Decrease In Vmax Research Articles

Identification and characterization of a unique cysteine residue proximal to the catalytic site of Arabidopsis thaliana carotenoid cleavage enzyme 1

AtCCD1 and AtNCED3 are related carotenoid cleavage enzymes from Arabidopsis thaliana that catalyze the oxidative cleavage of, respectively, the 9,10 (9',10') double bonds of carotenoid substrates such as beta-carotene, and the 11,12 double bond of 9-cis epoxycarotenoids. Although the cellular and cleavage functionalities of these enzymes have been reported, their mechanisms and related structural environments mediating these disparate specificities in homologous enzymes have not been well characterized. By relating the differences observed in UV and visible light absorption and Cu(II) electron paramagnetic signals to variations in sequence alignments and 3-D homology models of the two A. thaliana enzymes, we identified a putatively proximal cysteine residue (Cys352) in AtCCD1 that is not conserved in AtNCED3. Spectral analysis of the Cys to Ala mutant confirmed its uniqueness and proximity to the metal binding site, but precluded any role for the residue in the mediation of the observed metal binding affinity or associated steric constraint differences. Further analysis of kinetic substrate cleavage properties indicated a decrease in Vmax and a subtle increase in Km for the C352A mutant compared with those observed for the wild-type, thus confirming catalytic site proximity and suggesting possible roles for the unique cysteine in the modulation of substrate affinity and (or) the reaction rate of AtCCD1.

Dissociation between changes in muscle Na+-K+-ATPase isoform abundance and activity with consecutive days of exercise and recovery

The early plasticity of vastus lateralis Na(+)-K(+)-ATPase to the abrupt onset of prolonged submaximal cycling was studied in 12 untrained participants (Vo(2 peak) 44.8 +/- 2.0 ml x kg(-1) x min(-1), mean +/- SE) using a 6-day protocol (3 days of exercise plus 3 days of recovery). Tissue samples were extracted prior to (Pre) and following exercise (Post) on day 1 (E1) and day 3 (E3) and on each day of recovery (R1, R2, R3) and analyzed for changes in maximal protein (beta(max)) (vanadate-facilitated [(3)H]ouabain binding), alpha- and beta-isoform concentration (quantitative immunoblotting) and maximal Na(+)-K(+)-ATPase activity (V(max)) (3-O-methylfluorescein K(+)-stimulated phosphatase assay). For beta(max) (pmol/g wet wt), an increase (P < 0.05) of 11.8% was observed at R1 compared with E1-Pre (340 +/- 14 vs 304 +/- 17). For the alpha-isoforms alpha(1), alpha(2), and alpha(3), increases (P < 0.05) of 46, 42, and 31% were observed at R1, respectively. For the beta-isoform, beta(1) and beta(2) increased (P < 0.05) by 19 and 28% at R1, whereas beta(3) increased (P < 0.05) by 18% at R2. With the exception of alpha(2) and alpha(3), the increases in the isoforms persisted at R3. Exercise resulted in an average decrease (P < 0.05) in V(max) by 14.3%. No differences were observed in V(max) at E1 - Pre and E3 - Pre or between R1, R2, and R3. It is concluded that 3 days of prolonged exercise is a powerful stimulus for the rapid upregulation of the Na(+)-K(+)-ATPase subunit isoforms. Contrary to our hypothesis, the increase in subunit expression is not accompanied by increases in the maximal catalytic activity.

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.

Crystal structure of the endopolygalacturonase from the phytopathogenic fungus Colletotrichum lupini and its interaction with polygalacturonase‐inhibiting proteins

Crystal structure of the endopolygalacturonase from the phytopathogenic fungus <i>Colletotrichum lupini</i> and its interaction with polygalacturonase‐inhibiting proteins

Role of α-Asp181, β-Asp192, and γ-Asp190 in the Distinctive Subunits of Human NAD-Specific Isocitrate Dehydrogenase

Human NAD-dependent isocitrate dehydrogenase (IDH) is allosterically activated by ADP by lowering the Km for isocitrate. The enzyme has three subunit types with distinguishable sequences present in the approximate ratio 2alpha:1beta:1gamma and, per tetramer, binds 2 mol of each ligand. To evaluate whether the subunits also have distinct functions, we replaced equivalent aspartates, one subunit at a time, by asparagines; each expressed, purified enzyme was composed of one mutant and two wild-type subunits. The aspartates were chosen because beta-Asp192 and gamma-Asp190 had previously been affinity labeled by a reactive ADP analogue and alpha-Asp181 is equivalent based on sequence alignments. The alpha-D181N IDH mutant exhibits a 2000-fold decrease in Vmax, with increases of 15-fold in the Kms for Mn(II) and NAD and a much smaller change in the Km for isocitrate. In contrast, the Vmax values of the beta-D192N and gamma-D190N IDHs are only reduced 4-5-fold as compared to wild-type enzyme. The Km for NAD of the beta-D192N enzyme is 9 times that of the normal enzyme with little or no effect on the affinity for Mn(II) or isocitrate, while the Kms for coenzyme and for Mn(II) of the gamma-D190N enzyme are 19 and 72 times, respectively, that of the normal enzyme with a much smaller effect on the Km for isocitrate. Finally, all three mutant enzymes fail to respond to ADP by lowering the Km for isocitrate, although they do bind ADP. Thus, these aspartates are close to but not in the ADP site and are required for communication between the ADP and isocitrate sites. These results demonstrate that alpha-Asp181 is the only one of these aspartates essential for catalysis. Beta-Asp192 is a determinant of the enzyme's affinity for NAD, as is gamma-Asp190, while gamma-Asp190 also influences the enzyme's affinity for metal ion. We conclude that the NAD and ADP sites are shared between alpha- and beta- and alpha- and gamma-subunits, and the Mn(II) site is shared between alpha- and gamma-subunits, while the alpha-subunit is essential for catalysis. Although alpha-Asp181, beta-Asp192, and gamma-Asp190 may have derived from a common progenitor, these aspartates of the three subunits have evolved distinct functions.

Alterations in kinetic and thermotropic properties of cerebral membrane-bound acetylcholineesterase during thioacetamide-induced hepatic encephalopathy: Correlation with membrane lipid changes

Acetylcholinesterase (AchE) is an important peripheral membrane-bound enzyme, crucial for cholinergic neurotransmission. Changes in AchE activity, kinetic and thermotropic properties were studied in hepatic encephalopathy (HE) associated with acute liver failure induced experimentally by the administration of the hepatotoxin thioacetamide (TAA). Activity of AchE decreased significantly following TAA administration. AchE from cerebral cortex membrane isolates of TAA-treated rats also showed a decrease in Vmax and an increase in Km. Arrhenius plots revealed considerable changes in the thermotropic behavior of AchE from membrane isolates obtained from TAA-treated rats as evident from the decreased transition temperature. A positive correlation was observed between changes in membrane cholesterol ( r 2 = 0.987), sphingomyelin ( r 2 = 0.99) levels and AchE activity, thus indicating that membrane lipid changes could underlie the observed changes in kinetic and thermotropic properties of membrane-bound AchE during TAA-induced HE.

Plasma serotonin levels and the platelet serotonin transporter

Serotonin (5HT) is a platelet-stored vasoconstrictor. Altered concentrations of circulating 5HT are implicated in several pathologic conditions, including hypertension. The actions of 5HT are mediated by different types of receptors and terminated by a single 5HT transporter (SERT). Therefore, SERT is a major mechanism that regulates plasma 5HT levels to prevent vasoconstriction and thereby secure a stable blood flow. In this study, the response of platelet SERT to the plasma 5HT levels was examined within two models: (i) in subjects with chronic hypertension or normotension; (ii) on platelets isolated from normotensive subjects and pretreated with 5HT at various concentrations. The platelet 5HT uptake rates were lower during hypertension due to a decrease in Vmax with a similar Km; also, the decrease in Vmax was primarily due to a decrease in the density of SERT on the platelet membrane, with no change in whole cell expression. Additionally, while the platelet 5HT content decreased 33%, the plasma 5HT content increased 33%. Furthermore, exogenous 5HT altered the 5HT uptake rates by changing the density of SERT molecules on the plasma membrane in a biphasic manner. Therefore, we hypothesize that in a hypertensive state, the elevated plasma 5HT levels induces a loss in 5HT uptake function in platelets via a decrease in the density of SERT molecules on the plasma membrane. Through the feedback effect of this proposed mechanism, plasma 5HT controls its own concentration levels by modulating the uptake properties of platelet SERT.

Manganese and molybdenum affect acid phosphatases from potatoes

The activity and catalytic properties of two preparations of acid phosphatases from potato and sweet potato in the presence of different levels of manganese (Mn) and molybdenum (Mo) were determined. Preliminarily, two different methods usually suggested in literature for the assay of acid phosphatase activity were compared. Since the methodology using acetate buffer gave a higher transformation of the substrate, it was selected for investigating the effect of the metals on the enzymes. The two plant acid phosphatases showed significant differences in their catalytic properties and behaviour in response to the presence of Mn and Mo. Sweet potato acid phosphatase displayed higher activity levels and a greater substrate affinity than potato enzyme. Furthermore, a significant decrease of Vmax and a small increment of Km values were measured in the presence of 0.58 and 1.17 mM of Mn ions. No significant effects were observed for the potato enzyme. The two enzymes showed the greater difference in their activity and kinetic behaviour when evaluating the effect of increasing levels of Mo (0.001 to 0.01 mM). Both the enzymes were competitively inhibited by the anion but with a different intensity at all Mo levels. The different number of protein subunits and purity levels of the two enzymes as ascertained by electrophoretic studies may account for the observed differences.

Effect of Chronic Iron Ingestion on the Development of Brush Border Enzymes in Rat Intestine

ABSTRACTIron is an essential element for biological systems. There is increasing evidence that excess iron supplementation results in the deposition of iron in the duodenum and enhances mucosal injury and cell proliferation in the colon and cecum. In the present study we examined whether chronic exposure to high levels of iron fortification affects the functional integrity of the small intestine, especially the activities of various brush border enzymes. Wistar rats were fed iron 29 mg/kg body weight (or 6.58 mg/kg Fe) daily in the form of FeSO4.7H2O for 39 days. The activities of brush border alkaline phosphatase (AP) (p < 0.001), sucrase (p < 0.01), maltase (p < 0.05), lactase (p < 0.05), and trehalase (p < 0.001) were reduced in purified membranes in iron-fed animals compared to controls. However, the activities of leucine amino peptidase (LAP) and γ-glutamyl transpeptidase (γ-GTP) were unaffected under these conditions. Analysis of alkaline phosphatase activity across the crypt–villus unit revealed a significant decrease (p < 0.05) all across the crypt–villus length, while sucrase activity was reduced (p < 0.01) only in the midvillus axis in iron-exposed animals. Kinetic studies showed a decrease in Vmax of AP from 1.11 to 0.83 units/mg protein and for sucrase from 0.77 to 0.43 units/mg protein in iron-fed rats, with no change in the apparent Km of the enzymes (AP, 8 mM; sucrase, 10 mM). Western blot analysis corroborated these findings. These results indicate that chronic iron exposure alters the activities of brush border enzymes, resulting in intestinal dysfunctions.

A typical N-terminal Extensions Confer Novel Regulatory Properties on GTP Cyclohydrolase Isoforms in Drosophila melanogaster

The cofactor tetrahydrobiopterin plays critical roles in the modulation of the signaling molecules dopamine, serotonin, and nitric oxide. Deficits in cofactor synthesis have been associated with several human hereditary diseases. Responsibility for the regulation of cofactor pools resides with the first enzyme in its biosynthetic pathway, GTP cyclohydrolase I. Because organisms must be able to rapidly respond to environmental and developmental cues to adjust output of these signaling molecules, complex regulatory mechanisms are vital for signal modulation. Mammalian GTP cyclohydrolase is subject to end-product inhibition via an associated regulatory protein and to positive regulation via phosphorylation, although target residues are unknown. GTP cyclohydrolase is composed of a highly conserved homodecameric catalytic core and non-conserved N-terminal domains proposed to be regulatory sites. We demonstrate for the first time in any organism that the N-terminal arms of the protein serve regulatory functions. We identify two different modes of regulation of the enzyme mediated through the N-terminal domains. The first is end-product feedback inhibition, catalytically similar to that of the mammalian enzyme, except that feedback inhibition by the cofactor requires sequences in the N-terminal arms rather than a separate regulatory protein. The second is a novel inhibitory interaction between the N-terminal arms and the active sites, which can be alleviated through the phosphorylation of serine residues within the N termini. Both mechanisms allow for acute and highly responsive regulation of cofactor production as required by downstream signaling pathways.

Subcellular Redistribution of the Serotonin Transporter by Secretory Carrier Membrane Protein 2

The serotonin transporter (SERT) belongs to the SLC6 family of sodium- and chloride-dependent neurotransmitter transporters responsible for uptake of amino acids and biogenic amines from extracellular spaces. Their activities and subcellular distributions are regulated by various cellular mechanisms, including interactions with other proteins. Using the yeast two-hybrid approach we screened a human brain cDNA library and identified secretory carrier membrane protein 2 (SCAMP2) as a novel SERT-interacting protein. GST-pulldown assays confirmed the physical interaction between SCAMP2 and the N-terminal domain of SERT. In addition, SERT was found to form a complex with SCAMP2 as demonstrated by co-immunoprecipitation from a heterologous expression system and from rat brain homogenate. Co-expression of SERT and SCAMP2 in mammalian cells results in the subcellular redistribution of SERT with a decrease in cell surface SERT and a concomitant reduction in 5-HT uptake activity. Using confocal microscopy we show that in neuronal cells endogenous SERT co-localizes with SCAMP2 in discrete structures also containing the lipid raft marker flotillin-1 and the SNARE protein syntaxin 1A. In contrast, SERT immunoreactivity is clearly segregated from transferrin receptor-containing endosomes. A single amino acid mutation, cysteine-201 to alanine, within the conserved cytoplasmic E peptide of SCAMP2, abolished SCAMP2-mediated down-regulation of SERT, although this mutation had no effect on the physical interaction between SERT and SCAMP2. Taken together, our results suggest that SCAMP2 plays an important role in the regulation of the subcellular distribution of SERT.

Cytoskeleton/Endoplasmic Reticulum Collapse Induced by Prostaglandin J2 Parallels Centrosomal Deposition of Ubiquitinated Protein Aggregates

Many neurodegenerative disorders, such as Parkinson disease, exhibit inclusion bodies containing ubiquitinated proteins. The mechanisms implicated in this aberrant protein deposition remain elusive. In these disorders signs of inflammation are also apparent in the affected central nervous system areas. We show that prostaglandin J2 (PGJ2), an endogenous product of inflammation, disrupts the cytoskeleton in neuronal cells. Furthermore, PGJ2 perturbed microtubule polymerization in vitro and decreased the number of free sulfhydryl groups on tubulin cysteines. A direct effect of PGJ2 on actin was not apparent, although actin filaments were altered in cells treated with PGJ2. This cyclopentenone prostaglandin triggered endoplasmic reticulum (ER) collapse and the redistribution of ER proteins, such as calnexin and catechol-O-methyltransferase, into a large centrosomal aggregate containing ubiquitinated proteins and alpha-synuclein. The PGJ2-dependent cytoskeletal rearrangement paralleled the development of the large centrosomal aggregate. Both of these events were replicated by treating cells with colchicine, which disrupts the microtubule/ER network, but not with brefeldin A, which impairs ER/Golgi transport. PGJ2 also perturbed 26 S proteasome assembly and activity, which preceded the accumulation of ubiquitinated proteins as detergent/salt-insoluble aggregates. Our data support a mechanism by which, upon PGJ2 treatment, cytoskeleton/ER collapse coincides with the relocation of ER proteins, other potentially neighboring proteins, and ubiquitinated proteins into centrosomal aggregates. Development of these large perinuclear aggregates is associated with disruption of the microtubule/ER network. This aberrant protein deposition, triggered by a product of inflammation, may be common to other compounds that disrupt microtubules and induce protein aggregation, such as MPP+ and rotenone, found to be associated with neurodegeneration.

A propionitrile-induced nitrilase of Rhodococcus sp. NDB 1165 and its application in nicotinic acid synthesis

Rhodococcus sp. NDB 1165, a nitrile-transforming organism was isolated from temperate forest soil of Himalayas. The nitrilase (EC 3.5.5.2) activity of this organism had higher substrate specificity toward aromatic nitriles (benzonitrile, 3-cyanopyridine and 4-cyanopyridine) and unsaturated aliphatic nitrile (acrylonitrile) in comparison to saturated aliphatic nitriles (acetonitrile, propionitrile, butyronitrile and isobutyronitrile) nitrile and arylacetonitrile (phenylacetonitrile and indole-3-acetonitrile). The nitrilase of Rhodococcus sp. NDB 1165 was inducible in nature and propionitrile proved to be an efficient inducer. However, the salts of ferrous and cobalt ions had an inhibitory effect. Under optimized reaction conditions (pH 8.0 and temperature 45°C) the nitrilase activity of this organism was 2.39 ± 0.07 U/mg dry cell mass (dcm). The half-life of this enzyme was 150 min and 40 min at 45°C and 50°C respectively. However, it was quite stable at 40°C and around 58 % activity was retained even after 6 h at this temperature. The V max and K m value of this nitrilase were 1.67 µmol/ml min and 0.1 M respectively using 3-cyanopyridine as substrate. However, the decrease in V max and K m values (0.56 µmol/ml min and 0.02 M, respectively) were ␣observed at >0.05 M 3-cyanopyridine which revealed that this enzyme experienced uncompetitive inhibition at higher substrate concentrations. Under optimized reaction conditions, 1.6 M 3-cyanopyridine was successfully converted in to nicotinic acid using 2.0 mg resting cells (dcm)/ml reaction mixture in 11 h. This is the highest production of nicotinic acid i.e. 8.95 mg/mg resting cells (dcm)/h as compared to nitrilase systems reported hitherto.

Physiological Consequences of Loss of Allosteric Activation of Yeast NAD+-specific Isocitrate Dehydrogenase

Based on allosteric regulatory properties, NAD+-specific isocitrate dehydrogenase (IDH) is believed to control flux through the tricarboxylic acid cycle in vivo. To distinguish growth phenotypes associated with regulatory dysfunction of this enzyme in Saccharomyces cerevisiae, we analyzed strains expressing well defined mutant forms of IDH or a non-allosteric bacterial NAD+-specific isocitrate dehydrogenase (IDHa). As previously reported, expression of mutant forms of IDH with severe catalytic defects but intact regulatory properties produced an inability to grow with acetate as the carbon source and a dramatic increase in the frequency of generation of petite colonies, phenotypes also exhibited by a strain (idh1Deltaidh2Delta) lacking IDH. Reduced growth rates on acetate medium were also observed with expression of enzymes with severe regulatory defects or of the bacterial IDHa enzyme, suggesting that allosteric regulation is also important for optimal growth on this carbon source. However, expression of IDHa produced no effect on petite frequency, suggesting that the intermediate petite frequencies observed for strains expressing regulatory mutant forms of IDH are likely to correlate with the slight reductions in catalytic efficiency observed for these enzymes. Finally, rates of increase in oxygen consumption were measured during culture shifts from medium with glucose to medium with ethanol as the carbon source. Strains expressing wild-type or catalytically deficient mutant forms of IDH exhibited rapid respiratory transitions, whereas strains expressing regulatory mutant forms of IDH or the bacterial IDHa enzyme exhibited much slower respiratory transitions. This suggests an important physiological role for allosteric activation of IDH during changes in environmental conditions.

Effect of Neonatal Hypothyroidism on the Kinetic Properties of Na+, K+‐ATPase from Rat Brain Microsomes

The effects of neonatal hypothyroidism on the kinetic properties of Na+, K+ -ATPase from rat brain microsomes were examined. Neonatal hypothyroidism resulted in decreased Na+, K+ -ATPase activity compared to control samples (7.4 +/- 1.48 and 29.8 +/- 2.30 micromol Pi/h/mg protein, respectively, P < 0.001). Substrate kinetics studies with ATP, Na+ and K+ revealed that there were generalised decreases in Vmax. For ATP, Na+ and K+, activities resolved into two kinetic components in the control group. In hypothyroid animals, the low-affinity component for ATP was absent. The opposite pattern (i.e. an absence of the high-affinity component) was noted for Na+. For K+, although both kinetic components were discernible in neonatal hypothyroid brain microsomes, the Km of the high-affinity component was significantly higher (P < 0.001) compared to control samples. In the control group, the enzyme displayed allosteric behaviour at high concentrations of Mg2+; in hypothyroid animals, the pattern was completely allosteric. The Na+, K+ -ATPase enzyme from the hypothyroid brain microsomes bound two molecules of ATP rather than one, unlike in the control animals. Our results thus indicate that neonatal hypothyroidism results in an impairment of microsomal Na+, K+ -ATPase activity in the rat brain, together with subtle alterations in the kinetic properties of the enzyme.

CHARACTERIZATION &amp; FUNCTIONAL INVESTIGATION OF HUMAN PHOSPHOMEVALONATE KINASE

Phosphomevalonate kinase (PMK) catalyzes a key step in isoprenoid/sterol biosynthesis, converting mevalonate 5-phosphate and ATP to mevalonate 5-diphosphate and ADP. cDNA encoding human PMK has been used to construct expression plasmids encoding his-tagged enzyme. Expression of N-his-tagged PMK is much more efficient than C-his-tagged PMK, thus N-tagged enzyme has been selected for detailed study. Kinetic constants of human PMK have been determined for both forward (formation of mevalonate 5-diphosphate) and reverse (formation of mevalonate 5-phosphate) reactions. A homology model for the structure of human PMK has been generated. The model conforms to a nucleoside monophosphate kinase family fold. This result, together with sequence comparisons of animal and invertebrate PMKs, suggest an N-terminal basic residue rich sequence as an unusual “Walker A”ATP site motif. The functions of four basic (K17, R18, K19, K22) and one acidic (D23) residue in the conserved sequence have been tested by mutagenesis and characterization of isolated mutant proteins. Substrate Km values for K17M, R18Q, K19M, and D23N have been measured for forward and reverse reactions; in comparison with wild-type PMK values, only modest (<12-fold) changes are observed. In contrast R18Q exhibits a Vmax decrease of 100/300-fold (forward/reverse reaction). K22M activity is too low for measurement at nonsaturating substrate concentration; specific activity is decreased by >10,000-fold in both forward/reverse reactions, suggesting an active site location and an important role in phosphoryl transfer. DK53766

Characterization of regulatory mechanisms and states of human organic cation transporter 2

Polyspecific organic cation transporters (OCTs) have a large substrate binding pocket with different interaction domains. To determine whether OCT regulation is substrate specific, suitable fluorescent organic cations were selected by comparing their uptake in wild-type (WT) human embryonic kidney (HEK)-293 cells and in HEK-293 cells stably transfected with hOCT2. N-amidino-3,5-diamino-6-chloropyrazine-carboxamide (amiloride) and 4-[4-(dimethylamino)-styryl]-N-methylpyridinium (ASP) showed concentration-dependent uptake in hOCT2 at 37 degrees C. After subtraction of unspecific uptake determined in WT at 37 degrees C or in hOCT2 at 8 degrees C saturable specific uptake of both substrates was measured. Km values of hOCT2-mediated uptake of 95 microM amiloride and 24 microM ASP were calculated. Inhibition of amiloride and ASP uptake by several organic cations was also measured [IC50 (in microM) for amiloride and ASP, respectively, tetraethylammonium (TEA) 98 and 30, cimetidine 14 and 26, and tetrapentylammonium (TPA) 7 and 2]. Amiloride and ASP uptake were significantly reduced by inhibition of Ca2+/CaM complex (-55 +/- 5%, n = 10 and -63 +/- 2%, n = 15, for amiloride and ASP, respectively) and stimulation of PKC (-54 +/- 5%, n = 14, and -31 +/- 6%, n = 26) and PKA (-16 +/- 5%, n = 16, and -18 +/- 4%, n = 40), and they were increased by inhibition of phosphatidylinositol 3-kinase (+28 +/- 6%, n = 8, and +55 +/- 17%, n = 16). Inhibition of Ca2+/CaM complex resulted in a significant decrease of Vmax (160-99 photons/s) that can be explained in part by a reduction of the membrane-associated hOCT2 (-22 +/- 6%, n = 9) as determined using FACScan flow cytometry. The data indicate that saturable transport by hOCT2 can be measured by the fluorescent substrates amiloride and ASP and that transport activity for both substrates is regulated similarly. Inhibition of the Ca2+/CaM complex causes changes in transport capacity via hOCT2 trafficking.

Hemodialysis reduces inhibitory effect of plasma ultrafiltrate on LDL oxidation and subsequent endothelial reactions

Oxidative modification of low-density lipoprotein (LDL) and its deleterious effect on endothelium is implicated in the pathogenesis of atherosclerosis. Endothelium responds to such an insult by upregulating the synthesis of heme oxygenase-1 (HO-1) and ferritin. Endothelial cell damage and dysfunction have been observed in patients with chronic kidney disease (CKD) on maintenance hemodialysis (HD). We studied the effect of low-molecular-weight components of uremic plasma on LDL oxidation and LDL-oxidation-provoked endothelial cell reactions, such as the induction of cytotoxicity and the upregulation of cell-protective HO-1 and ferritin. Plasma ultrafiltrate (molecular weight<5000 Da) from CKD patients on HD or when treated conservatively exhibited a pronounced inhibition on heme-mediated oxidative modification of LDL. Endothelial cell cytotoxicity provoked by LDL oxidation was also attenuated by plasma ultrafiltrate from CKD patients. During HD treatment, a dramatic drop occurred in the retardation of oxidative reactions, and a loss of endothelial cytoprotection exerted by plasma ultrafiltrate was noted. The upregulation of HO-1 and ferritin in response to oxidative stress of LDL was blunted by uremic plasma ultrafiltrate that was released by the end of HD. The decreased antioxidant capacity of ultrafiltrate after HD occurred as a consequence of the intradialytic removal of L-ascorbic acid, uric acid, bilirubin, 3-indoxyl sulfate, indoxyl-beta-D-glucuronide, p-cresol, and phenol. Intradialytic removal of L-ascorbic acid, uric acid, bilirubin, 3-indoxyl sulfate, indoxyl-beta-D-glucuronide, p-cresol, and phenol increases the risk of LDL oxidation and subsequent endothelial cell damage, which underlines the importance of activation of cytoprotective HO-1 and ferritin in endothelium.

Characterization of a Thermoacidophilic l -Arabinose Isomerase from Alicyclobacillus acidocaldarius : Role of Lys-269 in pH Optimum

The araA gene encoding L-arabinose isomerase (AI) from the thermoacidophilic bacterium Alicyclobacillus acidocaldarius was cloned, sequenced, and expressed in Escherichia coli. Analysis of the sequence revealed that the open reading frame of the araA gene consists of 1,491 bp that encodes a protein of 497 amino acid residues with a calculated molecular mass of 56,043 Da. Comparison of the deduced amino acid sequence of A. acidocaldarius AI (AAAI) with other AIs demonstrated that AAAI has 97% and 66% identities (99% and 83% similarities) to Geobacillus stearothermophilus AI (GSAI) and Bacillus halodurans AI (BHAI), respectively. The recombinant AAAI was purified to homogeneity by heat treatment, ion-exchange chromatography, and gel filtration. The purified enzyme showed maximal activity at pH 6.0 to 6.5 and 65 degrees C under the assay conditions used, and it required divalent cations such as Mn2+, Co2+, and Mg2+ for its activity. The isoelectric point (pI) of the enzyme was about 5.0 (calculated pI of 5.5). The apparent Km values of the recombinant AAAI for L-arabinose and D-galactose were 48.0 mM (Vmax, 35.5 U/mg) and 129 mM (Vmax, 7.5 U/mg), respectively, at pH 6 and 65 degrees C. Interestingly, although the biochemical properties of AAAI are quite similar to those of GSAI and BHAI, the three AIs from A. acidocaldarius (pH 6), G. stearothermophilus (pH 7), and B. halodurans (pH 8) exhibited different pH activity profiles. Based on alignment of the amino acid sequences of these homologous AIs, we propose that the Lys-269 residue of AAAI may be responsible for the ability of the enzyme to act at low pH. To verify the role of Lys-269, we prepared the mutants AAAI-K269E and BHAI-E268K by site-directed mutagenesis and compared their kinetic parameters with those of wild-type AIs at various pHs. The pH optima of both AAAI-K269E and BHAI-E268K were rendered by 1.0 units (pH 6 to 7 and 8 to 7, respectively) compared to the wild-type enzymes. In addition, the catalytic efficiency (kcat/Km) of each mutant at different pHs was significantly affected by an increase or decrease in Vmax. From these results, we propose that the position corresponding to the Lys-269 residue of AAAI could play an important role in the determination of the pH optima of homologous AIs.

Nongenomic regulation by aldosterone of the epithelial NHE3 Na+/H+ exchanger

The relevance of nongenomic pathways to regulation of epithelial function by aldosterone is poorly understood. Recently, we demonstrated that aldosterone inhibits transepithelial HCO(3)(-) absorption in the renal medullary thick ascending limb (MTAL) through a nongenomic pathway. Here, we examined the transport mechanism(s) responsible for this regulation, focusing on Na(+)/H(+) exchangers (NHE). In the MTAL, apical NHE3 mediates H(+) secretion necessary for HCO(3)(-) absorption; basolateral NHE1 influences HCO(3)(-) absorption by regulating apical NHE3 activity. In microperfused rat MTALs, the addition of 1 nM aldosterone rapidly decreased HCO(3)(-) absorption by 30%. This inhibition was unaffected by three maneuvers that inhibit basolateral Na(+)/H(+) exchange and was preserved in MTALs from NHE1 knockout mice, ruling out the involvement of NHE1. In contrast, exposure to aldosterone for 15 min caused a 30% decrease in apical Na(+)/H(+) exchange activity over the intracellular pH range from 6.5 to 7.7, due to a decrease in V(max). Inhibition of HCO(3)(-) absorption by aldosterone was not affected by 0.1 mM lumen Zn(2+) or 1 mM lumen DIDS, arguing against the involvement of an apical H(+) conductance or apical K(+)-HCO(3)(-) cotransport. These results demonstrate that aldosterone inhibits HCO(3)(-) absorption in the MTAL through inhibition of apical NHE3, and identify NHE3 as a target for nongenomic regulation by aldosterone. Aldosterone may influence a broad range of epithelial transport functions important for extracellular fluid volume and acid-base homeostasis through direct regulation of this exchanger.