bluuebunny/crossref_metadata_2025 huggingface dataset:
Studies on the structural and functional aspects of Rhodotorula gracilis
d-amino acid oxidase by limited trypsinolysis
Authors: Pollegioni L , Ceciliani F , Curti B , Ronchi S , Pilone M S
Published: August 2015

The structure-function relationships of purified Rhodotorula gracilis D-amino acid oxidase (in its holo-, apo- and holo-enzyme-benzoate complex forms) was analysed by digestion with trypsin. In all cases trypsin cleaves this 80 kDa dimeric enzyme at the C-terminal region, since the peptide bonds sensitive to proteinase attack are clustered in this region. Digestion of native enzyme with trypsin produced a nicked and truncated form of 38.3 kDa containing two polypeptides of 34 and 5 kDa starting from Met1 and Ala319 respectively, and with detachment of the Thr306-Arg318 and Glu365-Leu368 peptides. Our results show that this *#x2018;core’, folded into a compact structure, is catalytically competent. The acquisition of this nicked form was marked by a shift from a dimeric to a monomeric active enzyme, a result never previously obtained. The deleted sequences, Thr306-Arg318 and Glu365-Leu368, are essential for the monomer-monomer interaction, and, in particular, the region encompassing Thr306-Arg318 should play an essential role in the dimerization process. interestingly, the Ser308-Lys321 sequence present in the lost peptide corresponds to a sequence not present in other known D-amino acid oxidases [Faotto, Pollegioni, Ceciliani, Ronchi and Pilone (1995) Biotechnol. Lett. 17, 193-198]. A role of the cleaved-off region for the thermostabilization of the enzyme is also discussed.
Covalent modification of engineered cysteines in the nicotinic acetylcholine receptor agonist-binding domain inhibits receptor activation
Authors: McLaughlin J T , Hawrot E , Yellen G
Published: August 2015

We constructed and characterized a series of nicotinic receptor mutants with a cysteine substituted for one of the amino acid residues in the alpha-subunit between positions 183 and 198. This region of the receptor is known to participate in agonist binding and channel activation. The goal of this ‘cysteine scanning mutagenesis’ is to introduce the reactivity of a free thiol group into functionally important protein domains; modification of the introduced cysteines can then be used to probe the structure and function of the targeted region. Mutants were examined by coexpression with the beta-, gamma- and delta-subunits in Xenopus oocytes using two-microelectrode voltage clamp recording. Twelve of fourteen mutants expressed receptors with properties comparable with the wild-type, including sensitivity to reduction by dithiothreitol (DTT). This indicates that introduction of an additional cysteine within this region of the receptor did not interfere with formation of the native disulphide between alpha Cys-192 and alpha Cys-193. Only one mutation, alpha Y198C, caused dramatic changes in the EC50 for acetylcholine (ACh) and in the sensitivity to DTT. We then examined the effects of the thiol modification and found two mutants, alpha H186C and alpha V188C, that showed significant decreases in responsiveness to ACh after exposure to methylmethanethiosulphonate (MMTS). Dose-response measurements show that exposure of alpha H186C mutants to MMTS causes a shift in apparent agonist affinity without changing the peak response, and this is not reversible by DTT. In contrast, the MMTS-treated alpha V188C mutants show changes in both apparent affinity and peak response which are readily reversed by DTT. Together, our data show that these two nearby residues occupy markedly different environments relative to the contact points for ACh. They also demonstrate that cysteine-substitution mutagenesis can be successfully applied to protein domains that include functionally important disulphides.
Identification of a functional promoter element in the 5′-flanking region of the rat cMG1/TIS11b gene
Authors: Corps A N , Pascall J C , Hadfield K M , Brown K D
Published: August 2015

The cMG1 gene was originally identified as a mitogen-stimulated primary response gene. However, in contrast to genes such as c-fos and TIS11, cMG1 is also expressed at significant levels before and after the transient elevation induced by agonists. We have sequenced a 1.3 kb rat genomic cMG1 clone, which includes 931 bp upstream of the transcription start site identified by primer-extension analysis. A 1033 bp fragment, including this 5′-flanking sequence, directed the expression of the reporter gene chloramphenicol acetyltransferase (CAT) in transfected NIH-3T3 cells. Progressive 5‘-to-3’ deletion indicated that an element located between -138 and -114 was responsible for most of this basal CAT expression. DNA mobility-shift assays showed that the sequence between -143 and -105 contained binding sites for cellular proteins, the principal complexes involving nucleotides between -119 and -105. We conclude that these complexes may represent the transcription factor-DNA element interactions that determine basal cMG1 expression.
The mRNAs for the three chains of human collagen type XI are widely distributed but not necessarily co-expressed: implications for homotrimeric, heterotrimeric and heterotypic collagen molecules
Authors: Lui V C H , Kong R Y C , Nicholls J , Cheung A N Y , Cheah K S E
Published: August 2015

In cartilage collagen type XI exists as heterotrimeric molecules composed of alpha 1(XI), alpha 2(XI) and alpha 3(XI) subunits. Messenger RNAs for some of the alpha chains of collagen type XI have also been found in non-chondrogenic tissues but the chain composition of the molecule in these sites is not known. Some non-chondrogenic tissues also contain heterotrimers containing collagen alpha 2(V) and alpha 1(XI) chains. We have explored the possibility that collagen type XI could exist in differing trimeric forms in non-chondrogenic tissues and aimed to predict the subunit composition of this collagen in those tissues. The distribution and relative levels of expression of collagen alpha 1(XI), alpha 2(XI) and alpha 3(XI)/alpha 1(II) mRNAs in different human fetal tissues were studied. Expression of mRNAs for all three genes of collagen type XI is not restricted to cartilage but is widespread. However, in some non-chondrogenic tissues, the mRNAs for all three alpha chains of collagen type XI were not co-expressed, but collagen alpha 1(XI) and alpha 2(XI) mRNAs were found either singly or without collagen alpha 3(XI) transcripts. Collagen type XI may therefore exist as homotrimers and/or heterotrimers composed of two collagen alpha(XI) chains in some tissues. The distribution of mRNAs for collagen alpha 2(V) and alpha 1(I) were also studied. Co-expression of collagen type XI, alpha 2(V) and alpha 1(I) mRNAs was found for many tissues. These findings have implications for the possibility of additional chain associations for collagen types XI and V in cross-type heterotrimers within heterotypic fibrils.
Biological fate of amino acid, peptide and protein hydroperoxides
Authors: Fu S , Gebicki S , Jessup W , Gebicki J M , Dean R T
Published: August 2015

In the course of searching for a suitable marker for studying protein oxidation, we have successfully elucidated the structures of three valine hydroperoxides, i.e. beta-hydroperoxyvaline, (2S,3S)-gamma-hydroperoxyvaline and (2S,3R)-gamma-hydroperoxyvaline, which are novel products of protein oxidation. The corresponding valine hydroxides were obtained by sodium borohydride reduction [Fu, Hick, Sheil and Dean (1995) Free Rad. Biol. Med. 19, 281-292]. We hypothesized that valine hydroxides might be the major biological degradation products of valine hydroperoxides and, as such, could be useful markers for the study of protein oxidation in vivo. The aim of this study was to investigate the fate of valine hydroperoxide in selected biological systems by the use of chemiluminescence detection of hydroperoxides and HPLC analysis of O-phthaldialdehyde derivatives of amino acid residues. The degradation of hydroperoxides present on gamma-radiolysed solutions of valine, Pro-Val-Gly, or BSA occurred in the presence of: (1) transition metals (Fe2+, Fe3+, or Cu2+), (2) the detoxifying enzyme GSH peroxidase, (3) human plasma, and (4) J774 mouse monocyte macrophage cells. The major degradation product of valine hydroperoxide recovered in each case was found to be a valine hydroxide. These results suggest that valine hydroxide (derived from the hydroperoxide) may well be a useful in vivo marker for studying protein damage under oxidative stress.
Prolonged elevation of intracellular cyclic AMP levels in U937 cells increases the number of receptors for and the responses to formylmethionyl-leucylphenylalanine, independently of the differentiation process
Authors: Fischer T , Zumbihl R , Armand J , Casellas P , Rouot B
Published: August 2015

The effects of elevated levels of cyclic AMP induced by cholera toxin (CTx) were investigated on the differentiated promyelomonocytic cell line U937. After CTx treatment, the initial inhibition of the oxidative burst induced by N-formylmethionyl-leucyl-phenylalanine (FMLP) was followed by a progressive increase over 20 h, resulting in 4-6-fold potentiation of the initial burst. Various cyclic-AMP-elevating agents produced similar potentiation of the FMLP- or C5a-induced oxidative burst, but the phorbol 12-myristate 13-acetate-induced oxidative burst was not affected by CTx pretreatment of cells. Furthermore, the increase in arachidonate release and intracellular Ca2+ triggered by FMLP were amplified after CTx treatment. ADP-ribosylation of Gi alpha subunits catalysed by pertussis toxin was slightly increased after CTx treatment, despite similar immunoreactivity of the alpha subunit of Gi2. FMLP binding sites present in CTx-treated membranes were 3-6 times more abundant than in control membranes. Expression of mRNAs encoding the FMLP receptor and one of its related receptors were enhanced after CTx treatment of both undifferentiated and undifferentiated U937 cells. In parallel, after undifferentiated cells were treated with CTx, they were able to increase intracellular Ca2+, but not the oxidative burst, in response to FMLP. These data demonstrate that CTx, by increasing cyclic AMP, enhances the expression of chemotactic receptors independently of U937 cell differentiation.
Inhibition of cytochrome c oxidase in turnover by nitric oxide: mechanism and implications for control of respiration
Authors: Torres J , Darley-Usmar V , Wilson M T
Published: August 2015

Binding of nitric oxide (NO) to isolated cytochrome c oxidase in turnover was investigated by static and kinetic spectroscopic methods. These studies indicate that cytochrome c oxidase rapidly binds NO when the enzyme enters turnover. Our results show that NO binds to ferrocytochrome a3, competing with oxygen for this binding site. However, the main features of the binding process, in particular the rapid onset of inhibition, cannot be fully explained on this basis. We suggest, therefore, that there is a second binding site for NO, which has lower affinity but nevertheless plays an important role in the inhibitory process. A likely possibility is that CuB+ constitutes this second binding site. The fast onset of inhibition observed in the presence of NO, along with the dependence on the oxygen concentration, suggests that under physiological conditions, where the oxygen concentration is low, nanomolar concentrations of NO can effectively act as a regulator of the mitochondrial respiratory chain.
The interaction of methanol dehydrogenase and its cytochrome electron acceptor
Authors: Dales S L , Anthony C
Published: August 2015

A fluorescence method is described for direct measurement of the interaction between methanol dehydrogenase (MDH) and its electron acceptor cytochrome cL. This has permitted a distinction to be made between factors affecting electron transfer and those affecting the initial binding or docking process. It was confirmed that the initial interaction is electrostatic, but previous conclusions with respect to the mechanism of EDTA inhibition have been modified. It is proposed that the initial ‘docking’ of MDH and cytochrome cL is by way of ionic interactions between lysyl residues on its surface and carboxylate groups on the surface of cytochrome cL. This interaction is not inhibited by EDTA, which we suggest acts by binding to nearby lysyl residues, thus preventing movement of the ‘docked’ cytochrome to its optimal position for electron transfer, which probably involves interaction with the hydrophobic funnel in the surface of MDH.
An investigation of the membrane topology of the ionotropic glutamate receptor subunit GluR1 in a cell-free system
Authors: Seal A J , Collingridge G L , Henley J M
Published: August 2015

We have utilized cell-free translation in rabbit-reticulocyte lysate supplemented with canine pancreatic microsomal membranes to study the processing and membrane topology of the rat ionotropic glutamate receptor subunit GluR1. In vitro-synthesized RNA encoding GluR1 was translated to yield a primary translation product with an apparent molecular mass of 99 kDa. In the presence of microsomal membranes this protein was processed to give a band of 107 kDa. Treatment with peptide-N-glycosidase F showed that this increase in molecular mass was due to N-linked glycosylation. Incubation of the processed receptor with proteinase K revealed the presence of a 68 kDa protease-resistant band which decreased to 56 kDa following deglycosylation. A deletion mutant (GluR1M1) lacking the predicted transmembrane domains was fully translocated across the microsomal membrane and protected from the action of the protease. The mutant and wild-type receptor could be immunoprecipitated by anti-peptide antibodies directed against the C-terminus. Following translocation of the wild-type and mutant receptor across the microsomal membrane and treatment with proteinase K the antibody binding to GluR1 was abolished, but was retained for GluR1M1. These data allow identification of the orientation of the N- and C-termini of GluR1 within the microsome; results which are consistent with an extracellular N-terminal and intracellular C-terminal localization following incorporation into the plasma membrane.
Functional expression and characterization of the Trypanosoma brucei procyclic glucose transporter, THT2
Authors: Barrett M P , Tetaud E , Seyfang A , Bringaud F , Baltz T
Published: August 2015

The gene encoding THT2, one of two hexose-transporter isoforms present in Trypanosoma brucei, has been expressed in both Xenopus laevis oocytes and a stably transfected line of Chinese hamster ovary (CHO) cells. The heterologously expressed gene encodes a protein with pharmacological and kinetic parameters similar to those of the hexose transporter measured in procyclic-culture-form trypanosomes. The substrate recognition of the THT2 transporter differed from that of the THT1 isoform, which is expressed only in bloodstream forms, in that: (i) it has a relatively high affinity for substrate with a Km of 59 microM for 2-deoxy-D-glucose (2-DOG) and a similar high affinity for D-glucose (compared with Km of 0.5 mM for 2-DOG in bloodstream forms); (ii) the affinity for 6-deoxy-D-glucose (6-DOG) is two orders of magnitude lower than that for D-glucose, whereas the bloodstream-form transporter recognizes D-glucose and its 6-DOG analogue with similar affinity; (iii) the bloodstream-form transporter, but not THT2, recognizes 3-fluoro-3-deoxy-D-glucose. D-Fructose-transport capacity and insensitivity to D-galactose was also found in THT2-expressing CHO cells and procyclic trypanosomes. We conclude from these cumulative results that the THT2 gene encodes the transporter responsible for hexose transport in procyclic trypanosomes. The transport of 2-DOG in procyclic organisms was inhibited by both the protonophore, carbonyl cyanide 4-trifluoromethoxy phenylhydrazone (FCCP), and KCN, suggesting a requirement for a protonmotive force. However, sensitivity to these reagents depended on the external substrate concentration, with uptake being unaffected at substrate concentrations higher than 2 mM. THT2 expressed in CHO cells behaved as a facilitated transporter, and was unaffected by FCCP or KCN over the whole substrate concentration range tested.
Cross-talk between glucagon- and adenosine-mediated signalling systems in rat hepatocytes: effects on cyclic AMP-phosphodiesterase activity
Authors: Robles-Flores M , Allende G , Piña E , García-Sáinz J A
Published: August 2015

The effect of adenosine analogues on glucagon-stimulated cyclic AMP accumulation in rat hepatocytes was explored. N6-Cyclopentyladenosine (CPA), 5′-N-ethylcarboxamidoadenosine and N6-(R-phenylisopropyl)adenosine inhibited in a dose-dependent manner the cyclic AMP accumulation induced by glucagon. This effect seems to be mediated through A1 adenosine receptors. Pertussis toxin completely abolished the effect of CPA on glucagon-stimulated cyclic AMP accumulation in whole cells which suggested that a pertussis-toxin-sensitive G-protein was involved. On the other hand, this action of adenosine analogues on glucagon-induced cyclic AMP accumulation was reverted by the selective low-Km cyclic AMP-phosphodiesterase inhibitor Ro 20-1724. Analysis of cyclic AMP-phosphodiesterase activity in purified hepatocyte plasma membranes showed that glucagon in the presence of GTP inhibited basal PDE activity by 45% and that CPA reverted this inhibition in dose-dependent manner. In membranes derived from pertussis-toxin-treated rats, we observed no inhibition of cyclic AMP-phosphodiesterase activity by glucagon in the absence or presence of CPA. Our results indicate that in hepatocyte plasma membranes, stimulation of adenylate cyclase activity and inhibition of a low-Km cyclic AMP phosphodiesterase activity are co-ordinately regulated by glucagon, and that A1 adenosine receptors can inhibit glucagon-stimulated cyclic AMP accumulation by blocking glucagon's effect on phosphodiesterase activity.
The human platelet ADP receptor activates Gi2 proteins
Authors: Ohlmann P , Laugwitz K L , Nürnberg B , Spicher K , Schultz G , Cazenave J P , Gachet C
Published: August 2015

We have previously shown that platelet ADP receptors are coupled to G-proteins by measuring the binding of [35S]guanosine-5′-[gamma-thio]triphosphate ([35S]GTP gamma S) to human platelet membranes stimulated with ADP. In order to identify the activated G-proteins, we used an approach which combines photolabelling of receptor-activated G-proteins with 4-azidoanilido-[alpha-32P]GTP and immunoprecipitation of the G-protein alpha-subunits with subtype-specific antibodies. Stimulation of human platelet membranes with ADP resulted in an increase in 4-azidoanilido-[alpha-32P]GTP incorporation into the immunoprecipitates of G alpha i but not of G alpha q proteins, whereas stimulation with the thromboxane analogue U46619 resulted in an increase in 4-azidoanilido-[alpha-32P]GTP incorporation into the immunoprecipitates of G alpha q but not of G alpha i proteins, and thrombin activated both G-proteins. This effect of ADP was concentration dependent and inhibited by the class P2 purinoceptor (P2T) antagonist ATP. Using specific antisera against subtypes of Gi proteins, we found that ADP stimulated labelling of the G alpha 12 immunoprecipitate, but not of the G alpha 13 precipitate. G alpha i1 was not detectable by immunoblotting of platelet membrane proteins. These data suggest that ADP inhibits cAMP formation by activation of G alpha 12 proteins and add evidence in support of the hypothesis that human platelet ADP receptors do not activate PLC through Gq activation.
Polymorphism of the yeast pyruvate carboxylase 2 gene and protein: effects on protein biotinylation
Authors: Val D L , Chapman-Smith A , Walker M E , Cronan J E , Wallace J C
Published: August 2015

In Saccharomyces cerevisiae there are two isoenzymes of pyruvate carboxylase (Pyc) encoded by separate genes designated PYC1 and PYC2. We report the isolation and sequencing of a PYC2 gene, and the localization of both genes on the physical map of S. cerevisiae. Comparison with the previously reported sequence [Stucka, Dequin, Salmon and Gancedo (1991) Mol. Gen. Genet. 229, 307-315] revealed significant differences within the open reading frame. The most notable difference was near the 3′ end, where we found a single base deletion reducing the open reading frame by 15 bases. We have confirmed the C-terminus of Pyc2 encoded by the gene isolated here by expressing and purifying an 86-amino-acid biotin-domain peptide. In addition, we investigated the effects of the two changes in the Pyc2 biotin domain (K1155R substitution and Q1178P/five-amino-acid extension) on the extent of biotinylation in vivo by Escherichia coli biotin ligase, and compared the biotinylation of peptides containing these changes with that of two different-length Pyc1 biotin-domain peptides. The K1155R substitution had very little effect on biotinylation, but the five-amino-acid C-terminal extension to Pyc2 and the N-terminal extension to Pycl both improved biotinylation in vivo.
Spermine uptake is necessary to induce haemoglobin synthesis in murine erythroleukaemia cells
Authors: Clément S , Delcros J G , Feuerstein B G
Published: August 2015

To determine whether intracellular uptake of spermine is necessary to induce haemoglobin synthesis in murine erythroleukaemia (MEL) DS 19 cells, we used single-step selection for resistance to N1,N12-bis(ethyl)spermine (BESM), a cytotoxic spermine analogue, to isolate clones deficient in polyamine transport. The cells were approximately 500-fold more resistant to BESM than parental cells and were unable to accumulate BESM, putrescine, spermidine or spermine. Addition of spermine to the polyamine-transport-deficient cells failed to induce haemoglobin synthesis. Hexamethylene-1,6-bisacetamide, a well-known differentiating agent, induced haemoglobin synthesis in both parental and resistant cells. Polyamine-transport-deficient cells transfected with DNA purified from the parental cell line were further selected for their ability to grow in the presence of alpha-difluoromethylornithine and putrescine. The transfectants had an active transport system for polyamines, and spermine added to their culture medium accumulated inside the cells and induced haemoglobin production. These findings indicate that intracellular spermine uptake is required to induce haemoglobin production in MEL cells.
A cytosolic sperm factor triggers calcium oscillations in rat hepatocytes
Authors: BERRIE Christopher P. , CUTHBERTSON K. S. Roy , PARRINGTON J. , LAI F. Anthony , SWANN Karl
Published: August 2015

Previously it has been shown that injecting a cytosolic sperm protein factor into mammalian eggs induces sustained repetitive transients of cytosolic free Ca2+ ([Ca2+]i), or [Ca2+]i oscillations [Swann (1990) Development 110, 1295-1302]. These sperm-factor (SF)-induced [Ca2+]i oscillations are similar to those seen at fertilization. Here we demonstrate that injecting the same cytosolic extracts of mammalian sperm into single rat hepatocytes induces a series of [Ca2+]i oscillations, as measured by aequorin luminescence. SF injection into hepatocytes induced [Ca2+]i oscillations that were of longer duration, lower frequency and greater amplitude than those seen with the Ins(1,4,5)P3-generating agonist phenylephrine. The SF-induced [Ca2+]i responses appeared to be due to internal release of Ca2+, since transients could occur in Ca2+-free media. Addition of the phorbol ester phorbol 12,13-dibutyrate (PDBu) at low concentrations did not inhibit the SF-induced [Ca2+]i oscillations; high concentrations of PDBu led to a sustained increase in [Ca2+]i concentrations. These data demonstrate that sperm contain a protein factor capable of inducing a characteristic series of [Ca2+]i oscillations in a somatic cell, the hepatocyte. Along with previous observations in dorsal root ganglion neurons, the data suggest a widespread efficacy of the factor in triggering Ca2+ oscillations.
Purification by cobalamin-Sepharose affinity chromatography and intrinsic factor-binding activity of an extramembrane proteolytic product from pig ileal mucosa
Authors: YERIMA Alibada , SAFI Amal , GASTIN Isabelle , MICHALSKI Jean-Claude , SAUNIER Monique , GUEANT Jean-Louis
Published: August 2015

We have purified a cobalamin-binding protein obtained by papain digestion of pig intestine by cobalamin-AH-Sepharose affinity chromatography, with a purification factor of 17300, a yield of 63% and a cobalamin-binding activity of 11260 pmol/mg of protein. The protein contained 3.8% carbohydrate and was O- and N-glycosylated. Its molecular mass was 69 kDa on SDS/PAGE and its isoelectric point was 5.1. It had a binding activity for both [57Co]cobalamin and [57Co]cobalamin-intrinsic factor in native PAGE autoradiography and it inhibited the binding of intrinsic factor to the intact intestinal receptor with an IC50 of 49.31 nmol/l in a radioisotope assay. In conclusion, the purified protein shared a binding activity for both cobalamin and intrinsic factor-cobalamin complexes and could correspond to the extracellular domain of the ileal intrinsic factor receptor.
Identification of a multienzyme complex of the tricarboxylic acid cycle enzymes containing citrate synthase isoenzymes from Pseudomonas aeruginosa
Authors: MITCHELL Colin G.
Published: August 2015

A multienzyme complex of tricarboxylic acid cycle enzymes, catalysing the consecutive reactions from fumarate to 2-oxoglutarate, has been identified in extracts of Pseudomonas aeruginosa prepared by gentle osmotic lysis of the cells. The individual enzyme activities of fumarase, malate dehydrogenase, citrate synthase, aconitase and isocitrate dehydrogenase can be used to reconstitute the complex. The citrate synthase isoenzymes, CSI and CSII, from this organism can be used either together or as the individual activities to reconstitute the complex. No complex can be reformed in the absence of CSI or CSII. Which CS isoenzyme predominates in the complex depends on the phase of growth at which the cells were harvested and the extract prepared. More CSI was found in the complex during exponential growth, whereas CSII predominated during the stationary phase. The results support the idea of a ‘metabolon’ in this organism, with the composition of the CS component varying during the growth cycle.
Soluble N-ethylmaleimide-sensitive-factor attachment protein and N-ethylmaleimide-insensitive factors are required for Ca2+-stimulated exocytosis of insulin
Authors: KIRALY-BORRI Catherine E. , MORGAN Alan , BURGOYNE Robert D. , WELLER Ulrich , WOLLHEIM Claes B. , LANG Jochen
Published: August 2015

Ca2+ stimulates exocytosis in permeabilized insulin-secreting cells. To investigate the putative cytosolic components involved in the Ca2+ response, HIT-T15 cells (a pancreatic B-cell line) were permeabilized with streptolysin-O, a procedure that allows rapid exchange of soluble components including macromolecules. We found that in this cell preparation the secretory response to Ca2+ but not to guanosine 5´-[γ-thio]triphosphate was lost as a function of time and could be restored by rat brain cytosol in a concentration-dependent manner. Reconstitutive activity of rat brain cytosol was found in a high-molecular-mass heat-labile partially N-ethylmaleimide (NEM)-sensitive fraction. The NEM-sensitive factor (NSF) and the soluble NSF attachment protein (α-SNAP) were found to be expressed in HIT-T15 cells and largely lost (about 30% remaining) from porated cells. Recombinant α-SNAP partially reconstituted the Ca2+ response when added to the permeabilized cells. Moreover, α-SNAP restored the effect of NEM-treated cytosol to the level observed for untreated cytosol. In contrast, NSF was ineffective when preincubated alone or with NEM-treated cytosol. Our results indicate that both α-SNAP and NEM-insensitive cytosolic factors are involved in Ca2+-mediated exocytosis from endocrine HIT-T15 cells.
The endopeptidase activity and the activation by Cl− of angiotensin-converting enzyme is evolutionarily conserved: purification and properties of an an angiotensin-converting enzyme from the housefly, Musca domestica
Authors: LAMANGO Nazarius S. , SAJID Mohammed , ISAAC R. Elwyn
Published: August 2015

A soluble 67 kDa angiotensin-converting enzyme (ACE) has been purified by lisinopril-Sepharose affinity column chromatography from adult houseflies, Musca domestica. The dipeptidyl carboxypeptidase activity towards benzoyl-Gly-His-Leu was inhibited by captopril (IC50 50 nM) and fosinoprilat (IC50 251 nM), two inhibitors of mammalian ACE, and was activated by Cl- (optimal Cl- concentration 600 mM). Musca ACE removed C-terminal dipeptides from angiotensin I, bradykinin, [Leu5]enkephalin and [Met5]enkephalin and also functioned as an endopeptidase by hydrolysing dipeptideamides from [Leu5]enkephalinamide and [Met5]enkephalinamide, and a dipeptideamide and a tripeptideamide from substance P. Musca ACE was also able to cleave a tripeptide from both the N-terminus and C-terminus of luteinizing hormone-releasing hormone, with C-terminal hydrolysis predominating. Maximal N-terminal tripeptidase activity occurred at 150 mM NaCl, whereas the C-terminal tripeptidase activity continued to rise with increasing concentration of Cl- (0–0.5 M). Musca ACE displays properties of both the N- and C-domains of human ACE, indicating a high degree of conservation during evolution of the substrate specificity of ACE and its response to Cl-.
Biochemical characterization of mouse brain necdin
Authors: MARUYAMA Etsuko
Published: August 2015

Necdin is a protein encoded by neural differentiation-specific mRNA derived from embryonal carcinoma cells (P19). Necdin of mouse brain was characterized by Western blotting and silver-staining analysis by using affinity purified antibodies to 17 synthetic peptides of deduced C-terminal amino acids. Necdin exhibits a molecular mass of 51 kDa on SDS/PAGE, and is localized in the S1 and S2 nucleosomal fractions. Sonicated necdin is found in all fractions of Sephacryl S-300 gel filtration chromatography, with a peak at 700 kDa. Necdin is released on micrococcal nuclease digestion, which is essential for electrophoretic migration on acetic acid/urea/Triton gels, suggesting that it could be a DNA-binding protein. Nucleosomal necdin shows two peaks at approx. 10 S and approx. 20 S on sucrose gradient centrifugation in the presence of 0.6 M NaCl, and a single peak in the presence of 2.0 M NaCl. Necdin forms a huge complex through chemical cross-linking with glutaraldehyde or dimethyl sulphate. The silver-staining intensity of the 51 kDa band corresponds to the decrease in the immuno-staining in a reagent concentration-dependent manner. Necdin binds tightly to a double-stranded DNA affinity chromatography column, and can be eluted from it with 2.0 M NaCl after washing with 0.6 M NaCl (approx. 100 ng per ml of gel). This purified necdin exhibits a pI of 9.1 on isoelectric focusing. The nucleosomal necdin complex (> 200 kDa) was adsorbed on an organomercurial agarose affinity chromatography column and was eluted with 10 mM DTT, revealing that necdin is possibly involved in the transactive nucleosomal complex. These data show that necdin is a nuclear basic DNA-binding protein that associates with other molecules to regulate transcriptionally active genes and nuclear function.
Time-dependent inhibition of phospholipase Cβ-catalysed phosphoinositide hydrolysis: a comparison of different assays
Authors: JAMES Stephen R. , SMITH Sheila , PATERSON Andrew , Kendall HARDEN T. , Peter DOWNES C.
Published: August 2015

The properties of three different β-isoforms of phospholipase C (PLC) were analysed using substrate lipids dispersed in phospholipid vesicles, phospholipid–detergent mixed micelles and phospholipid monolayers spread at an air–water interface. Phosphatidylinositol 4,5-bisphosphate hydrolysis went virtually to completion in monolayers, but inositol trisphosphate production was curtailed prematurely in vesicular and micellar assays. Assays were linear for less than 2 min with vesicles; the linear portion could be significantly extended in micelles by increasing the ratio of micelles to enzyme molecules. However, onset of a second lower rate of substrate hydrolysis always occurred when ⩽ 10% of PtdIns(4,5)P2 had been utilized. This was not due to enzyme inactivation in the micellar interface, determined by addition of fresh substrate or fresh enzyme after the slow phase of activity had started, nor was it due to overt product inhibition of PLC or apparent entrapment of PLC at the micelle surface. These results are similar to those seen in assays using bacterial PLC and we suggest that the biphasic kinetics may be due to product-dependent changes in the presentation of substrate lipid to PLC in lamellar assays, leading to reduced activity.
Evidence for an inhibitory feedback loop regulating simian virus 40 large T-antigen fusion protein nuclear transport
Authors: SEYDEL Ulrich , JANS David A.
Published: August 2015

Nuclear protein import is central to eukaryotic cell function. It is dependent on ATP, temperature and cytosolic factors, and requires specific targeting sequences called nuclear localization signals (NLSs). Nuclear import kinetics was studied in vitro using digitonin-permeabilized cells of the HTC rat hepatoma cell line and a fluorescently labelled β-galactosidase fusion protein carrying amino acids 111–135 of the simian virus 40 large T-antigen (T-ag), including the NLS. Nuclear accumulation was rapid, reaching steady-state after about 80 min at 37 °C (t1/2 at about 17 min). Surprisingly, maximal nuclear concentration was found to be directly proportional to the concentration of the cytosolic extract and of cytoplasmic T-ag protein. Neither preincubation of cells for 1 h at 37 °C before the addition of T-ag protein nor the addition of fresh transport medium after 1 h and continuation of the incubation for another hour affected the maximal nuclear concentration. If cells were allowed to accumulate T-ag protein for 1 h before the addition of fresh transport medium containing different concentrations of T-ag protein and incubated for a further hour, the maximal nuclear concentration did not change unless the concentration of T-ag protein in the second transport mixture exceeded that in the first, in which case the nuclear concentration increased. Nuclear import of T-ag thus appeared (i) to be strictly unidirectional over 2 h at 37 °C and (ii) to be regulated by an inhibitory feedback loop, whereby the cytosolic concentration of protein appears to determine directly the precise end point of nuclear accumulation. This study represents the first characterization of this previously undescribed mechanism of regulation of nuclear protein import.
Overexpression of neuronal nitric oxide synthase in insect cells reveals requirement of haem for tetrahydrobiopterin binding
Authors: LIST Barbara M. , KLATT Peter , WERNER Ernst R. , SCHMIDT Kurt , MAYER Bernd
Published: August 2015

Nitric oxide synthase (NOS) catalyses the conversion of L-arginine into L-citrulline and nitric oxide. Recently we have developed a method for expression of recombinant rat brain NOS in baculovirus-infected Sf9 cells and purification of the enzymically active enzyme [Harteneck, Klatt, Schmidt and Mayer (1994) Biochem. J. 304, 683–686]. To study how biosynthetic manipulation of the NOS cofactors haem, FAD/FMN, and tetrahydrobiopterin (H4biopterin) affects the properties of the isolated enzyme, Sf9 cells were infected in the absence and presence of haemin chloride (4 μg/ml), riboflavin (0.1 mM), and the inhibitor of H4biopterin biosynthesis 2,4-diamino-6-hydroxypyrimidine (10 mM). In the absence of haemin, NOS was expressed to a very high level but remained predominantly insoluble. Purification of the soluble fraction of the expressed protein showed that it had poor activity (0.35 μmol of citrulline·mg-1·min-1) and was haem-deficient (0.37 equiv. per monomer). Supplementing the culture medium with haemin resulted in pronounced solubilization of the expressed enzyme, which had a specific activity of ~1 μmol of citrulline·mg-1·min-1 and contained 0.95 equiv. of haem per monomer under these conditions. Unexpectedly, the amount of H4biopterin endogenously present in the different NOS preparations positively correlated with the amount of enzyme-bound haem (y = 0.066+0.430x; r = 0.998). Radioligand binding experiments demonstrated that haem-deficient enzyme preparations containing 30–40% of the holoenzyme bound only ~40% of H4biopterin as compared with haem-saturated controls. These results suggest that the prosthetic haem group is essentially involved in the correct folding of NOS that is a requisite for solubilization of the protein and tight binding of H4biopterin.
Characterization of the hydroxymethylglutaryl-CoA lyase precursor, a protein targeted to peroxisomes and mitochondria
Authors: ASHMARINA Lyudmila I. , ROBERT Marie-France , ELSLIGER Marc-André , MITCHELL Grant A.
Published: August 2015

We previously showed that human liver hydroxymethylglutaryl-CoA (HMG-CoA) lyase (HL; EC 4.1.3.4) is found in both mitochondria and peroxisomes. HL contains a 27-residue N-terminal mitochondrial targeting sequence which is cleaved on mitochondrial entry, as well as a C-terminal Cys-Lys-Leu peroxisomal targeting motif. Because peroxisomal HL has a greater molecular mass and more basic pI value than mitochondrial HL, we predicted that peroxisomal HL retains the mitochondrial leader. To test this hypothesis, we expressed both the precursor (pHL) and mature (mHL) peptides in Escherichia coli and studied their properties. pHL purified by ion-exchange and hydrophobic chromatography had a pI of 7.6 on FPLC chromatofocusing and a molecular mass of 34.5 kDa on SDS/PAGE, similar to our findings for peroxisomal HL. For purified mHL, pI (6.2) and molecular mass (32 kDa) values resemble those of mitochondrial HL. Purified pHL is similar to mHL in Km for HMG-CoA (44.8 μM), kcat (6.3 min-1) and pH optimum (9.0–9.5). However, the quaternary structures of pHL and mHL differ. On Superose 12 FPLC gel filtration and also on ultrafiltration, both in the presence and in the absence of HMG-CoA, pHL behaves as a monomer whereas mHL migrates as a dimer. We conclude that the HL precursor is probably identical to peroxisomal HL, that its catalytic properties resemble those of mature mitochondrial HL, and that the mitochondrial leader peptide prevents dimerization of pHL.
Heparin enhances the catalytic activity of des-ETW-thrombin
Authors: GOODWIN Christopher A. , DEADMAN John J. , BONNIEC Bernard F. LE , ELGENDY Said , KAKKAR Vijay V. , SCULLY Michael F.
Published: August 2015

The thrombin mutant, des-ETW-thrombin, lacking Glu146, Thr147 and Trp148 within a unique insertion loop located at the extreme end of the primary specificity pocket, has been shown previously to exhibit reduced catalytic activity with respect to macromolecular and synthetic thrombin substrates and reduced or enhanced susceptibility to inhibition. Investigation of the hydrolysis of peptidyl p-nitroanilide substrates by des-ETW-thrombin showed increased activity in the presence of heparin and other sulphated glycosaminoglycans. No effect was observed upon the activity of wild-type thrombin. Heparin was found to decrease the Km for cleavage of four thrombin-specific substrates by des-ETW-thrombin, by 3–4-fold. Similarly, pentosan polysulphate (PPS) decreased the Km with these substrates by 8–10-fold. Heparin also increased the rate of inhibition of des-ETW-thrombin by antithrombin III and D-phenylalanyl-prolyl-arginylchloromethane (PPACK). The inhibition of des-ETW-thrombin by a number of thrombin-specific peptide boronic acids also showed significant reduction in the final Ki in the presence of heparin, due to reduction in the off-rate. A peptide analogue of a sequence of hirudin which binds thrombin tightly to exosite 1 (fibrinogen recognition site) potentiated the activity of des-ETW-thrombin against peptide p-nitroanilide substrates in a manner similar to heparin. The Ki for the inhibition of des-ETW-thrombin by p-aminobenzamidine was decreased by these ligands from 9.7 mM to 7.5 mM, 5.1 mM and 2.5 mM in the presence of heparin, hirudin peptide and PPS respectively, suggesting the increased catalytic activity is due to enhanced access to the primary specificity pocket. The positive influence of these ligands on des-ETW-thrombin was reversed in the presence of ATP or ADP; the latter has previously been shown to inhibit thrombin activity by blocking initial interaction with fibrinogen at exosite 1. Because the effect of heparin and PPS is similar to that of hirudin peptide, it is proposed that the most likely mechanism is that binding at the heparin-binding site (thrombin exosite 2) facilitates interaction at exosite 1 causing a conformational change which partially corrects the defective ground-state binding of the mutant thrombin. Although no effect was observed upon the activity of wild-type thrombin, our findings do provide further evidence of an allosteric property of thrombin which may control the geometry of, and access to, the primary specificity pocket.
Peptidyl vinyl sulphones: a new class of potent and selective cysteine protease inhibitors: S2P2 specificity of human cathepsin O2 in comparison with cathepsins S and L
Authors: Brömme D , Klaus J L , Okamoto K , Rasnick D , Palmer J T
Published: August 2015

Peptidyl vinyl sulphones are a novel class of extremely potent and specific cysteine protease inhibitors. They are highly active against the therapeutically important cathepsins O2, S and L. The highest kinact/Ki values exceed 107 M-1·s-1 for cathepsin S and 105 M-1·s-1 for cathepsins O2 and L. To study the primary specificity site of the novel human cathepsin O2 and the effectiveness of this novel class of inhibitors, a series of peptidyl vinyl sulphones with variations in the P2 residue was synthesized. Leucine in the P2 position was proven to be the most effective residue for cathepsin O2 and also for cathepsins S and L. Cathepsins O2 and S share a decreased accessibility towards P2 hydrophobic non-branched residues such as aminohexanoic acid (norleucine), methionine and oxidized methionine, but are distinguished by their different affinity towards phenylalanine in the P2 position. In contrast, cathepsin S accepts a broader range of hydrophobic residues in its S2 subsite than cathepsins O2 and L. The primary specificity-determining subsite pocket S2 in cathepsin O2 appears to be spatially more restricted than those of cathepsins S and L.
A limited upstream region of the human sucrase-isomaltase gene confers glucose-regulated expression on a heterologous gene
Authors: RODOLOSSE Annie , CHANTRET Isabelle , LACASA Michel , CHEVALIER Guillemette , ZWEIBAUM Alain , SWALLOW Dallas , ROUSSET Monique
Published: August 2015

We have previously shown that glucose can exert a repressive effect on the transcription of the sucrase–isomaltase (SI) gene in the differentiated enterocyte-like human colon carcinoma cell lines HT-29 and Caco-2. To characterize the region through which glucose exerts this effect, three different-length fragments of the 5´-flanking region of the human SI gene were linked to the reporter gene luciferase in an episomal vector carrying a hygromycin resistance gene. These fragments were used for transfection into a clone of the Caco-2 cell line, PF11, which has high glucose consumption and only expresses SI at high levels when cultured in the presence of a low supply of glucose. By using the stably transformed PF11 cells grown either in standard high glucose (25 mM) or in low glucose (1 mM) it was possible to show that the smallest fragment of the SI promoter, extending from bases -370 to +30, contains all the information required for the glucose repression of the reporter gene luciferase.
Requirement for phosphoinositide 3-OH kinase in growth hormone signalling to the mitogen-activated protein kinase and p70s6k pathways
Authors: KILGOUR Elaine , GOUT Ivan , ANDERSON Neil G.
Published: August 2015

Pituitary growth hormone (GH) co-ordinately stimulates three distinct signalling pathways in 3T3-F442A preadipocytes, the STAT (signal transducer and activator of transcription) pathway, the mitogen-activated protein (MAP) kinase cascade and p70s6k. The mechanisms linking the GH receptor to these signals have not been fully identified. In this study we have examined the role of phosphoinositide 3-OH kinase (PI 3-kinase). Pretreatment of cells with wortmannin, a specific inhibitor of PI 3-kinase, prevented the activation of p70s6k and partially inhibited the activation of p42 and p44 MAP kinases by GH. In contrast, wortmannin failed to appreciably affect the GH-stimulated tyrosyl phosphorylation of JAK-2 or STAT-1. GH transiently increased the activity of PI 3-kinase recovered in anti-phosphotyrosine immunoprecipitates. In addition, several tyrosyl-phosphorylated proteins were specifically adsorbed from lysates of cells exposed to GH by a glutathione S-transferase fusion protein containing the 85 kDa regulatory subunit of PI 3-kinase. GH also induced an increase in the PI 3-kinase activity associated with both JAK-2 and insulin receptor substrate-1 (IRS-1) immunoprecipitates. These results establish PI 3-kinase as an important mediator of GH signalling to the MAP kinase and p70s6k pathways and suggest that PI 3-kinase is activated by a mechanism involving JAK-2 and IRS-1.
Structure of a group C streptococcal protein that binds to fibrinogen, albumin and immunoglobulin G via overlapping modules
Authors: TALAY Susanne R. , GRAMMEL Melanie P. , CHHATWAL Gursharan S.
Published: August 2015

Pathogenic streptococci express surface proteins that bind to host serum proteins. A novel multiple-ligand-binding protein has now been identified in a species belonging to serotype C streptococci. This protein binds to fibrinogen, albumin and IgG and was therefore designated FAI protein. The structure of the fai gene has been determined, and deletion analysis and expression of FAI fusion polypeptides revealed that the binding domain for fibrinogen and IgG is located within the non-repetitive N-terminal half of the protein. A 93-amino acid peptide retained the ability to bind both proteins, whereas a 56-amino acid subpeptide only bound fibrinogen. IgG-binding activity required the complete fibrinogen-binding domain and an additional 37 amino acids C-terminal to it, and albumin-binding activity was only obtained with a polypeptide reflecting the complete surface-exposed region of FAI protein indicating that the binding sites for each ligand were located on overlapping modules. Signal sequence, C repeat region and C-terminus revealed high homology to group A streptococcal M proteins whereas the N-terminal region containing the fibrinogen/IgG-binding domains is completely different and exhibits no similarity to any other previously characterized protein. Thus FAI protein exhibits a framework structure that might have evolved in group C streptococci via fusion of unrelated sequences, thereby generating an albumin-binding domain in the functional context of multiple-ligand-binding activity.
Dye-affinity labelling of bovine heart mitochondrial malate dehydrogenase and study of the NADH-binding site
Authors: LABROU Nikolaos E. , ELIOPOULOS Elias , CLONIS Yannis D.
Published: August 2015

The ability of the reactive dichlorotriazine dye Vilmafix Blue A-R (VBAR) to act as an affinity label for bovine heart L-malate dehydrogenase (MDH) was studied. VBAR binds specifically and irreversibly to MDH (k3 0.16 min-1; KD 14.4 μM). The inactivation of the NADH-dependent enzyme by VBAR is competitively inhibited by NAD+, NADH and ADP. Quantitatively inhibited MDH contained approx. 1 mol of dye per mol of active site. The inhibition is irreversible and activity cannot be recovered either on incubation with 10 mM NAD+, 10 mM NADH or 10 mM ADP, or by extensive dialysis or gel-filtration chromatography. Data obtained from high-performance gel-filtration chromatography and analysed by Scatchard plot suggested the presence of two coenzyme-binding sites per MDH dimer. Tryptic digestion of VBAR-labelled MDH followed by reverse-phase HPLC analysis revealed one VBAR-labelled peptide. It appears that each subunit features the same peptide bearing the modifying residue involved in MDH labelling. The pKa of the modifying residue is 8.05. Both total acid hydrolysis of VBAR-labelled MDH followed by HPLC and TLC analysis, and molecular-modelling studies suggest that the modifying residue is Lys-81 and/or Lys-217.