bluuebunny/crossref_metadata_2025 huggingface dataset:
Zn2+ depletion blocks endosome fusion
Authors: Aballay A , Sarrouf M N , Colombo M I , Stahl P D , Mayorga L S
Published: August 2015

Fusion among endosomes is an important step for transport and sorting of internalized macromolecules. Working in a cell-free system, we previously reported that endosome fusion requires cytosol and ATP, and is sensitive to N-ethylmaleimide. Fusion is regulated by monomeric and heterotrimeric GTP-binding proteins. We now report that fusion can proceed at very low Ca2+ concentrations, i.e. < 30 nM. Moreover, fusion is not affected when intravesicular Ca2+ is depleted by preincubation of vesicles with calcium ionophores (5 microM ionomycin or A23187) in the presence of calcium chelators (5 mM EGTA or 60 mM EDTA). The results indicate that fusion can proceed at extremely low concentrations of intravesicular and extravesicular Ca2+. However, BAPTA [1,2-bis-(o-aminophenoxy)ethane-N,N,N′,N′-tetra-acetic acid], a relatively specific Ca2+ chelator, inhibits fusion. BAPTA binds other metals besides Ca2+. We present evidence that BAPTA inhibition is due not to Ca2+ chelation but to Zn2+ depletion. TPEN [N,N,N′,N′-tetrakis-(2-pyridylmethyl) ethylenediamine], another metal-ion chelator with low affinity for Ca2+, also inhibited fusion. TPEN- and BAPTA-inhibited fusions were restored by addition of Zn2+. Zn(2+)-dependent fusion presents the same characteristics as control fusion. In intact cells, TPEN inhibited transport along the endocytic pathway. The results indicate that Zn2+ depletion blocks endosome fusion, suggesting that this ion is necessary for the function of one or more factors involved in the fusion process.
Annexin II up-regulates cellular levels of p11 protein by a post-translational mechanisms
Authors: PUISIEUX Alain , JI Jingwei , OZTURK Mehmet
Published: August 2015

Annexin II (p36) and p11, which belong to two different families of calcium-binding proteins, are able to form a heterotetrameric protein complex (p36)2(p11)2 called calpactin I. As these proteins were detectable only in the presence of each other in a variety of cell lines, we studied the mechanisms of regulation of cellular levels of annexin II and p11. In cells expressing p11 messenger RNA, p11 protein is undetectable unless annexin II is also expressed. As an example, the hepatoblastoma HepG2 cell line displays no detectable annexin II nor p11 protein, although it expresses p11 mRNA. The overexpression of annexin II by gene transfer into HepG2 cells leads to the up-regulation of the cellular levels of p11 by a post-translational mechanism. In the presence of annexin II, there is no major change in the p11 transcript levels, but the half-life of the p11 protein is increased more than 6-fold. Thus, the degree of expression of annexin II, which varies according to different states of cellular differentiation and transformation, is an essential factor in the regulation of cellular levels of p11.
The mitogen-activated protein kinase pathway in rat islets of Langerhans: studies on the regulation of insulin secretion
Authors: PERSAUD Shanta J. , WHEELER-JONES Caroline P. D. , JONES Peter M.
Published: August 2015

The expression of mitogen-activated protein kinases (MAPKs) and MAPK kinases (MEKs) in rat islets of Langerhans and the involvement of MAPKs in regulated insulin secretion were examined. Two major isoforms of both MEK (45 and 46 kDa) and MAPK (42 and 44 kDa) were detected in rat islets and shown to be localized to insulin-secreting β cells by detection of their expression in the β cell line MIN6. The tyrosine phosphatase inhibitor sodium pervanadate, and, to a lesser extent, the serine/threonine phosphatase inhibitor okadaic acid, stimulated MAPK phosphorylation, as assessed by a shift in its electrophoretic mobility and by increased phosphotyrosine immunoreactivity of immunoprecipitated MAPK. The increase in MAPK phosphorylation stimulated by sodium pervanadate was not coupled to an increase in MAPK activity, but okadaic acid, either alone or in the presence of sodium pervanadate, caused an increase in myelin basic protein phosphorylation by MAPK. Neither okadaic acid nor sodium pervanadate, either individually or combined, stimulated insulin secretion. 4β-phorbol myristate acetate stimulated an increase in phosphorylation of the 42 kDa isoform of MAPK (erk2) in human umbilical vein endothelial cells, but neither it nor glucose affected either the phosphorylation state of islet erk2 or the activities of immunoprecipitated islet MAPKs. These results provide evidence for the presence of a regulated MAPK pathway in adult rat islets, but our data suggest that MAPK activation alone is not a sufficient stimulus for insulin secretion.
The organization of the human GSTP1-1 gene promoter and its response to retinoic acid and cellular redox status
Authors: XIA Chulin , HU Jiangting , KETTERER Brian , TAYLOR John B.
Published: August 2015

High levels of expression of GSTP1-1 are associated with cell proliferation, embryogenesis and malignancy. Given the role of glutathione S-transferase (GST) in detoxication, it is possible that GSTP1-1 evolved specifically to protect proliferating cells and share regulatory mechanisms with other cellular genes which are involved in cell division and tumorigenesis. We have previously shown that the expression of GSTP1 is suppressed by retinoic acid (RA) in the presence of the retinoic acid receptor (RAR) as a result of decreased transcription from its promoter. Through deletion analysis, we show here that the RA-RAR-dependent repression is mediated by the region -73 to +8. Further mutation analysis of this region indicates that the DNA sequence required for RA-RAR-dependent repression co-localizes with a consensus activator protein-1 (AP1) site essential for the promoter activity. The degree of repression correlates with the residual activity of the AP1 site. There are two adjacent G/C boxes. The one immediately downstream from the AP1 site is not essential for the promoter activity, but mutation of the second, further downstream, impairs the promoter. On the other hand, mutation of either of these two G/C boxes has little effect on RA-RAR suppression. We also show that the expression of GSTP1 is regulated by the redox status of the cell. Using the chloramphenicol acetyltransferase assay system, we have demonstrated that treatment with H2O2 induced transcription from the promoter and that this effect can be blocked by pre-incubation with N-acetylcysteine (NAC). It was shown that the induction by H2O2 is mediated by trans-acting factor NF-ĸB (nuclear factor ĸB), via a putative NF-ĸB site, ‘GGGACCCTCC’, located from -96 to -86. Co-transfection with an NF-ĸB (p65) expression construct increased the promoter activity, an effect which could be blocked by co-transfection with an IĸB (MAD-3) expression construct. Deletion of the NF-ĸB site abolished the effect of both H2O2 and co-transfection of NF-ĸB. Interestingly, NAC is also an inducer for GSTP1. The effect of NAC was shown to be mediated largely by the AP1 site, since mutation of this site abolished the induction by NAC.
The role of cell adhesion in retinoic acid-induced modulation of chondrocyte phenotype
Authors: SANCHEZ Massimo , ARCELLA Antonietta , PONTARELLI Gianfranco , GIONTI Elisa
Published: August 2015

Retinoic acid (RA) treatment of a suspension of quail chondrocytes inhibits the expression of cartilage collagens and induces cell adhesion along with fibronectin expression. We asked whether the RA-induced modulation of the chondrocyte phenotype was dependent on cell adhesion. Prevention of cell adhesion blocks cell growth and many of the effects associated with RA, such as collagen II inhibition, collagen I activation and fibronectin induction. The activity of the bone/tendon promoter of the α2(I) collagen gene was determined by measuring the transient expression of COL1A2-CAT, a chimaeric gene bearing 3500 bp from upstream of the transcription start site of the human α2(I) gene fused to the chloramphenicol acetyltransferase (CAT) gene. This promoter is activated only in permissive conditions for cell adhesion. The attachment activities of chondrocytes on protein substrates was studied by an in vitro cell adhesion assay. Untreated cells or cells maintained in suspension while undergoing RA treatment do not attach when replated on protein substrates. Chondrocytes treated with RA in permissive conditions for cell adhesion rapidly attach and spread instead on collagen-coated wells. Altogether the results suggest that cell adhesion plays a major role in RA-induced modulation of the chondrocyte phenotype.
Characterization of an insulin from the three-toed amphiuma (Amphibia: Urodela) with an N-terminally extended A-chain and high receptor-binding affinity
Authors: CONLON J. Michael , CAVANAUGH Elizabeth S. , MYNARCIK Dennis C. , WHITTAKER Jonathan
Published: August 2015

Insulin was isolated from an extract of the pancreas of a urodele, the three-toed amphiuma (Amphiuma tridactylum), and its primary structure established as Ala-Arg-Gly-Ile-Val-Glu-Gln-Cys-Cys-His10-Asn-Thr-Cys-Ser-Leu-Asn-Gln-Leu-Glu-Asn20-Tyr-Cys-Asn for the A-chain and Ile-Thr-Asn-Gln-Tyr-Leu-Cys-Gly-Ser-His10-Leu-Val-Glu-Ala-Leu-Tyr-Leu-Val-Cys-Gly20-Asp-Arg-Gly-Phe-Phe-Tyr-Ser-Pro-Lysfor the B-chain. The N-terminus of the A-chain is extended by two amino acids (Ala-Arg) relative to all other known insulins suggesting an anomalous pathway of post-translational processing in the region of the C-peptide/A-chain junction of proinsulin. In common with chicken and Xenopus insulins, which contain a HisA8, amphiuma insulin was more potent (approx. 5-fold) than porcine insulin in inhibiting the binding of [125I-TyrA14]insulin to the soluble human insulin receptor from transfected 293EBNA cells (an adenovirus-transformed human kidney cell line). This result is consistent with previous data showing that insulin analogues extended at GlyA1 by uncharged groups have reduced binding affinity whereas high affinity is preserved in analogues extended by basic amino acid residues.
Molecular mechanism for catalysis by a new zinc-enzyme, dopachrome tautomerase
Authors: SOLANO Francisco , JIMÉNEZ-CERVANTES Celia , MARTÍNEZ-LIARTE José H. , GARCÍA-BORRÓN José C. , JARA José R. , LOZANO José A.
Published: August 2015

Dopachrome tautomerase (DCT; EC 5.3.3.12) catalyses the conversion of L-dopachrome into 5,6-dihydroxyindole-2-carboxylic acid in the mammalian eumelanogenic biosynthetic pathway. This enzyme, also named TRP2, belongs to a family of three metalloenzymes termed the tyrosinase-related proteins (TRPs). It is well known that tyrosinase has copper in its active site. However, the nature of the metal ion in the active site of DCT is under discussion. Whereas theoretical predictions based on similarity between the protein sequences of the TRPs suggest the presence of copper, the different inhibition pattern of DCT with some metal chelators compared with that of tyrosinase suggests that the nature of the metal ion could differ. Direct estimations of the metal content in purified DCT preparations show the presence of around 1.5 Zn atoms/molecule and the absence of copper. Apoenzyme preparation by treatment of DCT with cyanide or o-phenanthroline followed by reconstitution experiments of tautomerase activity in the presence of different ions confirmed that the metal cofactor for the DCT active site is zinc. Our results are consistent with Zn2+ chelation by the highly conserved histidine residues homologous to the histidines at the classical copper-binding sites in tyrosinase. This finding accounts for the reaction catalysed by DCT, i.e. a tautomerization, versus the copper-mediated oxidations catalysed by tyrosinase. Based on the predicted tetrahedrical co-ordination of the zinc ions in the enzyme active site, a molecular mechanism for the catalysis of L-dopachrome tautomerization is proposed. From the present data, the existence of additional ligands for metal ions other than zinc in the DCT molecule, such as the proposed cysteine iron-binding sites, cannot be completely ruled out. However, if such sites exist, they could be subsidiary binding sites, whose function would be likely to stabilize the protein.
Phospholipase D activation by P2Z-purinoceptor agonists in human lymphocytes is dependent on bivalent cation influx
Authors: GARGETT Caroline E. , CORNISH E. Jean , Wiley James S.
Published: August 2015

The role of bivalent cations in ATP-stimulated phospholipase D (PLD) activity was investigated in human leukaemic lymphocytes. Cells were labelled with [3H]oleic acid and incubated with extracellular ATP or benzoylbenzoic ATP in the presence of 1 mM Ca2+ and butanol, and PLD activity was assayed by the accumulation of [3H]phosphatidylbutanol ([3H]PBut). ATP stimulated PLD activity in a dose-dependent manner, and the inhibitory effects of suramin, oxidized ATP and extracellular Mg2+ suggested that the effect of ATP was mediated by P2Z purinoceptors known to be present on lymphocytes. Thapsigargin increased cytosolic [Ca2+] but did not stimulate PLD activity, whereas preloading cells with a Ca2+ chelator reduced cytosolic [Ca2+] and, paradoxically, potentiated ATP-stimulated [3H]PBut accumulation. ATP-stimulated [3H]PBut formation was supported by both Ba2+ and Sr2+ when they were substituted for extracellular Ca2+. Addition of EGTA to block bivalent cation influx inhibited the majority of ATP-stimulated PLD activity. Furthermore ATP-stimulated PLD activity showed a linear relationship to extracellular [Ba2+], and ATP-induced 133Ba2+ influx also had a linear dependence on extracellular [Ba2+]. These results suggest that ATP stimulates PLD activity in direct proportion to the influx of bivalent cations through the P2z-purinoceptor ion channel and that this PLD activity is insensitive to changes in bulk cytosolic [Ca2+].
Interleukin-13 inhibits inducible nitric oxide synthase expression in human mesangial cells
Authors: SAURA Marta , MARTÍNEZ-DALMAU Rosa , MINTY Adrian , PÉREZ-SALA Dolores , LAMAS Santiago
Published: August 2015

The synthesis of nitric oxide in inflammatory situations requires the expression of an inducible isoform of nitric oxide synthase (iNOS). Human mesangial cells (HMC) express an iNOS enzyme after exposure to multiple co-stimuli. In this study we have observed that while tumour necrosis factor-α, interleukin (IL)-1β, interferon-γ and bacterial lipopolysaccharide (LPS) were unable to significantly induce NO synthesis when used alone, they induced an evident stimulation of NO synthesis when used in various combinations. A mixture of the three cytokines (CM) and LPS resulted in a 10-15-fold stimulation of NO synthesis over control values which started to be significant after 16 h. The addition of IL-13, a cytokine with anti-inflammatory properties, inhibited CM/LPS-induced NO synthesis in a concentration-dependent manner. A marked inhibitory effect (60-65%) could be observed when HMC were treated with IL-13 (10 ng/ml) 24 h before, at the same time as, or even 4 h after the addition of CM/LPS. This inhibitory effect was still significant (25%) when IL-13 was added 16 h after CM/LPS. Northern analysis showed that IL-13-mediated iNOS inhibition was closely correlated with the suppression of iNOS mRNA expression. These results identify IL-13 as a powerful regulatory tool for the inhibition of NO synthesis in human cells, a property which may be pathophysiologically relevant in NO-related inflammatory processes.
Measurement of tissue transglutaminase activity in a permeabilized cell system: its regulation by Ca2+ and nucleotides
Authors: SMETHURST Peter A. , GRIFFIN Martin
Published: August 2015

Electropermeabilized human endothelial cells (ECV-304) were used to study the regulation of tissue transglutaminase (tTGase) activity in the intracellular environment. An ELSA (enzyme-linked sorbent assay) plate assay was developed for intracellular tTGase activity, using the incorporation of a biotinylated primary amine, 5-{[(N-biotinoylamino)hexanoyl]amino}pentylamine (biotin-x-cadaverine; BTC), into endogenous protein substrates of tTGase. This incorporation process was inhibited by competitive inhibitors of tTGase, cystamine and monodansylcadaverine, in a dose-dependent manner. Over a 30 min period tTGase and its protein substrates did not leak out of the cell, and no incorporation of BTC occurred in unpermeabilized cells, indicating the reaction to be intracellular. In the presence of 10 nM or 10 μM Ca2+, when nucleotides ATP and GTP were added at concentrations mimicking cytosolic levels, tTGase activity was decreased virtually to zero. Only at 100 μM Ca2+, when nucleotides were low or absent was tTGase activity observed. Under these conditions a variety of proteins was labelled by the enzyme, with the major labelling found in a protein of molecular mass around 51 kDa when analysed by SDS/PAGE/Western blotting.
Mechanism of indole-3-acetic acid oxidation by plant peroxidases: anaerobic stopped-flow spectrophotometric studies on horseradish and tobacco peroxidases
Authors: GAZARYAN Irina G. , LAGRIMINI L. Mark , ASHBY Gillian A. , THORNELEY Roger N. F.
Published: August 2015

Indole-3-acetic acid (IAA) is a powerful plant growth regulator. The oxidative decarboxylation of IAA by plant peroxidases is thought to be a major degradation reaction involved in controlling the in vivo level of IAA. Horseradish peroxidase isoenzyme C and an anionic tobacco peroxidase isolated from transgenic Nicotiana sylvestris have been used in experiments in vitro designed to determine the mechanism of IAA oxidation. In particular, the initial reduction of ferric to ferrous enzyme, a key step in previously proposed mechanisms, has been investigated by rapid-scan stopped-flow spectrophotometry under strictly anaerobic conditions and at defined oxygen concentrations. The data provide the first evidence for a ternary complex comprising peroxidase, IAA and oxygen that is kinetically competent both at the initiation stage and during the catalytic cycle of IAA oxidation. A general scheme describing the oxidative cycles of both anionic and cationic peroxidases is proposed that includes native ferric enzyme and compound II as kinetically competent intermediates. For anionic peroxidases, addition of hydrogen peroxide switches on the oxidative cycle thereby promoting IAA oxidation. 2-Methyl-IAA is not a substrate of the oxidase reaction, suggesting a specific interaction between plant peroxidases and IAA.
Control of 6-(d-threo-1′,2′-dihydroxypropyl) pterin (dictyopterin) synthesis during aggregation of Dictyostelium discoideum. Involvement of the G-protein-linked signalling pathway in the regulation of GTP cyclohydrolase I activity
Authors: GÜTLICH Markus , WITTER Klaus , BOURDAIS Julie , VERON Michel , RÖDL Wolfgang , ZIEGLER Irmgard
Published: August 2015

6-(D-threo-1´,2´-Dihydroxypropylpterin (dictyopterin) has been identified in extracts of growing Dictyostelium discoideum cells [Klein, Thiery and Tatischeff (1990) Eur. J. Biochem. 187, 665–669]. We demonstrate that it originates from GTP by de novo biosynthesis and that the first committed step is catalysed by GTP cyclohydrolase I, yielding dihydroneopterin triphosphate [neopterin is 6-(D-erythro-1´,2´,3´-trihydroxypropyl) pterin]. The GTP cyclohydrolase I activity is found in the cytosolic fraction and in a membrane-associated form. The level of a 0.9 kb mRNA coding for GTP cyclohydrolase I decreases to about 10% of its initial value within 2 h after Dictyostelium cells start development induced by starvation. In the cytosolic fraction, the specific activities of GTP cyclohydrolase I, as well as the concentrations of (6R/S)-5,6,7,8-tetrahydrodictyopterin (H4dictyopterin), follow this decline of the mRNA level. In the particulate fraction, however, the specific activities of GTP cyclohydrolase I and, in consequence, H4dictyopterin synthesis, transiently increase and reach a maximum after 4–5 h of development. The time-course of H4dictyopterin concentrations in the starvation medium closely correlates with its production in the membrane fraction. The activity of membrane-associated GTP cyclohydrolase I can be increased by pre-incubation of the cell lysate with guanosine 5´-[γ-thio]triphosphate and Mg2+. This GTP analogue does not serve as a substrate and has no direct effect on the enzyme activity, indicating that a G-protein-linked signalling pathway is involved in the regulation of GTP cyclohydrolase I activity and thus in H4dictyopterin production during early development of D. discoideum.
Evidence that the specificity of iron incorporation into homopolymers of human ferritin L- and H-chains is conferred by the nucleation and ferroxidase centres
Authors: SANTAMBROGIO Paolo , LEVI Sonia , COZZI Anna , CORSI Barbara , AROSIO Paolo
Published: August 2015

Mammalian ferritins are iron-storage proteins made of 24 subunits of two types: the H- and L-chains. L-chains, in contrast with H-chains, lack detectable ferroxidase activity. When ferritins were subjected to iron loading in vitro with increments near the saturation limit of 4000 Fe atoms per molecule, the homopolymers of human H-chains formed insoluble aggregates, caused by non-specific iron hydrolysis, whereas the homopolymers of L-chains remained soluble and incorporated most of the available iron. To analyse the molecular reasons for the difference, Glu-57 and Glu-60, which are conserved and exposed on the cavity of L-chains, were substituted with His, as in H-chains. The double substitution made the L-homopolymers as sensitive as the H-homopolymers to the iron-induced aggregation, whereas the opposite substitution in the H-chain increased homopolymer resistance to the aggregation only marginally. Millimolar concentrations of citrate and phosphate increased iron incorporation in H-homopolymers by reducing non-specific iron hydrolysis, but inhibited that in L-homopolymers by sequestering available iron. The data indicate that the specific iron incorporation into L-homopolymers is mainly due to the iron-nucleation capacity of Glu-57, Glu-60 and other carboxyl groups exposed on the cavity; in contrast, the specificity of iron incorporation into H-homopolymers is related to its ferroxidase activity, which determines rapid Fe(III) accumulation inside the cavity. The finding that ferroxidase centres are essential for the incorporation of iron in the presence of likely candidates of cellular iron transport, such as phosphate and citrate, confirms their importance in ferritin function in vivo.
A novel tool for the investigation of glutamate release from rat cerebrocortical synaptosomes: the toxin Tx3-3 from the venom of the spider Phoneutria nigriventer
Authors: PRADO Marco A. M. , GUATIMOSIM Cristina , GOMEZ Marcus V. , DINIZ Carlos R. , CORDEIRO Marta N. , ROMANO-SILVA MarcoA.
Published: August 2015

The present experiments investigated the effect of some of the toxic components present in the venom of the spider Phoneutria nigriventer on the release of neurotransmitter. The toxic fraction, Phoneutria nigriventer toxin-3 (PhTx3), abolished Ca2+-dependent glutamate release, but did not alter Ca2+-independent secretion of glutamate when rat brain cortical synaptosomes were depolarized with 33 mM KCl. This effect was most likely due to interference with the entry of calcium through voltage-gated calcium channels, because PhTx3 reduced by 50% the increase in intrasynaptosomal free calcium induced by membrane depolarization, and did not affect the release of glutamate evoked by a calcium ionophore (ionomycin). A polypeptide (Tx3-3) present in the PhTx3 fraction reproduced the effects of the PhTx3 fraction on transmitter release and intrasynaptosomal free calcium in the low nanomolar range. We compared the alterations produced by the Tx3-3 with the actions of toxins known to block calcium channels coupled to exocytosis: the results indicated that the Tx3-3 inhibition of glutamate release and intrasynaptosomal calcium resemble that observed with ω-conotoxin MVIIC. We suggest that the Tx3-3 is a calcium-channel antagonist that blocked glutamate exocytosis.
High-density lipoprotein 3 physicochemical modifications induced by interaction with human polymorphonuclear leucocytes affect their ability to remove cholesterol from cells
Authors: COGNY Anne , ATGER Véronique , PAUL Jean-Louis , SONI Théophile , MOATTI Nicole
Published: August 2015

1. We have recently reported that a short incubation (60 min) in vitro of high-density lipoprotein (HDL) 3 with human polymorphonuclear leucocytes (PMNs) leads to a proteolytic cleavage of apolipoprotein (apo) AII and to a change in the distribution of apo AI isoforms [Cogny, Paul, Atger, Soni and Moatti (1994) Eur. J. Biochem. 222, 965–973]. Since PMNs have been observed to be present in the earliest atherosclerotic lesions for a number of days, we investigated the HDL3 physicochemical modifications induced by in vitro interaction for a long period of time (24 h) with PMNs and the consequences of the changes on the ability of HDL3 to remove cholesterol from cells. 2. The stimulated PMN modification of HDL3 over 24 h resulted in a partial loss of protein with no variation in lipid molar ratio and a loss of 50% of HDL α-tocopherol content. The decrease in total protein was due first to a complete degradation of apo AII, and secondly to a partial loss of apo AI. The apo AI remaining on the particles was in part hydrolysed and the apo AI-1 isoform was completely shifted to the apo AI-2 isoform. These apo changes were accompanied by a displacement of the native HDL3 apparent size toward predominantly larger particles. 3. The ability of PMN-modified HDL3 to remove 3H-labelled free cholesterol from cells was measured in two cell lines: Fu5AH rat hepatoma cells and J774 mouse macrophages. HDL3 which had only a limited contact with PMNs (60 min) showed only a small non-significant reduction in the efficiency of cholesterol efflux. On the other hand, compared with native HDL3, HDL3 modified by PMNs for 24 h had a markedly reduced ability to remove cholesterol from cells, regardless of the type of cell. 4. The results suggest that PMN-modified HDL3, if occurring in vivo, could contribute to acceleration of the atherogenic process by decreasing the cholesterol efflux from cells.
Development of an aqueous-space mixing assay for fusion of granules and plasma membranes from human neutrophils
Authors: BLACKWOOD R. Alexander , SMOLEN James E. , HESSLER Ronald J. , HARSH Donna M. , TRANSUE Amy
Published: August 2015

Several models have been developed to study neutrophil degranulation. At the most basic level, phospholipid vesicles have been used to investigate the lipid interactions occurring during membrane fusion. The two major forms of assays used to measure phospholipid vesicle fusion are based either on the dilution of tagged phospholipids within the membrane of the two fusing partners or the mixing of the aqueous contents of the vesicles. Although problems exist with both methods, the latter is considered to be more accurate and representative of true fusion. Using 8-aminonaphthalene-1,3,6-trisulphonic acid (ANTS) as a fluorescent marker, we have taken advantage of the quenching properties of p-xylenebispyridinium bromide (‘DPX’) to develop a simple aqueous-space mixing assay that can be used with any sealed vesicle. We compared our new assay with more conventional assays using liposomes composed of phosphatidic acid (PA) and phosphatidylethanolamine (PE), obtaining comparable results with respect to Ca2+-dependent fusion. We extended our studies to measure the fusion of neutrophil plasma-membrane vesicles as well as azurophil and specific granules with PA/PE (1:3) liposomes. Both specific granules and plasma-membrane vesicles fused with PA/PE liposomes at [Ca2+] as low as 500 μM, while azurophil granules showed no fusion at [Ca2+] as high as 12 mM. These differences in the ability of Ca2+ to induce fusion may be related to differences observed in whole cells with respect to secretion.
The human leukotriene A4 hydrolase gene is expressed in two alternatively spliced mRNA forms
Authors: JENDRASCHAK Ellen , KAMINSKI Wolfgang E. , KIEFL Rosemarie , von SCHACKY Clemens
Published: August 2015

We identified a novel short (s-) mRNA of the human leukotriene-A4 (LTA4) hydrolase using sequential reverse transcriptase PCR mapping. The s-mRNA is generated by skipping an 83 bp exon located in the 3´ coding region and suggests the expression of an LTA4 hydrolase isoform with a calculated molecular mass of 59 kDa and a distinct C-terminus. Both LTA4 hydrolase mRNAs are constitutively expressed in blood cells, endothelial cells, smooth muscle cells, fibroblasts and tumour cells. The ratios of their mRNA expression levels are cell specific, with relatively high s-form mRNA expression in reticulocytes. Our data strongly suggest that the novel mRNA codes for the structurally related but distinct LTA4 hydrolase isoenzyme that has been postulated.
Paradoxical control properties of enzymes within pathways: can activation cause an enzyme to have increased control?
Authors: KHOLODENKO Boris N. , BROWN Guy C.
Published: August 2015

It is widely assumed that within a metabolic pathway inhibition of an enzyme causes the control exerted by that enzyme over the flux through its own reaction to increase, whereas activation causes its control to decrease. This assumption forms the basis of a number of experimental methods. For a pathway conceptually divided into two enzyme groups connected via a single metabolite we have derived a general condition under which this assumption is false, and thus the pathway shows paradoxical control behaviour, i.e. increased control with activation and decreased control with inhibition of an enzyme or group of enzymes. Paradoxical control behaviour occurs widely when enzyme activity is altered by changing Km (if an enzyme is already close to saturation by its substrate), but may also occur with changes in Vmax. when the elasticity to the linking metabolite increases with its concentration (as in some cases of sigmoidal and exponential kinetics or for reactions catalysed by isoenzymes). These findings suggest that enzymes with sigmoidal kinetics may have low control in the absence of activation, but may gain control with activation, and thus have beneficial regulatory properties.
Human 17 β-hydroxysteroid dehydrogenase type 1 and type 2 isoenzymes have opposite activities in cultured cells and characteristic cell- and tissue-specific expression
Authors: MIETTINEN Minna M. , MUSTONEN Mika V. J. , POUTANEN Matti H. , ISOMAA Veli V. , VIHKO Reijo K.
Published: August 2015

17β-Hydroxysteroid dehydrogenase (17HSD) isoenzymes catalyse the interconversion between highly active 17β-hydroxy-and low-activity 17-keto-steroids and thereby regulate the biological activity of sex steroids. The present study was carried out to characterize 17HSD activity and the expression of 17HSD type 1 and 2 isoenzymes in several human cell types and tissues. The data indicate that in cultured cells the direction of 17HSD activity is exclusively determined by the expression of these distinct isoenzymes. The intracellular environment could not modulate the direction of the enzyme activities in any of the cell types analysed. 17HSD type 1 acts as a reductase converting oestrone into oestradiol, whereas 17HSD type 2 possesses oxidative activity inactivating oestradiol by converting it into oestrone. The data, furthermore, suggest that of the two 17HSD type 1 mRNAs (1.3 and 2.3 kb), expression of the 1.3 kb mRNA is related to enzyme concentration in all the cell types studied. This mRNA is principally expressed in cells of placental and ovarian origin, but is also present in malignant breast epithelial cells. In contrast, 17HSD type 2 is more widely expressed. It is present in several oestradiol-metabolizing tissues as well as in some target cells of sex steroid action. The opposite reaction directions observed in the cultured cells, together with differences in the distribution of the isoenzymes, suggest that type 1 is involved in oestradiol production in females while type 2 plays a role in the inactivation of this sex steroid in peripheral tissues, both in females and in males. However, some examples exist of simultaneous expression of both enzymes in the same cell type or tissue.
Phosphotyrosine phosphatase associated with band 3 protein in the human erythrocyte membrane
Authors: ZIPSER Yehudit , KOSOWER Nechama S.
Published: August 2015

The anion-exchange band 3 protein is the main erythrocyte protein that is phosphorylated by tyrosine kinase. To study the regulation of band 3 phosphorylation, we examined phosphotyrosine phosphatase (PTP) activity in the human erythrocyte. We show that the human erythrocyte membrane contains a band 3-associated neutral PTP which is activated by Mg2+ and inhibited by Mn2+ and vanadate. The PTP is active in the intact cell and in the isolated membrane. A major fraction of the PTP is tightly bound to the membrane and can be extracted from it by Triton X-100; a minor part is associated with the Triton X-100-insoluble cytoskeleton. The behaviour of the PTP parallels that of band 3, the major fraction of which is extractable by detergents with a minor fraction being anchored to the cytoskeleton. Moreover, band 3 is co-precipitated when the PTP is immunoprecipitated from solubilized membranes, and PTP is co-precipitated when band 3 is immunoprecipitated. The PTP appears to be related to PTP1B (identified using an antibody to an epitope in its catalytic domain and by molecular mass). The system described here has a unique advantage for PTP research, since it allows the study of the interaction of a PTP with an endogenous physiological substrate that is present in substantial amounts in the cell membrane. The membrane-bound, band 3-associated, PTP may play a role in band 3 function in the erythrocyte and in other cells which have proteins analogous to band 3.
Inhibition studies on calf pregastric esterase: the enzyme has no functional thiol group
Authors: TIMMERMANS Miet Y. J. , REEKMANS Gunther , TEUCHY Henri J. H. , KUPERS Luc P. M.
Published: August 2015

Pregastric esterase (PGE) (EC 3.1.1.3) was purified to homogeneity from calf pharyngeal tissue. The enzyme had an apparent molecular mass of 50 kDa, as determined by SDS/PAGE. The serine-binding reagent diethyl p-nitrophenyl phosphate was a potent inhibitor of PGE. This is in accordance with the claim that a functional serine residue is necessary for the lipolytic activity of lipases. PGE was not inhibited by the thiol reagents 5,5´-dithiobis(2-nitrobenzoic acid) or 4,4´-dithiopyridine. A partial inhibition with dodecyldithio-5-(2-nitrobenzoic acid) was observed, but the same degree of inhibition was caused by the non-esterified fatty acid C12:0. PGE shows a great sequence similarity to gastric lipases. Gastric lipases have three cysteine residues, and two of these form a disulphide bridge. Blocking the remaining free cysteine with thiol reagents inactivates the gastric lipases. The fact that PGE is not inhibited by thiol reagents indicates that PGE has no functional free thiol group. The PGE cDNA codes only for two cysteines, and their involvement in the formation of a disulphide bridge was demonstrated.
Mechanisms by which the surface expression of the glycosyl-phosphatidylinositol-anchored complement regulatory proteins decay-accelerating factor (CD55) and CD59 is lost in human leukaemia cell lines
Authors: HATANAKA Michiyo , SEYA SEYA , MATSUMOTO Misako , HARA Tomoko , NONAKA Masaru , INOUE Norimitsu , TAKEDA Junji , SHIMIZU Akira
Published: August 2015

We have investigated the mechanisms of defects in the glycosyl-phosphatidylinositol (GPI)-anchored complement regulatory proteins delay-accelerating factor (DAF) and/or CD59 in a panel of human leukaemia cell lines that lack surface expression of these proteins: U937 (DAF+/CD59-), CEM (DAF-/CD59+), TALL (DAF-/CD59-) and a substrain of Ramos [Ramos(-)] (DAF-/CD59-). Northern blotting and reverse transcription-PCR revealed that the main cause of the DAF and/or CD59 deficiency is the failure of mRNA expression in most of the cell lines, except in Ramos(-) in which sufficient mRNA for DAF and CD59 was produced. U937, CEM and TALL cells were not defective in GPI anchor formation as assessed by the detection of other GPI-anchored proteins. No gene abnormality corresponding to DAF or CD59 was detected by Southern blotting. Thus the cause of the defects of DAF and/or CD59 in these leukaemia cell lines except for Ramos(-) is virtually undetectable steady-state levels of the relevant mRNA, most likely attributable to lack of transcription in these cell lines. On the other hand, Ramos(-) cells failed to generate a GPI anchor, whereas they normally expressed DAF and CD59 transcripts. The transfection of phosphatidylinositol-glycan class A (PIG-A) cDNA into Ramos(-) cells restored DAF and CD59 expression, indicating that the defective mechanism in GPI anchor formation is similar to that in paroxysmal nocturnal haemoglobinuria (PNH) cells, i.e. a deficiency of the PIG-A gene product. Thus the mechanisms of the defects of DAF and/or CD59 in human leukaemia cell lines are not uniform, and in most cases are different from that proposed to cause PNH.
Ability of a β-casein phosphopeptide to modulate the precipitation of calcium phosphate by forming amorphous dicalcium phosphate nanoclusters
Authors: HOLT Carl , WAHLGREN Magnus N. , DRAKENBERG Torbjorn
Published: August 2015

The ability of casein in the form of colloidal-sized casein micelles to modulate the phase separation of calcium phosphate during milk secretion is adapted to produce nanometre-sized particles of calcium phosphate stabilized by a casein phosphopeptide (nanoclusters). The nanoclusters were prepared from an undersaturated solution of salts and the peptide by raising the pH homogeneously from about 5.5 to 6.7 with urea plus urease. Chemical analysis and IR spectroscopy showed that they comprise an amorphous dicalcium phosphate bound to the phosphopeptide. Multinuclear NMR spectroscopy of the cluster solutions showed that the small ions and free peptide in the solution were in a state of dynamic exchange with the nanoclusters. The peptide is linked to the calcium phosphate through its sequence of phosphorylated residues, but, in a proportion of adsorbed conformational states, the termini retain the conformational freedom of the unbound peptide. The ability of casein to form nanoclusters in milk suggests a more general mechanism for avoiding pathological calcification and regulating calcium flow in tissues and biological fluids exposed to or containing high concentrations of calcium.
Characterization of rat 5α-reductases type 1 and type 2 expressed in Saccharomyces cerevisiae
Authors: POLETTI Angelo , CELOTTI Fabio , MOTTA Marcella , MARTINI Luciano
Published: August 2015

Two isoforms of the rat 5α-reductase (5α-R), the enzyme that converts testosterone into dihydrotestosterone (DHT), and other ∆4-3-keto steroids (e.g. progesterone and corticoids) into their 5α-reduced metabolites, have been cloned. In this study, a convenient and efficient system was developed to overexpress the two isoenzymes in Saccharomyces cerevisiae by using the ubiquitin-fusion expression system. Two yeast expression vectors have been prepared, YEpR1 and YEpR2, which code for 5α-R type 1 and 5α-R type 2 respectively; they contain the copper-responsive yeast metallothionein promoter (CUP1) upstream of the ubiquitin coding sequence, and the full-length rat 5α-R type 1 or 5α-R type 2 cDNAs in frame to the 3´ end of the ubiquitin cDNA. The activity of the two isoenzymes produced in yeast was determined in cell lysates at the enzyme pH optima (type 1, pH 7.5; type 2, pH 5.5) and a possible differential intracellular distribution was also evaluated. The kinetic parameters were: type 1, Km 4.6 μM, Vmax. 100.6 μg/h per mg of protein; type 2, Km 68.6 nM, Vmax. 0.84 μg/h per mg of protein. Yeast cell lysates were fractionated by differential centrifugation and the 5α-R type 1 activity was maximal in fractions containing nuclei (1000 g and 2500 g), whereas the maximal activity of 5α-R type 2 was present in subcellular fractions sedimenting at higher speeds (20000 g). The data indicate that yeasts overexpress the two 5α-R isoenzymes, maintaining their native biochemical properties, and that the two isoforms are probably differentially localized within the yeast cell.
Expression of lignin peroxidase H8 in Escherichia coli: folding and activation of the recombinant enzyme with Ca2+ and haem
Authors: DOYLE Wendy A. , SMITH Andrew T.
Published: August 2015

An engineered cDNA from Phanerochaete chrysosporium encoding both the mature and pro-sequence regions of Lip isoenzyme H8 (Lip) has been successfully overexpressed in Escherichia coli. The recombinant protein (LipP*) was sequestered in inclusion bodies. The reduced–denatured polypeptide has been purified by differential solubilization, and the active enzyme recovered after controlled in vitro refolding (albeit in low yield), by glutathione-mediated oxidation of disulphides, in a folding medium containing an intermediate concentration of urea, Ca2+ and haem. The procedure is analogous to that previously described for the production of active recombinant horseradish peroxidase (HRP-C*) from inclusion-body material. It is quite possible, therefore, that this type of procedure may be suitable for the recovery of most, if not all, active recombinant peroxidases. The resultant LipP* has spectral characteristics identical with that of the native enzyme as isolated from Phanerochaete chrysosporium. Its specific activity measured in the standard veratryl alcohol (VA) assay was 39 μmol of VA oxidized/min per mg of protein, a value which compares extremely favourably with that of the native enzyme (36 μmol of VA/min per mg). Although levels of active enzyme obtained are not yet as high as in the case of HRP-C* (1% conversion of crude inactive LipP* polypeptide into pure fully active Lip), it is envisaged that further refinement of the expression/folding/activation procedures will provide sufficient protein for biophysical characterization of both the wild-type and site-directed mutants.
Activation of chicken liver dihydrofolate reductase by urea and guanidine hydrochloride is accompanied by conformational change at the active site
Authors: FAN Ying-xin , JU Ming , ZHOU Jun-mei , TSOU Chen-lu
Published: August 2015

It has been reported that the activation of dihydrofolate reductase (DHFR) from L1210 mouse leukaemia cells by KCl or thiol modifiers is accompanied by increased digestibility by proteinases [Duffy, Beckman, Peterson, Vitols and Huennekens (1987) J. Biol. Chem. 262, 7028–7033], suggesting a loosening up of the general compact structure of the enzyme. In the present study, the peptide fragments liberated from the chicken liver enzyme by digestion with trypsin in dilute solutions of urea or guanidine hydrochloride (GuHCl) have been separated by FPLC and sequenced. The sequences obtained are unique when compared with the known sequence of DHFR and thus allow the points of proteolytic cleavage identified for the urea- and GuHCl-activated enzyme to be at or near the active site. It was also indicated by the enhanced fluorescence of 2-p-toluidinylnaphthalene 6-sulphonate that conformational changes at the active site in dilute GuHCl parallel GuHCl activation. The above results indicate that the activation of DHFR in dilute denaturants is accompanied by a loosening up of its compact structure especially at or near the active site, suggesting that the flexibility at its active site is essential for the full expression of its catalytic activity.
Effect of amino acid residues at the cleavable site of substrates on the remarkable activation of thermolysin by salts
Authors: INOUYE Kuniyo , LEE Soo-Bok , TONOMURA Ben'ichiro
Published: August 2015

The activity of thermolysin in the hydrolysis of N-[3-(2-furyl)acryloyl]-glycyl-L-leucine amide and N-carbobenzoxy-L-aspartyl-L-phenylalanine methyl ester is remarkably enhanced in the presence of high concentrations (1–5 M) of neutral salts [Inouye (1992) J. Biochem. (Tokyo) 112, 335–340]. In this study, the effect of salts on such activity has been examined using a series of substrates, furylacryloyl dipeptide amides, which have various hydrophobic amino acids at the cleavable bond. Although the enzyme activity varies widely depending on the substrate employed, the degree of activation at a given concentration of NaCl is considerably similar. This indicates that the degree of activation is not dependent on the hydrophobicity of the amino acid side chains at the scissile bond of the substrates. The molecular activity, kcat, and Michaelis constant, Km, were evaluated separately for substrates N-[3-(2-furyl)acryloyl]-L-leucyl-L-alanine amide and N-[3-(2-furyl)acryloyl]-L-phenylalanyl-L-alanine amide, and the activation was found to be brought about only by an increase in kcat. The effectiveness of monovalent cations on the increase of kcat was determined to follow the order of Na+ > K+ > Li+.
Expression of the rat GLUT1 glucose transporter in the yeast Saccharomyces cerevisiae
Authors: KASAHARA Toshiko , KASAHARA Michihiro
Published: August 2015

We expressed the rat GLUT1 facilitative glucose transporter in the yeast Saccharomyces cerevisiae with the use of a galactose-inducible expression system. Confocal immunofluorescence microscopy indicated that a majority of this protein is retained in an intracellular structure that probably corresponds to endoplasmic reticulum. Yeast cells expressing GLUT1 exhibited little increase in glucose-transport activity. We prepared a crude membrane fraction from these cells and made liposomes with this fraction using the freeze–thaw/sonication method. In this reconstituted system, D-glucose-transport activity was observed with a Km for D-glucose of 3.4±0.2 mM (mean±S.E.M.) and was inhibited by cytochalasin B (IC50 = 0.44±0.03 μM), HgCl2 (IC50 = 3.5±0.5 μM), phloretin (IC50 = 49±12 μM) and phloridzin (IC50 = 355±67 μM). To compare these properties with native GLUT1, we made reconstituted liposomes with a membrane fraction prepared from human erythrocytes, in which the Km of D-glucose transport and ICs of these inhibitors were approximately equal to those obtained with GLUT1 made by yeast. When the relative amounts of GLUT1 in the crude membrane fractions were measured by quantitative immunoblotting, the specific activity of the yeast-made GLUT1 was 110% of erythrocyte GLUT1, indicating that GLUT1 expressed in yeast is fully active in glucose transport.
Omega-3 polyunsaturated fatty acids increase purine but not pyrimidine transport in L1210 leukaemia cells
Authors: MARTIN Danielle , MECKLING-GILL Kelly A.
Published: August 2015

Here we show that in vitro supplementation of L1210 murine lymphoblastic leukaemia cells with n-3 polyunsaturated fatty acids results in considerable changes in the fatty acid composition of membrane phospholipids. Incubations for 48 h with 30 μM eicosapentaenoic acid (20:5, n-3; EPA) or docosahexaenoic acid (22:6, n-3; DHA) results primarily in substitution of long-chain n-6 fatty acids with long-chain n-3 fatty acids. This results in a decrease in the n-6/n-3 ratio from 6.9 in unsupplemented cultures to 1.2 or 1.6 for EPA and DHA supplemented cultures, respectively. Coincident with these changes in membrane fatty acid composition, we observed a 5-fold increase in the rate of adenosine (5 μM) uptake via the nitrobenzylthioinosine (NBMPR)-sensitive nucleoside transporter in EPA- and DHA- supplemented L1210 cells, relative to unsupplemented cells. This seemed to result from a decrease in the Km for adenosine from 12.5 μM in unsupplemented cultures to 5.1 μM in DHA-treated cultures. Guanosine (50 μM) transport was similarly affected by DHA with a 3.5-fold increase in the initial rate of uptake. In contrast, pyrimidine transport, as measured by uptake of thymidine and cytidine, was not similarly affected, suggesting that substrate recognition had been altered by fatty acid supplementation. Studies using [3H]NBMPR showed that there was no effect of EPA or DHA on either the number of NBMPR-binding sites or the affinity of these sites for NBMPR. This observation suggests that the increases in adenosine and guanosine transport were not due to increases in the number of transporter sites but rather that EPA and DHA directly or indirectly modulate transporter function.