bluuebunny/crossref_metadata_2025 huggingface dataset:
Elevated protein tyrosine phosphatase activity and increased membrane viscosity are associated with impaired activation of the insulin receptor kinase in old rats
Authors: Nadiv O , Shinitzky M , Manu H , Hecht D , Roberts C T , LeRoith D , Zick Y
Published: August 2015

Insulin resistance is very common in the elderly, and may be associated with glucose intolerance or frank diabetes. In previous studies we demonstrated that insulin resistance in old Wistar rats is associated with decreased autophosphorylation and activation of the hepatic insulin receptor kinase (IRK) in vivo. We now show that this defect can be reproduced in vitro, where the extent of insulin-induced activation of IRK in liver membranes of old rats was decreased by approximately 50% compared with young controls. The defect could be largely abolished after solubilization of the membranes with Triton X-100. We also show that: (a) the viscosity of membranes from the old rats was significantly (P < 0.001, n = 4) higher (by 15%) compared with young controls; (b) incubation of plasma membranes from old animals with lecithin liposomes, which lowered their cholesterol levels, partially abolished the defect in IRK activation; and (c) Triton extracts of liver membranes prepared from old rats did not interfere with the activation of IRK derived from young controls. Additionally, non-membrane components did contribute to the development of this defect. We observed a significant (approximately 30%) (P < 0.001, n = 18) elevation of cytosolic protein tyrosine phosphatase (PTP) activity directed against the beta subunit of the insulin receptor in livers of old rats. No such elevation of PTP activity could be demonstrated with synthetic substrates. Our findings are consistent with a model in which increased membrane viscosity as well as enhancement of a cytosolic PTP activity both markedly inhibit the activation in vivo of the hepatic IRK in old animals.
Further thermal characterization of an aspartate aminotransferase from a halophilic organism
Authors: Muriana F J G , Alvarez-Ossorio M C , Relimpio A M
Published: August 2015

Aspartate aminotransferase (AspAT, EC 2.6.1.1) from the halophilic archaebacterium Haloferax mediterranei was purified [Muriana, Alvarez-Ossorio and Relimpio (1991) Biochem. J. 278, 149-154] and further characterization of the effects of temperature on the activity and stability of the halophilic AspAT were carried out. The halophilic transaminase is most active at 65 degrees C and stable at high temperatures, under physiological or nearly physiological conditions (3.5 M KCl, pH 7.8). Thermal inactivation (60-85 degrees C) of the halophilic AspAT followed first-order kinetics, 2-oxoglutarate causing a shift of the thermal inactivation curves to higher temperatures. The salt concentration affected the thermal stability of the halophilic transaminase at 60 degrees C, suggesting that disruption of hydrophobic interactions may play an important role in the decreased thermal stability of the enzyme.
Radiation inactivation of proteins: temperature-dependent inter-protomeric energy transfer in ox liver catalase
Authors: Potier M , Villemure J F , Thauvette L
Published: August 2015

The radiation-inactivation method is widely used to determine the oligomeric structure of enzymes without need for solubilization or purification. We have used purified ox liver catalase, a tetrameric enzyme in solution, to study energy transfer between associated promoters responsible for oligomer inactivation. However, after freeze-drying the tetramer dissociates into an asymmetric dimer. In the present paper we compare both the radiation-inactivation size (obtained by following the activity decay) and the target size (obtained by measuring the amount of remaining protein by SDS/PAGE) of catalase under various states of aggregation and temperature. At −78 degrees C, only one promoter was fragmented after being hit by a gamma-ray and, as expected, this protomer was also inactivated. This result was obtained when either catalase was in tetrameric or in dimeric forms. However, at 38 degrees C, even though a single monomer was fragmented as at −78 degrees C, the whole dimer was inactivated. This result suggests that, at the higher temperature, there is a transfer of energy from the fragmented protomer to the other associated protomer, causing inactivation of the whole dimer. The inactivation of oligomeric enzymes is a two-step mechanism involving: (1) fragmentation of the hit monomer, followed by (2) temperature-dependent energy transfer from the fragmented towards the associated protomer. Thus we conclude that the radiation-inactivation size reflects the transfer of absorbed energy inside the oligomer which causes inactivation of one or several monomers.
Epidermal growth factor stimulates distinct diradylglycerol species generation in Swiss 3T3 fibroblasts: evidence for a potential phosphatidylcholine-specific phospholipase C-catalysed pathway
Authors: Pettitt T R , Zaqqa M , Wakelam M J
Published: August 2015

Stimulation of 3T3 fibroblasts with epidermal growth factor (EGF) results in an increase in 1,2-diacylglycerol (DAG) mass which is maximal at 25 s, declining at 1 min and returning to basal levels by 30 min. No changes in alkylacylglycerol or alkenylacylglycerol were detected. Three species account for most of this mass increase: 18:0/20:5,n-3, 18:0/20:4,n-6 and 18:0/20:3,n-9. These species are characteristic of the phosphoinositides; however, previous work failed to detect any EGF-stimulated rise in inositol phosphates in these cells [Cook and Wakelam (1992) Biochem. J. 285, 247-253]. This ruled out phosphoinositide hydrolysis by phospholipase C, but raised the possibility of phospholipase D/phosphatidate phosphohydrolase-catalysed hydrolysis of phosphatidylinositol. The inclusion of butanol in the incubation medium failed to block the diacylglycerol changes, indicating that the phospholipase D pathway is not involved and that DAG must be derived from another source, probably via phospholipase C-catalysed hydrolysis of a phosphatidylcholine pool that is particularly rich in these species. The tyrosine kinase inhibitor ST-271 almost abolished the elevation in 18:0/20:5,n-3, 18:0/20:4, n-6 and 18:0/20:3,n-9 at 25 s, but only reduced the rise in total DAG mass by about 50%. The protein kinase C (PKC) inhibitor Ro-31-8220 increased DAG levels at all time points but had no effect on the species profiles. This provides additional evidence for PKC-mediated regulation of cell-surface EGF receptors, since the inhibition of PKC would increase the availability and/or ligand binding affinity of receptors at the plasma membrane and hence increase and prolong the response to EGF.
Saponin-induced release of cell-surface-anchored Thy-1 by serum glycosylphosphatidylinositol-specific phospholipase D
Authors: Bergman A S , Carlsson S R
Published: August 2015

A glycosylphosphatidylinositol-specific phospholipase D (GPI-PLD) was purified from human serum and used for studies on the release of GPI-anchored Thy-1 glycoprotein from mouse T lymphoma cells Y191. Previous studies have shown that whereas GPI-PLD is highly active against detergent-solubilized GPI-anchored proteins, it is normally unable to release GPI-containing proteins anchored in a lipid bilayer. Confirming these findings, the addition of GPI-PLD to intact Y191 cells did not result in cleavage of Thy-1. However, pretreatment of cells with saponin, a cholesterol-sequestering agent, rendered Thy-1 susceptible to hydrolysis. Very little solubilization of GPI-containing Thy-1 occurred under these conditions. From experiments with reconstituted liposomes it was inferred that the effect of saponin on cells was to aid in the presentation of Thy-1 to GPI-PLD. Furthermore, it was concluded that cholesterol-saponin complexes formed in the membrane were not alone responsible for the effect. Rather, additional molecules in the plasma membrane are possibly involved in the presentation of Thy-1 on saponin-treated cells. This finding may have implications for a physiological role of circulating GPI-PLD in the regulation of GPI-anchored proteins on cells.
Fibrinogenolysis by a neutrophil membrane protease generates an A α 1–21 fragment
Authors: Kelly S L , Adams S A , Robson S C , Kirsch R E , Shephard E G
Published: August 2015

The 600 kDa neutrophil membrane neutral protease, which had been shown to generate bioactive peptides from the acute-phase reactant C-reactive protein, has now been shown to have fibrinogenolytic activity that is distinct from fibrinogenolysis by plasmin and neutrophil lysosomal enzymes. This protease gradually reduces the apparent molecular mass of fibrinogen (340 kDa) to non-clottable products and generates terminal products with apparent molecular mass values of 270 kDa, 200 kDa, 100 kDa and less than 40 kDa through cleavage of all three of the constituent chains. Characteristics of fibrinogenolysis by this neutrophil protease are cleavage of the bond between amino acids valine and glutamic acid at positions 21 and 22 respectively from the N-terminus of the A alpha chain to release an A alpha 1-21 peptide, digestion of the B beta chain at positions within the C-terminus, and proteolysis of the bond between amino acids isoleucine and glycine at positions 394 and 395 respectively from the N-terminus of the gamma chain. This generates products that lack anticoagulant activity. The thrombin clotting time of the product with an apparent molecular mass of 330 kDa was prolonged, although clot formation was still observed. Loss of coagulability and inability to clot was found with further degradation of fibrinogen to an apparent molecular mass of 290 kDa. Activity of this neutrophil membrane protease in vivo could be important for the regulation of fibrin deposition at sites of inflammation, and may contribute to the reported plasma levels of the A alpha 1-21 peptide.
Subcellular localization of a protein-tyrosine phosphatase: evidence for association with chromatin
Authors: Radha V , Nambirajan S , Swarup G
Published: August 2015

A non-transmembrane protein-tyrosine phosphatase, PTP-S, has been shown to bind to DNA in vitro through its C-terminal noncatalytic domain. The cellular PTP-S gene product was identified as 42-44 kDa polypeptides in the rat fibroblast cell lines Rat2 and F111, by immunoblotting using polyclonal and monoclonal antibodies. Indirect immunofluorescence-staining experiments showed that PTP-S was predominantly associated with the nuclear compartment in interphase cells, although some fluorescence was present in cytoplasm. This protein appeared to be uniformly distributed throughout the cell during mitosis. Subcellular fractionation followed by immunoblotting supports the immunofluorescence data showing that PTP-S was present in the nucleus as well as cytoplasm. PTP-S could be released from the isolated nuclei by moderate salt concentration (400 mM NaCl) or mild treatment with DNAase I, suggesting that at least part of this protein present in the nucleus was associated with chromatin. PTP-S extracted from nuclei of Rat2 cells binds to DNA, as determined by DNA affinity chromatography. The nuclear location of this phosphatase, and its association with chromatin, provide support to our previous suggestion that the DNA-binding property of this phosphatase may be physiologically relevant.
Pharmacological interference with tissue hypercatabolism in tumour-bearing rats
Authors: Tessitore L , Costelli P , Baccino F M
Published: August 2015

Marked loss of body weight and profound waste of both skeletal muscle and white adipose tissue occur in rats into which the ascites hepatoma Yoshida AH-130 has been transplanted, associated with marked perturbations in the hormonal homoeostasis and the presence of circulating tumour necrosis factor and high plasma levels of prostaglandin E2 [Tessitore, Costelli and Baccino (1993) Br. J. Cancer 67, 15-23]. On the basis of previous findings, the present study examined whether the development of cachexia in this model system could be significantly affected by adrenalectomy or by pharmacological treatments that may interfere with proximal or distal mediators of tissue hypercatabolism. In no instance was tumour growth modified. Medroxyprogesterone acetate, an anabolic-hormone-like drug, was completely ineffective. In adrenalectomized animals, although changes such as the elevation of plasma triacylglycerols and corticosterone were corrected, the general course of cachexia was not modified. A partial prevention of muscle waste was observed with acetylsalicylic acid, a non-steroidal anti-inflammatory drug, or with leupeptin, a proteinase inhibitor. Insulin afforded the most significant preservation of muscle protein and adipose-tissue mass, which were maintained close to control values even 10 days after transplantation. The effects of insulin on gastrocnemius muscle and liver protein content were exerted by slowing down protein turnover, mainly enhancing synthesis. Consistently, the total free amino acid concentration in the gastrocnemius of insulin-treated rats 10 days after tumour transplantation was close to that of controls. Although treatment with insulin decreased plasma corticosterone to normal values, it did not modify the circulating level of tumour necrosis factor. On the whole these data show that it seems possible to prevent, at least in part, the tissue waste that characterizes cancer cachexia by purely pharmacological means.
Angiotensin II stimulates phosphorylation of high-molecular-mass cytosolic phospholipase A2 in vascular smooth-muscle cells
Authors: Rao G N , Lassègue B , Alexander R W , Griendling K K
Published: August 2015

Phospholipase A2 (PLA2) may be one of the major components involved in cell signalling and proliferation, as suggested by recent studies. In this paper we show that the potent vasoconstrictor and hypertrophic agent angiotensin II (AngII) activates cytosolic PLA2 (cPLA2) in vascular smooth-muscle cells. AngII induced a rapid time-dependent release of [3H]arachidonic acid from prelabelled cells that was inhibited by mepacrine, a PLA2 inhibitor. AngII treatment of intact cells also activated a cPLA2, as measured in cell-free extracts by the release of radiolabelled arachidonic acid from exogenously added 1-stearoyl-2-[1-14C]arachidonoyl phosphatidylcholine. This AngII-stimulated cPLA2 activity was also significantly inhibited by mepacrine. AngII induced a rapid and time-dependent increase in cPLA2 phosphorylation. Protein kinase C (PKC) depletion inhibited both AngII-induced [3H]arachidonic acid release and cPLA2 phosphorylation. Together, these results suggest strongly that (1) AngII phosphorylates and activates cPLA2 in a PKC-dependent manner, and that (2) cPLA2 mediates the AngII-induced [3H]arachidonic acid release in vascular smooth-muscle cells.
Site-directed mutagenesis of β-lactamase I: role of Glu-166
Authors: Leung Y C , Robinson C V , Aplin R T , Waley S G
Published: August 2015

Two Glu-166 mutants of beta-lactamase I from Bacillus cereus 569/H were constructed: one with a lengthened side chain (E166Cmc, the S-carboxymethylcysteine mutant) and the other with the side chain shortened and made non-polar (E166A). Their kinetic properties were studied and compared with those of the wild-type and the E166D mutant (with a shortened side chain) previously made by Gibson, Christensen and Waley (1990) (Biochem. J. 272, 613-619). Surprisingly, with good penicillin substrates, Km, kcat. and kcat./Km of the two conservative mutants (E166Cmc and E166D) are similar to those of the non-conservative mutant E166A. Their kcat. values are 3000-fold lower than that of the wild-type enzyme, showing that Glu-166 is a very important residue. The acylenzyme intermediate of E166A and a good substrate, penicillin V, was trapped by acid-quench and observed by electrospray ionization mass spectrometry, suggesting that Glu-166 is more important in catalysing the deacylation step than the acylation step. The beta-lactamase I E166A mutant is about 200-fold more active than the Bacillus licheniformis E166A mutant with nitrocefin or 6 beta-furylacryloyl-amidopenicillanic acid as substrate. This suggested that other groups in the active site of the beta-lactamase I mutant may activate the catalytic water molecule for deacylation.
Reversibility of interleukin-1β-induced islet destruction and dysfunction by the inhibition of nitric oxide synthase
Authors: Corbett J A , McDaniel M L
Published: August 2015

We have examined the reversibility of NO-mediated islet dysfunction and destruction induced by interleukin-1 beta (IL-1 beta). Previous studies have shown that pretreatment of islets for 18 h with IL-1 beta results in an inhibition of glucose-stimulated insulin secretion that requires 4 days incubation in the absence of IL-1 beta to restore islet secretory function. In this study we use a sequential experimental design in which islets are first exposed to IL-1 beta for 18 h, and then treated with the NO synthase inhibitor NG-monomethyl-L-arginine (NMMA). Insulin secretion is inhibited by 98% after the 18 h incubation with IL-1 beta, and this inhibition is reversed in a time-dependent fashion by NMMA, with complete recovery of insulin secretion observed 8 h after the inhibition of NO synthase. Inhibition of NO synthase also restores IL-1 beta-induced inhibition of mitochondrial aconitase activity in a time-dependent fashion that mimics the recovery of glucose-stimulated insulin secretion by islets. Ferrous iron and the reducing agents cysteine and thiosulphate accelerate the rate of recovery of insulin secretion, and ferrous iron and thiosulphate stimulate the recovery of islet aconitase activity, suggesting that iron-sulphurcentre reconstitution may be involved in the recovery process. The recovery process also appears to require mRNA transcription, because the transcriptional inhibitor actinomycin D prevents the recovery of insulin secretion by islets after the inhibition of NO synthase. Although IL-1 beta induces the co-expression of NO synthase and cyclo-oxygenase by islets, cyclo-oxygenase is not involved in the recovery of glucose-stimulated insulin secretion. Inhibition of NO synthase also prevents IL-1 beta-induced islet destruction, which otherwise occurs during a 96 h continuous exposure to this cytokine. The destructive effects of IL-1 beta on islet viability are prevented if NMMA is added to islet cultures during the first 24 h of exposure to IL-1 beta, but islet destruction is not prevented if NMMA is added after the first 48 h exposure to IL-1 beta. These results show that IL-1 beta-induced islet dysfunction is reversed by the inhibition of NO synthase, that recovery of insulin secretion is stimulated by iron and reducing agents, and that the recovery process appears to require mRNA transcription. We also show that it is possible to rescue islets from the destructive effects of IL-1 beta if NO synthase is inhibited early after its induction.
Effect of cyclodextrins and undigested starch on the loss of chenodeoxycholate in the faeces
Authors: Abadie C , Hug M , Kübli C , Gains N
Published: August 2015

Starch that escapes digestion in the small intestine increases the elimination of chenodeoxycholate and its metabolites in the faeces of both mice and hamsters. In contrast, the elimination of cholate and its metabolites is not increased. In vitro, the affinity of starch for chenodeoxycholate is about 90-fold greater than for cholate. beta-Cyclodextrin, which approximates to one turn of the helical structures formed by the 1,4-linked glucose units of starch, shares these properties. It is proposed that these helical structures in starch act as binding sites for bile salts.
Bacterial morphine dehydrogenase further defines a distinct superfamily of oxidoreductases with diverse functional activities.
Authors: Bruce N C , Willey D L , Coulson A F , Jeffery J
Published: August 2015

Pseudomonas putida morphine dehydrogenase is shown to be closely homologous to 18 proteins, defining a superfamily within which morphine dehydrogenase particularly resembles two bacterial, 2,5-dioxo-D-gluconic acid reductases, and two eukaryotic proteins of unknown functions. Relationships within the superfamily are extensive and complex. Residue identities between protein pairs range from 29-90%. Three subgroups are proposed. Nevertheless, on the basis of residue conservations/exchanges it is suggested that the nicotinamide coenzyme binding and substrate reduction occur in all the enzymes by broadly analogous mechanisms, among which some probable differences are identified.
Synthesis, purification and kinetic properties of fluorescein-labelled penicillins
Authors: Lakaye B , Damblon C , Jamin M , Galleni M , Lepage S , Joris B , Marchand-Brynaert J , Frydrych C , Frere J M
Published: August 2015

The synthesis and properties of six fluorescein-labelled penicillins are reported. The two isomers of fluoresceyl-glycyl-6-amino-penicillanic acid are probably the best compounds to use for detection of all the penicillin-binding proteins (PBPs) present in a bacterial membrane preparation. However, the derivatives of ampicillin were much more efficient against Enterobacter aerogenes PBP3. The two isomers obtained when a commercial mixture of the two isomers of carboxyfluorescein was used most often exhibited similar properties, but the Streptomyces R61 extracellular DD-peptidase was only efficiently acylated by the 5′-carboxyfluorescein derivative of glycyl-6-aminopenicillanic acid.
Targeting of passenger protein domains to multiple intracellular membranes
Authors: Janiak F , Glover J R , Leber B , Rachubinski R A , Andrews D W
Published: August 2015

The role of passenger domains in protein targeting was examined by fusing previously characterized targeting motifs to different protein sequences. To compare the targeting requirements for a variety of subcellular compartments, targeting of the fusion proteins was examined for endoplasmic reticulum, mitochondria and peroxisomes in vitro and in yeast. Although most passenger domains were only partially passive to translocation, motif-dependent targeting via motifs positioned at either end of one passenger domain (gPA) was demonstrated for all of the subcellular compartments tested. The data presented extend earlier suggestions that translocation competence is an intrinsic property of the passenger protein. However, the properties that determine protein targeting are not mutually exclusive for the compartments tested. Therefore, although the primary determinant of specificity is the targeting motif, our results suggest that translocation competence of the targeted protein augments the fidelity of transport.
Exocytosis in chromaffin cells: evidence for a MgATP-independent step that requires a pertussis toxin-sensitive GTP-binding protein
Authors: Vitale N , Thiersé D , Aunis D , Bader M F
Published: August 2015

We have previously described that mastoparan, an amphiphilic tetradecapeptide that activates heterotrimeric G-proteins, inhibits Ca(2+)-induced MgATP-dependent secretion from streptolysin-O-permeabilized chromaffin cells [Vitale, Mukai, Rouot, Thiersé, Aunis and Bader (1993) J. Biol. Chem. 268, 14715-14723]. Our observations suggest the involvement of an inhibitory G(o)-protein, possibly located on the membrane of secretory granules, in the final stages of the exocytotic pathway in chromaffin cells. Here, we demonstrate that mastoparan is also able to stimulate the Ca(2+)-dependent secretion of catecholamines in the absence of MgATP in the medium. This MgATP-independent secretion is totally blocked by tetanus toxin, a potent inhibitor of exocytosis in all neurosecretory cells so far investigated, suggesting that the mastoparan target is a component of the exocytotic machinery. Mas17, a mastoparan analogue inactive on G-proteins, had no effect on catecholamine secretion whereas both Mas7, a highly active analogue of mastoparan, and AlF4-, which selectively activates trimeric G-proteins, triggered MgATP-independent secretion. Non-hydrolysable GTP analogues (GTP[S] and p[NH]ppG) mimicked the dual effects of mastoparan on secretion: they inhibited exocytosis in the presence of MgATP and stimulated MgATP-independent secretion. The different potencies displayed by these two analogues suggest the involvement of two distinct G-proteins. Accordingly, the mastoparan-induced MgATP-independent secretion is highly sensitive to pertussis toxin (PTX) whereas the inhibition by mastoparan of secretion in the presence of MgATP is resistant to PTX treatment. When permeabilized cells were incubated with mastoparan, the release of arachidonic acid increased in a PTX-sensitive manner. 7,7-Dimethyl-5,8-eicosadienoic acid, a potent inhibitor of intracellular phospholipase A2, inhibited both the arachidonate release and the MgATP-independent catecholamine secretion evoked by mastoparan. In contrast, neomycin, an inhibitor of phospholipase C, had no significant effect on either the release of arachidonic acid or the secretion of catecholamines provoked by mastoparan. We conclude that two distinct heterotrimeric G-proteins act in series in the exocytotic pathway in chromaffin cells: one controls an ATP-dependent priming step through an effector pathway that remains to be determined, and the second is involved in a late Ca(2+)-dependent step which does not require MgATP but possibly involves the generation of arachidonic acid.
The Ras-related protein R-ras interacts directly with Raf-1 in a GTP-dependent manner
Authors: Spaargaren M , Martin G A , McCormick F , Fernandez-Sarabia M J , Bischoff J R
Published: August 2015

R-ras is a member of the ras family of small GTPases that associates with the apoptosis-suppressing proto-oncogene product Bcl-2. Using the yeast two-hybrid system we provide evidence for an interaction between R-ras and the Raf-1 kinase. This interaction requires only the N-terminal regulatory domain (amino acids 1-256) of Raf-1, and is observed with both the wild type and a constitutively active R-ras mutant, but not with a deletion mutant that lacks the potential effector domain or a mutant of R-ras impaired for GTP binding. Moreover, using an in vitro binding assay we show a direct GTP-dependent interaction of purified R-ras with a purified Raf-1 fragment corresponding to the proposed 81-amino-acid H-Ras-binding domain of Raf-1 (amino acids 51-131). Taken together, these data indicate that R-ras may exert its biological effect by means of modulating the activity of the Raf-1 kinase as its direct downstream effector.
Surface-area cycling of different surfactant preparations: SP-A and SP-B are essential for large-aggregate integrity
Authors: Veldhuizen R A , Hearn S A , Lewis J F , Possmayer F
Published: August 2015

Surface-area cycling is an in vitro procedure for the conversion of large into small surfactant aggregates. In this procedure a tube containing a surfactant suspension is rotated end-over-end at 37 degrees C so that the surface area of the suspension changes twice each cycle. We have utilized this method to study the mechanisms involved in aggregate conversion. Several different surfactant preparations were analysed: (1) bovine natural surfactant, a sucrose-gradient-purified material containing surfactant phospholipid and surfactant-associated proteins (SP-) SP-A, SP-B and SP-C; (2) bovine lipid-extract surfactant, which contains the surfactant phospholipids and SP-B and SP-C; (3) mixtures of dipalmitoyl phosphatidylcholine and phosphatidylglycerol (7:3, w/w) reconstituted with one or more surfactant proteins. Aggregate conversion was measured by phosphorus analysis of a 40,000 g supernatant (small aggregate) and pellet (large aggregates) before and after surface-area cycling. Surface-area cycling of lipid extract surfactant or lipids plus SP-B or SP-C resulted in rapid aggregate conversion. Lipids alone were not converted. Only a small percentage of purified natural surfactant was converted into small aggregates. Addition of SP-A to lipid extract surfactant could inhibit aggregate conversion of this material, but this was only observed when an additional 1% (w/w) of SP-B was added to the lipid extract. It is concluded that SP-A is important for large-aggregate integrity. It appears that SP-A acts in conjunction with SP-B. The presence of SP-B and/or SP-C is required for aggregate conversion; it is proposed that this reflects the necessity for lipid adsorption in aggregate conversion.
Activation of rat brain ornithine decarboxylase by GTP
Authors: Kilpeläinen P T , Hietala O A
Published: August 2015

The activity of ornithine decarboxylase (ODC) measured in different regions of rat brain was highest in the hippocampus and lowest in the cerebellum. The ODC activity of a crude extract of the cerebellum was increased by the addition of GTP to the enzyme assay. Following dissociation of the ODC-antizyme complex by Sephadex G-75 chromatography in buffer containing 0.25 M NaCl, the GTP-activatable ODC was found in every brain region analysed. This GTP-activatable brain ODC has greater affinity for antizyme than the non-GTP-activatable brain ODC or the kidney ODC. The irreversible inhibitor of ODC, alpha-difluoromethylornithine (DFMO), inhibited approx. 60% of the ODC activity of all brain regions, whereas kidney ODC was inhibited totally by DFMO. When extracts of brain and kidney were incubated at 55 degrees C, kidney ODC was rapidly inactivated, but brain ODC was more heat-stable. Brain ODC, but not kidney ODC, was activated by GTP and ATP, and also by their deoxy forms. The K1/2 for activation of the enzyme was 2 microM for GTP and 40 microM for ATP. Using partially purified brain ODC, the activation by GTP was irreversible. These results demonstrate for the first time that the GTP-activatable ODC exists in the brain and is associated with the antizyme. The possible mechanisms of activation by GTP, the significance of this finding for the regulation of brain ODC, and the similarities to and differences from the GTP-activatable ODC found in certain rodent and human tumours are all discussed.
Inhibition of the translocation of GLUT1 and GLUT4 in 3T3-L1 cells by the phosphatidylinositol 3-kinase inhibitor, wortmannin
Authors: Clarke J F , Young P W , Yonezawa K , Kasuga M , Holman G D
Published: August 2015

Wortmannin is a potent and reversible inhibitor of insulin-stimulated PtdIns 3-kinase activity in 3T3-L1 cells (IC50 = 2.6 +/- 0.8 nM). Wortmannin inhibits the PtdIns 3-kinase activity which is precipitated with antibodies against insulin receptor substrate 1 and against the alpha-p85 subunit of PtdIns 3-kinase. These observations suggest that wortmannin inhibits at the p110 catalytic subunit of PtdIns 3-kinase. Insulin stimulation of glucose transport in permeabilized 3T3-L1 cells is also inhibited by wortmannin (IC50 = 6.4 +/- 1.4 nM). Wortmannin did not inhibit basal glucose transport activity. The close similarity of the IC50 values for wortmannin inhibition of insulin-stimulated PtdIns 3-kinase and glucose transport activities suggests that the PtdIns 3-kinase is a key intermediate in insulin signalling of glucose-transport stimulation. The wortmannin inhibitory effect on transport is associated with a reduction in the cell-surface, but not the total cellular, levels of both GLUT1 and GLUT4 glucose transporter isoforms that are accessible to the cell-impermeant photolabel, ATB-BMPA. These photolabelling results suggest that the glucose transporter translocation process is dependent upon PtdIns 3-kinase activity. The stimulatory effect of guanosine 5′-[gamma-thio]triphosphate (GTP gamma S) on glucose transport activity in permeabilized cells is only partially blocked by concentrations of wortmannin that completely inhibit the stimulatory effect of insulin. The residual stimulatory effect of GTP gamma S that occurs in the presence of wortmannin suggests that at least part of the GTP gamma S effect is mediated at a signalling site that is downstream of the site at which wortmannin inhibits the insulin stimulation of PtdIns 3-kinase and glucose transport activities.
Affinity of chaperonin-60 for a protein substrate and its modulation by nucleotides and chaperonin-10
Authors: Staniforth R A , Burston S G , Atkinson T , Clarke A R
Published: August 2015

The refolding of lactate dehydrogenase fully unfolded in 4 M guanidinium chloride was initiated by dilution into assay buffer, and the emergence of active enzyme was recorded. This was performed in the presence of the following chaperonin complexes in the refolding medium: chaperonin-60 (cpn60), cpn60-MgATP, cpn60-Mgp[NH]ppA, cpn60-MgADP in both the presence and absence of chaperonin-10 (cpn10). For each nucleotide-chaperonin complex studied, the effect of nucleotide concentration was measured. Dissociation constants (Kd) for unfolded LDH bound to the various chaperonin complexes were derived directly from the ability of the complexes to retard the folding of the enzyme. Dissociation constants for the different complexes were found to be in the order: cpn60 < cpn60-MgADP-cpn10 (formed at low [MgADP]) < cpn60-MgADP < cpn60-MgADP-cpn10 < cpn60-Mgp[NH]ppA < cpn60-Mgp[NH]ppA-cpn10 < cpn60-MgATP < cpn60-MgATP-cpn10; i.e. the tightest complex is with cpn60 and the weakest with cpn60-MgATP-cpn10. Only when MgATP is the nucleotide do we see the yield of native enzyme increased on the time scale of 1 h. The results provide estimates of the change in binding energy between the chaperonin and a substrate protein through the cycle of MgATP binding, hydrolysis and dissociation.
Nuclear localization of the ubiquitin-activating enzyme, E1, is cell-cycle-dependent
Authors: Grenfell S J , Trausch-Azar J S , Handley-Gearhart P M , Ciechanover A , Schwartz A L
Published: August 2015

The mechanisms that regulate ubiquitin-mediated degradation of proteins such as the mitotic cyclins at defined stages of the cell cycle are poorly understood. The initial step in the conjugation of ubiquitin to substrate proteins involves the activation of ubiquitin by the ubiquitin-activating enzyme, E1. Previously we have described the subcellular localization of this enzyme to both nuclear and cytoplasmic compartments. In the present study, we have used the 1C5 anti-E1 monoclonal antibody in immunofluorescent-microscopy and subcellular-fractionation techniques to examine the distribution of E1 during the HeLa cell cycle. E1 is both cytoskeletal and nuclear during the G1-phase. As the cells progress into S-phase, E1 is exclusively cytoskeletal and has a perinuclear distribution. During G2-phase, E1 reappears in the nucleus before breakdown of the nuclear envelope. In mitotic cells, E1 localizes to both the mitotic spindle and the cytosol, but is absent from the chromosomes. Immunoblot analysis reveals multiple forms of E1 in HeLa whole cell extract. This heterogeneity is not a result of polyubiquitination and may represent inactive pools of E1. Only the characteristic E1 doublet is able to activate ubiquitin. Cell-fractionation studies reveal a differential distribution of specific E1 isoforms throughout the cell cycle. Therefore we propose that the subcellular localization of E1 may play a role in regulating cell-cycle-dependent conjugation of ubiquitin to target proteins.
Characterization of binding and structural properties of rat liver fatty-acid-binding protein using tryptophan mutants
Authors: Thumser A E , Wilton D C
Published: August 2015

Rat liver fatty-acid-binding protein (FABP) does not contain tryptophan. Three mutant proteins have been produced in which a single tryptophan residue has been inserted by site-directed mutagenesis at positions 3 (F3W), 18 (F18W) and 69 (C69W). These tryptophans have been strategically located in order to provide fluorescent reporter groups to study the binding and structural characteristics of rat liver FABP. Two fluorescent fatty acid analogues, DAUDA (11-[(5-dimethylaminonaphthalene-1- sulphonyl)amino]undecanoic acid) and 3-[p-(6-phenyl)-hexa-1,3,5-trienyl]phenylpropionic acid, showed no significant difference in binding affinities for the different mutant proteins, although maximum fluorescence values were decreased for F3W and increased for C69W. These findings were confirmed by studies of DAUDA displacement by oleate. Protein-denaturation studies in the presence of urea indicated subtle differences for the three mutants which could be explained by multiple unfolding pathways. Fatty acid binding increased tryptophan fluorescence emission in the case of the F18W protein, but had no effect on the F3W and C69W proteins. Fluorescence quenching studies with 2-bromopalmitate showed that a fatty acid carboxylate is close to the tryptophan in the F18W protein. Energy-transfer studies showed that the fluorescent moiety of DAUDA is equidistant from the three mutated amino acids and is bound within the beta-clam solvent cavity of liver FABP. This interpretation of the fluorescence quenching and energy-transfer data supports the difference in ligand orientation between intestinal and liver FABP observed in previous studies.
Highly sensitive control of transcriptional activity by factor heterodimerization
Authors: Swillens S , Pirson I
Published: August 2015

Several molecular mechanisms have been proposed to explain highly sensitive controls of cellular functions by effector molecules. Here we study an equilibrium model describing the regulation of transcriptional activity through the heterodimerization of transcription factors. We demonstrate that this model involves a new type of biochemical control which accounts for a very high sensitivity.
Members of the CAAT/enhancer-binding protein, hepatocyte nuclear factor-1 and nuclear factor-1 families can differentially modulate the activities of the rat α-fetoprotein promoter and enhancer
Authors: Bois-Joyeux B , Danan J L
Published: August 2015

The promoter of the rat alpha-fetoprotein (AFP) gene, which makes the expression of the developmentally regulated AFP gene specific to the liver, is a putative target for transcription factors of the CAAT/enhancer-binding protein (C/EBP), hepatocyte nuclear factor-1 (HNF-1) and nuclear factor-1 (NF-1) families. We have evaluated the influence of these factors on the activity of the AFP promoter by transfection of HepG2 hepatoma cells with the appropriate expression vector plus a CAT plasmid under the control of the AFP promoter. A similar plasmid bearing the rat albumin promoter was used as a control. C/EBP alpha, C/EBP beta and D-binding protein (DBP) acted as trans-activators on the AFP promoter, whereas liver inhibitory protein (LIP), a truncated form of C/EBP beta, was a potent negative regulator of the promoter. C/EBP alpha also bound to and stimulated the activity of the AFP enhancer at -2.5 kb. Interestingly, HNF-1 beta was found to be more potent than HNF-1 alpha in activating the AFP promoter. This effect was specific, as it did not occur with the rat albumin promoter. HNF-1 beta, which is produced earlier than HNF-1 alpha during liver development, would thus have the greater influence on the AFP promoter in early development. Both HNF-1s allowed expression of the AFP promoter in cells of nonhepatic origin. Overexpression of NF-1 induced a specific decrease in the activity of the AFP promoter. This strongly suggests that competition between NF-1 and HNF-1 for binding to their overlapping binding sites on the AFP promoter is critical for modulating its activity. Thus changing combinations of these trans-acting factors may tightly modulate the AFP promoter activity in the course of liver development and carcinogenesis.
Temporal distribution, localization and metabolism of all-trans-retinol, didehydroretinol and all-trans-retinal during Xenopus development
Authors: Creech Kraft J , Schuh T , Juchau M R , Kimelman D
Published: August 2015

Recently, the temporal and spatial distribution patterns of the retinoid receptor ligands 9-cis-retinoic acid and all-trans-retinoic acid were described in Xenopus embryos during early development [Creech Kraft, Schuh, Juchau and Kimelman (1994) Proc. Natl. Acad. Sci. U.S.A., in the press]. The present study demonstrates the presence and distribution of their likely precursors, all-trans-retinol, didehydroretinol, didehydroretinal and all-trans-retinal, as well as the occurrence of 4-oxo metabolites, in Xenopus embryos. The temporal and spatial distribution patterns of all-trans-retinol, didehydroretinol and all-trans-retinal did not coincide with that observed for 9-cis-retinoic acid but, in certain regards, were similar to the patterns delineated for all-trans-retinoic acid and all-trans-retinoyl beta-glucuronide. Evidence is presented that 9-cis-retinoic acid can be synthesized from both all-trans-retinoic acid and all-trans-retinol in Xenopus embryos, suggesting that the difference between the distributions of 9-cis-retinoic acid and the other retinoids may be caused by selective synthesis and/or protein binding of the 9-cis isomer.
Insulin-induced translocation of the glucose transporter GLUT4 in cardiac muscle: studies on the role of small-molecular-mass GTP-binding proteins
Authors: Uphues I , Kolter T , Goud B , Eckel J
Published: August 2015

Subcellular fractions obtained from rat cardiac ventricular tissue were used to elucidate a possible functional relationship between small-molecular-mass G-proteins and the insulin-responsive glucose transporter GLUT4. Proteins were separated by SDS/PAGE and transferred to nitrocellulose membranes. Incubation with [alpha-32P]GTP revealed the presence of two major distinct GTP-binding protein bands of 24 and 26 kDa in both plasma and microsomal membranes. Immunoadsorption of microsomal membranes to anti-GLUT4 antibodies was used to isolate GLUT4-enriched membrane vesicles. This material was found to contain a much decreased amount of small G-proteins, with the exclusive presence of the 24 kDa species. Insulin treatment in vivo had no effect on the microsomal membrane content of small GTP-binding proteins, but significantly decreased the 24 kDa species in GLUT4-enriched vesicles by 36 +/- 5% (n = 3). This correlated with a decreased (30-40%) recovery of GLUT4-enriched vesicles from insulin-treated animals. Western-blot analysis of microsomal membranes with a panel of antisera against rab GTP-binding proteins indicated the presence of rab4A, with a molecular mass of 24 kDa, whereas rab1A, rab2 and rab6 were not observed. rab4A was barely detectable in GLUT4-enriched vesicles; however, insulin produced an extensive shift of rab4A from the cytosol and the microsomal fraction to the plasma membrane with a parallel increase in GLUT4. These data show that a small GTP-binding protein is co-localized with GLUT4 in an insulin-responsive intracellular compartment, and strongly suggest that this protein is involved in the exocytosis of GLUT4 in cardiac muscle. Furthermore, the observed translocation of rab4A is compatible with insulin-induced endosome recycling processes, possibly including the glucose transporters.
Identification of the vinculin-binding site in the cytoskeletal protein α-actinin
Authors: McGregor A , Blanchard A D , Rowe A J , Critchley D R
Published: August 2015

Using low-speed sedimentation equilibrium we have established that vinculin binds to alpha-actinin with a Kd of 1.3 x 10(-5) M. Electron microscopy of negatively stained preparations of vinculin revealed spherical particles (diameter 11.2 nm; S.D. 1.7 nm, n = 21), whereas alpha-actinin appeared as a rod-shaped particle (length 33 nm; S.D. 3.3 nm, n = 23). Mixtures of the two proteins contained both ‘lollipop’- and ‘dumbell’-shaped particles which we interpret as either one or two spherical vinculin molecules associated with the ends of the alpha-actinin rod. We have further defined the vinculin-binding site in alpha-actinin using 125I-vinculin and a gel-blot assay in which proteolytic fragments of alpha-actinin and fragments of alpha-actinin expressed in Escherichia coli were resolved by SDS/PAGE and blotted to nitrocellulose. 125I-vinculin bound to polypeptides derived from the spectrin-like repeat region of alpha-actinin, but did not bind to the actin-binding domain. Binding was inhibited by a 100-fold molar excess of unlabelled vinculin. Using a series of glutathione S-transferase fusion proteins we have mapped the vinculin-binding site to a region toward the C-terminal end of the molecule (alpha-actinin residues 713-749). 125I-vinculin also bound to fusion proteins containing this sequence which had been immobilized on glutathione-agarose beads. The vinculin-binding site is localized in a highly conserved region of the molecule close to the first of two EF-hand calcium-binding motifs.
Purification and characterization of glutathione-dependent dehydroascorbate reductase from rat liver
Authors: Maellaro E , Del Bello B , Sugherini L , Santucci A , Comporti M , Casini A F
Published: August 2015

GSH-dependent enzymic reduction of dehydroascorbic acid to ascorbic acid has been studied in rat liver cytosol. After gel filtration of cytosol on Sephadex G-100 SF, dehydroascorbate reductase activity was recovered in two distinct peaks, one corresponding to glutaredoxin (an enzyme already known for its dehydroascorbate reductase activity) and another, much larger one, corresponding to a novel enzyme different from glutaredoxin. The latter was purified to apparent homogeneity. The purification process involved (NH4)2SO4 fractionation, followed by DEAE-Sepharose, Sephadex G-100 SF and Reactive Red chromatography. SDS/PAGE of the purified enzyme in either the presence or absence of 2-mercaptoethanol demonstrated a single protein band of M(r) 31,000. The M(r) determined by both Sephadex G-100 SF chromatography and h.p.l.c. was found to be approx. 48,000. H.p.l.c. of the denatured enzyme gave an M(r) value identical with that obtained by SDS/PAGE (31,000). The apparent Km for dehydroascorbate was 245 microM and the Vmax. was 1.9 mumol/min per mg of protein; for GSH they were 2.8 mM and 4.5 mumol/min per mg of protein respectively. The optimal pH range was 7.5-8.0. Microsequence analysis of the electro-transferred enzyme band showed that the N-terminus is blocked. Data on internal primary structure were obtained from CNBr-and N-chlorosuccinimide-derived fragments. No significative sequence similarity was found to any of the protein sequences contained in the Protein Identification Resource database.
Expression of rat liver S-adenosylmethionine synthetase in Escherichia coli results in two active oligomeric forms
Authors: Alvarez L , Mingorance J , Pajares M A , Mato J M
Published: August 2015

A cDNA containing the complete coding sequence for rat liver S-adenosylmethionine synthetase was cloned into the prokaryotic expression vector pT7-7 and expressed in Escherichia coli BL21(DE3). A major additional band corresponding to a protein of 48 kDa was detected on SDS/PAGE after induction with isopropyl beta-D-thiogalactopyranoside. This protein was distributed in both the soluble and insoluble fractions and accounted for approx. 30% of the total bacterial protein. The soluble enzyme was fully active, as revealed by assays in vitro of S-adenosylmethionine synthetase activity. In addition, transformed bacteria exhibited highly increased levels of intracellular S-adenosylmethionine. Two active forms of the recombinant enzyme, with apparent molecular masses of 210 kDa and 110 kDa, were detected when cytosolic extracts of the transformed cells were fractionated by gel-filtration chromatography. It is concluded that the expressed S-adenosylmethionine synthetase polypeptide assemble as tetramers and dimers.