bluuebunny/crossref_metadata_2025 huggingface dataset:
Purification and functional characterization of a low-molecular-mass Ca2+,Mg2+- and Ca2+-ATPase modulator protein from rat brain cytosol
Authors: BHATTACHARYYA Dipankar , SEN C. Parimal
Published: August 2015

A low-molecular-mass modulator protein having a molecular mass of about 12 kDa has been purified from rat brain cytosol following gel filtration and FPLC/Mono Q anion-exchange chromatographic separation. A number of protein fractions were obtained from an FPLC column when eluted with a 0.1 M NaCl hold gradient. One fraction (peak no. 5) was found to stimulate Ca2+,Mg2+-ATPase but inhibit Ca2+-ATPase isolated from goat spermatozoa. The S50 (concentration producing 50% stimulation) and I50 were found to be in the nanomolar range. The modulator seems to bind to Ca2+, Mg2+- or Ca2+-ATPase at a site distal from the ATP binding site. The binding to both the ATPases is reversible and non-competitive in nature. The inhibitory activity is found to depend significantly on -SH or -NH2 group(s) of the modulator, whereas no appreciable dependency of the stimulatory effect was apparent. The study indicates that the modulator is not a glycoprotein. CD analysis suggests that the protein exists as an unordered secondary structure. An immuno-cross-reactivity study with specific antibody and inhibition by thapsigargin suggests that the Ca2+,Mg2+- and Ca2+-ATPases from goat testes microsomal membranes are two isoforms of the sarcoplasmic/endoplasmic-reticulum Ca2+-ATPase (SERCA) family. The modulator does not contain any Trp molecules, as evident from Trp fluorescence analysis. Amino acid analysis shows that glycine, serine, derivatives of tyrosine and phenylalanine are the predominant amino acids. The data suggest that the modulator is a negatively charged protein and is a good tool for distinguishing the regulation of Ca2+,Mg2+- and Ca2+-ATPase activities.
A dehydrogenase-mediated recycling system of NADPH in plant peroxisomes
Authors: CORPAS J. Francisco , BARROSO B. Juan , SANDALIO M. Luisa , DISTEFANO Stefania , PALMA M. José , LUPIÁÑEZ José A. , del RÍO A. Luis
Published: August 2015

The presence of the two NADP-dependent dehydrogenases of the pentose phosphate pathway has been investigated in plant peroxisomes from pea (Pisum sativum L.) leaves. Both enzymes, glucose-6-phosphate dehydrogenase (G6PDH; EC 1.1.1.49) and 6-phosphogluconate dehydrogenase (6PGDH; EC 1.1.1.44), were present in the matrix of leaf peroxisomes, and their kinetic properties were studied. G6PDH and 6PGDH showed a typical Michaelis-Menten kinetic saturation curve, and had specific activities of 12.4 and 29.6 mU/mg protein, respectively. The Km values of G6PDH and 6PGDH for glucose 6-phosphate and for 6-phosphogluconate were 107.3 and 10.2 μM, respectively. Dithiothreitol did not inhibit G6PDH activity. By isoelectric focusing of peroxisomal matrices, the G6PDH activity was resolved into three isoforms with isoelectric points of 5.55, 5.30 and 4.85. The isoelectric point of peroxisomal 6PGDH was 5.10. Immunoblot analyses of peroxisomal matrix with an antibody against yeast G6PDH revealed a single cross-reactive band of 56 kDa. Post-embedment, EM immunogold labelling of G6PDH confirmed that this enzyme was localized in the peroxisomal matrices, the thylakoid membrane and matrix of chloroplasts, and the cytosol. The presence of the two oxidative enzymes of the pentose phosphate pathway in plant peroxisomes implies that these organelles have the capacity to reduce NADP+ to NADPH for its re-utilization in the peroxisomal metabolism. NADPH is particularly required for the ascorbate-glutathione cycle, which has been recently demonstrated in plant peroxisomes [Jiménez, Hernández, del Río and Sevilla (1997) Plant Physiol. 114, 275-284] and represents an important antioxidant protection system against H2O2 generated in peroxisomes.
Chromatin remodelling of the cardiac β-myosin heavy chain gene
Authors: HUANG Weei-Yuarn , LIEW Choong-Chin
Published: August 2015

To investigate the role of chromatin structure in cardiac gene expression, we used the DNase I and micrococcal nuclease to probe the chromatin structure of the hamster cardiac β-MyHC gene. Two cardiac-specific DNase I hypersensitive sites (DHS) were identified, one of which was mapped to the -2.3 kb (β-2.3 kb) region and the other to the proximal promoter region of the β-MyHC gene. The two sites were readily detectable using nuclei from neonatal hamster heart; however, the proximal promoter site disappeared when adult hamster heart nuclei were used, and the -2.3 kb site decreased in intensity. We were able to demonstrate the gradual disappearance of this proximal promoter DHS by comparing heart nuclei isolated from animals at late-gestation and 1-day-old stages. Furthermore, injecting thyroid hormone caused the disappearance of the proximal promoter DHS in late gestational fetal ventricular nuclei. Digestion of nuclei from various tissues by micrococcal nuclease revealed that the β-MyHC gene proximal promoter exists in an array of three specifically-positioned nucleosomes only in fetal heart chromatin. The β-MyHC gene proximal promoter is DNase I hypersensitive within one of the nucleosomal particles. Our data suggest that chromatin structure may participate actively in cardiac gene expression.
The distribution and metabolism of arachidonate-containing phospholipids in cellular nuclei
Authors: SURETTE E. Marc , CHILTON H. Floyd
Published: August 2015

The cell nucleus has been identified as a location to which several arachidonic acid-metabolizing enzymes are located in stimulated cells. However, little information exists describing the distribution of arachidonate-containing phospholipids associated with the nucleus or the control of their composition. In this study, nuclei isolated from human monocyte-like THP-1 cells were found to have a distribution of arachidonyl-phospholipids which is markedly different from that of other cellular membranes. THP-1 nuclei which contained 22% of total cellular arachidonate, showed a near equal distribution of arachidonate in 1-acyl-2-arachidonoyl-glycero-3-phosphocholine, 1-acyl-2-arachidonyl-glycero-3-phosphoethanolamine, 1-acyl-2-arachidonoyl-glycero-3-phosphoinositol and 1-alk-1-enyl-2-arachidonoyl-glycero-3-phosphoethanolamine molecular species. In contrast in non-nuclear membranes, arachidonate was located primarily in 1-alk-1-enyl-2-arachidonoyl-glycero-3-phosphoethanolamine molecular species which accounted for approximately half of the arachidonate in all non-nuclear phospholipids. Isolated nuclei were incapable of initially acylating arachidonic acid into their phospholipids in the absence of cellular cytosol. However, they were capable of efficiently remodelling existing arachidonate between phospholipid classes and subclasses. Isolated nuclei contained 25-30% of the cellular activity of CoA-independent transacylase, the key enzyme responsible for arachidonate-phospholipid remodelling. This enzyme is also critical in the control of arachidonate availability following cell stimulation. Given that the cellular distribution of arachidonate is such that nuclei are enriched in donor substrates for the CoA-independent transacylase reaction, that non-nuclear membranes are enriched in acceptor substrates and that nuclei have the enzymatic machinery to remodel arachidonate efficiently, these results suggest that CoA-independent transacylase may be responsible for the remodelling of arachidonate not only between different phospholipid species within the same organelles but also between different sub-cellular compartments.
Oligomeric forms of the 90-kDa heat shock protein
Authors: NEMOTO Takayuki , SATO Nobuko
Published: August 2015

Two isoforms of the 90-kDa heat shock protein, HSP90α and HSP90β, are present in the cytosol of mammalian cells. Analysis by polyacrylamide gel electrophoresis under nondenaturing conditions (native PAGE) revealed that HSP90α predominantly exists as a homodimer and that HSP90β is present mainly as a monomer [Minami, Kawasaki, Miyata, Suzuki and Yahara (1991) J. Biol. Chem. 266, 10099-10103]. However, only the dimeric form has been observed under other analytical conditions such as gradient centrifugation. In this study, therefore, we investigated native forms of HSP90 by use of immunochemical techniques with isoform-specific monoclonal antibodies recently developed in our laboratory. Glycerol gradient centrifugation at the physiological salt concentration as well as native PAGE analysis of rat liver cytosol revealed oligomeric forms of HSP90α sedimenting at 8-10S as predominant ones. On the other hand, the glycerol gradient centrifugation revealed multiple forms of HSP90β oligomers sedimenting at 6-12S. All of the HSP90β oligomers, however, migrated at 100-kDa monomer and 190-kDa dimer positions on native PAGE. A novel two-dimensional double native PAGE revealed that the entity was converted from the HSP90β dimer to monomers during the electrophoresis. The same PAGE further revealed that the HSP90α oligomer also dissociated into dimers during the electrophoresis. Full-length form of bacterially-expressed human HSP90α migrated as dimers, but a considerable amount did not penetrate into the gel under native PAGE conditions, indicating the existence of oligomeric forms. Electrophoretic studies of deletion mutants of HSP90 demonstrated that the C-terminal 200 amino acids were capable of forming oligomers. Taken together, we conclude that both of the HSP90 isoforms predominantly exist as oligomeric forms in the cytosol even under unstressed conditions but that they artificially dissociate into smaller forms when subjected to native PAGE.
Bi-site activation occurs with the native and nucleotide-depleted mitochondrial F1-ATPase
Authors: MILGROM M. Yakov , MURATALIEV B. Marat , BOYER D. Paul
Published: August 2015

Experiments are reported on the uni-site catalysis and the transition from uni-site to multi-site catalysis with bovine heart mitochondrial F1-ATPase. The very slow uni-site ATP hydrolysis is shown to occur without tightly bound nucleotides present and with or without Pi in the buffer. Measurements of the transition to higher rates and the amount of bound ATP committed to hydrolysis as the ATP concentration is increased at different fixed enzyme concentrations give evidence that the filling of a second site can initiate near maximal turnover rates. They provide rate constant information, and show that an apparent Km for a second site of about 2 μM and Vmax of 10 s-1, as suggested by others, is not operative. Careful initial velocity measurements also eliminate other suggested Km values and are consistent with bi-site activation to near maximal hydrolysis rates, with a Km of about 130 μM and Vmax of about 700 s-1. However, the results do not eliminate the possibility of additional ‘hidden’ Km values with similar Vmax:Km ratios. Recent data on competition between TNP-ATP and ATP revealed a third catalytic site for ATP in the millimolar concentration range. This result, and those reported in the present paper, allow the conclusion that the mitochondrial F1-ATPase can attain near maximal activity in bi-site catalysis. Our data also add to the evidence that a recent claim, that the mitochondrial F1-ATPase does not show catalytic site cooperativity, is invalid.
Biotin synthase from Escherichia coli: isolation of an enzyme-generated intermediate and stoichiometry of S-adenosylmethionine use
Authors: SHAW M. Nicholas , BIRCH M. Olwen , TINSCHERT Andreas , VENETZ Veronika , DIETRICH Rüdiger , SAVOY Luc-Alain
Published: August 2015

A cell-free extract from Escherichia coli containing an E. coli biotin synthase that was expressed to approx. 1% of soluble cell protein by cloning the E. coli bioB gene was used to investigate the biotin synthase reaction. The pH optimum was between 8 and 8.5, and the reaction velocity was dependent on the concentrations of dethiobiotin, cysteine, S-adenosylmethionine and asparagine. The catalytic-centre activity of the enzyme in vitro was estimated to be 0.95 h-1, and each molecule of enzyme turned over less than one molecule of dethiobiotin, i.e. the enzyme was not acting catalytically. HPLC analysis of reaction mixtures revealed the presence of a compound with the characteristics of an intermediate: (1) it was labelled with 14C, and therefore derived from the [14C]dethiobiotin substrate; (2) it was present only in reaction mixtures containing biotin synthase; (3) it was not derived from [14C]biotin; (4) 35S from [35S]cystine was incorporated into the intermediate during the reaction; (5) its synthesis was dependent on the presence of S-adenosylmethionine, and was decreased when free cysteine was omitted from the reaction; (6) it could be isolated from the reaction mixture by chromatography and then re-introduced into an assay as the substrate, whereupon it was converted to biotin; (7) this conversion to biotin was S-adenosylmethionine-dependent. During the reaction S-adenosylmethionine was cleaved to methionine and presumably 5ʹ-deoxyadenosine. Observation of the intermediate allowed us to perform experiments to determine the stoichiometry of S-adenosylmethionine use. We propose that two molecules of S-adenosylmethionine are used to synthesize one molecule of biotin, i.e. one from dethiobiotin to the intermediate, and a second from the intermediate to biotin.
Effects of mutation of residue I67 on redox-linked protonation processes in yeast cytochrome c oxidase
Authors: MEUNIER Brigitte , ORTWEIN Claus , BRANDT Ulrich , RICH R. Peter
Published: August 2015

We describe effects of a mutation, Ile-67 → Asn, in subunit I of yeast cytochrome c oxidase on redox-linked protonation processes within the protein. The mutation lowers the midpoint potential of haem a and weakens its pH dependency, but has little effect on the potential of haem a3. The residue is close to a conserved glutamate (Glu-243) in the crystal structure. We propose that protonation of Glu-243 is redox-linked to haem a, that Asn-167 perturbs its pK and that redox-linked protonation in this location is essential for the catalytic reactions of the binuclear centre. These proposals are discussed in terms of a ‘glutamate trap’ mechanism for proton translocation in the haem/copper oxidases.
The gene structure and promoter sequence of mouse hyaluronan synthase 1 (mHAS1)
Authors: YAMADA Yoichi , ITANO Naoki , ZAKO Masahiro , YOSHIDA Mamoru , LENAS Petros , NIIMI Atsushi , UEDA Minoru , KIMATA Koji
Published: August 2015

The structure and organization of mouse hyaluronan synthase 1 gene, HAS1 were determined by direct sequencing of λ phage clones carrying the entire gene and by application of the long and accurate (LA)-PCR method to amplify regions encompassing the exon-intron boundaries and all of the exons. This gene spans about 11 kb of genomic DNA and consists of 5 exons and 4 introns. A similarity in the exon-intron organization was found between the genes of mouse HAS1 and Xenopus laevis DG42 which was recently identified as Xenopus hyaluronan synthase. The transcription initiation site was determined by rapid amplification of the cDNA ends (5ʹ-RACE). Position +1 is located 55 nucleotides upstream of the ATG initiation codon. The promoter region of the HAS1 gene has no typical TATA box, but contains a CCAAT box located 190 nucleotides upstream of the transcription initiation site. Further analysis of 1.4 kb of the 5ʹ flanking region revealed several potential binding motifs for transcription factors. This information about the gene structure may be useful for further studies on the promoter activity.
Expression, purification and characterization of a human serine-dependent phospholipase A2 with high specificity for oxidized phospholipids and platelet activating factor
Authors: RICE Q. J. Simon , SOUTHAN Christopher , BOYD F. Helen , TERRETT A. Jonathan , MACPHEE H. Colin , MOORES Kitty , GLOGER S. Israel , TEW G. David
Published: August 2015

Using expressed sequence tag (EST) homology screening, a new human serine dependent phospholipase A2 (HSD-PLA2) was identified that has 40% amino acid identity with human low density lipoprotein-associated phospholipase A2 (LDL-PLA2). HSD-PLA2 has very recently been purified and cloned from brain tissue but named PAF-AH II. However, because the homology with LDL-PLA2 suggested a broader substrate specificity than simply platelet activating factor (PAF), we have further characterized this enzyme using baculovirus-expressed protein. The recombinant enzyme, which was purified 21-fold to homogeneity, had a molecular mass of 44 kDa and possessed a specific activity of 35 μmol min-1 mg-1 when assayed against PAF. Activity could also be measured using 1-decanoyl-2-(4-nitrophenylglutaryl) phosphate (DNGP) as substrate. Like LDL-PLA2, HSD-PLA2 was able to hydrolyse oxidatively modified phosphatidylcholines when supplemented to human LDL prior to copper-stimulated oxidation. A GXSXG motif evident from sequence information and inhibition of its activity by 3,4,dichloroisocoumarin, diisopropyl fluorophosphate (DFP) and diethyl p-nitrophenyl phosphate (DENP) confirm that the enzyme is serine dependent. Moreover, sequence comparison indicates the HSD-PLA2 probable active site triad positions are shared with LDL-PLA2 and a C. elegans homologue, suggesting that these sequences comprise members of a new enzyme family. Although clearly structurally related with similar substrate specificities further work reported here shows HSD-PLA2 and LDL-PLA2 to be different with respect to chromosomal localization and tissue distribution.
Differential transcriptional regulation of the human thrombin receptor gene by the Sp family of transcription factors in human endothelial cells
Authors: WU Yaxu , RUEF Johannes , RAO N. Gadiparthi , PATTERSON Cam , RUNGE S. Marschall
Published: August 2015

The mitogenic effects of thrombin are mediated by a G-protein-coupled receptor. Because the effects of thrombin are strongly influenced by the expression of its receptor, an understanding of its regulatory mechanisms is essential. To identify mechanisms of human thrombin receptor (HTR) gene regulation, a series of HTR-promoter-luciferase constructs were made and transfected into human microvascular endothelial cells for analysis. Deletion from bp -303 to -164 abolished reporter gene expression. Dimethyl sulphate treatment in vivo and DNase I footprinting in vitro demonstrated that a cluster of three GC box consensus sites was occupied, and electrophoretic mobility-shift assays established that Sp1 and Sp3 both bind to this 3ʹ GC box cluster. We mutated each of the three GC boxes individually and all three collectively within this 3ʹ cluster. Basal promoter activity was decreased to 46%, 78% and 29% of control for each of the GC boxes mutated individually, and to 6% when the three were mutated collectively. To test the individual abilities of Sp1 and Sp3 to activate or repress HTR transcription, we conducted co-transfection experiments with wild-type or mutated HTR-promoter-luciferase constructs. Co-transfection with Sp1 significantly augmented wild-type HTR promoter activity. Sp3 alone did not affect activity, and inhibited Sp1-mediated activation. Competition for shared binding sites by Sp1 and Sp3 might differentially regulate HTR expression in vascular endothelial cells.
The C-terminus of factor H: monoclonal antibodies inhibit heparin binding and identify epitopes common to factor H and factor H-related proteins
Authors: PRODINGER Wolfgang M. , HELLWAGE Jens , SPRUTH Martin , DIERICH Manfred P. , ZIPFEL Peter F.
Published: August 2015

We have generated monoclonal antibodies (mAbs) specific for the C-terminus of factor H that can be used as inhibitory antibodies for heparin binding and for the specific detection of factor H and factor H-related proteins (FHRs) in plasma and triacylglycerol-rich lipoproteins. Four distinct mAbs were established: IXF9 (IgG1), VD3 (IgG2a), VIG8 (IgG1) and IIC5 (IgG1). Each reacts specifically with FHR-1 and factor H (and also with FHR-2 in the case of VIG8), but none binds to the related FHR-3 and FHR-4 proteins nor to factor H-like protein 1. By the use of deletion mutants of factor H and by comparing the reactivity with FHR-1 and FHR-2, the binding epitopes of the mAbs were identified and localized to different short consensus repeats (SCRs): mAbs IXF9 and VD3 bind to related or even identical sites within SCR 18 (factor H) and SCR 3 (FHR-1) respectively. mAbs VIG8 and IIC5 bind to different epitopes located within SCRs 19 to 20 of factor H and SCRs 4 to 5 of FHR-1 respectively. Only mAb VIG8 reacts with the corresponding SCRs 3 to 4 of FHR-2. These antibodies are useful for the detection of the corresponding proteins in biological specimens such as fractions of lipoproteins. In addition, mAb VIG8 has the unique feature of inhibiting binding of factor H to heparin. Given the recent identification of a heparin- and a C3b-binding domain within the C-terminus of factor H, these mAbs should provide useful tools for functional analysis and for the precise localization of the domain(s) required for this interaction.
Oxidation of high-density lipoprotein HDL3 leads to exposure of apo-AI and apo-AII epitopes and to formation of aldehyde protein adducts, and influences binding of oxidized low-density lipoprotein to type I and type III collagen in vitro
Authors: GREILBERGER Joachim , JÜRGENS Günther
Published: August 2015

The changes in the immunological properties of apolipoprotein AI (apo-AI) and AII (apo-AII) during the oxidation of the high-density lipoprotein HDL3 and its influence on the binding of heavily oxidized low-density lipoprotein (LDL) to type I and III collagen were investigated. Oxidation of HDL3 or Eu3+-labelled HDL3 was performed with CuSO4, varying the time of oxidation. Oxidation of HDL3 resulted in an increase in lipid hydroperoxides and enhanced the negative charge of this lipoprotein. Immunological studies with a solid-phase sandwich immunoassay revealed a strong increase in binding of Eu3+-labelled HDL3 to polyclonal antibodies against apo-AI and apo-AII within the first 4 h of oxidation. Neo-epitopes were also formed by interaction of the apolipoproteins with degradation products from the lipid peroxidation of polyunsaturated fatty acids, as evidenced by an immunoreaction of oxidized Eu3+-labelled HDL3 with antibodies to 4-hydroxynonenal (4-HNE)– and malondialdehyde (MDA)–protein adducts. Western blot analysis of oxidized HDL3 samples showed, as well as apo-AI and apo-AII bands, larger aggregated apolipoproteins, occurring after 0.5–2.5 h of oxidation. These aggregates were recognized by antibodies to apo-AI and apo-AII as well as by antibodies to 4-HNE- and MDA-protein adducts. Furthermore the original apo-AI monomers and apo-AII dimers decreased during the oxidation. The ability of native and oxidized HDL3 to prevent the binding of Eu3+-labelled 24 h-oxidized LDL to collagen on microtitration plates was estimated. Interestingly, 2 h-oxidized HDL3 competed most with the binding of 24 h-oxidized LDL on collagen type I and type III, followed by native HDL3. However, 24 h-oxidized HDL3 was a weaker competitor. Thus oxidative modification of HDL3 strongly alters the immunological properties of this lipoprotein and its binding affinity for collagen.
Utilization of phosphatidylcholine and production of diradylglycerol as a consequence of sphingomyelin synthesis
Authors: SILLENCE Daniel J. , ALLAN David
Published: August 2015

1. After the degradation of cell-surface sphingomyelin (SM) by exogenous sphingomyelinase (SMase), the resynthesis of SM by baby-hamster kidney (BHK) and human leukaemia-60 (HL-60) cells was examined in relation to utilization of substrate phosphatidylcholine (PtdCho) and generation of the expected product, diradylglycerol (DRG). Using [3H]choline-labelled BHK cells incubated in non-radioactive medium, SMase caused a release of phosphocholine, which was derived approximately equally from SM and PtdCho, consistent with the anticipated resynthesis of SM at the expense of PtdCho. However, with choline-labelled cells incubated in radioactive medium or [14C]acetate-labelled cells treated with SMase, no loss of radioactivity from PtdCho or accumulation of labelled DRG was observed, suggesting that any DRG produced as a consequence of SM synthesis must have been rapidly converted back into PtdCho. In contrast, SMase treatment of HL-60 cells caused more than a doubling of DRG levels at the expense of PtdCho, and this appears to be the first demonstration of a rise in DRG related to the synthesis of SM. The DRG produced consisted of about 80% 1,2-diacylglycerol and 18% 1-O-alkyl-2-acylglycerol species, a similar composition to that of the DRG backbone of total cell PtdCho. 2. The requirement for cell-surface PtdCho in the biosynthesis of SM by BHK cells was also investigated. Treatment of [3H]choline-labelled BHK cells with Bacillus cereus PtdCho-specific phospholipase C (PLC) rapidly degraded about 6% of the total PtdCho, which was assumed to represent the cell-surface pool. This did not appear to be the pool of PtdCho required for SM synthesis, since (a) the released phosphocholine was additional to that derived from PtdCho in cells treated with SMase and (b) treatment with PLC did not affect SM synthesis, either de novo or in response to degradation of cell-surface SM by SMase. These findings suggest either that there is no SM synthase in the plasma membrane or, if it is present, then it does not utilize cell-surface PtdCho as a substrate.
The reaction of halides with pulsed cytochrome bo from Escherichia coli
Authors: MOODY A. John , BUTLER Clive S. , WATMOUGH Nicholas J. , THOMSON Andrew J. , RICH Peter R.
Published: August 2015

Cytochrome bo forms complexes with chloride, bromide and iodide in which haem o remains high-spin and in which the ‘630 nm ’ charge-transfer band is red-shifted by 7–8 nm. The chloride and bromide complexes each have a characteristic set of integer-spin EPR signals arising from spin coupling between haem o and CuB. The rate and extent of chloride binding decreases as the pH increases from 5.5 to 8.5. At pH 5.5 the dissociation constant for chloride is 2 mM and the first-order rate constant for dissociation is 2×10-4 s-1. The order of rate of binding, and of affinity, at pH 5.5 is chloride (1) > bromide (0.3) > iodide (0.1). It is suggested that the halides bind in the binuclear site but, unlike fluoride, they are not direct ligands of the iron of haem o. In addition, both the stability of the halide complexes and the rate of halide binding seem to be increased by the co-binding of a proton.
The negative charge of glutamic acid-820 in the gastric H+,K+-ATPase α-subunit is essential for K+ activation of the enzyme activity
Authors: HERMSEN Harm P. H. , SWARTS Herman G. P. , KOENDERINK Jan B. , De PONT Jan Joep H. H. M.
Published: August 2015

To investigate the role of Glu820, located in transmembrane domain M6 of the α-subunit of gastric H+,K+-ATPase, a number of mutants was prepared and expressed in Sf9 cells using a baculovirus encoding for both H+,K+-ATPase subunits. The wild-type enzyme and the E820D (Glu820 → Asp) mutant showed a similar biphasic activation by K+ on the ATPase activity (maximum at 1 mM). The mutant E820A had a markedly decreased K+ affinity (maximum at 40–100 mM). The other mutants, E820Q, E820N, E820L and E820K, showed no K+-activated ATPase activity at all, whereas all mutants formed a phosphorylated intermediate. After preincubation with K+ before phosphorylation mutant E820D showed a similar K+-sensitivity as the wild-type enzyme. The mutants E820N and E820Q had a 10–20 times lower sensitivity, whereas the other three mutants were hardly sensitive towards K+. Upon preincubation with 3-(cyanomethyl)-2-methyl-8-(phenylmethoxy)imidazo[1,2a] pyridine (SCH 28080), all mutants showed similar sensitivity for this drug as the wild-type enzyme, except mutant E820Q, which could only partly be inhibited, and mutant E820K, which was completely insensitive towards SCH 28080. These experiments suggest that, with a relatively large residue at position 820, the binding of SCH 28080 is obstructed. The various mutants showed a behaviour in K+-stimulated-dephosphorylation experiments similar to that for K+-activated-ATPase-activity measurements. These results indicate that K+ binding, and indirectly the transition to the E2 form, is only fully possible when a negatively charged residue is present at position 820 in the α-subunit.
Iron loading of isolated rat hepatocytes inhibits asialoglycoprotein receptor dynamics and induces formation of rat hepatic leptin-1 (RHL-1) oligomers
Authors: McABEE Douglas D. , LING Yuan Yuan , STICH Charles
Published: August 2015

The major subunit [rat hepatic lectin-1 (RHL-1)] of the asialoglycoprotein (ASGP) receptor mediates endocytosis of the iron-binding protein lactoferrin (Lf) by isolated rat hepatocytes, yet iron loading of cultured adult rat hepatocytes increases the binding and endocytosis of Lf while greatly inhibiting the uptake of desialylated ligand. In the present study, we determined whether the iron-induced Lf-binding site is RHL-1 and examined the nature of the iron-induced block in ASGP receptor endocytic function. Isolated rat hepatocytes increased their non-haem iron content from 70 to 470 p.p.b. following incubation with ferric ammonium citrate (⩽ 100 µg/ml). These conditions blocked internalization of 125I-asialo-orosomucoid (ASOR) by ≈ 90% but increased 125I-Lf endocytosis by 40%. ASOR and anti-RHL-1 sera blocked the binding and endocytosis of 125I-Lf on control cells but not on iron-loaded cells, indicating that the iron-induced Lf-binding site on hepatocytes is not RHL-1. Iron-loading of hepatocytes in the presence or absence of excess ASOR did not significantly alter the number of active ASGP receptors on the cell surface. In contrast, iron-loading decreased the number of active intracellular receptors by 40% and blocked the uptake of 125I-ASOR prebound to the cells by ≈ 80%. Under these conditions, we found an iron-dependent evolution of 88 and 140 kDa RHL-1-containing, β-mercaptoethanol-sensitive multimers that constituted up to 34 and 23%, respectively, of total immunodetectable RHL-1. We propose that iron-induced formation of cystinyl-linked RHL-1-containing multimers inhibits ASGP receptor movement between cell surface and interior and disrupts acylation of intracellular receptors.
Thiol-independent interaction of protein disulphide isomerase with type X collagen during intra-cellular folding and assembly
Authors: McLAUGHLIN Stephen H. , BULLEID Neil J.
Published: August 2015

Protein disulphide isomerase (PDI) has been shown to be a multifunctional protein capable of catalysing disulphide-bond formation and isomerization, and of participating as a non-catalytic subunit of prolyl 4-hydroxylase (P4-H) and microsomal triacylglycerol transfer protein. It has also been proposed to function as a molecular chaperone during the refolding of denatured proteins in vitro. To investigate its potential role as a molecular chaperone within a cellular context, we studied the folding, modification and assembly of type X collagen in semi-permeabilized cells. Using this approach, we demonstrate that depletion of ATP has no effect on the rate or extent of helix formation, indicating that the individual triple helical regions do not interact with the molecular chaperone immunoglobulin heavy-chain binding protein (BiP). However, PDI was shown to interact transiently with type X during helix formation in a role related to its function as the β subunit of P4-H. Once the collagen triple helix was formed, PDI re-associated, indicating a role in preventing the premature assembly of this molecule into higher-order structures. This interaction was not thiol dependent, as a type X polypeptide that did not contain any cysteine residues was able to fold correctly and interact with PDI. Both PDI and the collagen-binding protein hsp47 showed a similar pH-dependent interaction with folded collagen, dissociating when the pH was lowered to pH 6.0. These results suggest a role for PDI in chaperoning type X collagen during its transport through the cell.
Reactions of nitric oxide with mitochondrial cytochrome c: a novel mechanism for the formation of nitroxyl anion and peroxynitrite
Authors: SHARPE Martyn A. , COOPER Chris E.
Published: August 2015

The aerobic reactions of nitric oxide with cytochrome c were analysed. Nitric oxide (NO) reacts with ferrocytochrome c at a rate of 200 M-1 s-1 to form ferricytochrome cand nitroxyl anion (NO-). Ferricytochrome c was detected by optical spectroscopy; NO- was detected by trapping with metmyoglobin (Mb3+) to form the EPR-detectable Mb–nitrosyl complex, and by the formation of dimers in yeast ferrocytochrome cvia cross-linking of the free cysteine residue. The NO- formed subsequently reacted with oxygen to form peroxynitrite, as measured by the oxidation of dihydrorhodamine 123. NO binds to ferricytochrome c to form the ferricytochrome c-NO complex. The on-rate for this reaction is 1.3±0.4×103 M-1·s-1, and the off-rate is 0.087±0.054 s-1. The dissociation constant (Kd)of the complex is 22±7 µM. These reactions of NO with cytochrome c are likely to be relevant to mitochondrial metabolism of NO. Ferricytochrome c can act as a reversible sink for excess NO in the mitochondria. The reduction of NO to NO- by ferrocytochrome cmay play a role in the irreversible inhibition of mitochondrial oxygen consumption by peroxynitrite. It is generally assumed that peroxynitrite would be formed in mitochondria via the reaction of NO with superoxide. The finding that NO- is formed from the reaction of NO and ferrocytochrome c provides a means of producing peroxynitrite in the absence of superoxide, via the reaction of NO- with oxygen.
Biochemical characterization of selenium-containing catalytic antibody as a cytosolic glutathione peroxidase mimic
Authors: DING Lan , LIU Zi , ZHU Zhenqi , LUO Guimin , ZHAO Daqing , NI Jiazuan
Published: August 2015

A selenium-containing catalytic antibody (Se-4A4), prepared by converting reactive serine residues of a monoclonal antibody (4A4) raised against a GSH derivative into selenocysteines, acts as a mimic of cytosolic glutathione peroxidase (cGPX). To clarify the mechanism of action of this catalytic antibody, detailed studies on kinetic behaviour and biological activity were carried out. A rate of acceleration (kcat/Km/kuncat) 107-fold that of the uncatalytic reaction is observed. Under similar conditions, the turnover number (kcat) of Se-4A4 is 42% of that of the natural rabbit liver cGPX. The Se-4A4 reaction involves a Ping Pong mechanism, which is the same as that of the natural cGPX. The selenocysteine residue is located in the binding site of the antibody and is shown to be crucial for this activity. Of the thiol compounds tested, only GSH is able to serve as substrate for Se-4A4. It was demonstrated, using the free-radical-damage system (hypoxanthine/xanthine oxidase) of cardiac mitochondria, that Se-4A4 can protect mitochondria from free-radical damage at least 104-fold more effectively than the natural cGPX.
Resistance to the apoptotic effect of aggregated amyloid-β peptide in several different cell types including neuronal- and hepatoma-derived cell lines
Authors: MAZZIOTTI Mark , PERLMUTTER David H.
Published: August 2015

There is a large body of literature indicating that aggregated amyloid-β peptide (Aβ) is toxic to neurons and suggesting that this neurotoxicity represents the final common pathway for neuronal degeneration in Alzheimer's disease. Previous studies have shown the outgrowth of a subclone of the rat neuronal cell line PC12 that is resistant to the toxic effect of aggregated Aβ peptide if the parent cell line is grown in the presence of aggregated Aβ peptide for a number of passages [Behl, Davis, Lesley and Schubert (1994) Cell 77, 817–827; Boland, Behrens, Choi, Manias and Perlmutter (1996) J. Biol. Chem. 271, 18032–18044]. To begin to characterize the mechanism by which PC12 cells become resistant to the apoptotic effect of Aβ peptide, in the present study we examined whether the resistance was specific to aggregated peptides, specific to an apoptotic form of cell death, and specific in cell type or was a general resistance to cell death that could be elicited in diverse cell types. The results show that the resistance is specific to compounds that have apoptotic effects through the generation of hydroxyl radical or H2O2, including aggregated Aβ-(25–35), Aβ-(1–40), Aβ-(1–42), Aβ-(1–43), amylin, 6-hydroxydopamine and H2O2 itself. The resistant subclones of PC12 were not resistant to other forms of apoptotic cell death or to necrotic cell death. The resistant state was also identified in a human hepatoma cell line, HepG2, when it was grown in the presence of aggregated Aβ-(25–35) for several passages, indicating that the mechanism(s) or molecule(s) responsible for this resistance are not restricted to neuronal cells and may be relevant to the pathobiology of oxidative injury in other cell types.
Genomic organization of the 3′-region of the human MUC5AC mucin gene: additional evidence for a common ancestral gene for the 11p15.5 mucin gene family
Authors: BUISINE Marie-Pierre , DESSEYN Jean-Luc , PORCHET Nicole , DEGAND Pierre , LAINE Anne , AUBERT Jean-Pierre
Published: August 2015

The human mucin gene MUC5AC is mapped clustered with MUC2, MUC5B and MUC6 on chromosome 11p15.5. We report here the isolation and characterization of a genomic cosmid clone, designated ELO9, spanning the 3´-region of MUC5AC and the 5´-region of MUC5B, allowing us to conclude that MUC5AC and MUC5B have the same transcriptional orientation. We determined the genomic organization and the entire sequence of the 3´-region of MUC5AC. The comparative molecular analysis of MUC5AC and MUC5B points to a remarkable similarity in the size and the distribution of exons, and in the type of splice sites, supporting the notion that MUC5AC and MUC5B have evolved from a single common ancestral gene. The derivation of the four genes of the 11p15.5 mucin gene family from a single ancestral gene is discussed.
Structural and functional effect of Trp-62→Gly and Asp-101→Gly substitutions on substrate-binding modes of mutant hen egg-white lysozymes
Authors: MAENAKA Katsumi , MATSUSHIMA Masaaki , KAWAI Gota , KIDERA Akinori , WATANABE Kimitsuna , KUROKI Ryota , KUMAGAI Izumi
Published: August 2015

In order to clarify the structural role of subsite B of hen egg-white lysozyme in hydrolytic activity towards a carbohydrate substrate, we analysed the structures of Trp-62 → Gly and Asp-101 → Gly mutant hen lysozymes, which have no side chain at positions 62 or 101, complexed with a substrate analogue, (N-acetyl-d-glucosamine)3 [(GlcNAc)3], using X-ray crystallography. The overall protein structures in the mutant lysozyme complexes were almost identical to those in the wild type. In the crystals of all the mutant complexes, the (GlcNAc)3 molecule, which is an inhibitor of wild-type lysozyme, had no inhibitory effect, but was hydrolysed as a substrate. One of the products, (GlcNAc)2, the reducing end of which is an α-anomer, was bound in an unproductive binding mode, protruding from the active-site cleft, and was able to act as an inhibitor. Hydrolysis of the synthetic substrate by the mutants occurred in a β-anomer-retaining manner, and so the α-anomer product was converted from the β-anomer product. Thus the interactions of Asp-101 and Trp-62 in subsite B are not essential for the catalytic mechanism, but co-operatively enhance the affinity of the substrate in the productive binding mode, other than the inhibitor in the unproductive mode.
Assembly of flammutoxin, a cytolytic protein from the edible mushroom Flammulina velutipes, into a pore-forming ring-shaped oligomer on the target cell
Authors: TOMITA Toshio , ISHIKAWA Dai , NOGUCHI Takayasu , KATAYAMA Eisaku , HASHIMOTO Yohichi
Published: August 2015

Flammutoxin has been previously isolated as a cardiotoxic and cytolytic polypeptide of 22 or 32 kDa from the fruiting bodies of the edible mushroom Flammulina velutipes. In the present study, we purified flammutoxin as a single haemolytic protein of 31 kDa and studied the mode of its cytolytic action. (1) Flammutoxin caused efflux of potassium ions from human erythrocytes and swelling of the cells before haemolysis. (2) Flammutoxin did not lyse human erythrocytes in the presence of non-electrolytes with hydrodynamic diameters of > 5.0 nm, although it caused leakage of potassium ions and swelling of the cells under the same conditions. (3) Experiments including solubilization of cell-bound toxin with 2% (w/v) SDS at 20 °C and subsequent Western immunoblots showed that flammutoxin formed a band corresponding to 180 kDa under the conditions where it lysed erythrocytes. (4) Electron microscopy of flammutoxin-treated human erythrocytes revealed the presence of a ring-shaped structure with outer and inner diameters of 10 and 5 nm, respectively, on the cells. (5) A ring-shaped toxin oligomer of the same dimensions was solubilized from the toxin-treated human erythrocytes with 2% (w/v) SDS at 20 °C and isolated by a sucrose-gradient ultracentrifugation. These data indicated that flammutoxin assembles into a ring-shaped oligomer possessing a hydrophilic pore of 4–5 nm on target cells.
Chaperonins
Authors: RANSON Neil A. , WHITE Helen E. , SAIBIL Helen R.
Published: August 2015

The molecular chaperones are a diverse set of protein families required for the correct folding, transport and degradation of other proteins in vivo. There has been great progress in understanding the structure and mechanism of action of the chaperonin family, exemplified by Escherichia coli GroEL. The chaperonins are large, double-ring oligomeric proteins that act as containers for the folding of other protein subunits. Together with its co-protein GroES, GroEL binds non-native polypeptides and facilitates their refolding in an ATP-dependent manner. The action of the ATPase cycle causes the substrate-binding surface of GroEL to alternate in character between hydrophobic (binding/unfolding) and hydrophilic (release/folding). ATP binding initiates a series of dramatic conformational changes that bury the substrate-binding sites, lowering the affinity for non-native polypeptide. In the presence of ATP, GroES binds to GroEL, forming a large chamber that encapsulates substrate proteins for folding. For proteins whose folding is absolutely dependent on the full GroE system, ATP binding (but not hydrolysis) in the encapsulating ring is needed to initiate protein folding. Similarly, ATP binding, but not hydrolysis, in the opposite GroEL ring is needed to release GroES, thus opening the chamber. If the released substrate protein is still not correctly folded, it will go through another round of interaction with GroEL.
Intestinal absorption of bile acids: paradoxical behaviour of the 14 kDa ileal lipid-binding protein in differential photoaffinity labelling
Authors: KRAMER Werner , CORSIERO Daniel , FRIEDRICH Michael , GIRBIG Frank , STENGELIN Siegfried , WEYLAND Claudia
Published: August 2015

Photoaffinity labelling of brush border membrane vesicles from rabbit ileum with radiolabelled 3,3-azo and 7,7-azo derivatives of taurocholate identified integral membrane proteins of molecular masses 93 and 46 kDa, as well as a 14 kDa peripheral membrane protein, as components of the ileal Na+/bile acid transport system [Kramer, Girbig, Gutjahr, Kowalewski, Jouvenal, Müller, Tripier and Wess (1993) J. Biol. Chem. 268, 18035–18046]. Differential photoaffinity labelling in the presence of non-radiolabelled bile acid derivatives led, as expected, to a concentration-dependent decrease in the extent of labelling of the 93 and 46 kDa transmembrane proteins, which are the monomeric and dimeric forms of the ileal bile acid transporter protein. The extent of labelling of the 14 kDa ileal lipid-binding protein (ILBP), however, increased on the addition of unlabelled bile acids, the increase being dependent on the structure of the bile acid added. The possibility of artifacts was excluded by photoaffinity labelling experiments in the frozen state as well as by model calculations. The experimental results suggest that the binding of bile acids to ILBP can increase the affinity of ILBP for bile acids. These results would be in accordance with a substrate-load modification of transport activity and a positive-feedback regulation mechanism for active uptake of bile acid in the ileum.
Multidrug resistance transporter P-glycoprotein has distinct but interacting binding sites for cytotoxic drugs and reversing agents
Authors: PASCAUD Claire , GARRIGOS Manuel , ORLOWSKI Stéphane
Published: August 2015

P-Glycoprotein, the plasma membrane protein responsible for the multidrug resistance of some tumour cells, is an active transporter of a number of structurally unrelated hydrophobic drugs. We have characterized the modulation of its ATPase activity by a multidrug-resistance-related cytotoxic drug, vinblastine, and different multidrug-resistance-reversing agents, verapamil and the dihydropyridines nicardipine, nimodipine, nitrendipine, nifedipine and azidopine. P-Glycoprotein ATPase activity was measured by using native membrane vesicles containing large amounts of P-glycoprotein, prepared from the highly multidrug-resistant lung fibroblasts DC-3F/ADX. P-Glycoprotein ATPase is activated by verapamil and by nicardipine but not by vinblastine. Among the five dihydropyridines tested, the higher the hydrophobicity, the higher was the activation factor with respect to the basal activity and the lower was the half-maximal activating concentration. The vinblastine-specific binding on P-glycoprotein is reported by the inhibitions of the verapamil- and the nicardipine-stimulated ATPase. These inhibitions are purely competitive, which means that the bindings of vinblastine and verapamil, or vinblastine and nicardipine, on P-glycoprotein are mutually exclusive. In contrast, verapamil and nicardipine display mutually non-competitive interactions. This demonstrates the existence of two distinct specific sites for these two P-glycoprotein modulators on which they can bind simultaneously and separately to the vinblastine site. The nicardipine-stimulated ATPase activity in the presence of the other dihydropyridines shows mixed-type inhibitions. These dihydropyridines have thus different binding sites that interact mutually to decrease their respective, separately determined affinities. This could be due to steric constraints between sites close to each other. This is supported by the observation that vinblastine binding is not mutually exclusive with nifedipine or nitrendipine binding, whereas it is mutually exclusive with nicardipine. Moreover, verapamil binding also interacts with the five dihydropyridines by mixed inhibitions, with different destabilization factors. On the whole our enzymic data show that P-glycoprotein has distinct but interacting binding sites for various modulators of its ATPase function.
Identification of a domain in apolipoprotein B-100 that inhibits the procoagulant activity of tissue factor
Authors: ETTELAIE Camille , JAMES Nicola J. , ADAM Jacqueline M. , NICOLA Kyriacos P. , WILBOURN Barry R. , BRUCKDORFER K. Richard
Published: August 2015

The ability of low-density lipoprotein (LDL) to inhibit the procoagulant activity of tissue factor is mediated by a direct protein–protein interaction involving apolipoprotein (apo) B-100. A lysine-rich sequence within apo B-100 (residues 3121–3217), which we have termed lysine-rich apo B-100-derived (KRAD)-98 peptide, may be responsible for its activity. Within this region, residues 3147–3160 (KRAD-14) contain an exceptionally high proportion of positive amino acids. Both recombinant KRAD-98 and KRAD-14 peptides inhibited the procoagulant activity of tissue factor by preventing the activation of factor VII. KRAD-14 also inhibited the prothrombinase components, factors Xa and V. In comparison with the parent protein (apo B-100), KRAD-14 peptide displayed a 20-fold enhancement in the rate of inhibition, whereas KRAD-98 peptide exhibited a rate closer to that of apo B-100. Mutational analysis of KRAD-14 peptide revealed three adjacent amino acids, alteration of which greatly reduced the inhibitory potential of this peptide. A peptide derived from tissue factor (residues 58–66) was found to act co-operatively with tissue factor itself, but also augmented the inhibition of tissue-factor activity by apo B-100. In conclusion, LDL may be a physiological regulator of haemostatic mechanisms through the interactions of lysine-rich domains of apo B-100 with tissue factor.