrafiltration) and by sedimentation equilibrium and velocity measurements in the ultracentrifuge. Binding values calculated from equilibria, where the ratio of free to bound zinc was varied in the range 0.01:1-10:1, indicated that proinsulin and insulin each contained two main orders of zinc binding with very different affinities for the metal. 2. In equilibria containing low concentrations of free zinc (free: bound ratios of 0.01-0.1:1) both insulin and proinsulin aggregated to form soluble hexamers containing firmly bound zinc (up to 0.284g-atom/monomer) with an apparent intrinsic association constant of 1.9x10(6)m(-1). 3. Higher concentrations of zinc (free: bound ratios of 0.1-10.0:1) resulted in a progressive difference in the zinc binding, aggregation and solubility properties of the metal complexes of insulin and proinsulin. At the highest concentration of free zinc, proinsulin bound a total of more than 5.0g-atom/monomer and aggregated to form a mixture of soluble polymers (mainly 5.1S). In contrast, insulin bound a total of only 1.0g-atom/monomer and was almost completely precipitated from solution. 4. These results would indicate that the presence of the peptide segment connecting the insulin moiety in proinsulin does not prevent the firm binding of zinc to the insulin moiety and the formation of hexamers of zinc-proinsulin. At the same time although the connecting peptide contains additional sites of lower affinity for zinc, which should facilitate inter- and intra-molecular cross-linking, the general conformation of the zinc-proinsulin hexamer must preclude the formation of very large and close-packed aggregates that are insoluble in solutions at equilibrium.     

Fungicidal Activity of Oxadiazolyl Sulfides

Glycyl-l-leucine is one of the best substrates for peptide hydrolases in the intestinal mucosa. Its absorption and hydrolysis were investigated in epithelial cells isolated from the rat intestine in the presence of bestatin, a specific inhibitor of certain peptide hydrolases, Bestatin competitively inhibited dipeptide hydrolase activities in isolated cells with a K_i value of 10^?8 m, but noncompetitively inhibited, and less significantly, the dipeptide absorption by isolated cells. At 10^?4 m bestatin inhibited half the dipeptide absorption, but only minimally inhibited the absorption of constituent amino acids. In the presence of bestatin at substantial concentrations the isolated cells took up a significant amount of the intact dipeptide, which otherwise appeared entirely in the form of free amino acids. These results are interpreted to substantiate a notion that a dual mechanism is operative for the absorption of readily-hydrolysable peptides: the peptide hydrolysis followed by uptake of thereby released amino acids, and the peptide transport followed by cytosolic hydrolysis.     

On the competition for available zinc

Extended x-ray absorption fine structure (EXAFS) spectroscopy was combined with thermodynamic and kinetic approaches to investigate zinc binding to a zinc finger (C2H2) and a tetrathiolate (C4) peptide. Both peptides represent structural zinc sites of proteins and rapidly bind a single zinc ion with picomolar dissociation constants. In competition with EDTA the transfer of peptide-bound zinc ions proved to be 6 orders of magnitude faster than predicted for a dissociation-association mechanism thus requiring ligand exchange mechanisms via peptide-zinc-EDTA complexes. EXAFS spectra of C2H2 showed the expected Cys2His2-ligand geometry when fully loaded with zinc. For a 2-fold excess of peptide, however, the existence of zinc-bridged peptide-peptide complexes with dominating sulfur coordination could be clearly shown. Whereas zinc binding kinetics of C2H2 appeared as a simple second order process, the suggested mechanism for C4 comprises a zinc-bridged Zn-(C4)2 species as well as a Zn-C4 species with less than 4 metal-bound thiolates, which is supported by EXAFS results. A rapid equilibrium of bound and unbound states of individual ligands might explain the kinetic instability of zinc-peptide complexes, which enables fast ligand exchange during the encounter of occupied and unoccupied acceptor sites. Depending on relative concentrations and stabilities, this results in a rapid transfer of zinc ions in the virtual absence of free zinc ions, as seen for the zinc transfer to EDTA, or in the formation of zinc-bridged complexes, as seen for both peptides with excess of peptides over available zinc.     

X-ray analysis of structural changes induced by reduced nicotinamide adenine dinucleotide when bound to cysteine-46-carboxymethylated liver alcohol dehydrogenase

The structure of the complex between Cys-46-carboxymethylated horse liver alcohol dehydrogenase (CM-LADH) and reduced nicotinamide adenine dinucleotide (NADH) has been determined by X-ray analysis. The complex represents NADH binding to the orthorhombic, "open" conformation of the enzyme. Coenzyme binding here induces a local structural change in the peptide loop 293-297, but there is no domain rotation, as observed for the "closed" conformation of the protein. This local movement of a few residues in the loop is sufficient to trap the nicotinamide ring of NADH within the active-site area close to a productive binding position. The carboxymethyl group on the zinc ligand cysteine-46 is oriented between the pyrophosphate bridge of NADH and the guanidinium group of arginine-369 and can occupy this position because the coenzyme binding cleft remains open and unchanged upon coenzyme binding. The zinc coordination sphere is distorted, and the position of the metal atom is shifted 1 A compared to native unliganded LADH. The distance between the zinc ion and the sulfur of the alkylated cysteine residue is of the order of 3 A. Alkylation experiments were performed at 0.15 and 10 mM iodoacetate, and peptide maps were examined. Gentle treatment with reagent yields an enzyme product which is substituted at only one of the two zinc binding sites per subunit of LADH (Cys-46). This enzyme species maintains its structural integrity; it binds coenzyme which induces conformational changes resolved into two steps. Thus, in addition to the orthorhombic complex, a crystalline NADH complex in the closed conformation of CM-LADH was obtained. These crystals showed enzymic activity, and single crystals were analyzed with microspectrophotometric methods. Formation of the stable crystalline abortive complex between CM-LADH-NAD+ and 4-trans-(N,N-dimethylamino)cinnamaldehyde (DACA) could be observed upon addition of excess aldehyde to the closed complex of CM-LADH-NADH. The CM-LADH-NAD+-DACA complex is characterized by an intense absorption band with a lambda max at 456 nm which corresponds to a shift in the spectrum of free DACA of approximately 60 nm. At the higher concentration of iodoacetate, three of the cysteine ligands to the second zinc atom (Cys-100, -103, and -111) are alkylated in addition to Cys-46. This enzyme product rapidly denatures and cannot be crystallized under our conditions. This is an experimental indication that the intact noncatalytic zinc binding site contributes to the structural stability of the protein.     

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.     

Binding of nitric oxide to reduced L-tryptophan-2,3-dioxygenase as studied by electron paramagnetic resonance.

Ferrous L-tryptophan-2,3-dioxygenase reacts with nitric oxide both in the presence and in the absence of L-tryptophan. Electron paramagnetic resonance studies suggest that the proximal ligand of the heme is a nitrogen atom, probably from an histidyl residue. The interaction of the protein with substrate changes both the symmetry of the paramagnetic center and the mode of interaction of the iron atom with its two axial ligands, NO and the proximal nitrogen atom. Optical absorption and EPR spectra suggest that the affinity of NO for tryptophan dioxygenase increases in the order: tryptophan dioxygenase, tryptophan dioxygenase + alpha-methyltryptophan, tryptophan diogenase " 5-hydroxytryptophan, tryptophan dioxygenase + L-tryptophan. A possible correlation between the number of superhyperfine lines in the EPR spectrum and the affinity of the enzyme for NO is discussed.     

Stimulation of L-tryptophan transport by di- and polysaccharides in the small intestine of chicks

In in vivo and in vitro experiments the effect of various carbohydrates on the absorption of L-tryptophan in the chick small intestine was investigated. On feeding the chicks with semisynthetic diet containing 58.5% of carbohydrates a stimulatory effect of glucose, particularly of starch and saccharose, on the entry of L-tryptophan into the portal vein from the chick gastrointestinal tract has been found. Using an in vitro technique the activating effect of starch and disaccharides (maltose, saccharose) on the intestinal transport of L-tryptophan was detected while monosaccharides (glucose, fructose) at different concentrations had no effect on this process or inhibited it. The possibility that energy of disaccharide hydrolysis is used to stimulate transport process is discussed.     

Metal binding properties of single amino acid deletion mutants of zinc finger peptides: studies using cobalt(II) as a spectroscopic probe.

Peptides corresponding to Cys2His2 zinc finger domains from which one amino acid has been deleted have been synthesized and their metal-binding properties characterized. In contrast to earlier reports (Párraga, G., S. Horvath, L. Hood, E. T. Young, and R. E. Klevit. 1990. Proc. Natl. Acad. Sci. USA. 87:137-141.), such peptides do bind metal ions such as cobalt(II). A peptide with the sequence ProTyrLysCysProGluCysLysSerPheSerGlnLysSerAspLeuValLysHisGlnArgThrHis ThrGly (which corresponds to a previously characterized consensus zinc finger sequence from which a Gly residue immediately following the second Cys residue has been deleted) was found to form a 1:1 peptide to cobalt(II) complex with an absorption spectrum quite similar to those previously observed for zinc finger peptide-cobalt(II) complexes. The dissociation constant for this complex is 6 x 10(-6)M, a factor of 100 times higher than that for the parent peptide. A peptide with the sequence LysProTyrProCysGlyLeuCysArgCysPheThrArgArgAspLeuLeulleArgHisAlaGln - LyslleHisSerGlyAsnLeu corresponding to a similar mutation of the peptide ADR1 was also characterized. Spectroscopic studies with cobalt(II) revealed that this peptide forms both 1:1 and 2:1 peptide to cobalt(II) complexes. The absorption spectra of the two forms and the dissociation constants were determined via deconvolution methods. In contrast, the parent peptide ADR1a was found to form only a 1:1 complex under comparable conditions and this 1:1 complex was found to be more stable than that for the mutant. These results reveal that deletion mutations do adversely affect the stability of zinc finger peptide-metal complexes but that the effects are not as drastic as had been previously described.     

N-Terminal domain of HTLV-I integrase. Complexation and conformational studies of the zinc finger.

The HTLV-I integrase N-terminal domain [50-residue peptide (IN50)], and a 35-residue truncated peptide formed by residues 9-43 (IN35) have been synthesized by solid-phase peptide synthesis. Formation of the 50-residue zinc finger type structure through a HHCC motif has been proved by UV-visible absorption spectroscopy. Its stability was demonstrated by an original method using RP-HPLC. Similar experiments performed on the 35-residue peptide showed that the truncation does not prevent zinc complex formation but rather that it significantly influences its stability. As evidenced by CD spectroscopy, the 50-residue zinc finger is unordered in aqueous solution but adopts a partially helical conformation when trifluoroethanol is added. These results are in agreement with our secondary structure predictions and demonstrate that the HTLV-I integrase N-terminal domain is likely to be composed of an helical region (residues 28-42) and a beta-strand (residues 20-23), associated with a HHCC zinc-binding motif. Size-exclusion chromatography showed that the structured zinc finger dimerizes through the helical region.     

Intraluminal chemistry of zinc in milks

The solubility of both free and low molecular weight ligand complexed calcium, magnesium, and zinc in skimmed human and bovine milks over intestinal luminal pH ranges (approximately 3-7) was measured using ultrafiltration techniques. Some of the experimental difficulties associated with the separation of labile metal ion ligand components from milks by ultrafiltration techniques are discussed. Experimental methods designed to minimize interferences in mineral ultrafiltrations from milks are outlined. Mineral solubilities in skimmed human and bovine milks are compared to data obtained in a previous study using milk models. The solubility of zinc in both skimmed bovine and bovine model milks is less than in human and human model milks at the higher pHs, characteristic of the luminal region where zinc absorption is thought to occur. The decrease in zinc solubility is caused by the coprecipitation of zinc with calcium phosphate, particularly in bovine milk samples. If solubility at the higher pHs is a requisite for zinc absorption then the enhanced bioavailability of zinc from human milk may be related to the detrimental element-compound interaction discussed in this study.     

Impaired peripheral zinc metabolism in patients with senile dementia of probable Alzheimer’s type as shown by low plasma concentrations of thymulin

Plasma concentrations of a zinc carrier peptide, namely thymulin, were measured according to a bioassay in young donors, healthy elderly, and patients with senile dementia of Alzheimer’s type (SDAT). Thymulin is a hormone released by thymic epithelial cells and its biological activity on cells of immune system is dependent on the presence of one molecule of zinc bound to the peptide. Plasma from different subjects were fractionated by gel filtration to yield protein-bound thymulin and free thymulin. The biological activity of the peptide was then assessed in the two different fractions. The activity of protein-bound thymulin was higher in young donors than in elderly of SDAT patients, being the lowest in SDAT. Addition of zinc ions to plasma fractions increased the thymulin activity of samples from elderly and SDAT patients to levels observed in young donors. Thymulin activity in free thymulin fractions was lower in the elderly than in the young and was practically undetectable in SDAT patients. The addition of zinc ions normalized the activity of thymulin in these fractions from both the elderly and SDAT patients. These findings confirm the presence of an altered zinc status in the elderly and suggest that an impaired zinc metabolism may be present in SDAT patients.     

Effect of alpha-tryptophan on conditioning and behavior in rats with experimental pathology of the thyroid gland

Effects of L-tryptophan on learning and behavior were studied in male rats with a deficit or excess of thyroid hormones. Learning was assessed in active avoidance paradigm, and behavior was estimated in the "open-field" test. It was found that L-tryptophan restored the capability of thyreoidectomized rats for acquisition and reproduction of the active avoidance reaction and increased the exploratory behavior of these rats in the open-field. In triiodothyronine treated rats, L-tryptophan eliminated a light stimulatory effects of thyroid hormones on the processes of formations and retention of the active avoidance reaction, increased the exploratory activity, but decreased grooming in the open-field.     

Effect of manganese on zinc-65 absorption by rice seedlings and its translocation within the plants

Summary Effect of manganese on absorption and translocation of zinc by rice seedlings was studied in a nutrient solution using radioactive zinc (Zn⁶⁵). With increase in manganese application, zinc uptake decreased in roots, but increased in shoots with an application upto 10 M manganese. Reduction in zinc absorption was more at low rates of zinc application. However, translocation of zinc from roots to shoots increased with manganese application upto 20 M but the increase was more pronounced with an application of 2M zinc only.