bluuebunny/crossref_metadata_2025 huggingface dataset:
Role of protein kinase C in the phosphorylation of cardiac myosin light chain 2
Authors: Venema R C , Raynor R L , Noland T A , Kuo J F
Published: August 2015

The role of protein kinase C (PKC) in the phosphorylation of myosin light chain 2 (MLC2) in adult rat heart cells has been investigated. PKC-mediated phosphorylation of MLC2 in adult rat cardiac myofibrils in vitro occurs with a stoichiometry (0.7 mol of phosphate/mol of protein) similar to that mediated by myosin light chain kinase (MLCK). Two-dimensional tryptic phosphopeptide mapping of MLC2 following phosphorylation by PKC or MLCK in vitro yields the same major phosphopeptides for each protein kinase. These sites are also 32P-labelled in situ when isolated cardiomyocytes are incubated with [32P]P(i). 32P labelling of MLC2 in cardiomyocytes is increased by 5-fold in 10 min upon incubation with the phosphatase inhibitor calyculin A, demonstrating the existence of a rapidly turning over component of MLC2 phosphorylation in these cells. 32P label is completely removed from MLC2 when myocytes are exposed to 2,3-butanedione monoxime, an inhibitor of cardiac contraction known to desensitize the myofilaments to activation by Ca2+. 32P labelling of MLC2 is also decreased by 50-100% following exposure to the PKC-selective inhibitors calphostin C and chelerythrine, suggesting that PKC, and not MLCK, is primarily responsible for incorporation of rapidly turning over phosphate into MLC2 in situ. Taken together, these data implicate PKC in the phosphorylation of MLC2 in heart cells and support the hypothesis that phosphorylation of cardiac MLC2 has a role in determining myofibrillar Ca2+ sensitivity.
Differential induction of phosphatidylcholine hydrolysis, diacylglycerol formation and protein kinase C activation by epidermal growth factor and transforming growth factor-α in normal human skin fibroblasts and keratinocytes
Authors: Reynolds N J , Talwar H S , Baldassare J J , Henderson P A , Elder J T , Voorhees J J , Fisher G J
Published: August 2015

We have investigated coupling between the epidermal growth factor (EGF) receptor and the phospholipase C (PLC)/protein kinase C (PKC) signal-transduction system in normal skin fibroblasts and keratinocytes, for which EGF and transforming growth factor alpha (TGF-alpha) are mitogenic. EGF and TGF-alpha induced a rapid increase in tyrosine phosphorylation of the EGF receptor, in both fibroblasts and keratinocytes, but failed to induce tyrosine phosphorylation of PLC-gamma 1 or detectable phosphoinositide hydrolysis, as measured by two sensitive assays. In fibroblasts, EGF induced phosphatidylcholine (PC) hydrolysis, resulting in increased diacylglycerol (DAG). In contrast, in keratinocytes, there was no detectable PC hydrolysis or elevation of DAG in response to EGF or TGF-alpha. EGF and TGF-alpha activated PKC in fibroblasts, as evidenced by increased phosphorylation of a specific cellular PKC substrate (myristoylated alanine-rich C-kinase substrate, ‘MARCKS’). In keratinocytes, TGF-alpha and EGF induced only a modest increase in MARCKS protein phosphorylation. This apparent modest activation of PKC, in the absence of detectable DAG formation, may have been mediated by arachidonic acid, which was released from keratinocytes in response to TGF-alpha, and has been shown to stimulate PKC activity in vitro. These data demonstrate that (1) in dermal fibroblasts and keratinocytes, which express normal levels of EGF receptors, EGF receptor activation is not coupled to tyrosine phosphorylation of PLC-gamma 1 or PtdIns hydrolysis, suggesting that these events are not required for the mitogenic activity of EGF or TGF-alpha in these cells, (2) coupling of EGF receptor to PC hydrolysis is cell-type specific, and (3) in skin fibroblasts, DAG, formed through EGF-induced PC hydrolysis, is capable of activating PKC.
Comparison of the active sites of the purified carnitine acyltransferases from peroxisomes and mitochondria by using a reaction-intermediate analogue
Authors: Nic a′ Bháird N , Kumaravel G , Gandour R D , Krueger M J , Ramsay R R
Published: August 2015

The carnitine acyltransferases contribute to the modulation of the acyl-CoA/CoA ratio in various cell compartments with consequent effects on many aspects of fatty acid metabolism. The properties of the enzymes are different in each location. The kinetic mechanisms and kinetic parameters for the carnitine acyltransferases purified from peroxisomes (COT) and from the mitochondrial inner membrane (CPT-II) were determined. Product-inhibition studies established that COT follows a rapid-equilibrium random-order mechanism, but CPT-II follows a strictly ordered mechanism in which acyl-CoA or CoA must bind before the carnitine substrate. Hemipalmitoylcarnitinium [(+)-HPC], a prototype tetrahedral intermediate analogue of the acyltransferase reaction, inhibits CPT-II 100-fold better than COT. (+)-HPC behaves as an analogue of palmitoyl-L-carnitine with COT. In contrast, with CPT-II(+)-HPC binds more tightly to the enzyme than do substrates or products, suggesting that it is a good model for the transition state and, unlike palmitoyl-L-carnitine, (+)-HPC can bind to the free enzyme. The data support the concept of three binding domains for the acyltransferases, a CoA site, an acyl site and a carnitine site. The CoA site is similar in COT and CPT-II, but there are distinct differences between the carnitine-binding site which may dictate the kinetic mechanism.
Spectroscopic characterization of cytochrome c peroxidase from Paracoccus denitrificans
Authors: Gilmour R , Goodhew C F , Pettigrew G W , Prazeres S , Moura I , Moura J J G
Published: August 2015

The cytochrome c peroxidase of Paracoccus denitrificans is similar to the well-studied enzyme from Pseudomonas aeruginosa. Like the Pseudomonas enzyme, the Paracoccus peroxidase contains two haem c groups, one high potential and one low potential. The high-potential haem acts as a source of the second electron for H2O2 reduction, and the low-potential haem acts as a peroxidatic centre. Reduction with ascorbate of the high-potential haem of the Paracoccus enzyme results in a switch of the low-potential haem to a high-spin state, as shown by visible and n.m.r. spectroscopy. This high-spin haem of the mixed-valence enzyme is accessible to ligands and binds CN- with a KD of 5 microM. The Paracoccus enzyme is significantly different from that from Pseudomonas in the time course of high-spin formation after reduction of the high-potential haem, and in the requirement for bivalent cations. Reduction with 1 mM ascorbate at pH 6 is complete within 2 min, and this is followed by a slow appearance of the high-spin state with a half-time of 10 min. Thus the process of reduction and spin state change can be easily separated in time and the intermediate form obtained. This separation is also evident in e.p.r. spectra, although the slow change involves an alteration in the low-spin ligation at this temperature rather than a change in spin state. The separation is even more striking at pH 7.5, where no high-spin form is obtained until 1 mM Ca2+ is added to the mixed-valence enzyme. The spin-state switch of the low-potential haem shifts the midpoint redox potential of the high-potential haem by 50 mV, a further indication of haem-haem interaction.
Use of pyrenemethyl laurate for fluorescence-based determination of lipase activity in intact living lymphoblastoid cells and for the diagnosis of acid lipase deficiency
Authors: Nègre-Salvayre A , Dagan A , Gatt S , Salvayre R
Published: August 2015

Pyrenemethyl laurate (PMLes), a fluorogenic substrate for determining in vitro lipase activity [Nègre, Salvayre, Dagan and Gatt (1989) Biochim. Biophys. Acta 1006, 84-88], has been administered to cultured lymphoblastoid cells from normal subjects and from a patient affected with Wolman disease, which is characterized by a deficiency of lysosomal acid lipase. The intracellular degradation of PMLes was dependent on the mode of administration of the substrate into the cells, and occurred by two separate pathways involving lysosomal and extra-lysosomal hydrolases. PMLes incorporated into LDL was taken up by normal lymphoblastoid cells through the apolipoprotein-B/E-receptor-mediated pathway and degraded in the lysosomal compartment, as suggested by the degradation block in Wolman cells. In contrast, when PMLes dissolved in 2% dimethyl sulphoxide was added directly to the culture medium, its hydrolysis was similar in lymphoblastoid cells from controls and from patients affected with Wolman disease, neutral lipid storage disease or familial hypercholesterolaemia. This suggested that the administered PMLes was degraded by a non-lysosomal enzyme which is not deficient in Wolman cells. This enzyme also differs from the neutral lipase system which is deficient in lymphoblastoid cells from patients with neutral lipid storage disease. When pyrenemethanol was administered directly to the cell culture, it was only poorly acylated and was rapidly released into the culture medium. These results and the fluorescence properties of PMLes (‘monomeric’ emission in a hydrophobic environment and ‘excimeric’ emission in a hydrophilic environment) and pyrenemethanol (‘monomeric’ emission in a hydrophilic environment) allowed us to design a ‘direct reading’ procedure by monitoring (without any lipid extraction) the fluorescence of intact living cells and that of the culture medium during pulse-chase experiments. This method allowed the direct evaluation of the time course of in situ degradation of PMLes. In pulse-chase experiments with LDL-PMLes, the fluorescence of normal cells decreased relatively rapidly with time whereas the fluorescence of the culture medium increased concomitantly. With Wolman cells, the cellular fluorescence decreased only very slightly, whereas that of the culture medium remained at the basal level; this demonstrates the catabolic block in intact living cells from patients with Wolman disease. In vitro degradation of PMLes indicated the existence of two PMLes-degrading enzymes in lymphoblastoid cell homogenates: one is the acid lipase which is involved in PMLes degradation in the lysosomal compartment (and is deficient in Wolman cells), while the second is a cytoplasmic enzyme (not deficient in Wolman cells).
Continuous bioluminescent monitoring of cytoplasmic ATP in single isolated rat hepatocytes during metabolic poisoning
Authors: Koop A , Cobbold P H
Published: August 2015

We have devised a technique for monitoring cytoplasmic ATP continuously in single hepatocytes. Single isolated rat hepatocytes were injected with the ATP-dependent luminescent protein firefly luciferase, and then superfused with 45 microM luciferin in air-equilibrated medium. Signals of approx. 10-200 photoelectron counts per second could be recorded from individual healthy cells for up to 3 h. The response of the luminescent signal to chemical hypoxia (2-5 mM CN- and 5-10 mM 2-deoxyglucose) was monitored. We found a great cell-to-cell variability in the time course of the ATP decline in response to CN-, 2-deoxyglucose or to their combination; the time for the signal to fall to 10% of the original (corresponding to approx. 100 microM ATP) ranged from approx. 20 to 75 min. This resistance of the cytoplasmic ATP concentration to depletion after blockade of oxidative phosphorylation and glycolysis could be abolished by pretreatment of the cells with etomoxir, which blocks mitochondrial beta-oxidation. Etomoxir alone had no effect on the luciferase signal, but etomoxir-pre-treated cells showed a prompt fall in the luciferase signal starting within 1-2 min of application of cyanide and 2-deoxyglucose and falling to 10% of the original signal in approx. 6-10 min. The technique allows cytoplasmic ATP changes to be monitored in single hepatocytes at concentrations of 1 mM or lower, but more precise calibration of the signal will require correction for the effects of cytoplasmic pH changes.
The kinetics of non-stoichiometric bursts of β-lactam hydrolysis catalysed by class C β-lactamases
Authors: Page M G P
Published: August 2015

Class C beta-lactamases from Pseudomonas aeruginosa and several species of the Enterobacteriaceae have been observed to undergo a rapid burst in hydrolysis of beta-lactam antibiotics before relaxation to a steady-state rate of hydrolysis. The amplitude of the burst corresponds to the hydrolysis of between 1 and 10,000 mol of the substrate per mol of enzyme. The decay of the rate of hydrolysis in the burst phase comprises two exponential reactions, which indicates that there are three different reactive states of the enzymes. Examination of the kinetics of acylation by slowly reacting beta-lactams suggests that there are three forms of the free enzyme in slow equilibrium. Thus it would appear that the burst kinetics exhibited by class C enzymes can be attributed to redistribution of the enzyme between different conformations induced by the reaction with substrate.
Vectorial Ca2+ flux from the extracellular space to the endoplasmic reticulum via a restricted cytoplasmic compartment regulates inositol 1,4,5-trisphosphate-stimulated Ca2+ release from internal stores in vascular endothelial cells
Authors: Cabello O A , Schilling W P
Published: August 2015

Depletion of the Ins(1,4,5)P3-sensitive intracellular Ca2+ store of vascular endothelial cells after selective inhibition of the endoplasmic-reticulum (ER) Ca2+ pump by thapsigargin or 2,5-di-t-butylhydroquinone (BHQ) increases Ca2+ influx from the extracellular space in the absence of phosphoinositide hydrolysis. One model to account for these results suggests a close association between the internal store and the plasmalemma, allowing for the vectorial movement of Ca2+ from the extracellular space to the ER. Furthermore, recent evidence suggests that Ins(1,4,5)P3-induced Ca2+ release from intracellular stores is regulated by the free cytosolic Ca2+ concentration ([Ca2+]i). Thus agonist-induced Ca2+ entry may directly regulate Ca2+ release from internal stores. To test these hypotheses, we examined the effect of 1-(beta-[3-(4-methoxyphenyl)propoxy]-4-methoxyphenethyl)-1H-imidazole (SKF 96365), an inhibitor of Ca2+ influx, on unidirectional 45Ca2+ efflux (i.e. retrograde radioisotope flux via the influx pathway) and on [Ca2+]i as measured by fura-2. Bradykinin produced a transient increase in [Ca2+]i, reflecting release of Ca2+ from internal stores, and a sustained increase indicative of Ca2+ influx. In the absence of agonist, 45Ca2+ efflux was slow and monoexponential with time. Addition of BK dramatically increased 45Ca2+ efflux; 50-60% of the 45Ca2+ associated with the cell monolayer was released within 2 min after addition of bradykinin. Both the bradykinin-induced change in [Ca2+]i and the stimulation of 45Ca2+ efflux was completely blocked by loading the cells with the Ca2+ chelator BAPTA. At a supermaximal concentration of bradykinin (50 nM), SKF 96365 (50 microM) inhibited the rise in [Ca2+]i attributed to influx without affecting release from internal stores. At a threshold concentration of bradykinin (2 nM), SKF 96365 blocked influx, but stimulated Ca2+ release from internal stores, as indicated by increases in both the transient component of the fura-2 response and 45Ca2+ efflux. Thapsigargin (200 nM) and BHQ (10 microM) produced an increase in 45Ca2+ efflux that was completely blocked by SKF 96365 or by cytosolic loading with BAPTA. These results suggest the existence of a restricted sub-plasmalemmal space that is defined by an area of surface membrane which contains the Ca(2+)-influx pathway but is devoid of Ca2+ pumps, and by a section of ER that is rich in thapsigargin-sensitive Ca(2+)-pump units.(ABSTRACT TRUNCATED AT 400 WORDS)
Isoprenoid biosynthesis in bacteria: a novel pathway for the early steps leading to isopentenyl diphosphate
Authors: Rohmer M , Knani M , Simonin P , Sutter B , Sahm H
Published: August 2015

Incorporation of 13C-labelled glucose, acetate, pyruvate or erythrose allowed the determination of the origin of the carbon atoms of triterpenoids of the hopane series and/or of the ubiquinones from several bacteria (Zymomonas mobilis, Methylobacterium fujisawaense, Escherichia coli and Alicyclobacillus acidoterrestris) confirmed our earlier results obtained by incorporation of 13C-labelled acetate into the hopanoids of other bacteria and led to the identification of a novel biosynthetic route for the early steps of isoprenoid biosynthesis. The C5 framework of isoprenic units results most probably (i) from the condensation of a C2 unit derived from pyruvate decarboxylation (e.g. thiamine-activated acetaldehyde) on the C-2 carbonyl group of a triose phosphate derivative issued probably from dihydroxyacetone phosphate and not from pyruvate and (ii) from a transposition step. Although this hypothetical biosynthetic pathway resembles that of L-valine biosynthesis, this amino acid or its C5 precursors could be excluded as intermediates in the formation of isoprenic units.
Histone H4 stimulates glucose transport activity in rat skeletal muscle
Authors: Louters L L , Henriksen E J , Tipton C M
Published: August 2015

We investigated the effects of purified histone H4 on glucose transport activity in rat soleus and flexor digitorum brevis muscles. Histone H4, at concentrations up to 11.8 microM, increased 2-deoxyglucose (2-DG) uptake in a dose-dependent fashion. However, at concentrations higher than 11.8 microM, H4 caused a decrease in 2-DG uptake from the maximum, suggesting a secondary inhibitory action of this compound. The maximal effect of H4 on 2-DG uptake was not additive to the maximal effect of insulin. Moreover, 2-DG uptake in the presence of both H4 and insulin was significantly lower than the 2-DG uptake in the presence of insulin alone. The maximal effect of H4 on stimulation of 2-DG uptake was neither additive nor inhibitory to the maximal effects of the intracellularly acting insulin mimetics sodium vanadate or H2O2. It was, on the other hand, additive to the maximal effects of muscle contractions. Also, in contrast with the effects of H4 on insulin-stimulated 2-DG uptake, H4 did not inhibit insulin-like growth factor-I (IGF-I)-stimulated 2-DG uptake, as the maximal effects of H4 and IGF-I were additive. Scatchard analysis of the binding of 125I-insulin in the absence or presence of histone H4 revealed that H4 increased the specific binding of insulin without affecting receptor affinity. These data suggest that H4 interacts with the insulin, rather than the hypoxia/contraction, pathway for activation of glucose transport in muscle tissue, and that H4 acts either directly or indirectly to increase the number of insulin receptors at the surface of the muscle cell. This interaction does not appear to occur with the similar, although distinct, IGF-I receptor. These studies may provide additional insight into the complex signal-transduction systems of insulin action.
A quantitative analysis of the control of glutamine catabolism in rat liver cells. Use of selective inhibitors
Authors: Low S Y , Salter M , Knowles R G , Pogson C I , Rennie M J
Published: August 2015

1. At a physiological concentration of glutamine (0.5 mM), 87% of the total transport across the plasma membrane of liver cells isolated from fed rats involved the Na(+)-dependent system N; this was substantially inhibited by L-histidine. The residual Na(+)-independent component was attributed to system L on the basis of inhibition by 2-amino-2-norbornanecarboxylate and L-tryptophan. 2. Catabolism of glutamine by intact liver cells or by isolated mitochondria was inhibited by glutamate gamma-hydrazide with IC50 values of 13.7 +/- 3.5 microM and 22.6 +/- 3.8 microM respectively and a maximal inhibition of approx. 75%. The site of inhibition was identified as glutaminase; glutamate gamma-hydrazide inhibited this enzyme in cell-free extracts (IC50 37.8 +/- 7.7 microM) but had no activity against glutamate dehydrogenase or transport of glutamine, whether across mitochondrial or plasma membranes. 3. The major control site in cells from fed animals incubated with 0.5 mM L-glutamine was glutaminase (flux control coefficient 0.96). Appreciable control also resided in both plasma membrane transport systems, with coefficients of 0.51 for system N and -0.46 for system L, such that both interacted to provide a fine control of the intracellular concentration of the amino acid. Similar values were obtained by computer simulation based on theoretical determination of elasticities. 4. Previous controversy about the locus of regulation of hepatic glutamine metabolism is resolved by this distribution of control.
Molecular cloning of a rat κ opioid receptor reveals sequence similarities to the μ and δ opioid receptors
Authors: Chen Y , Mestek A , Liu J , Yu L
Published: August 2015

By screening a rat brain cDNA library using a cloned mu opioid receptor cDNA as probe, a clone was identified that is very similar to both the mu and delta opioid receptor sequences. Transient expression of this clone in COS-7 cells showed that it encodes a kappa opioid receptor, designated KOR-1, which is capable of high-affinity binding to kappa-selective ligands. Treatment of transfected cell membranes with bremazocine, a kappa-selective agonist, resulted in a 53% decrease in adenylate cyclase activity, indicating that this kappa opioid receptor displays inhibitory coupling to adenylate cyclase. Thus, one member from each of the three opioid receptor types, mu, kappa and delta, has been molecularly cloned. Analysis of sequence similarities among these opioid receptors, as well as between opioid receptors and other G-protein-coupled receptors, revealed regions of sequence conservation that may underlie the ligand-binding and functional specificities of opioid receptors.
Retinoic acid regulates ornithine decarboxylase gene expression at the transcriptional level
Authors: Mao Y , Gurr J A , Hickok N J
Published: August 2015

Retinoic acid (RA) is important for normal mammalian development and growth. Ornithine decarboxylase (ODC) is the first and rate-limiting enzyme in the biosynthesis of the polyamines, and we have previously shown that ODC mRNA levels are suppressed by RA in human skin cells. Using HeLa cells, we now show that treatment with 0.5 microM RA for 24 h suppresses endogenous ODC mRNA levels and the expression of a transfected ODC/chloramphenicol acetyltransferase plasmid (Kpn-ODCCAT), containing sequences from -1450 to +810 of the human ODC gene. Co-transfection with either the alpha-RA receptor (alpha-RAR) or a chimeric alpha-RA/oestrogen receptor (alpha-RAER) followed by treatment with the cognate hormone suppresses expression of Kpn-ODCCAT and Not-ODCCAT, which contains sequences from -250 to +514. Liganded alpha-RAR suppresses the activity of Kpn-ODCCAT more markedly than does liganded alpha-RAER (98% and 80% suppression, respectively), whereas both receptors have very similar effects on Not-ODCCAT expression (73% and 67% suppression, respectively). The unliganded alpha-RAR suppresses Kpn-ODCCAT by 76%, whereas unliganded alpha-RAER has no significant effect. These data show that RA regulates ODC-gene expression at the transcriptional level, and that alpha-RAR, but not alpha-RAER, can confer full hormonal responsiveness. This suggests that the activating function present in the alpha-RAR ligand-binding domain is required for full transcriptional regulation.
Inositol phospho-oligosaccharides from rat fibroblasts and adipocytes stimulate 3-O-methylglucose transport
Authors: Kellerer M , Machicao F , Berti L , Sixt B , Mushack J , Seffer E , Mosthaf L , Ullrich A , Häring H U
Published: August 2015

Inositol phospho-oligosaccharides (IPOs), which are released from liver membranes upon stimulation by insulin, mimic a wide spectrum of insulin effects in different cells, but not the stimulation of glucose transport. We investigated whether other insulin-sensitive tissues release glucose transport-stimulating IPOs and whether this is related to the human insulin receptor isoform-A or -B (HIR-A or HIR-B). Rat1 fibroblasts overexpressing HIR-A or -B (rat1-HIR cells) were labelled with [3H]glucosamine, [3H]mannose or myo-[3H]inositol. IPOs from the cell supernatant were partially purified by an AG1X2 anion-exchange column, and fractions were eluted at different pH values (pH 3, pH 2 and pH 1.3). The label from glucosamine, mannose and myo-inositol appeared predominantly in the pH 2 fraction. The biological activity of the fractions was determined by measuring 3-O-methylglucose transport and lipogenesis in fat cells. Using the pH 2 fraction from the supernatant of rat1-HIR fibroblasts, insulin increased the release of 3-O-methylglucose-transport-stimulating activity (HIR-A: without insulin, 22.4 +/- 5.4%; with insulin 54.0 +/- 8.4%; HIR-B: without insulin 21.6 +/- 7.5%, with insulin, 44.7 +/- 10.6%, given as a percentage of equilibrium glucose transport reached after 4 s) and lipogenesis-stimulating activity (HIR-A: without insulin, 1.24 +/- 0.17; with insulin, 4.69 +/- 0.2; HIR-B: without insulin, 1.34 +/- 0.18; with insulin, 4.98 +/- 0.31, given as nmol of [3H]glucose converted into lipids/min per 10(6) cells). Analogous experiments were performed with isolated rat fat cells expressing the physiological level of insulin receptors. Upon insulin stimulation of fat cells in the presence of 2.5 mM mannose, the release of 3-O-methylglucose-transport-stimulating activity was detected (for purified supernatant of adipocytes without insulin, 6.9 +/- 1.12%; with insulin, 41.0 +/- 3.6%) and lipogenesis-stimulating activity (without insulin, 0.93 +/- 0.17, with insulin 2.96 +/- 0.31 nmol/min per mg). These data suggest (1) that adipocytes and rat1-HIR fibroblasts release IPOs that are able to stimulate glucose transport, (2) that both insulin receptor isoforms (HIR-A and HIR-B) mediate the effect of insulin on IPO release, and (3) that overexpression of insulin receptors increases the basal release of IPOs.
Dissociation of the tetrameric phosphoglycerate mutase from yeast by a mutation in the subunit contact region
Authors: White M F , Fothergill-Gilmore L A , Kelly S M , Price N C
Published: August 2015

Phosphoglycerate mutases from different sources exhibit a variety of quaternary structures (tetramer, dimer and monomer). To perturb the tetrameric structure of yeast phosphoglycerate mutase we have prepared a mutant enzyme in which Lys-168 in the subunit-contact region has been replaced by proline. The K168P mutant enzyme undergoes dissociation to dimers at low concentrations; thus on lowering the concentration from 200 micrograms/ml to 5 micrograms/ml the proportion of tetramer falls from 85% to 53%. The tetrameric structure of the wild-type enzyme remains intact over this range of concentrations. The mutant enzyme has similar kinetic properties to the wild-type enzyme, with kcat. being reduced by 26%. Far-u.v. c.d. studies show that there has been a small loss of helical structure in the mutant. Compared with wild-type enzyme, the K168P mutant enzyme is slightly less stable towards proteolysis by trypsin, but significantly less stable towards denaturation by guanidinium chloride, with the midpoint concentration of guanidinium chloride some 50% lower. After denaturation, the mutant enzyme could regain activity and quaternary structure when the guanidinium chloride concentration was lowered to 0.05 M. The properties of the mutant enzyme are discussed in terms of other dimeric phosphoglycerate and bisphosphoglycerate mutases which contain proline at position 168.
Complement-induced Ca2+ influx in cultured fibroblasts is decreased by the calcium-channel antagonist nifedipine or by some bivalent inorganic cations
Authors: Newsholme P , Adogu A A , Soos M A , Hales C N
Published: August 2015

The effects of different extracellular cations or organic Ca(2+)-channel modulators on complement-induced changes in intracellular Ca2+ and cell death have been investigated in the transfected NIH-3T3 HIR 3.5 cell line, which overexpresses the human insulin receptor. Cells were incubated with mouse anti-(human insulin receptor) monoclonal antibodies before exposure to rabbit or human serum (sources of heterologous complement). Changes in intracellular Ca2+ were complement-dependent (measured by influx of 45Ca), as was cytotoxicity (monitored by leakage of lactate dehydrogenase into the culture supernatant). Addition of a dihydropyridine Ca(2+)-channel antagonist (nifedipine) or some bivalent inorganic cations caused inhibition of 45Ca entry via a novel channel distinct from endogenous voltage-gated Ca2+ channels. Nifedipine decreased, but conversely the addition of a phenylalkylamine Ca(2+)-channel antagonist (verapamil) or the inorganic Ca2+ agonists Ba2+ and Sr+ increased, complement-induced cytotoxicity. These agents had no effect on cell viability at the studied concentrations, in the absence of complement. It is concluded that complement-induced cytotoxicity is mediated by Ca2+ influx through novel specific transmembrane channels which are sensitive to the Ca(2+)-channel antagonist nifedipine, but otherwise show little resemblance to L- or T-type voltage-gated Ca2+ channels.
Substrate-induced inactivation of the OXA2 β-lactamase
Authors: Ledent P , Frère J M
Published: August 2015

The hydrolysis time courses of 22 beta-lactam antibiotics by the class D OXA2 beta-lactamase were studied. Among these, only three appeared to correspond to the integrated Henri-Michaelis equation. ‘Burst’ kinetics, implying branched pathways, were observed with most penicillins, cephalosporins and with flomoxef and imipenem. Kinetic parameters characteristic of the different phases of the hydrolysis were determined for some substrates. Mechanisms generally accepted to explain such reversible partial inactivations involving branches at either the free enzyme or the acyl-enzyme were inadequate to explain the enzyme behaviour. The hydrolysis of imipenem was characterized by the occurrence of two ‘bursts’, and that of nitrocefin by a partial substrate-induced inactivation complicated by a competitive inhibition by the hydrolysis product.
Lipoxin synthase activity of human platelet 12-lipoxygenase
Authors: Romano M , Chen X S , Takahashi Y , Yamamoto S , Funk C D , Serhan C N
Published: August 2015

Human platelets and megacaryocytes generate lipoxins from exogenous leukotriene A4 (LTA4). We examined the role of human 12-lipoxygenase (12-LO) in lipoxin generation with recombinant histidine-tagged human platelet enzyme (6His-12-LO), partially purified 12-LO from human platelets (HPL 12-LO) and, for the purposes of direct comparison, permeabilized platelets. Recombinant and HPL 12-LO catalysed the conversion of intact LTA4 into both lipoxin A4 (LXA4) and lipoxin B4 (LXB4). In contrast, only negligible quantities of LXA4 were generated when recombinant 12-LO was incubated with the non-enzymic hydrolysis products of LTA4.6His-12-LO also converted a non-allylic epoxide, 5(6)-epoxy-(8Z,11Z,14Z)-eicosatrienoic acid. The apparent Km and Vmax. for lipoxin synthase activity of 6His-12-LO were estimated to be 7.9 +/- 0.8 microM and 24.5 +/- 2.5 nmol/min per mg respectively, and the LXB4 synthase activity of this enzyme was selectively regulated by suicide inactivation. Aspirin gave a 2-fold increase in lipoxin formation by platelets but did not enhance the conversion of LTA4 by the recombinant 12-LO. These results provide direct evidence for LXA4 and LXB4 synthase activity of human platelet 12-LO. Moreover, they suggest that 12-LO is a dual-function enzyme that carries both oxygenase and lipoxin synthase activity.
Modulation of iron metabolism in monocyte cell line U937 by inflammatory cytokines: changes in transferrin uptake, iron handling and ferritin mRNA
Authors: Fahmy M , Young S P
Published: August 2015

We have investigated the effects of the pro-inflammatory cytokines interleukin 1 beta (IL-1 beta), tumour necrosis factor alpha (TNF alpha) and interferon gamma (IFN gamma) on the iron metabolism of the human monocytic cell line U937. Cells were treated with each cytokine for up to 24 h, and then iron uptake from diferric transferrin was determined. The intracellular distribution of this iron, the expression of the transferrin receptor and levels of mRNA for the two ferritin subunits were also studied. IL-1 beta, TNF alpha and IFN gamma all decreased transferrin-iron uptake into cells, and all three cytokines had effects on the proportion of iron associated with ferritin. With TNF alpha there was a marked enhancement of the fraction incorporated into ferritin. Transferrin-receptor expression was diminished by TNF alpha and IL-1 beta, but not IFN gamma, suggesting different effector mechanisms. Both TNF alpha and IFN gamma increased the amount of cellular mRNA for ferritin H-chain, but not the L-chain; IL-1 beta affected mRNA for neither ferritin. These data demonstrate that cytokines, which can be present at high concentrations in inflammation, have the capacity to affect macrophage iron uptake, transferrin receptor expression, intracellular iron handling and the relative abundance of ferritin-subunit mRNA, and may therefore be important mediators in the observed perturbations of iron metabolism in inflammatory diseases.
The human phenolsulphotransferase polymorphism is determined by the level of expression of the enzyme protein
Authors: Jones A L , Roberts R C , Coughtrie M W H
Published: August 2015

We have examined the expression of platelet phenolsulphotransferase (PST) in 60 individuals. Using an antibody which recognizes both forms of PST present in man (P-PST and M-PST), we determined that the polymorphism of platelet P-PST activity is determined by the level of expression of the enzyme protein. The implications for susceptibility to adverse drug reactions and chemical carcinogenesis are discussed.
Two distinct secretory ribonucleases from human cerebrum: purification, characterization and relationships to other ribonucleases
Authors: Yasuda T , Nadano D , Takeshita H , Kishi K
Published: August 2015

Two RNAases from human cerebrum were purified to an electrophoretically homogeneous state and their molecular masses were 22.0 kDa (tentatively called RNAase HB-1) and 19.0 kDa (RNAase HB-2). Analyses of the amino acid compositions, N-terminal amino acid sequences and catalytic properties of these enzymes provided strong evidence that they were strictly related to the secretory (sec) RNAases, such as the pancreatic enzyme, very similar immunologically to urinary sec RNAase, but clearly distinguishable from urinary non-secretory (nonsec) RNAase. There were several differences between HB-1 and HB-2, namely their immunological reactivities with specific antibodies, heat-stabilities, attached carbohydrate moieties and molecular masses. In particular, HB-2 appeared to be nonglycosylated, in view of its lack of affinity for several conjugated lectins, the absence of hexosamine and no change in electrophoretic mobility before and after peptide:N-glycosidase F digestion, whereas HB-1 and human sec RNAases purified from kidney, pancreas and urine all appeared to be glycosylated, as they moved to the same position as HB-2 when electrophoresed after glycosidase digestion. An antibody against urinary sec RNAase inhibited 75% and 20% of the total activity of the crude cerebral extract against RNA at pH 8.0 and 6.0 respectively, whereas an antibody against urinary nonsec RNAase had no such inhibitory effect. These findings suggest that yet another type(s) of cerebral RNAase, which is unable to cross-react immunologically with sec and nonsec RNAases, may exist. Two RNAases corresponding to HB-1 and HB-2 were identified in fresh cerebrospinal fluid.
Cysteine residues are not essential for uncoupling protein function
Authors: Arechaga I , Raimbault S , Prieto S , Levi-Meyrueis C , Zaragoza P , Miroux B , Ricquier D , Bouillaud F , Rial E
Published: August 2015

The uncoupling protein (UCP) of brown adipose tissue is a regulated proton carrier which allows uncoupling of mitochondrial respiration from ATP synthesis and, therefore, dissipation of metabolic energy as heat. In this article we demonstrate that, when UCP is expressed in Saccharomyces cerevisiae, it retains all its functional properties: proton and chloride transport, high-affinity binding of nucleotides and regulation of proton conductance by nucleotides and fatty acids. Site-directed mutagenesis demonstrates that sequential replacement by serine of cysteine residues in the UCP does not affect either its uncoupling activity or its regulation by nucleotides and fatty acids, and therefore establishes that none of the seven cysteine residues present in the wild-type UCP is critical for its activity. These data indicate that transport models involving essential thiol groups can be discounted and that chemical modification data require critical re-evaluation.
Regulation of matrix metalloproteinase expression in human vein and microvascular endothelial cells. Effects of tumour necrosis factor α, interleukin 1 and phorbol ester
Authors: Hanemaaijer R , Koolwijk P , le Clercq L , de Vree W J A , van Hinsbergh V W M
Published: August 2015

Matrix metalloproteinases (MMPs) play a role in tissue remodelling and angiogenesis. We have investigated the expression and regulation of MMP-1 (interstitial collagenase), MMP-2 (gelatinase A), MMP-3 (stromelysin 1), MMP-7 (matrilysin), MMP-9 (gelatinase B) and their inhibitors TIMP-1 and TIMP-2 in human umbilical vein, femoral vein and microvascular endothelial cells, and compared these data with those obtained with human synovial fibroblasts. Non-stimulated vein endothelial cells expressed the mRNAs for MMP-1, MMP-2, TIMP-1 and TIMP-2. MMP-3 mRNA and protein were undetectable or only weakly expressed, but could be stimulated by the inflammatory mediator tumour necrosis factor alpha (TNF alpha). The expression of MMP-3 and MMP-1 was further enhanced by phorbol 12-myristate 13-acetate (PMA). Phorbol ester also induced TIMP-1 and MMP-9, the expression of the latter being further enhanced by TNF alpha or interleukin 1 alpha (IL-1 alpha). Similar stimulatory effects were observed in microvascular endothelial cells. Hence the inflammatory mediator TNF alpha induces/enhances the production of several matrix metalloproteinases in human endothelial cells. On the other hand, MMP-2 and TIMP-2 were not affected or were affected in a variable way by TNF alpha and/or phorbol ester, suggesting a dissimilar regulation of these proteins. The cyclic AMP-enhancing agent forskolin affected the production of MMPs in a cell-type-specific way. In human vein endothelial cells it enhanced the PMA-mediated induction of MMP-9, whereas it suppressed this induction in human microvascular endothelial cells and in synovial fibroblasts. On the other hand, forskolin suppressed the PMA-mediated induction of MMP-1 and MMP-3 in synovial fibroblasts, while it enhanced or did not affect this induction in various types of human endothelial cells. These observations may have implications for future pharmacological intervention in angiogenesis.
Calponin phosphorylation in vitro and in intact muscle
Authors: Winder S J , Allen B G , Fraser E D , Kang H M , Kargacin G J , Walsh M P
Published: August 2015

Calponin, a thin-filament-associated protein implicated in the regulation of smooth-muscle contraction, is phosphorylated in vitro by protein kinase C and Ca2+/calmodulin-dependent protein kinase II [Winder and Walsh (1990) J. Biol. Chem. 265, 10148-10155] and dephosphorylated by a type 2A protein phosphatase [Winder, Pato and Walsh (1992) Biochem. J. 286, 197-203]. Unphosphorylated calponin binds to actin and inhibits the actin-activated myosin MgATPase; these properties are lost on phosphorylation. Although both serine and threonine residues in calponin are phosphorylated, the major site of phosphorylation by either kinase is Ser-175. Calponin also undergoes phosphorylation when bound to actin in synthetic thin filaments, in a reconstituted actomyosin system, in washed myofibrils and in tissue extracts; this results in dissociation of calponin from actin. Tryptic phosphopeptide mapping indicates that the same sites are phosphorylated in the bound as in the isolated protein. Toad stomach calponin exists in at least three isoforms which differ in charge but exhibit the same molecular mass on SDS/PAGE. In a toad stomach extract, all three isoforms are phosphorylated by protein kinase C or Ca2+/calmodulin-dependent protein kinase II as shown by two-dimensional gel electrophoresis (non-equilibrium pH-gradient gel electrophoresis and SDS/PAGE). Calponin phosphorylation also occurs in intact toad stomach smooth-muscle strips metabolically labelled with 32Pi and stimulated to contract with carbachol. These results support the hypothesis that calponin may be regulated in vivo by phosphorylation-dephosphorylation.
The peripheral subunit-binding domain of the dihydrolipoyl acetyltransferase component of the pyruvate dehydrogenase complex of Bacillus stearothermophilus: preparation and characterization of its binding to the dihydrolipoyl dehydrogenase component
Authors: Hipps D S , Packman L C , Allen M D , Fuller C , Sakaguchi K , Appella E , Perham R N
Published: August 2015

The peripheral subunit-binding domain of the dihydrolipoyl acetyltransferase polypeptide chain of the pyruvate dehydrogenase multienzyme complex of Bacillus stearothermophilus was released by limited proteolysis from a di-domain (lipoyl domain plus binding domain) encoded by a subgene over-expressed in Escherichia coli. The domain was characterized by N-terminal sequence analysis, electrospray m.s. and c.d. spectroscopy. It was found to be identical in all respects to a chemically synthesized peptide of the same sequence. The association of the di-domain and binding domain (both natural and synthetic) with dihydrolipoyl dehydrogenase was analysed in detail and a tight binding was demonstrated. As judged by several different techniques, it was found that only one peripheral subunit-binding domain is bound to one dimer of dihydrolipoyl dehydrogenase, implying that the association is highly anti-cooperative.
Phospholipase C in Dictyostelium discoideum. Identification of stimulatory and inhibitory surface receptors and G-proteins
Authors: Bominaar A A , Van Haastert P J M
Published: August 2015

A combined biochemical and genetic approach was used to show that phospholipase C in the cellular slime mould Dictyostelium is under dual regulation by the chemoattractant cyclic AMP (cAMP). This dual regulation involves stimulatory and inhibitory surface receptors and G-proteins. In wild-type cells both cAMP and guanosine 5′-[gamma-thio]triphosphate (GTP[S]) stimulated phospholipase C. In contrast, mutant fgd A, lacking the G-protein alpha-subunit G alpha 2, showed no stimulation by either cAMP or GTP[S], indicating that G alpha 2 is the stimulatory G-protein. In mutant fgd C cAMP did not stimulate phospholipase C, but stimulation by GTP[S] was normal, suggesting that the defect in this mutant is upstream of the stimulatory G alpha 2. Inhibition of phospholipase C was achieved in wild-type cells by the partial antagonist 3′-deoxy-3′-aminoadenosine 3′,5′-phosphate (3′NH-cAMP). This inhibition was no longer observed in transformed cell lines lacking either the surface cAMP receptor cAR1 or the G-protein alpha-subunit G alpha 1; in these cells the agonist cAMP still activated phospholipase C. These results indicate that Dictyostelium phospholipase C is regulated via a stimulatory and an inhibitory pathway. The inhibitory pathway is composed of the surface receptor cAR1 and the G-protein G1. The stimulatory pathway consists of an unknown cAMP receptor (possibly the fgd C gene product) and the G-protein G2.
Structural disruption of the trans-Golgi network does not interfere with the acute stimulation of glucose and amino acid uptake by insulin-like growth factor I in muscle cells
Authors: Hundal H S , Bilan P J , Tsakiridis T , Marette A , Klip A
Published: August 2015

The effects of insulin-like growth factor I (IGF-I) on glucose and amino acid uptake were investigated in fully differentiated L6 muscle cells, in order to determine whether the two processes are functionally related. Transport of both glucose and amino acid (methylaminoisobutyric acid, MeAIB) was activated rapidly in response to IGF-I. Stimulation reached a peak within 30 min and was sustained for up to 90 min. Maximal activation of either glucose or MeAIB transport was achieved at 3 nM IGF-I; the half-maximal activation (ED50) of glucose transport was at 107 pM and that of MeAIB transport was at 36 pM. Stimulation of amino acid uptake occurred in the absence or presence of glucose, suggesting that this response is not secondary to increased glucose intake. Incubation of cells for 1 h with Brefeldin A (5 micrograms/ml), which disassembles the Golgi apparatus and inhibits the secretory pathway in eukaryotic cells, had no effect on the acute IGF-I activation of glucose and MeAIB transport. Moreover, Brefeldin A caused wide redistribution of the trans-Golgi antigen TGN38, as assessed by subcellular fractionation, without affecting the distribution of glucose transporters. The finding that the degree of activation, time response and sensitivity to IGF-I and Brefeldin A were similar for both glucose and MeAIB transport suggests commonalities in the IGF-I mechanism of recruitment of glucose transporters and stimulation of amino acid transport through System A. An integral trans-Golgi network does not appear to be required for the acute IGF-I stimulation of glucose or amino acid transport, even though stimulation of glucose transport occurs through recruitment of glucose transporters from intracellular stores in these cells. We propose that the donor site of glucose transporters (and perhaps of amino acid transporters) involved in the acute response to IGF-I lies beyond the trans-Golgi network, perhaps in an endosomal compartment in close proximity to the plasma membrane.
Proteolytic degradation of the RGD-binding and non-RGD-binding conformers of human platelet integrin glycoprotein IIb/IIIa: clues for identification of regions involved in the receptor's activation
Authors: Calvete J J , Mann K , Schäfer W , Fernandez-Lafuente R , Guisán J M
Published: August 2015

The human integrin glycoprotein (GP)IIb/IIIa plays a central role in haemostasis as an inducible receptor for fibrinogen and other RGD-containing adhesive proteins at the platelet plasma membrane. Expression of the fibrinogen receptor on platelet activation involves conformational changes in the quaternary structure of GPIIb/IIIa. Little is known, however, about the nature of this conformational transition. Given that isolated GPIIb/IIIa contains a mixture of RGD-binding and non-RGD-binding heterodimers, we used limited proteolysis as a tool for investigating the structural differences between the two conformers. Comparison of their fragmentation patterns shows that, whereas in the non-RGD-binding form of GPIIb/IIIa the N-terminal half of the heavy chain of GPIIb (GPIIbH) and the central region of GPIIIa are cleaved by endoproteinase Arg-C, these domains associate tightly with one another in the RGD-binding GPIIb/IIIa and are thus protected from proteolysis. In addition, the C-terminal half of GPIIb becomes more susceptible to degradation in the non-RGD-binding GPIIb/IIIa conformer. Our interpretation, in the context of available structural and functional data, is that a major relative reorientation of the GPIIbH and GPIIIa extracellular domains takes place along the subunit interface during the conformational transition of the platelet integrin.