bluuebunny/crossref_metadata_2025 huggingface dataset:
Light-dependent GTP-binding proteins in squid photoreceptors
Authors: Robinson P R , Wood S F , Szuts E Z , Fein A , Hamm H E , Lisman J E
Published: August 2015

Previous biochemical and electrophysiological evidence suggests that in invertebrate photoreceptors, a GTP-binding protein (G-protein) mediates the actions of photoactivated rhodopsin in the initial stages of transduction. We find that squid photoreceptors contain more than one protein (molecular masses 38, 42 and 46 kDa) whose ADP-ribosylation by bacterial exotoxins is light-sensitive. Several lines of evidence suggest that these proteins represent distinct alpha subunits of G-proteins. (1) Pertussis toxin and cholera toxin react with distinct subsets of these polypeptides. (2) Only the 42 kDa protein immunoreacts with the monoclonal antibody 4A, raised against the alpha subunit of the G-protein of vertebrate rods [Hamm & Bownds (1984) J. Gen. Physiol. 84. 265-280]. (3) In terms of ADP-ribosylation, the 42 kDa protein is the least labile to freezing. (4) Of the 38 kDa and 42 kDa proteins, the former is preferentially extracted with hypo-osmotic solutions, as demonstrated by the solubility of its ADP-ribosylated state and by the solubility of the light-dependent binding of guanosine 5′-[gamma-thio]triphosphate. The specific target enzymes for the observed G-proteins have not been established.
Coupling of muscarinic receptors to the mobilization of intracellular Ca2+ stores in permeabilized SH-SY5Y human neuroblastoma cells
Authors: Wojcikiewicz R J H , Safrany S T , Challiss R A J , Strupish J , Nahorski S R
Published: August 2015

Intracellular stores of Ca2+ were mobilized transiently by carbachol in suspensions of electrically permeabilized SH-SY5Y cells. The kinetics and the dose-dependence of this mobilization paralleled carbachol-induced increases in inositol 1,4,5-trisphosphate (InsP3) mass [for both parameters EC50 (concn. giving half-maximal response) = 60-70 microM]. Guanosine 5′-[gamma-thio]triphosphate enhanced the maximal effect and the potency of carbachol on Ca2+ mobilization and InsP3 mass, but caused separation of the dose-response curves (EC50 = 0.6 microM and 5.6 microM respectively). These data show that functional coupling of muscarinic receptors to Ca2+ mobilization can be maintained after permeabilization, reveal major effects of guanine nucleotides on agonist-induced Ca2+ mobilization and provide a basis for explanation of discrepancies between agonist potency on InsP3 concentration and Ca2+ mobilization in intact cells.
Cyclic AMP agonists induce the phosphorylation of phospholipase C-τ and of a 76 kDa protein co-precipitated by anti-(phospholipase C-τ) monoclonal antibodies in BALB/c-3T3 cells. Relationship to inositol phosphate formation
Authors: Olashaw N E , Rhee S G , Pledger W J
Published: August 2015

Previous studies have demonstrated enhanced phosphorylation of phospholipase C-tau (PLC-tau), a key regulatory enzyme in phosphoinositide metabolism, in cells treated with platelet-derived growth factor (PDGF) and epidermal growth factor, both of which act via specific receptor tyrosine kinases. Our studies on BALB/c-3T3 cells show that agents that promote cellular cyclic AMP accumulation also increase the phosphorylation, specifically the serine phosphorylation, of this enzyme. Increased phosphorylation of PLC-t (2-3-fold) was evident within 5-10 min of addition of isobutylmethylxanthine (IBMX) and either cholera toxin or forskolin to cells, and persisted for at least 3 h. Treatment of cells with cyclic AMP agonists also enhanced, with similar kinetics, the phosphorylation of a 76 kDa protein co-precipitated by anti-PLC-tau monoclonal antibodies. Brief exposure of cells to cholera toxin/IBMX or forskolin/IBMX decreased inositol phosphate formation induced by the GTP-binding protein (G-protein) activator aluminium fluoride by approx. 50%, but was without effect on PDGF-stimulated inositol phosphate formation. These findings suggest that PLC-tau, and perhaps the 76 kDa co-precipitated protein, are substrates of cyclic AMP-dependent protein kinase in BALB/c-3T3 cells: however, the lack of effect of cyclic AMP elevation on PDGF-stimulated inositol phosphate formation indicates that the intrinsic activity of PLC-tau is unaltered by cyclic AMP-mediated phosphorylation.
Inositol 1,3,4,5-tetrakisphosphate-induced release of intracellular Ca2+ in SH-SY5Y neuroblastoma cells
Authors: Gawler D J , Potter B V , Nahorski S R
Published: August 2015

Inositol-polyphosphate-induced Ca2+ mobilization was investigated in saponin-permeabilized SH-SY5Y human neuroblastoma cells. Ins(1,4,5)P3 induced a dose-related release from intracellular Ca2+ stores with an EC50 (concn. giving half-maximal effect) of 0.1 microM and a maximal release of 70%. Ins(1,3,4)P3, DL-Ins(1,4,5,6)P4 and Ins(1,3,4,5,6)P5 did not evoke Ca2+ mobilization in these cells when used at concentrations up to 10 microM. However, Ins(1,3,4,5)P4 was found to release Ca2+ in a dose-related manner, but the response was dependent on the source of Ins(1,3,4,5)P4 used. When commercially available D-Ins(1,3,4,5)P4 was used, the EC50 and maximal response values were 1 microM and 50% respectively, compared with values for chemically synthesized DL-Ins(1,3,4,5)P4 of 2 microM and 25%. The enhanced maximal response of commercial D-Ins(1,3,4,5)P4 was decreased by pretreatment with rat brain crude Ins(1,4,5)P3 3-kinase and was therefore concluded to be indicative of initial Ins(1,4,5)P3 contamination of the Ins(1,3,4,5)P4 preparation. When metabolism of DL-Ins(1,3,4,5)P4 (10 microM) in these cells at 25 degrees C was investigated by h.p.l.c., substantial amounts of Ins(1,4,5)P3 (0.2 microM) and Ins(1,3,4)P3 (0.8 microM) were found to be produced within 3 min. Analysis of DL-Ins(1,3,4,5)P4 incubation with cells at 4 degrees C, however, indicated that metabolism had been arrested ([3H]Ins(1,4,5)P3 detection limits were estimated to be approx. 0.01 microM). When chemically synthesized DL-Ins(1,3,4,5)P4 and incubation conditions of low temperature were used, the Ca2(+)-releasing properties of this compound were established to be 1 microM and 19% for the EC50 and maximal response values respectively. The results obtained strongly suggest that Ins(1,3,4,5)P4 alone has the ability to release intracellular Ca2+. However, in the presence of sub-maximal concentrations of Ins(1,4,5)P3, Ca2+ release appears to be synergistic with Ins(1,3,4,5)P4, but at supramaximal concentrations not even additive effects are observed.
Human liver steroid sulphotransferase sulphates bile acids
Authors: Radominska A , Comer K A , Zimniak P , Falany J , Iscan M , Falany C N
Published: August 2015

The sulphation of bile acids is an important pathway for the detoxification and elimination of bile acids during cholestatic liver disease. A dehydroepiandrosterone (DHEA) sulphotransferase has been purified from male and female human liver cytosol using DEAE-Sepharose CL-6B and adenosine 3′,5′-diphosphate-agarose affinity chromatography [Falany, Vazquez & Kalb (1989) Biochem. J. 260, 641-646]. Results in the present paper show that the DHEA sulphotransferase, purified to homogeneity, is also reactive towards bile acids, including lithocholic acid and 6-hydroxylated bile acids, as well as 3-hydroxylated short-chain bile acids. The highest activity towards bile acids was observed with lithocholic acid (54.3 +/- 3.6 nmol/min per mg of protein); of the substrates tested, the lowest activity was detected with hyodeoxycholic acid (4.2 +/- 0.01 nmol/min per mg of protein). The apparent Km values for the enzyme are 1.5 +/- 0.31 microM for lithocholic acid and 4.2 +/- 0.73 microM for taurolithocholic acid. Lithocholic acid also competitively inhibits DHEA sulphation by the purified sulphotransferase (Ki 1.4 microM). No evidence was found for the formation of bile acid sulphates by sulphotransferases different from the DHEA sulphotransferase during purification work. The above results suggest that a single steroid sulphotransferase with broad specificity encompassing neutral steroids and bile acids exists in human liver.
Comparison of the effects of various amino acids on glycogen synthesis, lipogenesis and ketogenesis in isolated rat hepatocytes
Authors: Baquet A , Lavoinne A , Hue L
Published: August 2015

Several amino acids were found to stimulate glycogen synthesis and lipogenesis, and to inhibit ketogenesis in isolated rat hepatocytes. When hepatocytes were incubated in the presence of 20 mM-glucose, the amino acids could be classified in decreasing order of efficiency as follows: glutamine and proline, alanine, aminoisobutyric acid, asparagine and histidine for stimulation of glycogen synthesis; glutamine, proline and alanine for stimulation of lipogenesis; proline and glutamine for inhibition of ketogenesis. The study of the time course revealed that the rates were not linear and were preceded by a lag period. In all conditions studied, glutamine and proline were found to have similar quantitative effects on glycogen synthesis and lipid metabolism. However, their effects differ qualitatively. Indeed, the effects of proline on glycogen synthesis, lipogenesis and glutamate and aspartate content were faster. Moreover, proline increased the hydroxybutyrate/acetoacetate ratio, whereas glutamine did not change it. Incubation of hepatocytes with aminoisobutyric acid or under hypo-osmotic conditions, which increased cell volume and mimicked the amino acid-induced stimulation of glycogen synthesis, had little effect on lipogenesis. In hepatocytes incubated without glucose, ketogenesis was inhibited, in decreasing order of efficiency, by alanine, asparagine, glutamine and proline. Under these conditions, glutamine increased, alanine decreased and asparagine did not affect the concentration of malonyl-CoA. This indicates that the latter cannot be responsible for the inhibition of ketogenesis by alanine and asparagine.
Protein kinase C and cyclic AMP regulate reversible exposure of binding sites for fibrinogen on the glycoprotein IIB-IIIA complex of human platelets
Authors: van Willigen G , Akkerman J W N
Published: August 2015

Platelet aggregation is mediated via binding of fibrinogen to sites on the membrane glycoprotein IIB-IIIA complex which become exposed when the cells are stimulated. We report here evidence of a dynamic and reversible exposure of binding sites for fibrinogen. In the absence of fibrinogen, exposed sites (B*) gradually lose their capacity to bind fibrinogen and close (Bo). On stimulation with platelet-activating factor (PAF, 500 nM) at 22 degrees C, closing of B* is enhanced by agents that raise cyclic AMP levels (10 ng of prostaglandin I2/ml; 5 mM-theophylline), inhibit protein kinase C (PKC; 25 microM-sphingosine; 1 microM-staurosporine), or disrupt the energy supply (30 mM-2-deoxy-D-glucose + 1 mM-CN-), or by raising the temperature to 37 degrees C. Conversely, activation of PKC 1 microM-1,2-dioctanoyl-sn-glycerol; 55 nM-phorbol 12-myristate 13-acetate) and an increase in intracellular [Ca2+] (100 nM-ionomycin + extracellular Ca2+) oppose the disappearance of B*. Phosphorylation of the 47 kDa protein illustrates the tight coupling between PKC and B* under all conditions tested, except when the cyclic AMP level is raised, and B* is converted to Bo without affecting PKC activity. Although the increase in PKC activity is much smaller with ADP or even absent upon stimulation with adrenaline, the control of B* is equally sensitive to modulation of cyclic AMP and PKC activity. We conclude that PAF, ADP and adrenaline regulate exposure of fibrinogen binding sites through a common mechanism consisting of two independent pathways, one dominated by PKC and the other by an as yet unidentified cyclic AMP-sensitive step.
Solubilization and purification of a putative quisqualate-sensitive glutamate receptor from crustacean muscle
Authors: Gray S R , Batstone F R , Santiapillai N F , Richardson P J
Published: August 2015

Two high-affinity glutamate-binding proteins have been solubilized and purified from crustacean muscle membranes. L-[3H]Glutamate binding to intact membranes gave IC50 values (concn. giving 50% inhibition) of 0.35 microM for glutamate. 9.0 microM for quisqualate and 36.2 microM for ibotenic acid. Kainate, domoate, N-methyl-D-aspartate, alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionate, D-glutamate and L-aspartate were poor inhibitors of glutamate binding. The two protein were purified approx. 600-fold from muscle membranes by use of concanavalin A and gel-filtration column chromatography. One protein showed an apparent Stokes radius of 5.4 nm, as determined by gel filtration, with a subunit of Mr 74000 as shown by SDS/polyacrylamide-gel electrophoresis. Analysis of glutamate binding to this protein revealed a KD of 2.4 microM and a Bmax, of 15.5 nmol/mg, and a k1 for quisqualate inhibition of glutamate binding of 1.0 microM. The second protein was eluted anomalously from the gel-filtration column and showed a subunit of Mr 65000 on SDS/polyacrylamide gels. Glutamate binding to this protein showed a KD of 1.3 microM and a Bmax, of 14.5 nmol/mg, and was only slightly sensitive to the presence of quisqualate (Ki = 0.8 mM). Glutamate binding to both proteins was insensitive to kainate. It is concluded that the Mr-74000 protein is a prime candidate for the quisqualate-sensitive receptor of the crustacean neuromuscular junction.
Glucose utilization by interscapular brown adipose tissue in vivo during nutritional transitions in the rat
Authors: Holness M J , Liu Y L , Beech J S , Sugden M C
Published: August 2015

Glucose utilization indices (GUI) of interscapular brown adipose tissue (IBAT) declined by 84% after 48 h starvation. Two-thirds of the overall response was observed within 6 h, correlating with decreased insulin concentrations. Re-feeding 48 h-starved rats restored insulin concentrations and evoked a rapid 15-fold increase in IBAT GUI. GUI values after re-feeding were markedly higher than those observed at equivalent insulin concentrations in control post-absorptive rats.
Nitric and nitrous oxide reductases are active under aerobic conditions in cells of Thiosphaera pantotropha
Authors: Bell L C , Ferguson S J
Published: August 2015

Use of Clark-type electrodes has shown that, in cells of Thiosphaera pantotropha, the nitrous oxide reductase is active in the presence of O2, and that the two gases involved (N2O, O2) are reduced simultaneously, but with mutual inhibition. Reduction of nitrate, or nitrite, to N2O under aerobic conditions involves NO as an intermediate, as judged by trapping experiments with the ferric form of extracellular horse heart cytochrome c and the demonstration that the cells possess a nitric oxide reductase activity. The overall conversion of nitrate to N2, the process of denitrification, under aerobic conditions, is thus not prevented by reaction of NO with O2 and depends upon a nitrous oxide reductase system which differs from that in other organisms by being neither directly inhibited nor inactivated by O2.
Exocytosis in neurohypophysial nerve terminals is not coupled to protein kinase C translocation
Authors: Nordmann J J , Stuenkel E L , Malviya A N
Published: August 2015

Protein kinase C (PKC) has been implicated in the mechanism of exocytosis, although various studies have been unable to pinpoint actual translocation or activation of PKC during exocytosis. We have studied, in neurohypophysial nerve endings, intracellular Ca2+ levels, secretion of neuropeptides and PKC translocation. Neurohormone secretion was triggered by K(+)-induced or electrically induced depolarization in both the absence and the presence of phorbol esters. PKC was translocated from the cytosol to the membrane on electrical stimulation or K+ depolarization, but not to the extent obtained with phorbol ester. Data are presented clearly demonstrating that the translocation of PKC from cytosol to membrane is not required for exocytosis, nor does it alter in any way neuropeptide release from neurohypophysial nerve terminals.
A Fischer rat substrain deficient in dipeptidyl peptidase IV activity makes normal steady-state RNA levels and an altered protein. Use as a liver-cell transplantation model
Authors: Thompson N L , Hixson D C , Callanan H , Panzica M , Flanagan D , Faris R A , Hong W J , Hartel-Schenk S , Doyle D
Published: August 2015

Dipeptidyl peptidase IV (DPPIV) is a serine exoproteinase expressed at high levels in epithelial cells of kidney, liver and small intestine. Recently Watanabe, Kohima & Fujimoto [(1987) Experientia 43, 400-401] and Gossrau et al. [(1990) Histochem. J. 22, 172-173] reported that Fischer 344 rats are deficient in this enzyme. We have examined DPPIV expression in Fischer 344 rats available from U.S. and German suppliers and find that livers of the U.S. Fischer rats, in contrast with their German counterparts, express active DPPIV (D+). Northern analysis of liver RNA showed comparable levels of 3.4 kb and 5.6 kb DPPIV transcripts in both D+ rats from the U.S. and German (D-) rats. Monoclonal antibody (MAb) 236.3 to DPPIV immunoprecipitated at 150 kDa enzymically active (105 kDa, denatured) protein from surface-labelled D+ hepatocytes and reacted with canalicular and sinusoidal membranes (as shown by immunofluorescence microscopy). MAb 236.3 failed to immunoprecipitate a labelled peptide from D- cell extract or to stain D- liver sections. Polyclonal antibody (PAb) specific for DPPIV immunoprecipitated an enzymically active peptide from D+ hepatocyte extracts and a smaller, inactive peptide from D- hepatocyte extracts. Peptide maps of DPPIV immunoprecipitated from D+ extracts with MAb 236.3 and PAb were identical, but differed from that of the D- hepatocyte component recognized by PAb. The molecular basis of the DPPIV deficiency in the D- rats thus appears to be the translation of an enzymically inactive protein missing the epitope recognized by MAb 236.3. We have exploited these D- rats as hosts for syngeneic transplantation of liver cells from D+ Fischer rats. DPPIV expression is stable in the transplanted cells and allows them to be readily distinguished from the surrounding D- tissue.
Purification and properties of malonyl-CoA synthetase from Rhizobium japonicum
Authors: Kim Y S , Chae H Z
Published: August 2015

A novel malonyl-CoA synthetase was found in Rhizobium japonicum bacteroid of the soybean nodule. The levels of the enzyme in the free-living cells grown on a variety of carbon sources including glucose were similar, indicating that this enzyme is not inducible. The malonyl-CoA synthetase from glucose-grown Rhizobium japonicum was purified to homogeneity. The Mr of the enzyme was determined to be 58,000 by gel filtration on a Sephacryl S-300 and by SDS/PAGE respectively, indicating a single polypeptide enzyme. N-Terminal amino acid of the enzyme was methionine but the enzyme preparation contained about 40% de-methionylated protein. The enzyme catalyses the formation of malonyl-CoA, AMP and PPi directly from malonate, CoA and ATP in the presence of Mg2+. High substrate specificity on malonate and ATP was revealed, but Mn2+ could be substituted for Mg2+ without any difference in activity. Optimum pH was 7.9. Kinetic constants, Km and Vmax, for malonate, CoA and ATP were 200 microM and 21.3 mumol/min per mg, 87 microM and 41.7 mumol/min per mg, and 33.3 microM and 29.4 mumol/min per mg respectively. Succinate inhibited the enzyme noncompetitively, whereas AMP and ADP inhibited competitively. Diethylpyrocarbonate and pyridoxal-5′-phosphate severely inhibited the enzyme, but iodoacetamide, p-chloromercuriphenylsulphonate, N-acetylimidazole and phenylglyoxal did not.
Fibroblast adhesion to recombinant tropoelastin expressed as a protein A-fusion protein
Authors: Grosso L E , Parks W C , Wu L J , Mecham R P
Published: August 2015

A bovine tropoelastin cDNA encoding exons 15-36 that includes the elastin-receptor binding site was expressed in Escherichia coli as a fusion protein with Protein A from Staphylococcus aureus. After isolation of the fusion protein by affinity chromatography on Ig-Sepharose, the tropoelastin domain was separated from plasmid-pR1T2T-encoded Protein A (Protein A') by CNBr cleavage. Cell-adhesion assays demonstrated specific adhesion to the recombinant tropoelastin. Furthermore, the data indicate that interactions involving the bovine elastin receptor mediate nuchalligament fibroblast adhesion to the recombinant protein. In agreement with earlier studies of fibroblast chemotaxis to bovine tropoelastin, nuchal-ligament fibroblast adhesion demonstrated developmental regulation of the elastin receptor.
‘Slow-binding’ sixth-ligand inhibitors of cytochrome P-450 aromatase. Studies with 19-thiomethyl- and 19-azido-androstenedione
Authors: Wright J N , Slatcher G , Akhtar M
Published: August 2015

The progress curves for the inhibition of aromatase by 19-thiomethylandrostenedione and 19-azidoandrostenedione were found to be non-linear where the extent of inhibition increased with time. Further experiments enabled these compounds to be classified as ‘slow-binding’ inhibitors of aromatase. The phenomenon was attributed to the formation of an initial E.I complex that rearranged to another species (E.I*) in which the interaction between the enzyme and inhibitor had been maximized, giving rise to tighter binding. When 19-thiomethylandrostenedione was used as the inhibitor the t0.5 (half-time) for the dissociation of E.I* was calculated to be 12.6 min with Ki and Ki* values of 2.4 and 1.4 nM respectively. In the case of 19-azidoandrostenedione, the two separate dissociation constants were not determined, and a single Ki value of 5 nM was obtained. The conclusions drawn from kinetic studies were confirmed by absorption spectrometry, when time-dependent formation of complexes between aromatase and either 19-thiomethylandrostenedione or 19-azidoandrostenedione were observed by the formation of ‘Type II’ spectra. The two complexes respectively had maxima at 429 and 418 nm. The spectral data suggested that the two inhibitors interact with the haem iron of aromatase, forming hexaco-ordinated species for which structural models are presented.
A new fluorescent probe for the equilibrative inhibitor-sensitive nucleoside transporter. 5′-S-(2-aminoethyl)-N6-(4-nitrobenzyl)-5′-thioadenosine (SAENTA)-x2-fluorescein
Authors: Wiley J S , Brocklebank A M , Snook M B , Jamieson G P , Sawyer W H , Craik J D , Cass C E , Robins M J , McAdam D P , Paterson A R
Published: August 2015

The N6-(4-nitrobenzyl) derivative of adenosine is a tight-binding inhibitor of the equilibrative inhibitor-sensitive nucleoside transporter of mammalian cells. A fluorescent ligand for this transporter has been synthesized by allowing an adenosine analogue. 5′-S-(2-aminoethyl)-N6-(4-nitrobenzyl)-5′-thioadenosine (SAENTA), to react with fluorescein isothiocyanate. The purified adduct had a SAENTA/fluorescein molar ratio of 0.92:1 calculated from its absorption spectrum. The intensity of fluorescent emission from the SAENTA-chi 2-fluorescein adduct was 30% that of fluorescein isothiocyanate (chi 2 is the number of atoms in the linkage between fluorescein and SAENTA). SAENTA-chi 2-fluorescein inhibited the influx of nucleosides into cultured leukaemic cells with an IC50 (total concentration of inhibitor producing 50% inhibition) of 40 nM. The adduct inhibited the binding of [3H]nitrobenzylthioinosine ([3H]NBMPR) with half-maximal inhibition at 50-100 nM. Mass Law analysis of the competitive-binding data suggested the presence of two classes of sites for [3H]NBMPR binding, only one of which was accessible to SAENTA-chi 2-fluorescein. Flow cytometry was used to analyse equilibrium binding of SAENTA-chi 2-fluorescein to leukaemic cells and a Kd of 6 nM was obtained. SAENTA-chi 2-fluorescein is a high-affinity ligand for the equilibrative inhibitor-sensitive nucleoside transporter which allows rapid assessment of transport capacity by flow cytometry.
The potency of inducers of NAD(P)H:(quinone-acceptor) oxidoreductase parallels their efficiency as substrates for glutathione transferases. Structural and electronic correlations
Authors: Spencer S R , Xue L A , Klenz E M , Talalay P
Published: August 2015

Induction of glutathione transferases (EC. 2.5.1.18), NAD(P)H:(quinone-acceptor) oxidoreductase (EC 1.6.99.2; quinone reductase) and other detoxification enzymes is a major mechanism for protecting cells against the toxicities of electrophiles, including many carcinogens. Although inducers of these two enzymes belong to many different chemical classes, they nevertheless contain (or acquire by metabolism) electrophilic centres that appear to be essential for inclusive activity, and many inducers are Michael reaction acceptors [Talalay, De Long & Prochaska (1988) Proc. Natl. Acad. Sci. U.S.A., 85, 8261-8265]. The inducers therefore share structural and electronic features with glutathione transferase substrates. To define these features more precisely, we examined the inductive potencies (by measuring quinone reductase in murine hepatoma cells) of two types of glutathione transferase substrates: a series of 1-chloro-2-nitrobenzenes bearing para-oriented electron-donating or -withdrawing substituents and a wide variety of other commonly used and structurally unrelated glutathione transferase substrates. We conclude that virtually all glutathione transferase substrates are inducers, and their potencies in the nitrobenzene series correlate linearly with the Hammett sigma or sigma- values of the aromatic substituents, precisely as previously reported for their efficiencies as glutathione transferase substrates. More detailed information on the electronic requirements for inductive activity was obtained with a series of methyl trans-cinnamates bearing electron-withdrawing or -donating substituents on the aromatic ring, and in which the electronic densities at the olefinic and adjacent carbon atoms were measured by 13C n.m.r. Electron-withdrawing meta-substituents markedly enhance inductive potency in parallel with their increased non-enzymic reactivity with GSH. Thus, methyl 3-bromo-, 3-nitro- and 3-chloro-cinnamates are 21, 14 and 8 times more potent inducers than the parent methyl cinnamate. This finding permits the design of more potent inducers, which are important for elucidation of the molecular mechanisms of induction.
Metalloendopeptidase QG. Isolation from Escherichia coli and characterization
Authors: Polgár L , Szigetvári A , Löw M , Kóródi I , Balla E
Published: August 2015

A new proteinase, which preferentially cleaves the Gln-Gly bond, was isolated from Escherichia coli. Because of this narrow specificity, the enzyme was called metalloendopeptidase QG. The proteinase is a monomer and consists of a single polypeptide chain of Mr 67,000, which is significantly smaller than the other known metalloendopeptidases of E. coli. It is found in the cytoplasm, but not in the periplasm. The enzyme cleaves the substrate benzyloxycarbonyl-Gln-Gly-Pro 2-naphthylamide between the glutamine and glycine residues, as well as its extended homologues including a nonapeptide, but it does not hydrolyse either the oxidized A and B chains of insulin or azo-casein. The pH-dependence of substrate hydrolysis gives a bell-shaped curve with pK1 = 6.6 and pK2 = 8.8. The metallopeptidase is inhibited in Tris and imidazole buffers, the basic components of which are presumably liganded to the essential Zn2+ ion. 2-Aminobenzoyl-Gln-Gly-Pro 2-naphthylamide, designed as a fluorescent substrate for the metallopeptidase, proved to be a strong inhibitor. Bestatin, an inhibitor of aminopeptidases in the micromolar concentration range, inhibits the metalloendopeptidase only in the millimolar concentration range. Captopril, the widely used inhibitor of angiotensin-converting enzyme, is a fairly good inhibitor of the metalloendopeptidase. The simplest inhibitor that can be used to protect recombinant proteins from degradation by the metalloendopeptidase may be EDTA, which is effective at low millimolar concentration.
Characterization and organization of the genes encoding the A-, B- and C-chains of human complement subcomponent C1q. The complete derived amino acid sequence of human C1q
Authors: Sellar G C , Blake D J , Reid K B
Published: August 2015

A partial cDNA clone for the A-chain of human complement subcomponent C1q was isolated from a monocyte library. Use of the A-chain cDNA clone, and a previously characterized B-chain cDNA clone [Reid (1985) Biochem. J. 231, 729-735] allowed the isolation of overlapping cosmid clones that were shown to contain the genes encoding the A-, B- and C-chains of human C1q. The three genes were found to be aligned, 5′→3′, in the same orientation, in the order A-C-B on a 24 kb stretch of DNA on chromosome 1p. The A-, B- and C-chain genes are approx. 2.5, 2.6 and 3.2 kb long respectively, and each contains one intron, located within a codon for a glycine residue found half-way along the collagen-like region present in each chain. These glycine residues are located just before the point where the triple-helical portions of the C1q molecule appear to bend when viewed in the electron microscope. Southern-blot analyses indicated that there is only one gene per chain, and preliminary examination of genomic DNA from several C1q-deficient patients showed no evidence for major deletions or insertions within the A-, B- or C-chain genes. The DNA sequence of the coding region of the C-chain gene allows the completion of the entire derived amino acid sequence for the human C1q molecule. The globular, C-terminal, regions of the chains of C1q show a strong similarity in amino acid sequence to the non-collagen-like, C-terminal, regions of the type VIII and type X collagens, indicating structural and evolutionary relationships between these three molecules.
Effects of directed mutagenesis on conserved arginine residues in a human Class Alpha glutathione transferase
Authors: Stenberg G , Board P G , Carlberg I , Mannervik B
Published: August 2015

Glutathione transferase (GST) epsilon (also known as GST2 or GST B1B1), the major Class Alpha GST in human liver has been subjected to oligonucleotide-directed site-specific mutagenesis. Four arginine residues, R13, R20, R69 and R187, of which all but R69 are strictly conserved through GST Classes Alpha, Mu and Pi have been replaced by Ala. The mutant enzymes have been expressed in Escherichia coli, purified by affinity chromatography and characterised. Compared with the wild-type enzyme, all mutant GSTs had altered catalytic properties. All mutants had decreased specific activity with 1-chloro-2,4-dinitrobenzene (CDNB). Mutants R13A, R69A and R187A also showed decreased activities with other substrates such as cumene hydroperoxide (CuOOH) and androstenedione. In contrast, mutant R20A had an increased peroxidase activity and an isomerase activity essentially the same as that of the wild-type GST. With the substrates used, kcat./Km values were decreased for all mutant GSTs. Increases in the [S0.5] values were most significant for glutathione (GSH), while values for CDNB and CuOOH were less markedly affected. Thus, various kinetic data indicate that the GSH affinity has been reduced by the mutations and that this loss of affinity is linked to the decreased specific activities. Inhibition studies showed an increased sensitivity towards S-hexyl-GSH; this was particularly marked for mutant R69A. Mutant R20A had a lowered [I50] value but, in contrast, also the highest [I80] value as compared with the wild-type enzyme. Towards bromosulphophthalein, mutants R20A and R69A had a markedly increased sensitivity, about 35-fold in comparison with the wild-type. The inhibition properties of mutant R187A were similar to those of the wild-type enzyme and the properties of mutant R13A were in between. The increased sensitivity to S-hexyl-GSH, in contrast with the decreased affinity for GSH, was suggested to be due to an altered distribution between conformational states of the enzyme induced by the mutations. The arginine residues in positions 13, 20 and 69 all seem to be important for the catalytic properties of GST. Further, the inhibition studies indicate a role of arginine residues in the stabilisation of conformational states of the enzyme.
Measurement of picomole amounts of any inositol phosphate isomer separable by h.p.l.c. by means of a bioluminescence assay
Authors: Prestwich S A , Bolton T B
Published: August 2015

An assay is described which allows the determination of the mass of any individual inositol phosphate (InsP) isomer by combining a popular h.p.l.c. separation method with simple desalting, dephosphorylation and final measurement of Ins liberated using an inositol dehydrogenase-NADH-linked bioluminescence reaction. The limit of sensitivity of this assay is about 1 pmol of Ins. routinely 5 pmol. About 40 mg wet wt. of guinea pig small intestine longitudinal smooth muscle contains 5 pmol of Ins(1,4,5)P3. For Inst(1,3,4)P3 or Ins(1,3,4,5)P4 slightly more smooth muscle is needed, and for major isomers of InsP1 or InsP2 10 mg wet wt. or less of tissue can be used. A 35 mm tissue culture plate with a confluent layer of rat fibroblasts contains about 30 pmol of Ins(1,4,5)P3. The method was applied to the measurement of the masses of Ins1P1. Ins4P1, Ins(1,4)P2, Ins(1,3,4)P3, Ins(1,4,5)P3, Ins(1,3,4,5)P4 and InsP5. The h.p.l.c. elution profiles of radiolabelled InsPS generated from [32P]Pi-labelled human Erythrocytes, [3H]Ins-labelled cultured rat fibroblasts and [3H]Ins-labelled smooth muscle fragments from guinea pig small intestine were compared with the h.p.l.c. elution profiles of their masses.
Human plasma fibronectin. Demonstration of structural differences between the A- and B-chains in the III CS region
Authors: Tressel T , McCarthy J B , Calaycay J , Lee T D , Legesse K , Shively J E , Pande H
Published: August 2015

Fibronectins are a class of cell-adhesion proteins produced from a single gene. The soluble plasma form is synthesized by hepatocytes and the insoluble cellular form by fibroblasts and other cell types. The proteins possess multiple binding domains for macromolecules including collagen, fibrin and heparin along with at least one cell-binding domain. Cellular as well as plasma fibronectins are dimers of similar but not identical polypeptides. Their differences are the result of internal amino acid sequence variability due to alternative RNA splicing in at least three regions (ED-A, ED-B and III CS). We have been studying this polymorphism at the protein level in plasma fibronectin (pFn). Cathepsin D-digested pFn applied to a heparin-agarose column and eluted with an NaCl stepwise gradient (0.1 M, 0.25 M and 0.5 M) released two polypeptides (75 kDa and 65 kDa) in the 0.5 M-NaCl peak. Immunoblots with monoclonal antibodies IST-2 (specific for the C-terminal heparin-binding domain) and AHB-3 (specific for the III CS domain) suggest that both peptides contain the C-terminal heparin-binding (Hep-2) domain, but that only the larger fragment possesses the III CS region. These two polypeptides (75 kDa and 65 kDa) were digested with trypsin, and the resulting peptides were analyzed by fast-atom-bombardment mass spectrometry and compared with the known cDNA-derived peptide sequence. Peptides that were unique to the III CS region were further characterized by micro sequence analysis. The 75 kDa fragment is derived from the A-chain and contains the III CS region (89 amino acid residues) along with the C-terminal heparin-binding (Hep-2) domain and the fibrin-binding (Fib-2) domain. A single galactosamine-based carbohydrate group was detected at Thr-73/74 of the III CS region present in the 75 kDa fragment. The 65 kDa fragment is derived from the B-chain and lacks the entire III CS region but does contain the Hep-2 and Fib-2 domains.
Transport of ascorbic acid and dehydroascorbic acid by pancreatic islet cells from neonatal rats
Authors: Zhou A , Nielsen J H , Farver O , Thorn N A
Published: August 2015

Several amidated biologically active peptides such as pancreastatin, thyrotropin-releasing hormone, pancreatic polypeptide and amylin are produced in endocrine pancreatic tissue which contains the enzyme necessary for their final processing, i.e. peptidylglycine alpha-amidating mono-oxygenase (EC 1.14.17.3). The enzyme needs ascorbic acid for activity as well as copper and molecular oxygen. The present work shows that pancreatic islet cells prepared from overnight cultures of isolated islets from 5-7-day-old rats accumulate 14C-labelled ascorbic acid by a Na(+)-dependent active transport mechanism which involves a saturable process (estimated Km 17.6 microM). Transport was inhibited by ouabain, phloridzin, cytochalasin B, amiloride and probenecid. Glucose inhibited or stimulated uptake, depending on the length of incubation time of the cells. The uptake of dehydroascorbic acid was linearly dependent on concentration. Dehydroascorbic acid was converted to ascorbic acid by an unknown mechanism after uptake. The uptake of both ascorbic acid and dehydroascorbic acid was inhibited by tri-iodothyronine, and uptake of ascorbic acid, but not of dehydroascorbic acid, was inhibited by glucocorticoids. Isolated secretory granules contained a fairly low concentration of iron but a high concentration of copper.
Histidines, histamines and imidazoles as glycosidase inhibitors
Authors: Field R A , Haines A H , Chrystal E J T , Luszniak M C
Published: August 2015

This present study reports the ability of a range of derivatives of L-histidine, histamine and imidazole to act as inhibitors of sweet-almond β-glucosidase, yeast alpha-glucosidase and Escherichia coli β-galactosidase. The addition of a hydrophobic group to the basic imidazole nucleus greatly enhances binding to both the alpha- and β-glucosidases. L-Histidine (beta-naphthylamide (Ki 17 microM) is a potent competitive inhibitor of sweet-almond β-glucosidase as is omega-N-acetylhistamine (K1 35 microM), which inhibits the sweet-almond β-glucosidase at least 700 times more strongly than either yeast alpha-glucosidase or Escherichia coli β-galactosidase, and suggests potential for the development of selective reversible β-glucosidase inhibitors. A range of hydrophobic omega-N-acylhistamines were synthesized and shown to be among the most potent inhibitors of sweet-almond β-glucosidase reported to date.
Changes in insulin-receptor structure associated with trypsin-induced activation of the receptor tyrosine kinase
Authors: Clark S , Eckardt G , Siddle K , Harrison L C
Published: August 2015

The tyrosine kinase of the insulin receptor can be activated by trypsin treatment. The concomitant abolition of insulin binding has been postulated to result from proteolytic destruction of the receptor. A discrepancy between the decrease in insulin binding and receptor immunoreactivity after trypsin treatment led us to investigate more closely the structure of the trypsin-treated receptor. After trypsin treatment of the CHOT cell line, which over-expresses transfected human insulin receptors, insulin binding was significantly decreased, but reactivity with five alpha-subunit monoclonal antibodies was either unaffected or only moderately decreased, indicating that the alpha-subunit was substantially intact. Examination of receptor structure after trypsin treatment, receptor autophosphorylation and gel electrophoresis revealed a single band at 110 kDa in non-reduced gels, comprising a small fragment (21 kDa) of the alpha-subunit linked to the beta-subunit by class II disulphides. When the receptor was radio-labelled with 125I, two additional alpha-subunit bands of 142 kDa and 81 kDa (composed of identical reduced bands) were observed on non-reduced gels, which contained disulphide-linked (class I) fragments. All fragments could be precipitated by antibodies to both alpha- and beta-subunits. However, only antibodies directed towards the N-terminus of the receptor could immunoblot trypsin-treated fragments. Thus activation of the receptor tyrosine kinase by trypsin occurs after cleavage, but not loss of the alpha-subunit. This finding has implications for the mechanism of transmembrane activation of the receptor kinase by insulin.
Interaction of Mg2+ with F0.F1 mitochondrial ATPase as related to its slow active/inactive transition
Authors: Bulygin V V , Vinogradov A D
Published: August 2015

Bovine heart submitochondrial particles incubated with a low concentration of ADP in the presence of Mg2+ and passed through a Sephadex column equilibrated with EDTA exhibit sensitivity of their initial ATPase activity to preincubation with Mg2+. By using particles thus prepared, several characteristics of a Mg(2+)-specific inhibitory site on F0.F1 ATPase were studied. The inhibition was shown to be both time- and Mg(2+)-concentration-dependent, with an equilibrium constant (at infinite time) of 2 x 10(-6) M (25 degrees C, pH 7.5). The dependence of the pseudo-first-order rate constant for the inhibition process on Mg2+ concentration suggests the presence of a single Mg(2+)-binding site with K8 = 1.1 x 10(-4) M. The data obtained are consistent with a two-step mechanism of Mg(2+)-F0.F1 interaction which results in a loss of the ATPase activity; it includes rapid pH-dependent binding of Mg2+ at the site with K8 = 1.1 x 10(-4) M, followed by a slow interconversion of the Mg(2+)-F1 complex into inactive ATPase (kin. = 0.65 min-1, kact. = 0.01 min-1). The Mg(2+)-inhibited ATPase is very slowly (t1/2 approximately 90 min) re-activated in the presence of EDTA. The rate of EDTA-induced re-activation is pH-independent and can be dramatically increased by added ATP, Pi and sulphite. The dissociation constants for free ATP and P1 (5 x 10(-7) M and 1 x 10(-3) M respectively) and the maximal activation rates were determined by measuring the hyperbolic dependencies of the EDTA-induced re-activation of Mg(2+)-de-activated ATPase on the concentrations of the accelerating ligands. Taken together, the data obtained show two functionally detectable free nucleotide-specific binding sites, one site for Pi and one Mg(2+)-specific ATPase-inhibitory site on the F0.F1 mitochondrial ATP synthase complex.
Evidence for involvement of glycoprotein-CD45 phosphatase in reversing glycoprotein-CD3-induced microtubule-associated protein-2 kinase activity in Jurkat T-cells
Authors: Pollack S , Ledbetter J A , Katz R , Williams K , Akerley B , Franklin K , Schieven G , Nel A E
Published: August 2015

Ligation of CD3/TCR on T-cells induces transient activation of lymphoid MAP-2 kinase (MAP-2K), a 43 kDa serine kinase which itself is a substrate of an unidentified tyrosine kinase (pp43). The reversibility of the MAP-2K response agrees with removal of tyrosine phosphates from pp43. Since both activity as well as tyrosine phosphorylation of MAP-2K could be prolonged by Na3VO4, a phosphotyrosine phosphatase inhibitor, we studied the effect of the common CD45 isoform, which is a member of the CD45 phosphatase family, on MAP-2K activity in vivo and in vitro. We demonstrate the ability of purified CD45 phosphatase to remove tyrosine phosphates from partially purified lymphoid MAP-2K. Utilizing the approach of heterologous receptor aggregation, we also showed that CD45 could inhibit the induction of MAP-2K activity in intact Jurkat cells during CD3 or CD3 + CD4 stimulation. We therefore suggest that this phosphatase may control the activity of lymphoid MAP-2K in vivo.
Purification, crystallization and characterization of N-acetylneuraminate lyase from Escherichia coli
Authors: Aisaka K , Igarashi A , Yamaguchi K , Uwajima T
Published: August 2015

N-Acetylneuraminate lyase produced by Escherichia coli was purified and crystallized from a genetically engineered strain (E. coli SF8/pNAL1). The enzyme showed apparent molecular masses of 105,000 Da on gel filtration and 35,000 Da on SDS/PAGE, suggesting that the enzyme is a trimer. The apparent optimum pH and temperature were found to be 6.5-7.0 and 80 degrees C respectively. The Km values for N-acetylneuraminate and N-glycollylneuraminate were 3.3 and 3.3 mM respectively. The enzyme was inhibited by reduction with NaBH4 in the presence of the substrate, indicating that the enzyme belongs to the Schiff-base-forming Class I aldolases. The enzyme was strongly inhibited by Cu2+ ions, p-chloromercuribenzoate and N-bromosuccinimide, and also inhibited competitively by the reaction product, pyruvate, and its structurally related compounds, dihydroxyacetone and DL-glyceraldehyde.
Functional size measurements of the ubiquinol oxidase activity of the cytochrome o terminal oxidase complex of Escherichia coli
Authors: Williams H D , Hubbard J A M , Nugent J H A , Poole R K
Published: August 2015

The functional molecular mass for ubiquinol-1 oxidation by the Escherichia coli terminal oxidase, cytochrome o, was determined by radiation-inactivation analysis of membranes from a cytochrome d-deficient mutant. The functional molecular mass for ubiquinol-l oxidase activity was found to be 38.3 +/- 2.65 kDa.
Characterization of the binding sites for dicarboxylic acids on bovine serum albumin
Authors: Tonsgard J H , Meredith S C
Published: August 2015

Dicarboxylic acids are prominent features of several diseases, including Reye's syndrome and inborn errors of mitochondrial and peroxisomal fatty acid oxidation. Moreover, dicarboxylic acids are potentially toxic to cellular processes. Previous studies [Tonsgard, Mendelson & Meredith (1988) J. Clin. Invest. 82, 1567-1573] demonstrated that long-chain dicarboxylic acids have a single high-affinity binding site and between one and three lower-affinity sites on albumin. Medium-chain-length dicarboxylic acids have a single low-affinity site. We further characterized dicarboxylic acid binding to albumin in order to understand the potential effects of drugs and other ligands on dicarboxylic acid binding and toxicity. Progesterone and oleate competitively inhibit octadecanedioic acid binding to the single high-affinity site. Octanoate inhibits binding to the low-affinity sites. Dansylated probes for subdomain 2AB inhibit dodecanedioic acid binding whereas probes for subdomain 3AB do not. In contrast, low concentrations of octadecanedioic acid inhibit the binding of dansylated probes to subdomain 3AB and 2AB. L-Tryptophan, which binds in subdomain 3AB, inhibits hexadecanedioic acid binding but has no effect on dodecanedioic acid. Bilirubin and acetylsalicylic acid, which bind in subdomain 2AB, inhibit the binding of medium-chain and long-chain dicarboxylic acids. Our results suggest that long-chain dicarboxylic acids bind in subdomains 2C, 3AB and 2AB. The single low-affinity binding site for medium-chain dicarboxylic acids is in subdomain 2AB. These studies suggest that dicarboxylic acids are likely to be unbound in disease states and may be potentially toxic.
Hepatic caeruloplasmin-gene expression during development in the guinea-pig. Correlation with changes in hepatic copper metabolism
Authors: Bingle C D , Epstein O , Srai S K S , Gitlin J D
Published: August 2015

Decreased amounts of the serum copper-binding protein caeruloplasmin (hypocaeruloplasminaemia) are one of the characteristic abnormalities of copper metabolism that are found in all neonatal mammals. In the present study we have investigated the mechanism responsible for hypocaeruloplasminaemia found in neonatal guinea pigs. Northern-blot analysis of guinea-pig liver RNA revealed the presence of two caeruloplasmin mRNA species. A marked developmental change in expression was observed, with no detectable mRNA before birth, when hepatic copper is elevated. Expression was initiated soon after birth, when copper levels in the liver are falling, and was closely linked to the rise in enzymic activity and protein levels in serum. There was no differential regulation of the two mRNA species; however, a marked inter-animal variation in mRNA levels was observed. Using gel-filtration chromatography we were able to show that, before birth, serum copper was associated with a low-molecular-mass species and that, with the advent of expression of the caeruloplasmin gene after birth, copper was increasingly associated with caeruloplasmin.