bluuebunny/crossref_metadata_2025 huggingface dataset:
Formation of inositol phosphate isomers in GH3 pituitary tumour cells stimulated with thyrotropin-releasing hormone. Acute effects of lithium ions
Authors: Hughes P J , Drummond A H
Published: August 2015

With a h.p.l.c. system, the inositol mono-, bis- and tris-phosphate isomers found in [3H]inositol-labelled GH3 cells were resolved and identified. These cells possess at least ten distinct [3H]inositol-containing substances when acid-soluble extracts are analysed by anion-exchange h.p.l.c. These substances were identified by their co-elution with known inositol phosphate standards and, to a limited extent, by examining their chemical structure. Two major inositol monophosphate (InsP) isomers were identified, namely Ins1P and Ins4P, both of which accumulate after stimulation with the hypothalamic releasing factor (TRH) (thyrotropin-releasing hormone). Three inositol bisphosphate (InsP2) isomers were resolved, of which two were positively identified, i.e. Ins(1,4)P2 and Ins(3,4)P2. TRH treatment increases both of these isomers, with Ins(1,4)P2 being produced at a faster rate than Ins(3,4)P2. The third InsP2 isomer has yet to be fully identified, although it is co-eluted with an Ins(4,5)P2 standard. This third InsP2 is also increased after TRH stimulation. In common with other cell types, the GH3 cell contains two inositol trisphosphate (InsP3) isomers: Ins(1,4,5)P3, which accumulates rapidly, and Ins(1,3,4)P3, which is formed more slowly. The latter substance appears simultaneously with its precursor, inositol 1,3,4,5-tetrakisphosphate. We also examined the effects of acute Li+ treatment on the rates of accumulation of these isomers, and demonstrated that Li+ augments TRH-mediated accumulation of Ins1P, Ins4P, Ins(1,4)P2, the presumed Ins(4,5)P2 and Ins(1,3,4)P3. These results suggest that the effects of Li+ on inositol phosphate metabolism are more complex than was originally envisaged, and support work carried out by less sophisticated chromatographic analysis.
Identification of tonin in brain and exocrine tissues and in the cell-free translation products encoded by the mRNA of these tissues
Authors: Woodley-Miller C , Chao J , Chao L
Published: August 2015

Tissue-specific expression of the esteropeptidase tonin [EC 3.4.99.-] was investigated in rat brain, submandibular gland, pancreas and kidney. Specific polyclonal and monoclonal antibodies to purified rat tonin from the submandibular gland have been developed and characterized and have been purified via a tonin-agarose affinity column. Immunoreactive tonin was measured by a recently developed tonin direct radioimmunoassay using a rabbit tonin antiserum. Resulting tonin levels were found to be 105.27 +/- 2.71 micrograms/mg (of protein) in submandibular gland, 3.18 +/- 0.32 ng/mg in pancreas, 1.35 +/- 0.08 ng/mg in kidney and 0.12 +/- 0.01 ng/mg in brain (means +/- S.E.M.). Western-blot analysis shows that affinity-purified anti-tonin antibody binds to a 32,000-Mr protein from brain and submandibular-gland extracts. The protein, a tonin precursor, was identified from cell-free translation products directly by polyadenylated [Poly(A)+]mRNA species in a wheat-germ system. After the translation products were subjected to immunoprecipitation with affinity-purified tonin antibody, SDS/polyacrylamide-gel electrophoresis of these precipitates revealed two precursors of tonin, with Mr values of 30,000 and 29,000, which are encoded by brain and submandibular-gland mRNA; however, only the 30,000-Mr preprotonin was encoded by pancreas and kidney mRNA. Collectively, the data show that tonin exists in brain, submandibular gland, pancreas and kidney, and can be synthesized by the mRNA of these tissues.
Extracellular metabolites in suspensions of isolated hepatocytes
Authors: Van Schaftingen E , Hue L , Hers H G
Published: August 2015

The activity of lactate dehydrogenase and the concentration of several metabolites were measured in a suspension of isolated hepatocytes and in the extracellular medium, obtained after elimination of the cells by centrifugation for 15 s. The initial proportions of ATP, fructose 2,6-bisphosphate and glycogen present in the medium were similar to that of lactate dehydrogenase, and were therefore explained by unavoidable cell breakage occurring during resuspension of the hepatocytes. ATP disappeared from the medium in less than 10 min, being presumably destroyed by membrane nucleotidases. By contrast, the proportions of hexose 6-phosphates and of glycerol 3-phosphate in the medium were several-fold in excess over that of lactate dehydrogenase; under certain conditions, the extracellular value accounted for 80-90% of the metabolite present in the total suspension, and there was no relationship between the extra- and intracellular concentrations of these metabolites. A potential source of external glycerol 3-phosphate was the hydrolysis of glycerophosphocholine by membranous enzymes. The main conclusion of this work is that the measurement, in isolated hepatocytes, of hexose 6-phosphates, glycerol 3-phosphate and possibly other metabolites that were not investigated, requires the previous separation of the cells from the incubation medium. This conclusion may apply to other cellular suspensions.
Human β-adrenergic receptors. Simultaneous purification of β1- and β2-adrenergic-receptor peptides
Authors: Bahouth S W , Malbon C C
Published: August 2015

Beta-Adrenergic receptors from basal membranes of human placenta were purified from digitonin extracts by sequential rounds of affinity chromatography, hydrophobic chromatography, ion-exchange chromatography and steric-exclusion h.p.l.c. Basal membranes display both beta 1- and beta 2-adrenergic receptors, in the ratio 65:35. Affinity chromatography, hydrophobic chromatography on heptylamine-Sepharose and ion-exchange chromatography on DEAE-Sephacel removed most of the contaminating proteins, and final purification of the receptor to apparent homogeneity was achieved by steric-exclusion h.p.l.c. The purified receptors showed Mr 67000 on SDS/polyacrylamide-gel electrophoresis under reducing conditions. Specific binding of radioligand to the purified beta-adrenergic receptors displayed stereoselectivity, and the agonist competition profiles demonstrated the presence of both beta 1- and beta 2-receptors. By using the subtype-selective ligands CGP-20712A (beta 1-selective) and ICI-118,551 (beta 2-selective), the purified Mr-67000 species was shown to be composed of equivalent amounts of beta 1- and beta 2-adrenergic receptors. Affinity chromatography on Sepharose-alprenolol and sequential elution with 1 microM-CGP-20712A followed by 100 microM(-)-alprenolol permitted beta 1-adrenergic receptors to be resolved from the mixture of beta 1-/beta 2-adrenergic receptors. The pharmacologically distinct human beta 1 and beta 2-adrenergic receptors are shown to be structurally very similar peptides.
Regulation of aortic CuZn-superoxide dismutase with copper. Caeruloplasmin and albumin re-activate and transfer copper to the enzyme in culture
Authors: Dameron C T , Harris E D
Published: August 2015

Caeruloplasmin and albumin were compared as potential donors of copper to Cu2Zn2-superoxide dismutase (CuZn-SOD) in culture. Aortas from 15-day copper-deficient chicks were suspended in oxygenated, serum-free, Waymouth medium (752/1) for 24 h. SOD activity was restored when the medium was supplemented with CuCl2, a copper-albumin complex or caeruloplasmin, all present at a level equivalent to 5 microM-copper. Activation did not occur at 4 degrees C or with Cu-EDTA as the supplement. Mn2+ and Zn2+, alone or in combination, did not activate nor enhance the activation achieved by CuCl2. The activation with CuCl2 was not inhibited by cycloheximide or cordycepin. [67Cu]Caeruloplasmin and albumin when added to the medium transferred radioactive copper to at least three cytosolic protein fractions, one of which was determined by immunoprecipitation to be CuZn-SOD. The transfer of 67Cu from caeruloplasmin was inhibited by increasing amounts of unlabelled caeruloplasmin; disodium EDTA (1.0 mM) had no effect on the transfer of copper from caeruloplasmin. These data show that aortic SOD activity, suppressed in copper deficiency, can be restored by incubating the aortas in culture medium supplemented with copper salts. In this system, caeruloplasmin and Cu-albumin appear equally capable of activating aortic CuZn-SOD. Moreover, the transfer of copper into the enzyme structure appears to be the primary event restoring catalytic activity to the enzyme.
Kinetic heterogeneity of F-actin polymers. Further evidence that the elongation reaction may occur through condensation of the actin filaments with small aggregates
Authors: Grazi E , Magri E
Published: August 2015

We have shown that F-actin, polymerized in 50 mM-KCl at 20 degrees C and pH 8.0, can be resolved by centrifugation into two polymer populations, which differ morphologically as well as kinetically. The first population represents about 10% of the overall polymer and is composed of small amorphous aggregates. It rapidly exchanges the bound nucleotide with free ATP in the medium, either directly or through the monomers. The second population is composed of long actin filaments. These are labelled by free ATP in the medium only through condensation with labelled small amorphous aggregates.
Purification and properties of l-mandelate dehydrogenase and comparison with other membrane-bound dehydrogenases from Acinetobacter calcoaceticus
Authors: Hoey M E , Allison N , Scott A J , Fewson C A
Published: August 2015

L-Mandelate dehydrogenase was purified from Acinetobacter calcoaceticus by Triton X-100 extraction from a ‘wall + membrane’ fraction, ion-exchange chromatography on DEAE-Sephacel, (NH4)2SO4 fractionation and gel filtration followed by further ion-exchange chromatography. The purified enzyme was partially characterized with respect to its subunit Mr (44,000), pH optimum (7.5), pI value (4.2), substrate specificity and susceptibility to various potential inhibitors including thiol-blocking reagents. FMN was identified as the non-covalently bound cofactor. The properties of L-mandelate dehydrogenase are compared with those of D-mandelate dehydrogenase, D-lactate dehydrogenase and L-lactate dehydrogenase from A. calcoaceticus.
Polyamine inhibition of lipoperoxidation. The influence of polyamines on iron oxidation in the presence of compounds mimicking phospholipid polar heads
Authors: Tadolini B
Published: August 2015

Polyamines appear to inhibit peroxidation of vesicles containing acidic phospholipids. A correlation exists between polyamine binding to phospholipid vesicles and its protective effect. However, phosphatidylinositol-containing vesicles which bind spermine are not protected by the polyamine [Tadolini, Cabrini, Landi, Varani & Pasquali (1985) Biogenic Amines 3, 97-106]. In the present paper I tested the hypothesis that polyamines, in particular spermine, by forming a ternary complex with iron and the phospholipid polar head may change the susceptibility of Fe2+ to autoxidation and thus its ability to generate free oxygen radicals. Different compounds mimicking phospholipid polar heads were studied, namely AMP, mimicking phosphatidic acid, CDP-choline, mimicking phosphatidylcholine, and glycerophosphoinositol, mimicking phosphatidylinositol. The results support the proposed hypothesis. In the presence of CDP-choline or of glycerophosphoinositol, spermine poorly affects Fe2+ autoxidation, whereas a considerable inhibition is observed in the presence of AMP. The ability of other phosphorus-containing compounds (ATP, ADP, cyclic AMP, sodium phosphate) to affect Fe2+ autoxidation in the presence of polyamines was also evaluated to understand the molecular mechanism of this phenomenon. It is proposed that polyamines may be part of the passive cellular defence mechanism against the oxidative damage caused by Fe2+.
Synthesis of very long chain (up to 36 carbon) tetra, penta and hexaenoic fatty acids in retina
Authors: Rotstein N P , Aveldaño M I
Published: August 2015

The synthesis of very long chain (C24 to C36) polyunsaturated (four, five and six double bonds) fatty acids (VLCPUFA) is investigated in bovine retina using [14C]acetate. Saturates on the one hand (mainly palmitate), and polyenes on the other (mainly VLCPUFA), incorporate most of the label found in lipids. Phosphatidylcholine (PC) is the most highly labelled lipid class, since both types of 14C-labelled fatty acids, but especially this novel series of VLCPUFA, are concentrated in this phospholipid. Radioactivity from [14C]acetate is found in very long chain tetra, penta and hexaenoic fatty acids of PC. The labelling of 20:4(n − 6), 20:5(n − 3), 22:5(n − 6) and 22:6(n − 3) is much lower than that of longer polyenes of each of these series, indicating that VLCPUFA are synthesized in situ by successive elongations of the above polyenes, pre-existing in retina lipids. In various subcellular fractions isolated from retinas after incubations with [14C]acetate (including cytosol, microsomes, mitochondria and photoreceptor membranes), the labelling of the VLCPUFA of PC is very high, even at relatively short intervals of incubation. The results suggest that not only the synthesis but also the intracellular traffic among membranes of VLCPUFA-containing species of PC are very active processes in the retina.
Sequencing and overexpression of the Escherichia coli aroE gene encoding shikimate dehydrogenase
Authors: Anton I A , Coggins J R
Published: August 2015

The Escherichia coli aroE gene encoding shikimate dehydrogenase was sequenced. The deduced amino acid sequence was confirmed by N-terminal amino acid sequencing and amino acid analysis of the overproduced protein. The complete polypeptide chain has 272 amino acid residues and has a calculated Mr of 29,380. E. coli shikimate dehydrogenase is homologous to the shikimate dehydrogenase domain of the fungal arom multifunctional enzymes and to the catabolic quinate dehydrogenase of Neurospora crassa.
Different insulin-secretory responses to calcium-channel blockers in islets of lean and obese (ob/ob) mice
Authors: Black M A , Fournier L A , Heick H M , Bégin-Heick N
Published: August 2015

The purpose of these experiments was to determine whether the activity of the voltage-dependent Ca2+ channel was modulated in the same manner in islets of the ob/ob mouse as in islets of homozygous lean mice of the same strain. The effect of agents that are known to alter the concentrations and movements of intracellular Ca2+ were investigated in relation to glucose-stimulated insulin secretion and in relation to the effect of forskolin. In islets of obese mice, verapamil and nifedipine both inhibited glucose-induced insulin release, nifedipine being the more potent inhibitor. Forskolin-stimulated secretion was inhibited either not at all (verapamil) or much less (nifedipine) in islets of the ob/ob mouse compared with those of lean mice. At basal glucose concentrations, verapamil initiated insulin secretion in islets of the ob/ob mouse and acted synergistically with forskolin to evoke a secretory activity that was 3-fold greater than that evoked by 20 mM-glucose. Nifedipine also initiated secretion at basal glucose concentrations and acted synergistically with forskolin, but its effect was considerably smaller than that of verapamil. A comparison of the effect of forskolin in the presence of Ca2+-channel blockers and in the absence of Ca2+ suggests that, in the obese mouse, the operation of the voltage-dependent Ca2+ channel is impaired.
Possible physiological role of guanosine triphosphate and inositol 1,4,5-trisphosphate in Ca2+ release in macrophages stimulated with chemotactic peptide
Authors: Kimura Y , Hirata M , Hamachi T , Koga T
Published: August 2015

The release of Ca2+ induced by inositol 1,4,5-trisphosphate (InsP3) in the presence of GTP was examined by using saponin-permeabilized macrophages. The origin and the amount of mobilized Ca2+ in intact macrophages stimulated with chemotactic peptide were also examined to assess the physiological significance of GTP and InsP3 on Ca2+-releasing activities. The total amount of Ca2+ released by 20 microM-A23187 from the unstimulated intact macrophages was 1.4 nmol/4 x 10(6) cells, and the mitochondrial uncoupler did not cause an efflux of Ca2+ from the cells. The Ca2+ accumulation by the non-mitochondrial pool(s) was inhibited by the presence of GTP, and the total amount of releasable Ca2+ (1.4 nmol/4 x 10(6) cells) was comparable with that accumulated by the non-mitochondrial pool(s) in the presence of GTP at a free Ca2+ concentration of 0.14 microM. The mobilized and subsequently effluxed Ca2+ in cells stimulated with chemotactic peptide was estimated to be 0.3 nmol/4 x 10(6) cells. Much the same amounts were released by about the half-maximal dose of InsP3 from the non-mitochondrial pool(s) of saponin-treated macrophages that had accumulated Ca2+ at a free concentration of 0.14 microM in the presence of GTP. These results suggest that the Ca2+-releasing activity induced by GTP may play a role in the long-term regulation of Ca2+ content in the non-mitochondrial pool(s) of macrophages, and that released by InsP3 can explain, quantitatively, the chemotactic-peptide-induced mobilization of Ca2+.
Nucleoside transport in rat cerebral-cortical synaptosomes. Evidence for two types of nucleoside transporters
Authors: Lee C W , Jarvis S M
Published: August 2015

The transport of [U-14C]uridine was investigated in rat cerebral-cortical synaptosomes using an inhibitor-stop filtration method. Under these conditions the rapid efflux of uridine from the synaptosomes is prevented and uridine is not significantly metabolized in the synaptosome during the first 1 min of uptake. The dose-response curve for the inhibition of uridine transport by nitrobenzylthioinosine (NBMPR) was biphasic: approx. 40% of the transport activity was inhibited with an IC50 (concentration causing half-maximal inhibition) value of 0.5 nM, but the remaining activity was insensitive to concentrations as high as 1 microM. Similar biphasic dose-response curves were observed for dilazep inhibition, but both transport components were equally sensitive to dipyridamole inhibition. Uridine influx by both components was saturable (Km 300 +/- 51 and 214 +/- 23 microM, and Vmax. 12 +/- 3 and 16 +/- 3 pmol/s per mg of protein, for NBMPR-sensitive and NBMPR-insensitive components respectively), and inhibited by other nucleosides such as 2-chloroadenosine, adenosine, inosine, thymidine and guanosine with similar IC50 values for the two components. Inhibition of uridine transport by NBMPR was associated with high-affinity binding of NBMPR to the synaptosome membrane (Kd 58 +/- 15 pM). Binding of NBMPR to these sites was competitively blocked by uridine and adenosine and inhibited by dilazep and dipyridamole, with Ki values similar to those measured for inhibiting NBMPR-sensitive uridine influx. These results demonstrate that there are two components of nucleoside transport in our rat synaptosomal preparation that differ in their sensitivity to inhibition by NBMPR. Thus conclusions regarding nucleoside transport in rat brain based only on NBMPR-binding activity must be viewed with caution.
Formaldehyde adducts of glutathione. Structure elucidation by two-dimensional n.m.r. spectroscopy and fast-atom-bombardment tandem mass spectrometry
Authors: Naylor S , Mason R P , Sanders J K M , Williams D H , Moneti G
Published: August 2015

Aqueous mixtures of formaldehyde and glutathione react to form a variety of cyclized adducts in addition to S-hydroxymethylglutathione. The adducts are in labile equilibrium with each other and are not readily separated. The structures of two of the other major adducts were determined by concerted application of 13C-1H two-dimensional chemical-shift correlation, fast-atom-bombardment mass spectrometry and tandem mass spectrometry to the adduct mixtures in aqueous solution.
Primary structure of rat liver alkaline phosphatase deduced from its cDNA
Authors: Misumi Y , Tashiro K , Hattori M , Sakaki Y , Ikehara Y
Published: August 2015

Rat liver alkaline phosphatase (ALP) was markedly induced by treatment of rats by bile-duct ligation and colchicine injection. Taking this advantage for enrichment of ALP mRNA, we constructed a lambda gt11 liver cDNA library using polyadenylated RNA prepared from the treated rat liver, and isolated an ALP cDNA clone. The 2165 bp cDNA contained an open reading frame that encodes a 524-amino-acid-residue polypeptide with a predicted molecular mass of 57737 Da. The precursor protein contained a presumed signal peptide of 17 amino acid residues followed by 28 amino acid residues identical with the N-terminal sequence determined from the purified rat liver ALP. It was also confirmed that amino acid sequences of two CNBr-cleavage peptides obtained from liver ALP were contained within the cDNA-encoded protein. Five possible N-linked glycosylation sites were found in the molecule and a highly hydrophobic amino acid sequence at the C-terminus. The deduced polypeptide of rat liver ALP showed 88% homology to that of the human liver-type enzyme in osteosarcoma cells. RNA blot hybridization analysis identified a single species of ALP mRNA with 2.7 kb in both the control and the treated rat livers. An approx. 20-fold increase of the mRNA was detected in the treated liver at 12 h after the onset of stimulation, compared with that in the control liver.
Comparison of the effects of [leucine]enkephalin and angiotensin on hepatic carbohydrate and cyclic nucleotide metabolism
Authors: Hothi S K , Leach R P , Titheradge M A
Published: August 2015

The effects of [leucine]enkephalin and angiotensin on hepatic carbohydrate and cyclic nucleotide metabolism are compared. Both peptides stimulated glycogenolysis as a result of an increase in phosphorylase a activity and enhanced glucose synthesis from [2-14C]pyruvate, although neither had any significant effect on pyruvate kinase activity. Although the magnitudes of the effects of both peptides on glycogenolysis were comparable and unaffected by the presence of insulin. [Leu]enkephalin proved to be more efficacious in enhancing gluconeogenesis, the response being comparable with that to glucagon. Both effectors decreased the intracellular concentration of cyclic AMP in hepatocytes when incubated under control conditions and after addition of sub-optimal concentrations of glucagon. This was correlated with the ability of the two peptides to inhibit both basal and hormone-stimulated adenylate cyclase activity in purified liver plasma membranes.
γ-Glutamylamine derivatives in isolated rat hepatocyte proteins
Authors: Piacentini M , Beninati S
Published: August 2015

Freshly isolated rat hepatocytes were found to contain a 3-fold higher level of putrescine than perfused liver. The bulk of this diamine was recovered in the acid-insoluble fraction of the cell. In order to determine the nature of the amine binding, the levels of gamma-glutamylamine derivatives were measured. The method used involves exhaustive proteolytic digestion of the acid-insoluble fraction of hepatocytes, followed by ion-exchange chromatography. For N1-(gamma-glutamyl)putrescine, a combined ion-exchange chromatographic and reverse-phase h.p.l.c. procedure was adopted. This allowed for the direct detection of less than 50 pmol of this derivative in enzymic hydrolysates. Several of the gamma-glutamylamines reported previously [Beninati, Piacentini, Argento-Ceru', Russo-Caia & Autuori (1985) Biochim. Biophys. Acta 841, 120-126] in the whole organ were found in the isolated liver cells. The elevated level of N1-(gamma-glutamyl)putrescine and the absence of bis-(gamma-glutamyl)spermine was noteworthy. The results suggest that, in rat hepatocytes, both polyamine-dependent post-translational modification of some proteins and cross-linking between proteins involving the glutamine and lysine residues occurs.
Platelet-activating factor-induced hydrolysis of phosphatidylinositol 4,5-bisphosphate stimulates the production of reactive oxygen intermediates in macrophages
Authors: Huang S J , Monk P N , Downes C P , Whetton A D
Published: August 2015

To investigate the relationship between inositol lipid hydrolysis and reactive oxygen-intermediate (ROI) production in macrophages we have examined the effect of platelet-activating factor (PAF) on normal bone marrow-derived macrophages. Addition of PAF to macrophages prelabelled with [3H]inositol caused a marked and rapid increase in [3H]inositol trisphosphate levels. Similarly when PAF was added to [3H]-glycerol prelabelled macrophages there was a rapid increase in 1,2-diacyl[3H]glycerol levels. These events preceded any increase in the rate of PAF-stimulated ROI production by a discernible period of several seconds. Increasing concentrations of PAF led to a markedly similar increase in both ROI production and [3H]inositol lipid hydrolysis suggesting that inositol lipid hydrolysis may lead to the generation of ROI in macrophages. Further evidence that this is the case came from experiments in which pretreatment of macrophages with phorbol esters was shown to inhibit both PAF-stimulated [3H]inositol phosphate production and ROI production to a markedly similar degree. Similarly pertussis toxin inhibited both PAF-stimulated ROI production and [3H]inositol phosphate production. Phorbol esters were shown to activate ROI production in normal bone marrow-derived macrophages whereas the Ca2+ ionophore, A23187, did not. These experiments suggest that PAF stimulates a pertussis toxin-sensitive activation of inositol lipid hydrolysis leading to the formation of inositol trisphosphate and diacylglycerol. The diacylglycerol formed can then activate protein kinase C leading to the stimulation of ROI production in normal bone marrow-derived macrophages.
Tumour necrosis factor (cachectin) induces phospholipase A2 activity and synthesis of a phospholipase A2-activating protein in endothelial cells
Authors: Clark M A , Chen M J , Crooke S T , Bomalaski J S
Published: August 2015

Tumour necrosis factor (TNF) is an important mediator of endotoxin-induced vascular collapse and other inflammatory reactions. Eicosanoids have been implicated in the pathogeensis of these responses. In order to explore further the potential interactions between TNF and eicosanoid metabolism in eliciting vascular responses, we studied the effects of TNF on the bovine endothelial cell line CPAE. TNF induced cellular retraction observed by light microscope. This morphological change was monitored by the passage of iodinated protein A between adjacent cells and by release of [3H]arachidonic acid metabolites from cells. Both the morphological and functional responses were abrogated by inhibition of eicosanoid synthesis with BW755c. The release of [3H]arachidonic acid metabolites appeared to be mediated by a transient increase in phospholipase A2 activity. Phospholipase C activity was not affected by TNF. The maximal increase in phospholipase A2 activity occurred at 5 min following the addition of TNF. Phospholipase A2 activation, [3H]arachidonic acid-metabolite synthesis and passage of iodinated protein A, required both RNA and protein synthesis and were associated with an increase in the synthesis of a recently described phospholipase A2-activating protein. The Bordetella pertussis toxin, islet-activating protein, also inhibited the increase in phospholipase A2 activity, the release of [3H]arachidonic acid metabolites and the passage of iodinated protein A, suggesting that the TNF receptor-ligand interaction resulting in cellular retraction, phospholipase A2 activation and eicosanoid synthesis, is coupled through the Ni guanine nucleotide regulatory protein in these cells.
Evidence for an essential histidine residue in d-xylose isomerases
Authors: Vangrysperre W , Callens M , Kersters-Hilderson H , De Bruyne C K
Published: August 2015

Diethyl pyrocarbonate inactivated D-xylose isomerases from Streptomyces violaceoruber, Streptomyces sp., Lactobacillus xylosus and Lactobacillus brevis with second-order rate constants of 422, 417, 99 and 92 M-1.min-1 respectively (at pH 6.0 and 25 degrees C). Activity was completely restored by the addition of neutral hydroxylamine, and total protection was afforded by the substrate analogue xylitol in the presence of either Mg2+ or Mn2+ according to the genus studied. The difference spectra of the modified enzymes revealed an absorption maximum at 237-242 nm, characteristic for N-ethoxycarbonylhistidine. In addition, the spectrum of ethoxycarbonylated D-xylose isomerase from L. xylosus showed absorption minima at both 280 and 230 nm, indicative for modification of tyrosine residues. Nitration with tetranitromethane followed by diethyl pyrocarbonate treatment eliminated the possibility that modification of tyrosine residues was responsible for inactivation, and resulted in modification of one non-essential tyrosine residue and six histidine residues. Inactivation of the other D-xylose isomerases with diethyl pyrocarbonate required the modification of one (L. brevis), two (Streptomyces sp.) and four (S. violaceoruber) histidine residues per monomer. Spectral analysis and maintenance of total enzyme activities further indicated that either xylitol Mg2+ (streptomycetes) or xylitol Mn2+ (lactobacilli) prevented the modification of one crucial histidine residue. The overall results thus provide evidence that a single active-site histidine residue is involved in the catalytic reaction mechanism of D-xylose isomerases.
Characterization of the Mg2+-activated ATPase activity in smooth-muscle membranes. NADH oxidase and adenylate kinase interfere with the NADH-coupled enzyme assay
Authors: Missiaen L , Wuytack F , Casteels R
Published: August 2015

The apparent Mg2+-activated ATPase activity measured by the continuous NADH-coupled enzyme assay was studied in a number of microsomal preparations obtained from smooth muscle of the myometrium from pregnant or 17 beta-oestradiol-pretreated rats, the bovine aorta, the guinea-pig taenia coli, the rabbit ear artery and pig antrum. It was shown that this ATPase assay is prone to the effects of a number of artefacts that are tissue-dependent. The apparent Mg2+-ATPase activity in microsomes (microsomal fractions) from myometrium, aorta and taenia coli declines non-linearly during the assay. Its initial high rate gradually diminishes over 15-60 min, depending on the type of smooth muscle, to a constant value. This decline depends on the presence of ATP and can be partially prevented by concanavalin A. The non-linearity is limited in microsomes from rabbit ear artery. In microsomes from antrum the apparent Mg2+-ATPase activity actually increases with time, albeit gradually. Storage on ice of the microsomes of the aorta, and especially of myometrium of pregnant rats and of taenia coli, is accompanied over a few hours after their preparation by a gradual suppression of the component of the Mg2+-ATPase activity that is inhibited by ATP. The Mg2+-ATPase activity in microsomes from antrum remains constant. NADH oxidase activity accounts for 10% of the Mg2+-ATPase activity in microsomes from stomach smooth muscle. The apparent initial non-linearity of the Mg2+-ATPase activity in that tissue is due to a time-dependent decrease of a rotenone-sensitive NADH oxidase activity. The adenylate kinase activity, as deduced from the effect of the adenylate kinase inhibitor P1,P5-di(adenosine-5′) pentaphosphate, could account for 45.0, 35.0 and 31.0% respectively of the Mg2+-ATPase activity in microsomes from stomach, myometrium and aorta. No adenylate kinase activity could be detected in microsomes from ear artery and taenia coli. When microsomes from stomach smooth muscle were separated on a sucrose gradient, the contribution of adenylate kinase and NADH oxidase to the Mg2+-ATPase activity was most pronounced in the higher-density fractions. Part of the NADH oxidase activity and of the Mg2+-ATPase activity, and most of the adenylate kinase activity, are not sedimented at 224000 gmax. for 30 min and may therefore be present as soluble enzymes.(ABSTRACT TRUNCATED AT 400 WORDS)
Potassium-channel blockers inhibit inositol trisphosphate-induced calcium release in the microsomal fractions isolated from the rat brain
Authors: Shah J , Pant H C
Published: August 2015

The ionic mechanism of inositol trisphosphate (InsP3)-induced Ca2+ release was investigated in microsomes (microsomal fractions) isolated from rat brain. InsP3 stimulated Ca2+ release from microsomes incubated in media containing 100 mM-KCl. The InsP3-induced Ca2+ release was insensitive to a variety of Ca2+-channel blockers; however, the K+-channel blockers tetraethylammonium chloride (TEA; 1 mM) and 9-tetraethylammonium chloride (9-TEA; 1 mM) blocked InsP3-induced Ca2+ release. Moreover, addition of InsP3 increased 86Rb+ influx into the microsomes. The influx of 86Rb+ also was sensitive to TEA and 9-TEA. The above results suggest that InsP3-induced Ca2+ release requires an opposite flow of K+ ions, and modulation of K+ channels by TEA and 9-TEA may underlie the inhibition of InsP3-induced Ca2+ release from brain microsomes by these agents.
5′-Nucleotidase activities in human erythrocytes. Identification of a purine 5′-nucleotidase stimulated by ATP and glycerate 2,3-bisphosphate
Authors: Bontemps F , Van den Berghe G , Hers H G
Published: August 2015

A purine 5′-nucleotidase has been separated by DEAE-Trisacryl chromatography from other 5′-nucleotidase activities present in human haemolysates and purified approx. 30,000-fold by subsequent chromatography on Blue Sepharose. The enzyme has an Mr of around 250,000, displays hyperbolic substrate-saturation kinetics and hydrolyses preferentially IMP, GMP and their deoxy counterparts. It is much less active with AMP and dAMP. The purine 5′-nucleotidase is inhibited by Pi, and is strongly stimulated by ATP, dATP and GTP, and by glycerate 2,3-bisphosphate. Stimulators decrease Km and increase Vmax. Glycerate 2,3-bisphosphate is the most potent stimulator of the enzyme and, under physiological conditions, over-rides the influence of the other effectors. Glycerate 2,3-bisphosphate also influences the binding of the enzyme to DEAE-Trisacryl, as evidenced by the different elution profile obtained with fresh as compared with outdated blood. It is concluded that the glycerate 2,3-bisphosphate-stimulated purine 5′-nucleotidase is responsible for the dephosphorylation of IMP and GMP, but not of AMP, in human erythrocytes.
A cadmium-binding protein in rat liver identified as ornithine carbamoyltransferase
Authors: Aoki Y , Sunaga H , Suzuki K T
Published: August 2015

A cadmium-binding protein of Mr about 40,000 (40K Cd-BPa) was detected in rat liver by Western blotting [Aoki, Kunimoto, Shibata & Suzuki (1986) Anal. Biochem. 157, 117-122]. It was characterized and identified as ornithine carbamoyltransferase (OCTase, EC 2.1.3.3) on the basis of coincidence of their physicochemical and enzymological features. The amino acid sequence of the N-terminal and those of three tryptic digests in 40K Cd-BPa were identical with those of OCTase. The Mr values of the denatured and native forms of 40K Cd-BPa (39,000 and 110,000 respectively) were the same as those of OCTase. 40K Cd-BPa showed, as OCTase activity, a specific activity of 230 mumol/min per mg of protein and Km of 0.6 mM for ornithine, this value also being essentially the same as that for OCTase. A rabbit antibody against OCTase reacted with 40K Cd-BPa. The native form of 40K Cd-BPa bound to 0.8 molar equiv, of cadmium, with a dissociation constant of 7.6 x 10(-6) M.
Acute regulation of the branched-chain 2-oxo acid dehydrogenase complex by adrenaline and glucagon in the perfused rat heart
Authors: Hildebrandt E F , Buxton D B , Olson M S
Published: August 2015

Rates of transamination and decarboxylation of [1-14C]leucine at a physiological concentration (0.1 mM) were measured in the perfused rat heart. In hearts from fasted rats, metabolic flux through the branched-chain 2-oxo acid dehydrogenase reaction was low initially, but increased gradually during the perfusion period. The increase in 14CO2 production was accompanied by an increase in the amount of active branched-chain 2-oxo acid dehydrogenase complex present in the tissue. In hearts from rats fed ad libitum, extractable branched-chain dehydrogenase activity was low initially, but increased rapidly during perfusion, and high rates of decarboxylation were attained within the first 10 min. Infusion of glucagon, adrenaline, isoprenaline, or adrenaline in the presence of phentolamine all produced rapid, transient, inhibition (40-50%) of the formation of 4-methyl-2-oxo[1-14C]pentanoate and 14CO2 within 1-2 min, but the specific radioactivity of 4-methyl-2-oxo[14C]pentanoate released into the perfusate remained constant. Glucagon and adrenaline infusion also resulted in transient decreases (16-24%) in the amount of active branched-chain 2-oxo acid dehydrogenase. In hearts from fasted animals, infusion for 10 min of adrenaline, phenylephrine, or adrenaline in the presence of propranolol, but not infusion of glucagon or isoprenaline, stimulated the rate of 14CO2 production 3-fold, and increased 2-fold the extractable branched-chain 2-oxo acid dehydrogenase activity. These results demonstrate that stimulation of glucagon or beta-adrenergic receptors in the perfused rat heart causes a transient inhibition of branched-chain amino acid metabolism, whereas alpha-adrenergic stimulation causes a slower, more sustained, enhancement of branched-chain amino acid metabolism. Both effects reflect interconversion of the branched-chain 2-oxo acid dehydrogenase complex between active and inactive forms. Also, these studies suggest that the concentration of branched-chain 2-oxo acid available for decarboxylation can be regulated by adrenaline and glucagon.
Affinity purification and biochemical characterization of histolysin, the major cysteine proteinase of Entamoeba histolytica
Authors: Luaces A L , Barrett A J
Published: August 2015

We report a one-step method for the purification to homogeneity of a cysteine proteinase of Entamoeba histolytica (histolysin) by affinity chromatography of the soluble extract of the parasite on immobilized phenylalanyl(2-phenyl)aminoacetaldehyde semicarbazone. The enzyme has an apparent Mr of 26,000 by SDS/polyacrylamide-gel electrophoresis and 29,000 by gel chromatography. Its pH optimum varies widely, from 5.5 with azocasein to approx. 7 with other protein substrates and benzyloxycarbonylphenylalanyl-L-citrullylaminomethylcourmarin++ + (Z-Phe-Cit-NHMec), and to 9.5 with benzyloxycarbonylphenylalanylarginylaminomethylcoumarin (Z-Phe-Arg-NHMec) and benzyloxycarbonylarginylarginylaminomethylcourmarin (Z-Arg-Arg-NHMec). Values of Km, kcat. and kcat/Km are 1.5 microM, 130 s-1 and 87 X 10(6) M-1.s-1 for Z-Arg-Arg-NHMec, and 32 microM, 0.4 s-1 and 0.012 x 10(6) M-1.s-1 for Z-Phe-Arg-NHMec, respectively, at pH 7.5 and 37 degrees C. The enzyme is inhibited by leupeptin and such inhibitors of cysteine proteinases as L-transepoxysuccinyl-L-leucylamido-4-(guanidino)butane, peptidyldiazomethanes, iodoacetic acid and chicken cystatin. The tentative N-terminal amino acid sequence of the enzyme closely resembles that of papain. Histolysin does not degrade type I collagen or elastin, but it is active against cartilage proteoglycan and kidney glomerular basement-membrane collagen. It also detaches cells from their substratum in vitro, and could well play a role in tissue invasion.
Proteus mirabilis urease. Partial purification and inhibition by boric acid and boronic acids
Authors: Breitenbach J M , Hausinger R P
Published: August 2015

Urease was purified 800-fold and partially characterized from Proteus mirabilis, the predominant microorganism associated with urinary stones. Boric acid is a rapid reversible competitive inhibitor of urease. The pH-dependence of inhibition exhibited pKa values of 6.25 and 9.3, where the latter value is probably due to the inherent pKa of boric acid. Three boronic acids also were shown to inhibit urease competitively.
Phosphorylation of phospholipids in isolated guinea pig hearts stimulated with isoprenaline
Authors: Jakab G , Rapundalo S T , Solaro R J , Kranias E G
Published: August 2015

Phosphorylation of phospholipids was studied in Langendorff perfused guinea pig hearts subjected to beta-adrenergic stimulation. Hearts were perfused with Krebs-Henseleit buffer containing [32P]Pi and freeze-clamped in a control condition or at the peak of the inotropic response to isoprenaline. 32P incorporation into total phospholipids, individual phospholipids and polyphosphoinositides was analysed in whole tissue homogenates and membranes, enriched in sarcoplasmic reticulum, prepared from the same hearts. Isoprenaline stimulation of the hearts did not result in any significant changes in the levels of phosphate incorporation in the total phospholipid present in cardiac homogenates (11.6 +/- 0.4 nmol of 32P/g for control hearts and 12.4 +/- 0.5 nmol of 32P/g for isoprenaline-treated hearts; n = 6), although there was a significant increase in the degree of phospholipid phosphorylation in sarcoplasmic reticulum (3.5 +/- 0.3 nmol of 32P/mg for control hearts and 6.7 +/- 0.2 nmol of 32P/mg for isoprenaline-treated hearts; n = 6). Analysis of 32P incorporation into individual phospholipids and polyphosphoinositides revealed that isoprenaline stimulation of the hearts was associated with a 2-3-fold increase in the degree of phosphorylation of phosphatidylinositol monophosphate and bisphosphate as well as phosphatidic acid in both cardiac homogenates and sarcoplasmic reticulum membranes. In addition, there was increased phosphate incorporation into phosphatidylinositol in sarcoplasmic reticulum membranes. Thus, perfusion of guinea pig hearts with isoprenaline is associated with increased formation of polyphosphoinositides and these phospholipids may be involved, at least in part, in mediating the effects of beta-adrenergic agents in the mammalian heart.
Thrombin-stimulated elevation of human endothelial-cell cytoplasmic free calcium concentration causes prostacyclin production
Authors: Hallam T J , Pearson J D , Needham L A
Published: August 2015

Endothelial cells are known to release prostacyclin (PGI2) in response to agonists, and this has generally been assumed to be caused, at least in part, by activation of a phospholipase A2 by elevated concentrations of cytoplasmic free calcium ([Ca2+]i). However, it has been shown in the blood platelet that agonists can cause arachidonate release without elevating [Ca2+]i. In the present study, rigorous analysis is made of the [Ca2+]i-dependence of PGI2 production in the human umbilical-vein endothelial cell. Thrombin caused a rapid increase in [Ca2+]i from the resting basal value of 0.1 microM to a peak, within 10-15 s, of approx. 2 microM. In the absence of extracellular Ca2+, [Ca2+]i then declined back to the resting value within 2-3 min. In the presence of extracellular Ca2+, [Ca2+]i partly decreased to a new steady-state value of approx. 1 microM. The elevated [Ca2+]i was maintained while the stimulus and the source of extracellular Ca2+ were present, suggesting that it was dependent on influx of Ca2+ across the plasma membrane. Thrombin stimulated the production of PGI2 in the presence or in the absence of extracellular Ca2+. However, the production of PGI2 was more prolonged in the presence of extracellular Ca2+. Total accumulated amounts of 6-oxo-prostaglandin F1 alpha on stimulation with thrombin without extracellular Ca2+ were only 65% of those accumulated with extracellular Ca2+ present. Cells depleted of extracellular and intracellular sources of Ca2+ by incubation with 1 mM extracellular EGTA and exposing them to ionomycin to discharge intracellular stores produced no elevation of [Ca2+]i on stimulation with thrombin or production of PGI2. The threshold [Ca2+]i required to support the production of PGI2 was measured to be 0.8-1.0 microM by using different doses of ionomycin selectively to increase [Ca2+]i. This relationship between [Ca2+]i and PGI2 production was similar to that produced by using different doses of thrombin. Our results show that the major and probably exclusive intracellular stimulus for the production of PGI2 by the vascular endothelial cell in response to thrombin is the elevation of [Ca2+]i.
The induction, desensitization and de-induction of tyrosine aminotransferase by 8-bromo-cyclic AMP in rat hepatoma cells
Authors: Smith J D , Liu A Y C
Published: August 2015

Addition of 1-3 mM-8-bromo-cyclic AMP to monolayer cultures of H-4 rat hepatoma cells resulted in a rapid but short-lived increase in tyrosine aminotransferase (EC 2.6.1.5) activity. The transient nature of this induction is due to desensitization to 8-bromo-cyclic AMP. Throughout this time course of induction and desensitization, removal of 8-bromo-cyclic AMP resulted in a rapid and significant decrease in tyrosine aminotransferase activity, a process referred to as ‘de-induction’ in this study. We showed that the changes in tyrosine aminotransferase activity in its induction, desensitization and de-induction by 8-bromo-cyclic AMP were directly attributable to changes in the synthesis rate of the protein, and the amount of translatable and hybridizable mRNA encoding for tyrosine aminotransferase (mRNATAT). We further showed that this desensitization was specific to cyclic AMP. First, only active analogues of cyclic AMP and agents which increased cellular concentrations of cyclic AMP elicited this desensitization. Second, the desensitized cells were refractory only to the effects of 8-bromo-cyclic AMP; dexamethasone and insulin induced the tyrosine aminotransferase activity in the 8-bromo-cyclic AMP-desensitized cells in a manner similar to that of the controls. Studies on the metabolism of 8-bromo-cyclic AMP suggest that neither its degradation nor the accumulation of its primary metabolite, 8-bromoadenosine, played a significant role in modulating the expression of tyrosine aminotransferase during the time course of action of 8-bromo-cyclic AMP. These results provide evidence for a specific pretranslational mode of action of cyclic AMP in the control of tyrosine aminotransferase expression in its desensitization and de-induction, in addition to the early phase of induction.
Wheat-germ-agglutinin and Ricinus communis-agglutinin-binding sites of BHK cells compared with each other and with 140 kDa fibronectin receptors
Authors: Tuan T L , Grinnell F
Published: August 2015

We compared the wheat-germ agglutinin (WGA) and Ricinus communis agglutinin (RCA) binding sites of baby-hamster kidney (BHK) cells. There were 1.01 × 10(8) WGA-binding sites per cell (Kd = 0.027 nM) and 6 × 10(6) RCA-binding sites per cell (Kd = 0.014 nM). Binding of WGA or RCA to BHK cells resulted in more than 75% of the cell-surface binding sites becoming associated with the cytoskeleton (i.e. resistant to extraction with detergent), although no more than 10% of these sites were associated with the cytoskeleton before addition of the lectins. After binding of WGA to the cells, the cell surface was cross-linked so extensively that it remained intact even after detergent extraction of the treated cells, and could be observed by electron microscopy. A similar cross-linking effect did not occur after binding of RCA to cells, which may be because there were so many more binding sites for WGA than for RCA. The composition of WGA- and RCA-binding molecules was analysed by lectin affinity chromatography of metabolically radiolabelled BHK cells. We found that in the WGA-binding-molecule preparations there were eight major polypeptides, ranging in molecular mass from 93 to 340 kDa, and that the RCA-binding molecules were a subpopulation of the WGA-binding molecules. A polyclonal antibody against the 140 kDa fibronectin (FN) receptors of Chinese-hamster ovary (CHO) cells immunoblotted a 145 kDa polypeptide component in both WGA- and RCA-binding-molecule preparations. The results indicated that the 145 kDa component was present in at least two FN-receptor complexes that differed in glycosylation, only one of which was able to bind to RCA affinity columns. The oligomeric nature of the FN-receptor complex, which contained three polypeptides with molecular masses of 120-145 kDa, was demonstrated by using anti-(CHO-cell FN receptor) antibodies to immunoprecipitate extracts prepared from radioiodinated BHK cells.