Cofactor Activity in Factor VIIIa of the Blood Clotting Pathway Is Stabilized by an Interdomain Bond between His281 and Ser524 Formed in Factor VIII

The factor VIII (FVIII) crystal structure suggests a possible bonding interaction of His²⁸¹ (A1 domain) with Ser⁵²⁴ (A2 domain), although the resolution of the structure (∼4 Å) does not firmly establish this bonding. To establish that side chains of these residues participate in an interdomain bond, we prepared and examined the functional properties of a residue swap variant (H281S/S524H) where His²⁸¹ and Ser⁵²⁴ residues were exchanged with one another and a disulfide-bridged variant (H281C/S524C) where the two residues were replaced with Cys. The latter variant showed efficient disulfide bonding of the A1 and A2 domains. The swap variant showed WT-like FVIII and FVIIIa stability, which were markedly reduced for H281A and S524A variants in an earlier study. The disulfide-bridged variant showed ∼20% increased FVIII stability, and FVIIIa did not decay during the time course measured. This variant also yielded 35% increased thrombin peak values compared with WT in a plasma-based thrombin generation assay. Binding analyses of H281S-A1/A3C1C2 dimer with S524H-A2 subunit yielded a near WT-like affinity value, whereas combining the variant dimer or A2 subunit with the WT complement yielded ∼5- and ∼10-fold reductions, respectively, in affinity. Other functional properties including thrombin generation potential, FIXa binding affinity, K_m for FX of FXase complexes, thrombin activation efficiency, and down-regulation by activated protein C showed similar results for the two variants compared with WT FVIII. These results indicate that the side chains of His²⁸¹ and Ser⁵²⁴ are in close proximity and contribute to a bonding interaction in FVIII that is retained in FVIIIa.     

In rat dipeptidyl peptidase III,His is essential for catalysis,and Glu or Glu stabilizes the coordination bond between His or His and zinc ion

Dipeptidyl peptidase (DPP) III is a zinc-dependent exopeptidase that has a unique motif, “HELLGH,” as the zinc-binding site. In the present study, a three-dimensional (3D) model of rat DPP III was generated with the X-ray crystal structure of human DPP III (PDB: 3FVY [Dobrovetsky E. et al. (2009) SGC]) as a template. The replacement of the seven charged amino acid residues with a hydrophobic amino acid around the zinc ion did not cause any significant changes in K_m values or in the substrate specificity. However, the k_cat values of H568R and H568Y were remarkably reduced, by factors of 50 and 400, respectively. The His⁵⁶⁸ residue of rat DPP III is essential for enzyme catalysis. The k_cat values of the mutants E507A and E512A were 2.38 and 3.88 s^− 1 toward Arg-Arg-NA, and 0.097 and 0.59 s⁻¹ toward Phe-Arg-NA, respectively. These values were markedly lower than those of the wild-type DPP III. Furthermore, the zinc contents of E507A and E512A were 0.29 and 0.08 atom per mol of protein, respectively, and those mutations caused remarkable increases in the dissociation constants of the zinc ions from DPP III by factors of 5 × 10³ to 2 × 10⁴. The 3D model of the catalytic domain of rat DPP III showed that the carboxyl oxygen atoms of Glu⁵⁰⁷ and Glu⁵¹² form the hydrogen bonds to the nitrogen atoms of His⁴⁵⁵ and His⁴⁵⁰. All of these results showed that Glu⁵⁰⁷ or Glu⁵¹² stabilizes the coordination bond between the zinc ion and His⁴⁵⁵ or His⁴⁵⁰.     

A novel polymorphism of apolipoprotein A-IV is the result of an asparagine to serine substitution at residue 127

We have identified a hitherto genetic polymorphism of apolipoprotein A-IV (apo-IV). The molecular basis for this polymorphism is an A to G substitution at nucleotide 1687 resulting in an Asn to Ser change of amino acid 127. The frequencies of the two apoA-IV alleles (designated apoA-IV^127Asn and apoA-IV^127Ser), determined by Hinc II restriction analysis of PCR amplified exon three of the apoA-IV gene, were 0.788 and 0.212, respectively, in a Finnish population sample. Allele frequencies of another polymorphism due to a Thr to Ser substitution at amino acid 347 were determined using Hinf I restriction analysis. The allele frequencies were 0.823 for apoA-IV^347Thr and 0.177 for apoA-IV^247Ser. None of the apoA-IV polymorphisms (apoA-IV_{127:Asn→Ser}, apoA-IV_{347:Thr→Ser} and apoA-IV_{360:Gln→His}) had any effect of plasma lipid and lipoprotein concentrations in cohorts of dyslipidemic men and in a population sample of normolipidemic controls. There was also no association between the history of previous myocardial infarction and any of the apoA-IV alleles.     

Hereditary folate malabsorption: A positively charged amino acid at position 113 of the proton-coupled folate transporter (PCFT/SLC46A1) is required for folic acid binding

The proton-coupled folate transporter (PCFT/SLC46A1) mediates intestinal folate uptake at acidic pH. Some loss of folic acid (FA) transport mutations in PCFT from hereditary folate malabsorption (HFM) patients cluster in R113, thereby suggesting a functional role for this residue. Herein, unlike non-conservative substitutions, an R113H mutant displayed 80-fold increase in the FA transport Km while retaining parental Vmax, hence indicating a major fall in folate substrate affinity. Furthermore, consistent with the preservation of 9% of parental transport activity, R113H transfectants displayed a substantial decrease in the FA growth requirement relative to mock transfectants. Homology modeling based on the crystal structures of the Escherichia coli transporter homologues EmrD and glycerol-3-phosphate transporter revealed that the R113H rotamer properly protrudes into the cytoplasmic face of the minor cleft normally occupied by R113. These findings constitute the first demonstration that a basic amino acid at position 113 is required for folate substrate binding.     

Human proton coupled folic acid transporter is a monodisperse oligomer in the lauryl maltose neopentyl glycol solubilized state

The human proton coupled folic acid transporter PCFT is the major import route for dietary folates. Mutations in the gene encoding PCFT cause hereditary folic acid malabsorption, which manifests itself by compromised folate absorption from the intestine and also in impaired folate transport into the central nervous system. Since its recent discovery, PCFT has been the subject of numerous biochemical studies aiming at understanding its structure and mechanism. One major focus has been its oligomeric state, with some reports supporting oligomers and others a monomer. Here, we report the overexpression and purification of recombinant PCFT. Following detergent screening, n-Dodecyl β-D-maltoside (DDM) and lauryl maltose neopentyl glycol (LMNG) were chosen for further work as they exhibited the most optimal solubilization. We found that purified detergent solubilized PCFT was able to bind folic acid, thus indicating a functionally active protein. Size exclusion chromatography showed that PCFT in DDM was polydisperse; the LMNG preparation was clearly monodisperse but with shorter retention time than the major DDM peak. To assess the oligomeric state negative stain electron microscopy was performed which showed a particle with the size of a PCFT dimer.     

Delineating the Extracellular Water-Accessible Surface of the Proton-Coupled Folate Transporter

The proton-coupled folate transporter (PCFT) was recently identified as the major uptake route for dietary folates in humans. The three-dimensional structure of PCFT and its detailed interplay with function remain to be determined. We screened the water-accessible extracellular surface of HsPCFT using the substituted-cysteine accessibility method, to investigate the boundaries between the water-accessible surface and inaccessible buried protein segments. Single-cysteines, engineered individually at 40 positions in a functional cysteine-less HsPCFT background construct, were probed for plasma-membrane expression in Xenopus oocytes with a bilayer-impermeant primary-amine-reactive biotinylating agent (sulfosuccinimidyl 6-(biotinamido) hexanoate), and additionally for water-accessibility of the respective engineered cysteine with the sulfhydryl-selective biotinylating agent 2-((biotinoyl)amino)ethyl methanethiosulfonate. The ratio between Cys-selective over amine-selective labeling was further used to evaluate three-dimensional models of HsPCFT generated by homology / threading modeling. The closest homologues of HsPCFT with a known experimentally-determined three-dimensional structure are all members of one of the largest membrane protein super-families, the major facilitator superfamily (MFS). The low sequence identity - 14% or less – between HsPCFT and these templates necessitates experiment-based evaluation and model refinement of homology / threading models. With the present set of single-cysteine accessibilities, the models based on GlpT and PepT_St are most promising for further refinement.     

Role of Lys281 in the <Emphasis Type="Italic">Dunaliella salina</Emphasis> (6-4) Photolyase Reaction

His³⁵⁴ and His³⁵⁸, two highly conserved histidines in Xenopus laevis (6-4) photolyase [equivalent to His⁴⁰¹ and His⁴⁰⁵, in Dunaliella salina (6-4) photolyase], are critical for photoreactivation. They act as a base and an acid, respectively. However, the remaining high repair activity when the pH value is higher than the pKa of histidine suggests the involvement of other basic amino acids in photoreactivation. According to the results of in vivo enzyme assay and three-dimension structural model of Dunaliella salina (6-4) photolyase we hypothesized that Lys²⁸¹ might be involved in the photoreactivation over the pH range from 10.0 to 11.0. To test this, we generated two mutant forms of the (6-4) photolyase, K281G and K281R mutant, by overlap extension polymerase chain reaction, and performed the enzyme assay with these mutants. From these results we conclude that the Lys²⁸¹, which is highly conserved in (6-4) photolyases, participates in the photoreactivation and acts as an acid to donate a proton to His⁴⁰¹ when the environmental pH is higher than the pKa value of histidine.     

Irreversible inhibition of folate transport in Lactobacillus casei by covalent modification of the binding protein with carbodiimide-activated folate

Activated folate formed by reaction of folic acid and 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide irreversibly inhibits the folate transport system of Lactobacillus casei. Complete inhibition of both folate binding to the carrier protein and folate transport was achieved by pretreatment of the cells at low temperature (4 °C) and at neutral pH with 200 nm activated folate. Fifty percent inhibition of binding and transport occurred at 35 and 40 nm activated folate, respectively. Specificity was demonstrated by the fact that excess nonactivated folate added during the pretreatment step afforded complete protection of the binding protein against inhibition, and that activated folate had no effect on the binding or transport of thiamine. Rapid measurements at 4 °C were employed to show that, prior to the appearance of irreversible inhibition, activated folate (K_i = 15 nM) interacted reversibly with the binding site for folate (K_d = 0.8 nM). Cells treated with activated [³H]folate incorporated 1 mol of folate per mole of binding protein. Purification of the labeled protein followed by digestion with Pronase led to the isolation of a compound identified as ?-N-folyl lysine. The ?-amino group of a lysyl residue thus appears to be the nucleophilic group at the binding site that reacts with activated folate.     

Folate Production by Bifidobacteria as a Potential Probiotic Property

The ability of 76 Bifidobacterium strains to produce folate was investigated. In order to evaluate folic acid productivity, bifidobacteria were cultivated in the folate-free semisynthetic medium SM7. Most of the tested strains needed folate for growth. The production and the extent of vitamin accumulation were not a function of species but were distinctive features of individual strains. Six strains among the 17 that grew without folate produced significantly higher concentrations of vitamin (between 41 and 82 ng ml⁻¹). The effects of exogenous folate and p-aminobenzoic acid (PABA) concentrations on folate production were evaluated. In contrast to most of the other strains, the folate yield of B. adolescentis MB 239 was not negatively affected by either PABA or exogenous folic acid. Folate production by B. adolescentis MB 239 was studied in the pH range of the colonic environment, and a comparison of folate production on raffinose, lactose, and fructo-oligosaccharides, which belong to three important groups of fermentable intestinal carbon sources, was established. Differences in folate biosynthesis by B. adolescentis MB 239 were not observed as a function either of the pH or of the carbon source. Fecal culture experiments demonstrated that the addition of B. adolescentis MB 239 may increase the folate concentration in the colonic environment.     

Cation-Dependent Binding of Substrate to the Folate Transport Protein of Lactobacillus casei

Lactobacillus casei cells grown in the presence of limiting folate contained large amounts of a membrane-associated binding protein which mediates folate transport. Binding to this protein at 4°C was time and concentration dependent and at low levels (1 to 10 nM) of folate required 60 min to reach a steady state. The apparent dissociation constant (K_d) for folate was 1.2 nM at pH 7.5 in 100 mM K-phosphate buffer, and it varied by less than twofold when measured over a range of pH values (5.5 to 7.5) or in buffered salt solutions of differing ionic compositions. Conversely, removal of ions and their replacement with isotonic sucrose (pH 7.5) led to a 200-fold reduction in binding affinity for folate. Restoration of the high-affinity state of the binding protein could be achieved by the readdition of various cations to the sucrose medium. K_d measurements over a range of cation concentrations revealed that a half-maximal restoration of binding affinity was obtained with relatively low levels (10 to 50 μM) of divalent cations (e.g., Ca²⁺, Mg²⁺, and ethylenediammonium²⁺ ions). Monovalent cations (e.g., Na⁺, K⁺, and Tris⁺) were also effective, but only at concentrations in the millimolar range. The K_d for folate reached a minimum of 0.6 nM at pH 7.5 in the presence of excess CaCl₂. In cells suspended in sucrose, the affinity of the binding protein for folate increased 20-fold by decreasing the pH from 7.5 to 4.5, indicating that protons can partially fulfill the cation requirement. These results suggest that the folate transport protein of L. casei may contain both a substrate- and cation-binding site and that folate binds with a high affinity only after the cation-binding site has been occupied. The presence of these binding sites would support the hypothesis that folate is transported across the cell membrane via a cation-folate symport mechanism.     

CIDNP study of the aromatic side chain interactions in myotoxina

CIDNP and COSY measurements were applied to study aromatic side chain interactions and conformations in myotoxina, aCrotalus venom toxin which acts as blocker of the Ca²⁺ influx in the sarcoplasmic reticulum calcium pump. New evidence for the existence of a hydrophobic aromatic cluster at the amino terminus was obtained. This cluster consists of Tyr₁, His₅, His₁₀, and (possibly) F₁₂. The CIDNP data clearly establish that the usual order of the tyrosine 2, 6 and 3, 5 proton signals of Tyr, is inverted, because of the large diamagnetic shielding effects of one ring on the other. The lines of the 2, 6 ring protons of Tyr₁, and proton 4 in each of His₅ and His₁₀ are significantly broadened, an outcome of the side-chain hydrophobic interaction. The aromatic cluster could possibly present a hydrophobic sticky patch for binding of toxin by Ca²⁺ ATPase.     

Catalytic properties of thimet oligopeptidase H600A mutant

Thimet oligopeptidase (EC 3.4.24.15, TOP) is a metallo-oligopeptidase that participates in the intracellular metabolism of peptides. Predictions based on structurally analogous peptidases (Dcp and ACE-2) show that TOP can present a hinge-bend movement during substrate hydrolysis, what brings some residues closer to the substrate. One of these residues that in TOP crystallographic structure are far from the catalytic residues, but, moves toward the substrate considering this possible structural reorganization is His⁶⁰⁰. In the present work, the role of His⁶⁰⁰ of TOP was investigated by site-directed mutagenesis. TOP H600A mutant was characterized through analysis of S₁ and S₁′ specificity, pH-activity profile and inhibition by JA-2. Results showed that TOP His⁶⁰⁰ residue makes important interactions with the substrate, supporting the prediction that His⁶⁰⁰ moves toward the substrate due to a hinge movement similar to the Dcp and ACE-2. Furthermore, the mutation H600A affected both K_m and k_cat, showing the importance of His⁶⁰⁰ for both substrate binding and/or product release from active site. Changes in the pH-profile may indicate also the participation of His⁶⁰⁰ in TOP catalysis, transferring a proton to the newly generated NH₂-terminus or helping Tyr⁶⁰⁵ and/or Tyr⁶¹² in the intermediate oxyanion stabilization.     

Partial structure and properties of the ferredoxin from Rhodymenia palmata

A ferredoxin of MW 11 000 was isolated from the marine alga Rhodymenia palmata (Palmaria palmata). In its oxidised form the ferredoxin had absorption maxima at 276, sh 281, 328, 423 and 465 nm, and contained a single [2Fe-2S] cluster. The midpoint potential of the ferredoxin was ?400 mV and it effectively mediated electron transport in NADP⁺-photoreduction by higher plant chloroplasts, and pyruvate decarboxylation by the phosphoroclastic system of an anacrobic bacterium. The amino acid composition was Lys₃, His₁, Arg₁, Asx₁₂, Thr₉, Ser₈, Glx₁₃, Pro₄, Gly₈, Ala₇, Cys₅, Val₈, Ile₄, Leu₉, Tyr₄, Phe₂; tryptophan and methionine were absent from the molecule. The N-terminal amino acid region consisting of ca half the total amino acid sequence was determined using an automatic sequencer.     

Histidine 352 (His352) and Tryptophan 355 (Trp355) Are Essential for Flax UGT74S1 Glucosylation Activity toward Secoisolariciresinol

Flax secoisolariciresinol diglucoside (SDG) lignan is a natural phytoestrogen for which a positive role in metabolic diseases is emerging. Until recently however, much less was known about SDG and its monoglucoside (SMG) biosynthesis. Lately, flax UGT74S1 was identified and characterized as an enzyme sequentially glucosylating secoisolariciresinol (SECO) into SMG and SDG when expressed in yeast. However, the amino acids critical for UGT74S1 glucosyltransferase activity were unknown. A 3D structural modeling and docking, site-directed mutagenesis of five amino acids in the plant secondary product glycosyltransferase (PSPG) motif, and enzyme assays were conducted. UGT74S1 appeared to be structurally similar to the Arabidopsis thaliana UGT72B1 model. The ligand docking predicted Ser³⁵⁷ and Trp³⁵⁵ as binding to the phosphate and hydroxyl groups of UDP-glucose, whereas Cys³³⁵, Gln³³⁷ and Trp³⁵⁵ were predicted to bind the 7-OH, 2-OCH₃ and 17-OCH₃ of SECO. Site-directed mutagenesis of Cys³³⁵, Gln³³⁷, His³⁵², Trp³⁵⁵ and Ser³⁵⁷_, and enzyme assays revealed an alteration of these binding sites and a significant reduction of UGT74S1 glucosyltransferase catalytic activity towards SECO and UDP-glucose in all mutants. A complete abolition of UGT74S1 activity was observed when Trp³⁵⁵ was substituted to Ala³⁵⁵ and Gly³⁵⁵ or when changing His³⁵² to Asp³⁵²_, and an altered metabolite profile was observed in Cys335Ala, Gln337Ala, and Ser357Ala mutants. This study provided for the first time evidence that Trp³⁵⁵ and His³⁵² are critical for UGT74S1’s glucosylation activity toward SECO and suggested the possibility for SMG production in vitro.     

Rodent intestinal folate transporters (SLC46A1): secondary structure, functional properties, and response to dietary folate restriction

This laboratory recently identified a human gene that encodes a novel folate transporter [Homo sapiens proton-coupled folate transporter (HsPCFT); SLC46A1] required for intestinal folate absorption. This study focused on mouse (Mus musculus) PCFT (MmPCFT) and rat (Rattus norvegicus) PCFT (RnPCFT) and addresses their secondary structure, specificity, tissue expression, and regulation by dietary folates. Both rodent PCFT proteins traffic to the cell membrane with the NH(2)- and COOH-termini accessible to antibodies targeted to these domains only in permeabilized HeLa cells. This, together with computer-based topological analyses, is consistent with a model in which rodent PCFT proteins likely contain 12 transmembrane domains. Transport of [(3)H]folates was optimal at pH 5.5 and decreased with increasing pH due to an increase in K(m) and a decrease in V(max). At pH 7.0, folic acid and methotrexate influx was negligible, but there was residual (6S)5-methyltetrahydrofolate transport. Uptake of folates in PCFT-injected Xenopus oocytes was electrogenic and pH dependent. Folic acid influx K(m) values of MmPCFT and RnPCFT, assessed electrophysiologically, were 0.7 and 0.3 microM at pH 5.5 and 1.1 and 0.8 microM at pH 6.5, respectively. Rodent PCFTs were highly specific for monoglutamyl but not polyglutamyl methotrexate. MmPCFT mRNA was highly expressed in the duodenum, proximal jejunum, liver, and kidney with lesser expression in the brain and other tissues. MmPCFT protein was localized to the apical brush-border membrane of the duodenum and proximal jejunum. MmPCFT mRNA levels increased approximately 13-fold in the proximal small intestine in mice fed a folate-deficient vesus folate-replete diet, consistent with the critical role that PCFT plays in intestinal folate absorption.     

Effect of theophylline and Na+ on methionine influx in Na+-depleted intestine

The unidirectional influx of methionine into the brush border epithelium of chicken jejunum has been studied. Tissues leached of Na⁺ transport methionine from a medium devoid of Na⁺ with reduced apparent affinity (K_t) and maximal flux (J_max). Addition of Na⁺ to the medium during a 1-min incubation with substrate, or during a 30-min preincubation, restored K_t but affected J_max slightly. Theophylline was found to maintain J_max in the absence of Na⁺. Essentially complete restoration of K_t and J_max could be attained when theophylline-treated tissue was exposed to Na⁺ for 30 min. Influx from a Na⁺ medium was unaffected by theophylline pretreatment in Na⁺-containing buffer. K_t was increased without an effet upon J_max when influx was studied from choline medium following preincubation in Na⁺.Modifiers of tissue cyclic AMP levels were investigated in conjunction with theophylline. Histamine and carbachol were found to inhibit theophylline-stimulated transport. Secretin was found to stimulate influx in Na⁺-leached tissue, but did not potentiate the theophylline effect. Amino acids in the incubation medium inhibited theophylline-stimulated influx, whereas preloaded lysine or methionine had no effect.The results are interpreted in terms of a model which envisions roles for cellular and external Na⁺ and for cyclic AMP in the activation and regulation of amino acid transport in intestine.     

Folate Catabolites in Spot Urine as Non-Invasive Biomarkers of Folate Status during Habitual Intake and Folic Acid Supplementation

Background

Folate status, as reflected by red blood cell (RCF) and plasma folates (PF), is related to health and disease risk. Folate degradation products para-aminobenzoylglutamate (pABG) and para-acetamidobenzoylglutamate (apABG) in 24 hour urine have recently been shown to correlate with blood folate.

Aim

Since blood sampling and collection of 24 hour urine are cumbersome, we investigated whether the determination of urinary folate catabolites in fasted spot urine is a suitable non-invasive biomarker for folate status in subjects before and during folic acid supplementation.

Study Design and Methods

Immediate effects of oral folic acid bolus intake on urinary folate catabolites were assessed in a short-term pre-study. In the main study we included 53 healthy men. Of these, 29 were selected for a 12 week folic acid supplementation (400 µg). Blood, 24 hour and spot urine were collected at baseline and after 6 and 12 weeks and PF, RCF, urinary apABG and pABG were determined.

Results

Intake of a 400 µg folic acid bolus resulted in immediate increase of urinary catabolites. In the main study pABG and apABG concentrations in spot urine correlated well with their excretion in 24 hour urine. In healthy men consuming habitual diet, pABG showed closer correlation with PF (r_s = 0.676) and RCF (r_s = 0.649) than apABG (r_s = 0.264, ns and 0.543). Supplementation led to significantly increased folate in plasma and red cells as well as elevated urinary folate catabolites, while only pABG correlated significantly with PF (r_s = 0.574) after 12 weeks.

Conclusion

Quantification of folate catabolites in fasted spot urine seems suitable as a non-invasive alternative to blood or 24 hour urine analysis for evaluation of folate status in populations consuming habitual diet. In non-steady-state conditions (folic acid supplementation) correlations between folate marker (RCF, PF, urinary catabolites) decrease due to differing kinetics.     

Reduced levels of folate transporters (PCFT and RFC) in membrane lipid rafts result in colonic folate malabsorption in chronic alcoholism

We studied the effect of chronic ethanol ingestion on folate transport across the colonic apical membranes (CAM) in rats. Male Wistar rats were fed 1 g/kg body weight/day ethanol (20%) solution orally for 3 months and folate transport was studied in the isolated colon apical membrane vesicles. The folate transport was found to be carrier mediated, saturable, with pH optima at 5.0. Chronic ethanol ingestion reduced the folate transport across the CAM by decreasing the affinity of transporters (high K_m) for the substrate and by decreasing the number of transporter molecules (low V_max) on the colon luminal surface. The decreased transport activity at the CAM was associated with down‐regulation of the proton‐coupled folate transporter (PCFT) and the reduced folate carrier (RFC) which resulted in decreased PCFT and RFC protein levels in the colon of rats fed alcohol chronically. Moreover, the PCFT and the RFC were found to be distributed in detergent insoluble fraction of the CAM in rats. Floatation experiments on Optiprep density gradients demonstrated the association of the PCFT and the RFC protein with lipid rafts (LR). Chronic alcoholism decreased the PCFT and the RFC protein levels in the CAM LR in accordance with the decreased synthesis. Hence, we propose that downregulation in the expression of the PCFT and the RFC in colon results in reduced levels of these transporters in colon apical membrane LR as a mechanism of folate malabsorption during chronic alcoholism. J. Cell. Physiol. 226: 579–587, 2011. © 2010 Wiley‐Liss, Inc.     

Further Characterization on the Transport Property of Plasmalemma NADH Oxidation System in Isolated Corn Root Protoplasts

Recent experiments show that exogenous NADH increases the O₂ consumption and uptake of inorganic ions into isolated corn (Zea mays L. Pioneer Hybrid 3320) root protoplasts (Lin 1982, Proc Natl Acad Sci USA 79: 3773-3776). A mild treatment of protoplasts with trypsin released most of the NADH oxidation system from the plasmalemma (Lin 1982 Plant Physiol 70: 326-328). Further studies on this system showed that exogenous NADH (1.5 millimolar) tripled the proton efflux from the protoplasts thus generating a greater electrochemical proton gradient across the plasmalemma. Trypsin also released ubiquinone (11.95 nanomoles per milligrams protein) but not flavin or cytochrome from the system. Kinetic analyses showed that 1.5 millimolar NADH quadrupled V_max of the mechanism I (saturable) component of K⁺ uptake, while K_m was not affected. Diethylstibestrol and vanadate inhibited basal (ATPase-mediated) K⁺ influx and H⁺ efflux, while NADH-stimulated K⁺ uptake was not or only slightly inhibited. p-Chloromercuribenzene-sulfonic acid, N,N′-dicyclohexylcarbodiimide, ethidium bromide, and oligomycin inhibited both ATPase- and NADH-mediated H⁺ and K⁺ fluxes. A combination of 10 millimolar fusicoccin and 1.5 millimolar NADH gave an 11-fold increase of K⁺ influx and a more than 3-fold increase of H⁺ efflux. It is concluded that a plasmalemma ATPase is not involved in the NADH-mediated ion transport mechanism. NADH oxidase is a -SH containing enzyme (protein) and the proton channel is an important element in this transport system. Fusicoccin synergistically stimulates the effect of NADH on K⁺ uptake.     

Histidine Residues in the Na+-coupled Ascorbic Acid Transporter-2 (SVCT2) Are Central Regulators of SVCT2 Function,Modulating pH Sensitivity,Transporter Kinetics,Na+ Cooperativity,Conformational Stability,and Subcellular Localization

Na⁺-coupled ascorbic acid transporter-2 (SVCT2) activity is impaired at acid pH, but little is known about the molecular determinants that define the transporter pH sensitivity. SVCT2 contains six histidine residues in its primary sequence, three of which are exofacial in the transporter secondary structure model. We used site-directed mutagenesis and treatment with diethylpyrocarbonate to identify histidine residues responsible for SVCT2 pH sensitivity. We conclude that five histidine residues, His¹⁰⁹, His²⁰³, His²⁰⁶, His²⁶⁹, and His⁴¹³, are central regulators of SVCT2 function, participating to different degrees in modulating pH sensitivity, transporter kinetics, Na⁺ cooperativity, conformational stability, and subcellular localization. Our results are compatible with a model in which (i) a single exofacial histidine residue, His⁴¹³, localized in the exofacial loop IV that connects transmembrane helices VII-VIII defines the pH sensitivity of SVCT2 through a mechanism involving a marked attenuation of the activation by Na⁺ and loss of Na⁺ cooperativity, which leads to a decreased V_max without altering the transport K_m; (ii) exofacial histidine residues His²⁰³, His²⁰⁶, and His⁴¹³ may be involved in maintaining a functional interaction between exofacial loops II and IV and influence the general folding of the transporter; (iii) histidines 203, 206, 269, and 413 affect the transporter kinetics by modulating the apparent transport K_m; and (iv) histidine 109, localized at the center of transmembrane helix I, might be fundamental for the interaction of SVCT2 with the transported substrate ascorbic acid. Thus, histidine residues are central regulators of SVCT2 function.