bluuebunny/crossref_metadata_2025 huggingface dataset:
The protein oxidation product 3,4-dihydroxyphenylalanine (DOPA) mediates oxidative DNA damage
Authors: MORIN Bénédicte , DAVIES J. Michael , DEAN T. Roger
Published: August 2015

A major product of hydroxy-radical addition to tyrosine is 3,4-dihydroxyphenylalanine (DOPA) which has reducing properties. Protein-bound DOPA (PB-DOPA) has been shown to be a major component of the stable reducing species formed during protein oxidation under several conditions. The aim of the present work was to investigate whether DOPA, and especially PB-DOPA, can mediate oxidative damage to DNA. We chose to generate PB-DOPA using mushroom tyrosinase, which catalyses the hydroxylation of tyrosine residues in protein. This permitted us to study the reactions of PB-DOPA in the virtual absence of other protein-bound oxidation products. The formation of two oxidation products of DNA, 8-oxo-7,8-dihydro-2ʹ-deoxyguanosine (8oxodG) and 5-hydroxy-2ʹ-deoxycytidine (5OHdC), were studied with a novel HPLC using gradient elution and an electrochemical detection method, which allowed the detection of both DNA modifications in a single experiment. We found that exposure of calf thymus DNA to DOPA or PB-DOPA resulted in the formation of 8oxodG and 5OHdC, with the former predominating. The formation of these DNA oxidation products by either DOPA or PB-DOPA depended on the presence of oxygen, and also on the presence and on the concentration of transition metal ions, with copper being more effective than iron. The yields of 8oxodG and 5OHdC increased with DOPA concentration in proteins. Thus PB-DOPA was able to promote further radical-generating events, which then transferred damage to other biomolecules such as DNA.
CD38 binding to human myeloid cells is mediated by mouse and human CD31
Authors: HORENSTEIN L. Alberto , STOCKINGER Hannes , IMHOF A. Beat , MALAVASI Fabio
Published: August 2015

Soluble forms of membrane receptors are emerging candidates as physiological regulators of leukocyte trafficking. In the present study, we found that the soluble form of the CD38 antigen (sCD38) bears a binding domain of low affinity for a cellular receptor on U937 cells. Cross-linking and peptide-mapping studies confirmed the physical association and the identification of the U937 receptor as a 130 kDa protein. The binding of sCD38 to the receptor was differentially inhibited by several monoclonal antibodies against the CD31 cell-adhesion molecule. Thus the interaction was analysed through direct association of soluble and membrane CD38 with soluble recombinant murine CD31 with three N-terminal and with all six extracellular Ig domains. Cross-linking experiments on U937 intact cells, and ligand blot assays of the immunoprecipitated CD38 molecule, indicated that (i) the recognized epitope is determined by the tertiary structure of the molecule, and that (ii) the binding domain involved resides in the ectocellular portion of the CD31 molecule, more precisely in the first three N-terminal domains. A comparative functional activity between murine and human CD31 was also explored. The data presented suggest that (i) human CD31 bears a highly functional similarity with its murine counterpart, as it is a receptor in myeloid cells with more than one ligand (the αvβ3 integrin and the CD38 molecule), and that (ii) the activity of sCD38 as decoy molecule for CD31 may play an important role in cell-cell interactions in physiological and pathological conditions.
Interaction of two proline-rich sequences of cell adhesion kinase β with SH3 domains of p130Cas-related proteins and a GTPase-activating protein, Graf
Authors: OHBA Takeaki , ISHINO Masaho , AOTO Hiroshi , SASAKI Terukatsu
Published: August 2015

Cell adhesion kinase β (CAKβ) is a protein tyrosine kinase closely related to focal adhesion kinase (FAK) in structure. CAKβ contains two proline-rich sequences within its C-terminal region. Since proline-rich sequences present in the corresponding region of FAK are known to mediate protein-protein interactions by binding to SH3 domains, we investigated binding of CAKβ to a panel of SH3 domains. Affinity precipitation from rat brain lysate revealed selective interactions of CAKβ with glutathione S-transferase (GST)-fused SH3 domains of p130Cas(Cas)-related proteins and Graf. Mutational analysis indicated that the proline-rich sequences of CAKβ mediate this interaction. Each of the two proline-rich sequences fused to GST bound directly to these SH3 domains in dot blot analysis. A competitive binding assay revealed that the first proline-rich sequence of CAKβ preferentially associated with the SH3 domain of Cas. The second proline-rich sequence of CAKβ bound to the SH3 domain of Graf with higher specificity than the corresponding proline-rich sequence of FAK. Finally, we showed co-immunoprecipitation of CAKβ with Graf from rat brain lysate. These results indicate that CAKβ associates in vivo with Graf through its SH3 domain.
The inhibitory effect of novel triterpenoid compounds, fomitellic acids, on DNA polymerase β
Authors: MIZUSHINA Yoshiyuki , TANAKA Nobukazu , KITAMURA Akitoshi , TAMAI Katsuyuki , IKEDA Masako , TAKEMURA Masaharu , SUGAWARA Fumio , ARAI Takao , MATSUKAGE Akio , YOSHIDA Shonen , SAKAGUCHI Kengo
Published: August 2015

We previously found new triterpenoid compounds, designated fomitellic acid A and B, which selectively inhibit the activities of mammalian DNA polymerase α and β in vitro. On DNA polymerase β, the fomitellic acids acted by competing with both the substrate and the template primer, but on DNA polymerase α, they acted non-competitively. At least on DNA polymerase β, the evidence suggests that each of the fomitellic acids bind to the active region competing with the substrate and/or template primer, and subsequently inhibits the catalytic activity. We therefore further investigated the enzyme-binding properties by using DNA polymerase β and its proteolytic fragments. The 39 kDa enzyme was proteolytically separated into two fragments of the template-primer-binding domain (8 kDa) and the catalytic domain (31 kDa). The fomitellic acids bound tightly to the 8 kDa fragment, but not to the 31 kDa fragment. The immuno-precipitation by antibodies against the enzyme or each of the fragments also proved the binding. These results suggest that the fomitellic acid molecule competes with the template-primer molecule on its 8 kDa binding site, binds to the site, and the fomitellic acid molecule simultaneously disturbs the substrate incorporation into the template primer.
Guanine nucleotide-dependent translocation of RhoA from cytosol to high affinity membrane binding sites in human erythrocytes
Authors: BOUKHAROV A. Andrey , COHEN M. Carl
Published: August 2015

The translocation of the small GTP-binding protein Rho from the cytosolic to membrane-bound form is an early step in many cellular signal-transduction events, but little is known regarding the mechanism of Rho association with the plasma membrane. We have used membranes from human erythrocytes to uncover a novel class of integral membrane components involved in the Rho-membrane association. Membranes of human erythrocytes contain several proteins of the Ras superfamily. Using specific antibodies and C3 exoenzyme of Clostridium botulinum we have identified one of them as RhoA. This protein was detected in both cytosol and membrane fractions of hypotonically lysed erythrocytes. We found that cytosolic Rho bound specifically to the cytoplasmic surface of the erythrocyte membrane and that the translocation of Rho to the membrane was absolutely dependent on the prior incubation of the cytosol with guanosine 5ʹ-[γ-thio]triphosphate (1-50 μM) at low Mg2+ concentration. Rho binding sites could not be extracted from the membrane using conditions that extracted all other peripheral proteins and were unaffected by heat treatment and protease digestion. Rho binding was saturable, with a Kd in the range 1-5.0 nM, and the number of binding sites was estimated to be approx. (1-2)×103 sites per cell. This is the first report of Rho binding to integral membrane components. The identity of these components may reveal novel aspects of the mechanism by which Rho exerts its multiple biochemical effects.
Metabolism of agmatine in macrophages: modulation by lipopolysaccharide and inhibitory cytokines
Authors: SASTRE Magdalena , GALEA Elena , FEINSTEIN Douglas , REIS J. Donald , REGUNATHAN Soundararajan
Published: August 2015

Agmatine is an amine derived from the decarboxylation of arginine by arginine decarboxylase (ADC) and metabolized to putrescine by agmatinase. While prevalent in bacteria and plants, agmatine and its metabolic enzymes have been recently identified in mammalian tissues. In the present study we sought to determine: (a) whether macrophages (cell line RAW 264.7) express ADC and agmatinase, and (b) if the enzymes are regulated by lipopolysaccharide (LPS), and/or by the inhibitory cytokines transforming growth factor-β (TGF-β), interleukin-10 (IL-10) and interleukin-4 (IL-4). LPS induced a dose-dependent stimulation of agmatinase, while it decreased ADC, the effect in both cases being maximum at 20 h. As expected, LPS dose-dependently stimulated the inducible nitric oxide synthase activity (iNOS). A strong correlation was observed between the effects of LPS on the agmatine-related enzymes and iNOS. By contrast, exposure to IL-10 and TGF-β caused a reduction in ADC and agmatinase, whereas IL-4 was ineffective on ADC, but reverted the LPS-induced increase of agmatinase. We conclude that the agmatine pathway may be an alternative metabolic route for arginine in macrophages, suggesting a regulatory role of agmatine during inflammation.
DNA polymerase β: effects of gapped DNA substrates on dNTP specificity, fidelity, processivity and conformational changes
Authors: AHN Jinwoo , KRAYNOV Vadim S. , ZHONG Xuejun , WERNEBURG Brian G. , TSAI Ming-Daw
Published: August 2015

Pre-steady-state kinetic analysis was used to compare the catalytic properties of DNA polymerase β (Pol β) for single-base gap-filling and regular duplex DNA synthesis. The rate of polymerization (kpol) and the apparent equilibrium dissociation constant of dNTP (Kd) were determined with single-nucleotide gapped DNA substrates for all four possible correct base pairs and twelve possible incorrect base pairs, and the results were compared with those obtained previously with non-gapped primer/template duplex DNA substrates. For correct dNTP incorporation, the use of single-nucleotide gapped DNA led to significant decreases in the Kd of dNTP. Although kpol was little affected, the catalytic efficiency kpol/Kd increased significantly owing to the decreases in Kd. In contrast, for incorrect dNTP incorporation, the use of single-nucleotide gapped DNA substrates did not affect the Kd of dNTP appreciably but caused the kpol (and thus kpol/Kd) for incorrect dNTP incorporation to increase. As a consequence the fidelity of Pol β was not significantly affected by the use of single-nucleotide gapped DNA substrates. In addition we show that under processive polymerization conditions the processivity of Pol β increases in the gap-filling synthesis owing to a decreased rate of DNA dissociation. Finally, with a single-nucleotide gapped DNA substrate the rate-limiting conformational change step before chemistry was also observed. However, the preceding fast conformational change observed with duplex DNA substrates was not clearly detected. A possible cause is that in the complex with the gapped DNA, the 8 kDa N-terminal domain of Pol β already exists in a closed conformation. This interpretation was supported by tryptic digestion experiments.
ATP-dependent transport of unconjugated bilirubin by rat liver canalicular plasma membrane vesicles
Authors: PASCOLO Lorella , BAYON Enrique J. , CUPELLI Felicia , OSTROW J. Donald , TIRIBELLI Claudio
Published: August 2015

The transport of highly purified 3H-labelled unconjugated bilirubin (UCB) was investigated in rat liver plasma membrane vesicles enriched in the canalicular domain and found to be stimulated (more than 5-fold) by the addition of ATP. Other nucleotides, such as AMP, ADP, GTP and a non-hydrolysable ATP analogue (adenosine 5´-[α,β-methylene] triphosphate), did not stimulate [3H]UCB transport, indicating that ATP hydrolysis was necessary for the stimulatory effect. [3H]UCB uptake occurred into an osmotically sensitive space. At an unbound bilirubin concentration ([Bf]) below saturation of the aqueous phase (no more than 70 nM UCB), the ATP-dependent transport followed saturation kinetics with respect to [Bf], with a Km of 26±8 nM and a Vmax of 117±11 pmol per 15 s per mg of protein. Unlabelled UCB inhibited the uptake of [3H]UCB, indicating that UCB was the transported species. Inhibitors of ATPase activity such as vanadate or diethyl pyrocarbonate decreased the ATP effect (59±11% and 100% respectively). Daunomycin, a known substrate for multidrug resistance protein-1, and taurocholate did not inhibit the ATP-dependent [3H]UCB transport, suggesting that neither mdr-1 nor the canalicular bile acid transporter is involved in the canalicular transport of UCB. [3H]UCB uptake (both with and without ATP) in canalicular vesicles obtained from TR- rats was comparable to that in vesicles obtained from Wistar rats, indicating that the canalicular multispecific organic anion transporter, cMOAT, does not account for UCB transport. These results indicate that UCB is transported across the canalicular membrane of the liver cell by an ATP-dependent mechanism involving an as yet unidentified transporter.
Membrane integration of Sec61α: a core component of the endoplasmic reticulum translocation complex
Authors: KNIGHT Bruce C. , HIGH Stephen
Published: August 2015

The Sec61 complex is a central component of the endoplasmic reticulum (ER) translocation site. The complex consists of three subunits: Sec61α, Sec61β and Sec61γ, at least two of which (α and β) are adjacent to nascent proteins during membrane insertion. Another component of the translocation machinery is the translocating chain-associating membrane (TRAM) protein, which is also adjacent to many nascent proteins during membrane insertion. Sec61α functions as the major component of a transmembrane channel formed by oligomers of the Sec61 complex. This channel is the site of secretory protein translocation and membrane protein integration at the ER membrane. Sec61α is a polytopic integral membrane protein, and we have studied its biosynthesis and membrane integration in vitro. Using a cross-linking approach to analyse the environment of a series of discrete Sec61α membrane-integration intermediates, we find: (i) newly synthesized Sec61α is adjacent to known components of the ER membrane-insertion site, namely Sec61α, Sec61β and TRAM, and thus the integration of Sec61α appears to require a pre-existing Sec61 complex; (ii) a site-specific cross-linking analysis indicates that the first transmembrane domain of Sec61α remains adjacent to protein components of the ER-insertion site (specifically TRAM and Sec61β) during the insertion of at least three subsequent transmembrane domains; and (iii) the membrane integration of Sec61α requires ER targeting by the signal-recognition particle.
Mutational analysis of histidine residues in the rabbit Na+/dicarboxylate co-transporter NaDC-1
Authors: PAJOR Ana M. , SUN Ning , VALMONTE Heidi G.
Published: August 2015

Succinate transport by the rabbit Na+/dicarboxylate co-transporter, NaDC-1, expressed in Xenopusoocytes was inhibited by the histidyl-selective reagent diethyl pyrocarbonate (DEPC). Therefore the role of histidine residues in the function of NaDC-1 was examined by site-directed mutagenesis. All 11 histidine residues in NaDC-1 were converted to alanine, but only mutant H106A exhibited a decrease in succinate transport. Additional mutations of NaDC-1 at position 106 showed that aspartic acid and asparagine, but not arginine, can substitute for histidine. Examination of succinate and citrate kinetics of H106A revealed a decrease in Vmax with no change in Km. Cell surface biotinylation experiments showed that the transport activity of all four mutants at position 106 was correlated with the amount of cell surface expression, suggesting a role of His-106 in membrane expression rather than function. Two of the histidine mutants, H153A and H569A, exhibited insensitivity to inhibition by DEPC, indicating that these residues are involved in binding DEPC. Neither of these residues is required for transport activity; thus DEPC probably inhibits NaDC-1 function by hindrance of the mobility of the carrier. We conclude that histidine residues are not critical for transport function in NaDC-1, although His-106 might be involved in determining protein expression or stability in the membrane.
Characterization of a spleen sulphotransferase responsible for the 6-O-sulphation of the galactose residue in sialyl-N-acetyl-lactosamine sequences
Authors: SPIRO Robert G. , BHOYROO Vishnu D.
Published: August 2015

An enzyme which catalyses the transfer of sulphate from 3´-phosphoadenosine 5´-phosphosulphate (PAPS) to C-6 of galactose in the NeuAcα2-3Galβ1-4GlcNAc (3´SLN) sequence has been found in rat spleen microsomes and its specificity indicates that it is well suited to participate in the assembly of 3´-sialyl-6´-sulpho-LacNAc [NeuAcα2-3Gal(6-SO4)β1-4GlcNAc] and 3´-sialyl-6´-sulpho-LewisX [NeuAcα2-3Gal(6-SO4)β1-4(Fucα1-3)GlcNAc] saccharide groups which have been implicated as selectin ligands. This sulphotransferase has a strict requirement for oligosaccharide acceptors which are capped by an α2-3-linked sialic acid residue, although GlcNAc in 3´SLN can be substituted by Glc, and Galβ1-4GlcNAc can be replaced by Galβ1-3GlcNAc without loss of activity. The finding that 3´-sialyl LewisX was inert as an acceptor suggested that fucosylation, in contrast with sialylation, follows the addition of the sulphate group. Since fetuin glycopeptides containing the NeuAcα2-3Galβ1-4GlcNAc sequence had a similar affinity for the enzyme as the unattached 3´SLN, it would appear that the acceptor determinants reside primarily in the peripheral trisaccharide constellation. The position of the sulphate on C-6 of galactose was elucidated by Smith periodate oxidation, hydrazine/nitrous acid/NaBH4 treatment and elder (Sambucus nigra)bark lectin chromatography of the desialylated [35S]sulphate-labelled products of the enzyme. Assays carried out with 3´SLN as acceptor indicated that the sulphotransferase had a pH optimum between 6.5 and 7.0 and a dependence on a bivalent cation best met by Mn2+ (12–25 mM); Triton X-100 (0.02 to 0.35%) brought about maximal stimulation. Tentative Km values determined for this enzyme were 4.7 µM for PAPS, and 0.72 mM and 1.16 mM for 3´SLN and fetuin glycopeptides respectively. A survey of several rat organs indicated that the PAPS:3´SLN-6-O-sulphotransferase is selectively distributed with maximal activity occurring in spleen which was substantially greater than thymus or lymph nodes. In contrast, other enzymes (i.e. PAPS:Gal-3-O-and GlcNAc-6-O-sulphotransferases) involved in the sulphation of sialyl-lactosamine and lactosamine sequences, which in the sulphated form are believed to also be selectin ligands, were more evenly distributed in lymphoid tissues. Relatively high activities for all three enzymes were found in brain.
Measurement of oxidative DNA damage by gas chromatography–mass spectrometry: ethanethiol prevents artifactual generation of oxidized DNA bases
Authors: JENNER Andrew , ENGLAND Timothy G. , ARUOMA Okezie I. , HALLIWELL Barry
Published: August 2015

Analysis of oxidative damage to DNA bases by GC-MS enables identification of a range of base oxidation products, but requires a derivatization procedure. However, derivatization at high temperature in the presence of air can cause ‘artifactual ’ oxidation of some undamaged bases, leading to an overestimation of their oxidation products, including 8-hydroxyguanine. Therefore derivatization conditions that could minimize this problem were investigated. Decreasing derivatization temperature to 23 °C lowered levels of 8-hydroxyguanine, 8-hydroxyadenine, 5-hydroxycytosine and 5-(hydroxymethyl)uracil measured by GC–MS in hydrolysed calf thymus DNA. Addition of the reducing agent ethanethiol (5%, v/v) to DNA samples during trimethylsilylation at 90 °C also decreased levels of these four oxidized DNA bases as well as 5-hydroxyuracil. Removal of guanine from hydrolysed DNA samples by treatment with guanase, prior to derivatization, resulted in 8-hydroxyguanine levels (54–59 pmol/mg of DNA) that were significantly lower than samples not pretreated with guanase, independent of the derivatizationconditions used. Only hydrolysed DNA samples that were derivatized at 23 °C in the presence of ethanethiol produced 8-hydroxyguanine levels (56±8 pmol/mg of DNA) that were as low as those of guanase-pretreated samples. Levels of other oxidized bases were similar to samples derivatized at 23 °C without ethanethiol, except for 5-hydroxycytosine and 5-hydroxyuracil, which were further decreased by ethanethiol. Levels of 8-hydroxyguanine, 8-hydroxyadenine and 5-hydroxycytosine measured in hydrolysed calf thymus DNA by the improved procedures described here were comparable with those reported previously by HPLC with electrochemical detection and by GC–MS with prepurification to remove undamaged base. We conclude that artifactual oxidation of DNA bases during derivatization can be prevented by decreasing the temperature to 23 °C, removing air from the derivatization reaction and adding ethanethiol.
Glutathione transferase Zeta catalyses the oxygenation of the carcinogen dichloroacetic acid to glyoxylic acid
Authors: TONG Zeen , BOARD Philip G. , ANDERS M. W.
Published: August 2015

Dichloroacetic acid (DCA), a common drinking-water contaminant, is hepatocarcinogenic in rats and mice, and is a therapeutic agent used clinically in the management of lactic acidosis. DCA is biotransformed to glyoxylic acid by glutathione-dependent cytosolic enzymes in vitro and is metabolized to glyoxylic acid in vivo. The enzymes that catalyse the oxygenation of DCA to glyoxylic acid have not, however, been identified or characterized. In the present investigation, an enzyme that catalyses the glutathione-dependent oxygenation of DCA was purified to homogeneity (587-fold) from rat liver cytosol. SDS/PAGE and HPLC gel-filtration chromatography showed that the purified enzyme had a molecular mass of 27–28 kDa. Sequence analysis showed that the N-terminus of the purified protein was blocked. An internal sequence of 30 amino acid residues was obtained that matched the recently discovered human glutathione transferase Zeta well [Board, Baker, Chelvanayagam and Jermiin (1997) Biochem. J. 328, 929–935]. Western-blot analysis showed that the purified rat-liver enzyme cross-reacted with rabbit antiserum raised against recombinant human glutathione transferase Zeta. The apparent Km and Vmax values of the purified enzyme with DCA as the variable substrate were 71.4 µM and 1334 nmol/min per mg of protein, respectively; the Km for glutathione was 59 µM. Both the purified rat-liver enzyme and the recombinant human enzyme showed high activity with DCA as the substrate. These results demonstrate that the glutathione-dependent oxygenation of DCA to glyoxylic acid is catalysed by a Zeta-class glutathione transferase.
Phosphinic peptides, the first potent inhibitors of astacin, behave as extremely slow-binding inhibitors
Authors: YIALLOUROS Irene , VASSILIOU Stamatia , YIOTAKIS Athanasios , ZWILLING Robert , STÖCKER Walter , DIVE Vincent
Published: August 2015

A series of phosphinic pseudo-peptides varying in length and composition have been designed as inhibitors of the crayfish zinc endopeptidase astacin, the prototype of the astacin family and of the metzincin superfamily of metalloproteinases. The most efficient phosphinic peptide, fluorenylmethyloxycarbonyl-Pro-Lys-PheΨ(PO2CH2)Ala-Pro-Leu-Val, binds to astacin with a Ki value of 42 nM, which is about three orders of magnitude below the corresponding values for previously used hydroxamic acid derivatives. However, the rate constants for association (kon = 96.8 M-1·s-1) and dissociation (koff = 4.1×10-6 s-1) are evidence for the extremely slow binding behaviour of this compound. N-terminally or C-terminally truncated phosphinic analogues of this parent molecule are much less potent, indicating a critical role of the peptide size on the potency. In particular, omission of the N-terminal proline residue leads to a 40-fold increase in Ki which is mostly due to a 75-fold higher koff value. These findings are consistent with the previously solved crystal structure of astacin complexed with one of the phosphinic peptides, benzyloxycarbonyl-Pro-Lys-PheΨ(PO2CH2)Ala-Pro-O-methyl, Ki = 14 µM [Grams, Dive, Yiotakis, Yiallouros, Vassiliou, Zwilling, Bode and Stöcker (1996) Nature Struct. Biol. 3, 671–675]. This structure also reveals that the phosphinic group binds to the active site as a transition-state analogue. The extremely slow binding behaviour of the phosphinic peptides is discussed in the light of the conformational changes involving a unique ‘tyrosine switch ’ in the structure of astacin upon inhibitor binding. The phosphinic peptides may provide a rational basis for the design of drugs directed towards other members of the astacin family which, like bone morphogenetic protein 1 (BMP1; i.e. the procollagen C-proteinase), have become targets of pharmacological research.
Triplet structure of human von Willebrand factor
Authors: FISCHER Bernhard E. , THOMAS Kathy B. , SCHLOKAT Uwe , DORNER Friedrich
Published: August 2015

Human von Willebrand factor (hp-vWF) is a high-molecular- mass protein found in plasma as a series of multimers. It consists of subunits comprising 2050 amino acids linked by disulphide bonds into multimers of various size ranging in molecular mass up to greater than 10000 kDa. Partial proteolysis at position Tyr842–Mer843 of the subunit [Dent et al. (1990) Proc. Natl. Acad. Sci. U.S.A. 87, 6306–6310] by a vWF-specific protease [Furlan et al. (1993) Proc. Natl. Acad. Sci. U.S.A. 90, 7503–7507] results in the generation of an N-terminal and a C-terminal fragment and the appearance of hp-vWF triplet bands. It has been suggested [Furlan et al. (1993) Proc. Natl. Acad. Sci. U.S.A. 90, 7503–7507] that (i) the intermediate triplet band of the primary dimer represents a dimer of two C-terminal fragments, (ii) the slower migrating satellite band of the primary dimer represents an asymmetric structure composed of a mature subunit to which one N-terminal and one C-terminal fragment are linked by disulphide bonds, and (iii) the faster migrating satellite band of the primary dimer contains two N-terminal fragments. Here we used recombinant vWF (r-vWF) for structural analysis of hp-vWF multimers. r-vWF exhibited no proteolytic degradation and all multimers contained mature subunits. High-resolution agarose-gel electrophoresis and two-dimensional electrophoresis demonstrated that (i) r-vWF multimers and hp-vWF intermediate triplet bands exhibited identical molecular mass and electrophoretic mobilities, (ii) the faster and slower migrating satellite bands of hp-vWF differ by less than the molecular mass of one subunit from the corresponding intermediate triplet band, and (iii) the triplet bands of hp-vWF are composed of mature and degraded subunits. The results support a structural model of hp-vWF triplet bands according to which the intermediate triplet bands represent multiple numbers of symmetric and/or asymmetric dimers, the slower migrating satellite bands have one extra N-terminal fragment, and the faster migrating satellite band lacks one N-terminal fragment respectively in comparison with the corresponding intermediate triplet band.
A novel assay in vitro of human islet amyloid polypeptide amyloidogenesis and effects of insulin secretory vesicle peptides on amyloid formation
Authors: KUDVA Yogish C. , MUESKE Cheryl , BUTLER Peter C. , EBERHARDT Norman L.
Published: August 2015

Human islet amyloid polypeptide (IAPP) is a 37-residue peptide that is co-secreted with insulin by the β-cell and might be involved in the pathogenesis of non-insulin-dependent diabetes mellitus. We developed an improved assay in vitrobased on the fluorescence of bound thioflavin T to study factors affecting amyloidogenesis. Monomeric IAPP formed amyloid fibrils, as detected by increased fluorescence and by electron microscopy. Fluorimetric analysis revealed that the initial rate of amyloid formation was: (1) proportional to the peptide monomer concentration, (2) maximal at pH 9.5, (3) maximal at 200 mM KCl, and (4) proportional to temperature from 4 to 37 °C. We found that 5-fold and 10-fold molar excesses of proinsulin inhibited fibril formation by 39% and 59% respectively. Insulin was somewhat more potent with 5-fold and 10-fold molar excesses inhibiting fibril formation by 69% and 73% respectively, whereas C-peptide had no effect at these concentrations. Thus at physiological ratios of IAPP to insulin, insulin and proinsulin, but not C-peptide, can retard amyloidogenesis. Because insulin resistance or hyperglycaemia increase the IAPP-to-insulin ratio, increased intracellular IAPP compared with insulin expression in genetically predisposed individuals might contribute to intracellular amyloid formation, β-cell death and the genesis of non-insulin-dependent diabetes mellitus.
Multiple endosomal recycling pathways in rat adipose cells
Authors: KANDROR Konstantin V. , PILCH Paul F.
Published: August 2015

Adipose and skeletal-muscle cells can translocate several membrane proteins from intracellular compartment(s) to the cell surface in an insulin-dependent fashion. Among these proteins is Glut4, a physiologically important glucose transporter which mediates insulin's effect on blood glucose clearance. Under basal conditions, Glut4 is localized in uniform, intracellular membrane vesicles with an average diameter of 50–70 nm and a sedimentation coefficient of 100–120 S. The nature of this compartment and its trafficking pathway to the plasma membrane is still unresolved. We show here that, in addition to Glut4, the aminopeptidase gp160 or insulin-responsive aminopeptidase (‘IRAP’), sortilin, and an acutely recycling population of the insulin-like growth factor-II/mannose 6-phosphate receptor, this compartment includes 60% of the intracellular population of the transferrin receptor. We used subcellular fractionation, cell-surface biotinylation, and radioactive-ligand (125I-transferrin) uptake to demonstrate that the transferrin receptor recycles between this compartment and the plasma membrane in response to insulin along with Glut4 and other protein components of these vesicles. The co-localization of Glut4 and several endosomal markers in the terminally differentiated fat-cells during several stages of their cycling pathways suggests that the ‘Glut4 pathway ’ may derive from the hormone-insensitive endosomes of undifferentiated preadipocytes. The insulin receptor is excluded from Glut4-containing vesicles in both insulin-stimulated and unstimulated adipocytes, and thus it is likely to traffic independently from Glut4 through different intracellular compartments. Our data show that, in adipose cells, the ligand-dependent recycling pathway of the insulin receptor is structurally separated from the ligand-independent pathway of the transferrin receptor, and that Glut4 is specifically targetted to the latter.
Osmotic swelling-induced activation of the extracellular-signal-regulated protein kinases Erk-1 and Erk-2 in Intestine 407 cells involves the Ras/Raf-signalling pathway
Authors: WIJK Thea van Der , DORRESTIJN Jannette , NARUMIYA Shuh , MAASSEN J. Antonie , JONGE Hugo R. De , TILLY Ben C.
Published: August 2015

Human Intestine 407 cells respond to hypo-osmotic stress with a rapid stimulation of compensatory ionic conductances accompanied by a transient increase in the activity of the extracellular-signal-regulated protein kinases Erk-1 and Erk-2. In this study, we examined the upstream regulators of hypotonicity-induced Erk-1/Erk-2 activation and their possible role in cell-volume regulation. The hypotonicity-provoked Erk-1/Erk-2 activation was greatly reduced in cells pretreated with the specific mitogen-activated/Erk-activating kinase inhibitor PD098059 and was preceded by a transient stimulation of Raf-1. Pretreatment of the cells with PMA, GF 109203X, wortmannin or Clostridium botulinum C3 exoenzyme did not appreciably affect the hypotonicity-provoked Erk-1/Erk-2 stimulation, suggesting the osmosensitive signalling pathway to be largely independent of protein kinase C and p21rho. In contrast, expression of dominant negative RasN17 completely abolished the hypotonicity-induced Erk-1/Erk-2 activation. Stimulation of the swelling-induced ion efflux was independent of activation of these mitogen-activated protein kinases, as revealed by hypotonicity-provoked isotope efflux from 125I-- and 86Rb+-loaded cells after pretreatment with PD098059 and after expression of RasN17. In addition, the epidermal-growth-factor-induced potentiation of the hypotonicity-provoked anionic response did not depend on the increase in Erk-1/Erk-2 activity but, instead, was found to depend on Ca2+ influx. Taken together, these results indicate that hypotonic stress induces Erk-1/Erk-2 activation through the Ras/Raf-signalling pathway, and argue against a direct role for this pathway in cell-volume control.
Cloning and expression of a cDNA encoding a new neurocalcin isoform (neurocalcin α) from bovine brain
Authors: KATO Masumi , WATANABE Yasuo , IINO Satoshi , TAKAOKA Yuri , KOBAYASHI Shigeru , HAGA Tatsuya , HIDAKA Hiroyoshi
Published: August 2015

Neurocalcin (NC), a neuron-specific EF-hand Ca2+-binding protein, purified from bovine brain [Terasawa, Nakano, Kobayashi and Hidaka (1992) J. Biol. Chem. 267, 19596–19599] contains multiple isoforms. We previously cloned NCδ from bovine brain and showed high expression in neuronal tissues [Okazaki, Watanabe, Ando, Hagiwara, Terasawa and Hidaka (1992) Biochem. Biophys. Res. Commun. 185, 147–153]. We report here the molecular cloning and expression of a cDNA encoding bovine brain NCα. The translated bovine protein is 191 amino acids long and shares 69.1% of its amino acid sequence with NCδ. Recombinant NCα migrates as a single 23 kDa band and exhibits a Ca2+-dependent mobility shift on SDS/PAGE. Analysis of fluorescence emission spectra showed the Ca2+-induced peak at 337 nm. Interestingly, the mobility shift and the fluorescence intensity at 337 nm were larger for NCα than for NCδ. In Ca2+-overlay experiments, however, the apparent affinity of NCα for 45Ca2+ was similar to that of NCδ. Immunohistochemical analysis revealed NCα expression in the granular layer of the rat cerebellar cortex whereas NCδ was found in the Purkinje cell layer. In the rat olfactory bulb, NCα was located in external tufted cells, and NCδ was found in the periglomerular cells. These data demonstrate that NC isoforms differ in their tissue distribution and conformational changes induced by Ca2+ binding. Thus differential regulation of the two NC isoforms may be involved in control of neuron function.
Molecular cloning and expression of a cDNA encoding an olfactory-specific mouse phenol sulphotransferase
Authors: TAMURA Hiro-omi , HARADA Yuki , MIYAWAKI Atsushi , MIKOSHIBA Katsuhiko , MATSUI Michio
Published: August 2015

Previously we demonstrated the presence of phenol sulphotransferase (P-ST) in mouse nasal cytosols and identified its zonal location in mouse nasal cavity by staining with an antiserum raised against a rat liver P-ST isoenzyme, PSTg. In the present study a cDNA was isolated from a mouse olfactory cDNA library by immunological screening with the antiserum. The isolated cDNA consisted of 1347 bp with a 912 bp open reading frame encoding a 304-residue polypeptide. Both the nucleotide and deduced amino acid sequences of the cDNA were 94% identical with those of a rat liver P-ST isoenzyme, ST1C1. The expressed enzyme in Escherichia coli displayed high P-ST activity towards phenolic odorants such as eugenol and guaiacol, and it showed a high N-hydroxy-2-acetylaminofluorene sulphation activity in comparison with the rat ST1C1 enzyme. These results indicate that the olfactory P-ST encoded by the cDNA is a mouse orthologue of rat ST1C1; however, expression of the olfactory P-ST mRNA is specific for nasal tissues as revealed by reverse transcriptase-mediated PCR (RT–PCR).
Activation of gelatinase–tissue-inhibitors-of-metalloproteinase complexes by matrilysin
Authors: BREDOW Dorothea C. von , CRESS Anne E. , HOWARD Eric W. , BOWDEN Timothy G. , NAGLE Raymond B.
Published: August 2015

Matrilysin, gelatinase A and gelatinase B are matrix metalloproteinases (MMPs) implicated in normal and pathological processes that require remodelling of the extracellular matrix. In human prostate tissue, matrilysin is synthesized in ducts surrounded by inflammatory cells, and focally in prostate carcinoma, but not in normal glands. Gelatinase B expression is restricted to inflammatory cells. Gelatinase A can be found in both benign and malignant prostate tissue. MMP activities are regulated by their transition from latent to activated forms, as well as by the presence of tissue inhibitors of metalloproteinases (TIMPs). We investigated whether matrilysin can activate progelatinases A and B in the presence of their bound inhibitors TIMP2 and TIMP1 respectively. Incubation of progelatinase B–TIMP1 complex with active matrilysin resulted in 78 and 68 kDa active forms, as measured by SDS–PAGE and enzyme activity assays. TIMP-free gelatinase B was also activated by matrilysin. In addition, activation of progelatinase B by matrilysin was demonstrated in the conditioned medium of phorbol ester-treated HT1080 cells, confirming the results obtained in the in vitro experiments. In contrast, matrilysin did not proteolytically cleave gelatinase A–TIMP2 complex, but led to a transient increase in gelatinolytic activity of the proenzyme. Matrilysin did not enhance the autocatalytic conversion of its own proform. The data presented here suggest that matrilysin participates in a proteolytic cascade and can activate gelatinases in the presence of TIMPs.
Characterization of the UDP-glucuronosyltransferase isoenzyme expressed in rat ovary and its regulation by gonadotropins
Authors: BECEDAS Luisa , LUNDGREN Bo , PIERRE Joseph W. De
Published: August 2015

Earlier studies have demonstrated that phenol UDP-glucuronosyltransferase (UGT) activity is up-regulated by pregnant mare's serum gonadotropin (PMSG) in rat ovary, but not liver. This phenomenon was investigated in more detail in the present study. Ovaries and livers of immature rats, rats synchronized with respect to their preovulatory and corpus lutealphases by treatment with PMSG, and mature rats hyperstimulated with PMSG were compared. Under all of these conditions, only one immunoreactive band of UGT, shown to be phenol UGT, was detected in the rat ovary. The effects of oestradiol, progesterone and/or human chorionic gonadotropin (hCG) on the level of phenol UGT in immature rat ovary were also examined. Partial up-regulation was caused by progesterone or oestradiol, together with hCG, whereas progesterone or oestradiol alone had no up-regulating effect. Follicle-stimulating hormone also seemed to be required for the up-regulation in ovaries enriched in corpus luteum. The present findings demonstrate that progesterone is involved in the regulation of phenol UGT in rat ovary by gonadotropins. Regulation by both progesterone and oestradiol was dependent on induction of ovulation and steroidogenesis by luteinizing hormone.
The iron ligand sphere geometry of mammalian 15-lipoxygenases
Authors: KUBAN Ralf Jürgen , WIESNER Rainer , RATHMAN Jörg , VELDINK Gerrit , NOLTING Hans , SOLÉ V. A. , KÜHN Hartmut
Published: August 2015

We investigated the geometry of the iron ligand sphere of the native rabbit 15-lipoxygenase (15-LOX) by X-ray absorption spectroscopy using synchrotron radiation. The soybean LOX-1 was used as a reference compound because its iron ligand sphere is well characterized. For structural information the X-ray absorption spectra were evaluated using the Excurve Program (CCLRC Daresbury Laboratory, Warrington, U.K.). From the positions of the absorption edges and from the intensities of the 1s–3d pre-edge transition peaks a six-coordinate ferrous iron was concluded for the rabbit 15-LOX. Evaluation of the extended region of the absorption spectra suggested six nitrogen and/or oxygen atoms as direct iron ligands, and the following binding distances were determined (means±S.D.; estimated accuracy is ±0.001 nm for bond distances, on the basis of more than 22 X-ray absorption spectra): 0.213±0.001 nm, 0.213±0.001 nm, 0.236±0.001 nm, 0.293±0.001 nm, 0.189±0.001 nm and 0.242±0.001. Lyophilization of the LOX altered the binding distances but did not destroy the octahedral iron ligand sphere. For construction of a structural model of the iron ligand sphere the binding distances extracted from the X-ray spectra were assigned to specific amino acids (His-360, -365, -540, -544 and the C-terminal Ile-662) by molecular modelling using the crystal coordinates of the soybean LOX-1 and of a rabbit 15-LOX–inhibitor complex.
Isolation of a Chinese hamster fibroblast variant defective in dihydroxyacetonephosphate acyltransferase activity and plasmalogen biosynthesis: use of a novel two-step selection protocol
Authors: NAGAN Narasimhan , HAJRA Amiya K. , LARKINS Leslie K. , LAZAROW Paul , PURDUE Edward , RIZZO William B. , ZOELLER Raphael A.
Published: August 2015

We have developed a two-step selection protocol to generate a population of Chinese hamster ovary (CHO) cell variants that are plasmalogen-deficient, but contain intact, functional peroxisomes (plasmalogen-/peroxisome+). This involved sequential exposures of a mutagenized cell population to photodynamic damage by using two different pyrene-labelled sensors, 9-(1´-pyrene)nonanol and 12-(1´-pyrene)dodecanoic acid. By this procedure we generated several isolates, all except one of which displayed a severe decrease in plasmalogen biosynthesis. Further characterization of one of the plasmalogen-deficient isolates, NRel-4, showed that it contained intact, functional peroxisomes. Whole-cell homogenates from NRel-4 displayed severely decreased dihydroxyacetone phosphate acyltransferase, which catalyses the first step in plasmalogen biosynthesis. NRel-4 and another, recently described, plasmalogen-deficient cell line, NZel-1 [Nagan, Hajra, Das, Moser, Moser, Lazarow, Purdue and Zoeller (1997) Proc. Natl. Acad. Sci. U.S.A. 94, 4475–4480] were hypersensitive to singlet oxygen, supporting the notion of plasmalogens as radical oxygen scavengers. Wild-type-like resistance could be conferred on NRel-4 upon restoration of plasmalogen content by supplementation with a bypass compound, sn-1-hexadecylglycerol. NRel-4 and other plasmalogen-/peroxisome+ strains will allow us to examine further the role of ether lipids in cellular functions without complications associated with peroxisome deficiency, and might serve as an animal cell model for certain forms of the human genetic disorder rhizomelic chondrodysplasia punctata.
Intermediates in the catalytic cycle of lentil (Lens esculenta) seedling copper-containing amine oxidase
Authors: MEDDA Rosaria , PADIGLIA Alessandra , BELLELLI Andrea , SARTI Paolo , SANTANCHÈ Stefano , FINAZZI AGRÒ Alessandro , FLORIS Giovanni
Published: August 2015

Spectrophotometry and rapid-scanning stopped-flow spectroscopy have been used to investigate the visible absorbance changes that occur in the course of the reduction of lentil (Lens esculenta) seedling amine oxidase by substrate. The catalytic cycle of the enzyme employs several intermediates but, owing to kinetic limitations, some of them were not identified in previous studies. In this study we have examined several substrates, either rapidly reacting (e.g. putrescine) or slowly reacting (e.g. γ-aminobutanoic acid). Two forms of the enzyme, namely the Cu(I)-aminoresorcinol and quinone ketimine derivatives, whose characterization was elusive in previous studies, have been identified and assigned an optical spectrum. Moreover the reduced form of the enzyme is shown to be an equilibrium mixture of two species, the Cu(I)-semiquinolamine radical and Cu(II)-aminoresorcinol; these have been resolved by pH dependence and assigned spectra as well as a second-order rate constant for the reaction with oxygen. Thus the results presented here identify all the catalytic intermediates suggested by the chemical nature of the coenzyme and define their spectroscopic and reactivity properties.
Oxidative scission of plant cell wall polysaccharides by ascorbate-induced hydroxyl radicals
Authors: FRY Stephen C.
Published: August 2015

Scission of plant cell wall polysaccharides in vivo has generally been assumed to be enzymic. However, in the presence of l-ascorbate, such polysaccharides are shown to undergo non-enzymic scission under physiologically relevant conditions. Scission of xyloglucan by 1 mM ascorbate had a pH optimum of 4.5, and the maximum scission rate was reached after a 10–25-min delay. Catalase prevented the scission, whereas added H2O2 (0.1–10 mM) increased the scission rate and shortened the delay. Ascorbate caused detectable xyloglucan scission above approx. 5 µM. Dehydroascorbate was much less effective. Added Cu2+ (> 0.3 µM) also increased the rate of ascorbate-induced scission; EDTA was inhibitory. The rate of scission in the absence of added metals appeared to be attributable to the traces of Cu (2.8 mg·kg-1) present in the xyloglucan. Ascorbate-induced scission of xyloglucan was inhibited by radical scavengers; their effectiveness was proportional to their rate constants for reaction with hydroxyl radicals (•OH). It is proposed that ascorbate non-enzymically reduces O2 to H2O2, and Cu2+ to Cu+, and that H2O2 and Cu+ react to form •OH, which causes oxidative scission of polysaccharide chains. Evidence is reviewed to suggest that, in the wall of a living plant cell, Cu+ and H2O2 are formed by reactions involving ascorbate and its products, dehydroascorbate and oxalate. Systems may thus be in place to produce apoplastic •OH radicals in vivo. Although •OH radicals are often regarded as detrimental, they are so short-lived that they could act as site-specific oxidants targeted to play a useful role in loosening the cell wall, e.g. during cell expansion, fruit ripening and organ abscission.
Binding of human α-thrombin to platelet GpIb: energetics and functional effects
Authors: CRISTOFARO Raimondo de , CANDIA Erica de , CROCE Giovanni , MOROSETTI Roberta , LANDOLFI Raffaele
Published: August 2015

Thrombin interaction with platelet glycocalicin (GC), the 140 kDa extracytoplasmic fragment of the membrane glycoprotein Ib, was investigated by using a solid-phase assay. Thrombin bound to GC-coated polystyrene wells was detected by measuring the hydrolysis of a chromogenic substrate. The monoclonal antibody LJ-Ib10, which specifically binds to the thrombin-binding site of GC, could displace thrombin from immobilized GC, whereas the monoclonal antibody LJ-Ib1, which interacts with the von Willebrand factor-binding domain of GC, did not affect thrombin binding to GC. Competitive inhibition of thrombin binding to immobilized GC was also observed using GC in solution or ligands that bind to the thrombin heparin-binding site, such as heparin and prothrombin fragment 2. Furthermore functional experiments demonstrated that GC binding to thrombin competes with heparin for thrombin inactivation by the antithrombin III–heparin complex as well. Thrombin–GC interaction was also studied as a function of temperature over the range 4–37 °C. A large negative heat capacity change (ΔCp), of -4.14±0.8 kJ·mol-1·K-1, was demonstrated to dominate the thermodynamics of thrombin–GC complex-formation. Finally it was demonstrated that GC binding to thrombin can allosterically decrease the enzyme affinity for hirudin via a simultaneous decrease in association rate and increase in the dissociation velocity of the enzyme–inhibitor adduct. Together these observations indicate the GC binding to the heparin-binding domain of thrombin is largely driven by a hydrophobic effect and that such interaction can protect the enzyme from inhibition by the heparin–anti-thrombin III complex.