bluuebunny/crossref_metadata_2025 huggingface dataset:
Purification of a 100 kDa phospholipase A2 from spleen, lung and kidney: antiserum raised to pig spleen phospholipase A2 recognizes a similar form in bovine lung, kidney and platelets, and immunoprecipitates phospholipase A2 activity
Authors: Kim D K , Bonventre J V
Published: August 2015

Phospholipase A2 (PLA2) plays a key role in the production of intracellular and extracellular chemical mediators such as arachidonic acid, eicosanoids and platelet-activating factor, which modulate membrane channel activity, signal transduction, are vasoactive and chemotactic, and are implicated in many pathophysiological mechanisms of inflammation and tissue injury. We previously identified, purified and characterized an arachidonic acid-selective cytosolic 100-110 kDa PLA2 from bovine platelets and rat kidney that is activated during cell stimulation. The purification schemes previously published resulted in low yields of enzyme, insufficient for extensive biochemical characterization. We report the purification of a large-molecular-mass (100 kDa) PLA2 from pig spleen, bovine kidney and bovine lung, using a novel large-scale purification scheme. The enzyme was purified to near homogeneity from an acidified extract obtained from 4.8 kg of pig spleen by sequential use of DEAE-cellulose anionic exchange, Butyl-Toyopearl hydrophobic chromatography and DEAE-5PW h.p.l.c., and further purified by non-denaturing PAGE. This purification scheme will permit the preparation of quantities of purified native enzyme sufficient to study its properties and regulation. To generate antiserum against the PLA2 enzyme, the 100 kDa protein was excised and electroeluted from SDS/PAGE gels of the active fractions after DEAE-5PW h.p.l.c., and this was used as antigen. This polyclonal antibody against pig spleen 100 kDa PLA2 protein reacted with 100 kDa bands in preparations partially purified from bovine platelets, kidney and lung as well as pig spleen, and immunoprecipitated PLA2 activity from these sources. The antibody also immunoprecipitated a 100 kDa protein from cytosolic fractions of cultured renal mesangial cells, human erythroleukaemia cells and human monocytic U937 cells. Considerable PLA2 activity was present in the immunoprecipitates. To our knowledge this antibody is unique in its ability to permit measurement of PLA2 activity in the immunoprecipitate itself, and will be a useful tool for the study of the regulation and the activation mechanisms of the native PLA2 enzyme.
Lack of stereospecificity of suid pseudorabies virus thymidine kinase
Authors: Maga G , Verri A , Bonizzi L , Ponti W , Poli G , Garbesi A , Niccolai D , Spadari S , Focher F
Published: August 2015

We have partially purified suid pseudorabies virus (PRV) thymidine kinase from infected thymidine kinase- mouse cells, and cytosolic swine thymidine kinase from lymphatic glands, and we have found that PRV thymidine kinase, unlike the host enzyme, shows no stereospecificity for D- and L-beta-nucleosides. In vitro, unnatural L-enantiomers, except L-deoxycytidine, function as specific inhibitors for the viral enzyme in the order: L-thymidine >> L-deoxyguanosine > L-deoxyuridine > L-deoxyadenosine. Contrary to human and swine thymidine kinases and like herpes simplex virus-1 and -2 thymidine kinases, PRV thymidine kinase phosphorylates both the natural (D-) and the unnatural (L-) thymidine enantiomers to their corresponding monophosphates with comparable efficiency. The kinetic parameters Vmax/Km for D- and L-thymidine are 3.7 and 2.3 respectively. Our results demonstrate that the lack of stereospecificity might be a common feature of the thymidine kinases that are encoded by human and animal herpes viruses. These observations could lead to the development of a novel class of antiviral drugs.
Expression of the calsequestrin gene in chicken cerebellum Purkinje neurons
Authors: Volpe P , Gorza L , Brini M , Sacchetto R , Ausoni S , Clegg D O
Published: August 2015

Intracellular rapidly exchanging Ca2+ stores are identified and defined in terms of intralumenal low-affinity, high-capacity Ca(2+)-binding proteins, of which calsequestrin (CS) is the prototype in striated muscles. In chicken striated muscles, there is a single gene for CS [Choi and Clegg (1990) Dev. Biol. 142, 169-177]. In the chicken brain, the gene for CS was found to be selectively expressed in Purkinje neurons, as judged by Northern blotting, in situ hybridization and immunocytochemistry. The synthetic machinery for CS was found to be restricted to the cell body, i.e. excluded from dendrites and axon.
Stimulation of tyrosine phosphorylation and mitogen-activated-protein (MAP) kinase activity in human SH-SY5Y neuroblastoma cells by carbachol
Authors: Offermanns S , Bombien E , Schultz G
Published: August 2015

Activation of the G-protein-coupled muscarinic (M3) receptor in human neuroblastoma SH-SY5Y cells is known to lead to phosphoinositol hydrolysis and noradrenaline release. In this study, the effect of carbachol on tyrosine phosphorylation and mitogen-activated protein (MAP) kinase activity in SH-SY5Y cells was examined. Carbachol concentration-dependently induced tyrosine phosphorylation of several proteins, including one of 42 kDa. This tyrosine-phosphorylated 42 kDa protein co-eluted from a Mono Q anion-exchange column with MAP kinase activity and with immunologically detected MAP kinase. Stimulation of tyrosine phosphorylation and activation of MAP kinase were also observed after incubation of cells with phorbol 12-myristate 13-acetate (PMA) and epidermal growth factor (EGF). Down-regulation or inhibition of protein kinase C (PKC) abolished the stimulatory effects of both carbachol and PMA on MAP kinase activity, whereas EGF-stimulated MAP kinase activity remained unaffected. Thus carbachol acting through the muscarinic (M3) receptor PKC-dependently induced tyrosine phosphorylation and activation of a 42 kDa MAP kinase in SH-SY5Y cells, whereas EGF-induced MAP kinase activation occurred independently of PKC.
Overexpression of N-acetylgalactosamine-4-sulphatase induces a multiple sulphatase deficiency in mucopolysaccharidosis-type-VI fibroblasts
Authors: Anson D S , Muller V , Bielicki J , Harper G S , Hopwood J J
Published: August 2015

High-titre stocks of an amphotropic retrovirus, constructed so as to express a full-length cDNA encoding the human lysosomal enzyme N-acetylgalactosamine-4-sulphatase (4-sulphatase) from the cytomegalovirus immediate early promoter, were used to infect skin fibroblasts from a clinically severe mucopolysaccharidosis type VI (MPS VI) patient. The infected MPS VI cells showed correction of the enzymic defect with the enzyme being expressed at high levels and in the correct subcellular compartment. Surprisingly this did not result in correction of glycosaminoglycan turnover as measured by accumulation of 35S in metabolically labelled cells. We demonstrate that this is apparently caused by an induced reduction of the activities of other lysosomal sulphatases, presumably due to competition for a sulphatase-specific processing mechanism by the over-expressed 4-sulphatase. The level of steroid sulphatase, which is a microsomal sulphatase, was also reduced. Infection of skin fibroblasts from a second, clinically mildly affected, MPS VI patient with the same virus also resulted in no significant change in the level of glycosaminoglycan storage. However, in this case the cause of the observed phenomenon was less clear. These results are of obvious practical importance when considering gene therapy for a sulphatase deficiency such as MPS VI and also provide possible new avenues for exploration of the processes involved in sulphatase synthesis and genetically determined multiple sulphatase deficiency.
Enhancement of pertussis-toxin-sensitive Na+-dependent uridine transporter activity in HL-60 granulocytes by N-formylmethionyl-leucyl-phenylalanine
Authors: Goh L B , Sokoloski J A , Sartorelli A C , Lee C W
Published: August 2015

N-Formyl-Met-Leu-Phe (FMLP), at concentrations as low as 5 nM, caused an increase in intracellular uridine pools in dimethyl sulphoxide (Me2SO)-differentiated HL-60 cells. Intracellular uridine pools were elevated rapidly and reached a maximum within 10 min of exposure to 10 microM FMLP, followed by a gradual decline. This enhancement by FMLP was a consequence of a 3-fold increase in the Vmax of pertussis-toxin-sensitive Na(+)-dependent uridine transport system, with no change in the apparent Km. Km values of 2.67 +/- 0.45 and 3.85 +/- 0.52 microM and Vmax. values of 0.046 +/- 0.017 and 0.125 +/- 0.020 microM/s were obtained for untreated and FMLP-treated Me2SO-differentiated cells respectively. The effect of FMLP on the Na(+)-dependent transport of uridine in Me2SO-differentiated HL-60 cells was specific, as the facilitated transport of uridine was unaffected. Furthermore, this phenomenon was not observed in undifferentiated, phorbol 12-myristate 13-acetate (PMA)-differentiated or pertussis-toxin-treated Me2SO-differentiated HL-60 cells. Removal of extracellular Ca2+ with EGTA abolished the FMLP enhancement of uridine transport in a reversible manner, suggesting the involvement of Ca2+. However, the Ca2+ ionophore A23187 only partially mimicked the effect of FMLP. Similarly, with PMA the transport was sub-optimally enhanced, but a full activation was observed in cells treated with both A23187 and PMA. These findings suggest that activation of the Na(+)-dependent uridine transporter by FMLP in Me2SO-differentiated HL-60 cells involves a pertussis-toxin-sensitive G-protein with a bifurcating signal-transduction pathway.
Is pyridoxal 5′-phosphate an affinity label for phosphate-binding sites in proteins?: The case of bovine glutamate dehydrogenase
Authors: Valinger Z , Engel P C , Metzler D E
Published: August 2015

The effects of pyridoxal 5′-phosphate (PalP) on ox liver glutamate dehydrogenase (94% inactivation by 1.8 mM reagent at pH 7 and 25 degrees C) have been compared with those of three analogues, 5′-deoxypyridoxal (96% inactivation), pyridoxal 5′-sulphate (97%) and pyridoxal 5-methylsulphonate (94%), in order to establish whether PalP acts as an affinity label for this enzyme. Like PalP and unlike pyridoxal, which is a much less potent inactivator, none of the analogues has a free 5′-OH group to cyclize with the aldehyde function. The result with 5′-deoxypyridoxal shows that a negative charge, such as that of the phosphate group, is not required for efficient inactivation. With all four reagents, addition of an excess of cysteine or lysine led to 90-100% re-activation over 3-20 h. Dialysis also caused reactivation to a similar extent. A combination of 2.15 mM NADH, 1 mM GTP and 10 mM 2-oxoglutarate gave complete protection against PalP, but only partial protection against the analogues. 5′-Deoxypyridoxal still caused 20-25% inactivation in the presence of the protection mixture. Absorbance measurements after reduction with NaBH4 show the characteristic features of a reduced Schiff's base and allowed estimation of the extent of reaction. With all the reagents the protection mixture decreased incorporation by about 1 mol/mol, but levels of incorporation without protection varied from about 2 mol/mol for PalP up to about 5 mol/mol for 5′-deoxypyridoxal. The labelling at additional sites may explain the residual inactivation in the presence of potent protecting agents.
Induction of peroxisomal β-oxidation by a microbial catabolite of cholic acid in rat liver and cultured rat hepatocytes
Authors: Nishimaki-Mogami T , Takahashi A , Toyoda K , Hayashi Y
Published: August 2015

The capability of (4R)-4-(2,3,4,6,6a beta,7,8,9,9a alpha,9b beta-decahydro-6a beta-methyl-3-oxo-1H-cyclopental[f]quinolin-7 beta-yl)valeric acid (DCQVA), a catabolite of cholic acid produced by enterobacteria, to induce peroxisome proliferation in vivo and in vitro was studied. Rats given 0.3% DCQVA in the diet for 2 weeks showed marked increases in peroxisomal beta-oxidation, mitochondrial 2,4-dienoyl-CoA reductase and microsomal laurate omega-oxidation activities in the liver compared with control rats given the diet without DCQVA. Cultured rat hepatocytes treated with DCQVA for 72 h also exhibited greatly enhanced beta-oxidation activity. The increased activity was concentration-dependent and the effective concentrations were comparable with those of clofibric acid that produced the same degree of induction in the assay. The results demonstrate that DCQVA is a potent peroxisome proliferator that occurs naturally in rat intestine.
Modification of 1 α,25-dihydroxyvitamin D3 metabolism by introduction of 26,26,26,27,27,27-hexafluoro atoms in human promyelocytic leukaemia (HL-60) cells: isolation and identification of a novel bioactive metabolite, 26,26,26,27,27,27-hexafluoro-1 α,23(S),25-trihydroxyvitamin D3
Authors: Honda A , Nakashima N , Shida Y , Mori Y , Nagata A , Ishizuka S
Published: August 2015

To study whether the introduction of fluoro atoms into C-26 and C-27 positions on the 1 alpha,25-dihydroxyvitamin D3 [1 alpha,25(OH)2D3] molecule could affect metabolism in human promyelocytic leukaemia (HL-60) cells, we compared the metabolism of 26,26,26,27,27,27-hexafluoro-1 alpha,25-dihydroxyvitamin D3 [26,27-F6-1 alpha,25(OH)2D3] and 1 alpha,25(OH)2D3 in HL-60 cells. 26,27-F6-1 alpha,25(OH)2D3 was mainly converted into a new bioactive metabolite, 26,26,26,27,27,27-hexafluoro-1 alpha,23(S),25- trihydroxyvitamin D3 [26,27-F6-1 alpha,23(S),25(OH)3D3], but not into 26,26,26,27,27,27-hexafluoro-1 alpha,24(R),25-trihydroxyvitamin D3 [26,27-F6-1 alpha,24(R),25(OH)3D3] in HL-60 cells. 26,27-F6-1 alpha,23(S),25(OH)3D3 was identified by combinations of h.p.l.c., u.v. spectroscopy and g.c.-mass spectrometry. Evidence is presented that 26,27-F6-1 alpha,25(OH)2D2 was metabolized to 26,27-F6-1 alpha,23(S),25(OH)3D3 by C-23 hydroxylation as a first step of the metabolism, and the 23-hydroxylated bioactive metabolite was accumulated in the cells, whereas 1 alpha,25(OH)2D3 was initially deactivated and metabolized to 1 alpha,24(R),25(OH)3D3 by C-24 hydroxylation through a side-chain oxidation pathway resulting in C23-C24 cleavage, yielding 24,25,26,27-tetranor-1 alpha,23(OH)2D3 in HL-60 cells. These results show that 26,27-F6-1 alpha,25(OH)2D3 and 1 alpha,25(OH)2D3 are metabolized by different metabolic pathways in HL-60 cells.
Inositol lipid signalling occurs in brush-border membranes during initiation of compensatory renal growth in the rat
Authors: Banfić H , Vuica M , Knotek M , Moslavac S , Divecha N
Published: August 2015

Using highly specific mass assays, concentrations of inositol lipids and 1,2-diacylglycerol (DAG) were determined in plasma membranes isolated from rat kidney cortex. Significantly higher concentrations of inositol lipids were determined in brush-border (BBM) than in basal-lateral (BLM) plasma membranes, although DAG concentrations were similar in both. After unilateral nephrectomy, a decrease in PtdIns(4,5)P2 and PtdIns4P, with a concomitant increase in DAG and translocation of protein kinase C (PKC), were observed in BBM but not in BLM isolated from the remaining kidney. On the other hand, stimulation of renal cortical slices with insulin-like growth factor II (IGF-II) or phenylephrine caused similar effects in BLM but not in BBM. Stimulation of phospholipase C activity with translocation of PKC only to BBM in one kidney was also induced by occlusion of blood flow through the contralateral kidney for 15 min. At 30 min after the occlusion was removed and reflow established, DAG concentration and the amount of PKC in BBM returned to control values. These results suggest that an early signal after unilateral nephrectomy is transmitted to cells through BBM and can be switched on and off by blood occlusion and reflow through the contralateral kidney, while hormonal signals caused by IGF-II and phenylephrine are transmitted to cells through BLM.
Spontaneously hypertensive rats and platelet Ca2+-ATPases: specific up-regulation of the 97 kDa isoform
Authors: Papp B , Corvazier E , Magnier C , Kovàcs T , Bourdeau N , Lévy-Tolédano S , Bredoux R , Lévy B , Poitevin P , Lompré A M , Wuytack F , Enouf J
Published: August 2015

The use of platelets instead of smooth muscle cells (SMC) to study the abnormal Ca2+ handling found in hypertension was investigated using spontaneously hypertensive rats (SHR). We studied the regulation of platelet Ca(2+)-ATPases, as we have recently demonstrated that human platelets, like SMC, contain the Ca(2+)-ATPase isoform termed SERCA2-b (sarco-endoplasmic reticulum Ca(2+)-ATPase). In mixed membranes isolated from platelets of normotensive Wistar-Kyoto (WKY) rats and SHR, total Ca(2+)-ATPase activity was found to be 43% higher in SHR than in WKY rats. By the use of autophosphorylation of rat platelet Ca(2+)-ATPases with [gamma-32P]ATP, followed by SDS/PAGE and Western blotting, we found that rat platelets express two distinct Ca(2+)-ATPases: a 100 kDa isoform, recognized by a SERCA2-b-specific anti-peptide antibody, and a 97 kDa isoform, specifically recognized by a polyclonal anti-SERCA antibody. Comparative analysis of platelet membrane Ca(2+)-ATPases from WKY rats and SHR demonstrated that the expression of the SERCA2-b isoform did not change significantly (128 +/- 22%), whereas that of the 97 kDa isoform reached 300 +/- 35% in SHR when compared with WKY rats. We concluded that the upregulation of total platelet Ca(2+)-ATPases in SHR is not due to the 100 kDa SERCA2-b isoform found in SMC, but is specific to the 97 kDa Ca(2+)-ATPase isoform which is not present in SMC. Therefore platelets should be used with extreme caution as a surrogate model of vascular smooth muscle Ca2+ homeostasis.
Epidermal growth factor and phorbol myristate acetate increase expression of the mRNA for cytosolic phospholipase A2 in glomerular mesangial cells
Authors: Maxwell A P , Goldberg H J , Tay A H N , Li Z G , Arbus G S , Skorecki K L
Published: August 2015

We have previously shown that phospholipase A2 (PLA2) activity is rapidly activated by epidermal growth factor (EGF) and phorbol 12-myristate 13-acetate (PMA) in renal mesangial cells and other cell systems in a manner that suggests a covalent modification of the PLA2 enzyme(s). This PLA2 activity is cytosolic (cPLA2) and is distinct from secretory forms of PLA2, which are also stimulated in mesangial cells in response to cytokines and other agonists. However, longer-term regulation of cPLA2 in renal cells may also occur at the level of gene expression. Cultured rat mesangial cells were used as a model system to test the effects of EGF and PMA on the regulation of cPLA2 gene expression. EGF and PMA both produced sustained increases in cPLA2 mRNA levels, with a parallel increase in enzyme activity over time. Inhibition of protein synthesis by cycloheximide increased basal cPLA2 mRNA accumulation in serum-starved mesangial cells, and the combination of EGF and cycloheximide resulted in super-induction of cPLA2 gene expression compared with EGF alone. Actinomycin D treatment entirely abrogated the effect of EGF on cPLA2 mRNA accumulation. These findings suggest that regulation of cPLA2 is achieved by factors controlling gene transcription and possibly mRNA stability, in addition to previously characterized posttranslational modifications.
Stimulation by paraquat of microsomal and cytochrome P-450-dependent oxidation of glycerol to formaldehyde
Authors: Clejan L A , Cederbaum A I
Published: August 2015

Glycerol can be oxidized to formaldehyde by microsomes in a reaction that is dependent on cytochrome P-450. An oxidant derived from the interaction of H2O2 with iron was responsible for oxidizing the glycerol, with P-450 suggested to be necessary to produce H2O2 and reduce non-haem iron. The effect of paraquat on formaldehyde production from glycerol and whether paraquat could replace P-450 in supporting this reaction were studied. Paraquat increased NADPH-dependent microsomal oxidation of glycerol; the stimulation was inhibited by glutathione, catalase, EDTA and desferrioxamine, but not by superoxide dismutase or hydroxyl-radical scavengers. The paraquat stimulation was also inhibited by inhibitors, substrate and ligand for P-4502E1 (pyrazole-induced P-450 isozyme), as well as by anti-(P-4502E1) IgG. These results suggest that P-450 still played an important role in glycerol oxidation, even in the presence of paraquat. Purified NADPH-cytochrome P-450 reductase did not oxidize glycerol to formaldehyde; some oxidation, however, did occur in the presence of paraquat. Reductase plus P-4502E1 oxidized glycerol, and a large stimulation was observed in the presence of paraquat. Rates in the presence of P-450, reductase and paraquat were more than additive than the sums from the reductase plus P-450 and reductase plus paraquat rates, suggesting synergistic interactions between paraquat and P-450. These results indicate that paraquat increases oxidation of glycerol to formaldehyde by microsomes and reconstituted systems, that H2O2 and iron play a role in the overall reaction, and that paraquat can substitute, in part, for P-450 in supporting oxidation of glycerol. However, cytochrome P-450 is required for elevated rates of formaldehyde production even in the presence of paraquat.
Ionomycin activates electrogenic Ca2+ influx in rat thymic lymphocytes
Authors: Mason M J , Grinstein S
Published: August 2015

Increases in intracellular Ca2+ concentration ([Ca2+]i) after addition of the carboxylic Ca(2+)-transporting ionophore ionomycin have been reported to be inhibited by depolarization in mouse T- and B-lymphocytes, a result attributed to effects on the plasma-membrane Ca2+ pump. The present experiments were undertaken to test the hypothesis that activation of electrogenic Ca2+ uptake, due to release of Ca2+i from intracellular pools, underlies the membrane-potential (Em)-sensitivity of ionomycin-mediated Ca2+ uptake in T-lymphocytes. To address this proposal we have compared the characteristics of Ca2+ influx induced by ionomycin with those of Ca2+ influx activated by release of Ca2+ from intracellular stores (store-regulated Ca2+ uptake, SRCU). The endosomal Ca(2+)-ATPase inhibitor thapsigargin was used to bring about loss of Ca2+ from intracellular pools. In support of this hypothesis, we report that: (1) ionomycin releases Ca2+ from a thapsigargin-sensitive endosomal pool, a pool previously demonstrated to regulate electrogenic SRCU in rat thymic lymphocytes, (2) Ca2+ and Mn2+ uptake mediated by low doses of ionomycin are inhibited by compounds previously reported to inhibit SRCU, and (3) in the absence of SRCU, ionomycin-mediated Ca2+ uptake displays no sensitivity to Em. We conclude that activation of electrogenic SRCU can adequately account for the Em-sensitivity of Ca2+ flux previously attributed to alterations in Ca(2+)-pump activity. Such a mechanism of action may underlie previous reports of electrogenic Ca2+ transport mediated by ionomycin in other tissues.
Studies of the active site of m-calpain and the interaction with calpastatin
Authors: Crawford C , Brown N R , Willis A C
Published: August 2015

Calpain autolyses in the presence of Ca2+. In the case of m-calpain (80 + 30 kDa) the first product is an 80 + 18 kDa species which has an intact large subunit and the C-terminal Ca(2+)-binding domain of the small subunit. It was possible to bind E64 into the active site of calpain in the presence of Ca2+ before cleavage of either calpain subunit. This suggests that the active site is functional before any autolysis has occurred and that calpain is not a proenzyme. Prolonged autolysis generates several fragments including a 42 kDa active-site domain fragment that showed no proteolytic activity and Ca(2+)-binding domain fragments. Some of the Ca(2+)-binding domain fragments were found to exist as heterodimers (23 + 18 kDa and 22 + 18 kDa), with the Ca(2+)-binding domain of the large subunit interacting with the Ca(2+)-binding domain of the small subunit. These species were true heterodimers, as they showed co-elution of the two Ca(2+)-binding domains on ion-exchange and gel-filtration chromatography, and immunoprecipitation of both polypeptides with an antiserum specific for the small-subunit Ca(2+)-binding domain. The generation of the dimer species after only 15 min autolysis suggests that the interaction between the Ca(2+)-binding domains is present in the native calpain structure. The interaction of calpain with calpastatin was investigated using an assay based on binding to calpastatin-Sepharose and a competitive binding assay. Calpain, active-site-blocked calpain and calpain fragments generated by autolysis were studied. Calpain bound to calpastatin in the presence of Ca2+; however, the isolated active-site-containing 80 kDa large subunit (proteolytically inactive), a 42 kDa active-site-containing fragment (proteolytically inactive) and Ca(2+)-binding domain fragments of calpain did not. Active-site-blocked calpain bound to calpastatin, but with an affinity reduced by approximately two orders of magnitude when compared with native calpain.
Activation of phosphatidylinositol 4,5-bisphosphate supply by agonists and non-hydrolysable GTP analogues
Authors: Stephens L , Jackson T R , Hawkins P T
Published: August 2015

PtdIns(4,5)P2 serves as a precursor of a diverse family of signalling molecules, including diacylglycerol (and hence phosphatidic acid), Ins(1,4,5)P3 [and hence Ins(1,3,4,5)P4] and PtdIns(3,4,5)P3. The production of these messengers can be activated by agonists, and therefore the rate of utilization of PtdIns(4,5)P2 can vary dramatically. Although cells can only meet these large changes in demand for PtdIns(4,5)P2 by increasing its synthesis and/or by continuously cycling it at a rate that exceeds its potential consumption (avoiding the need for a co-ordinated activation mechanism), no satisfactory explanation for how this is achieved in agonist-stimulated cells has yet been provided. We show here that, in streptolysin-O-permeabilized neutrophils, N-formylmethionyl-leucyl-phenylalanine (FMLP), platelet-activating factor (PAF) and non-hydrolysable GTP analogues can cause large activations of PtdIns4P 5-kinase, suggesting that cells can accommodate agonist-activated rates of consumption of PtdIns(4,5)P2 without having to sustain continuous, comparably rapid and energetically expensive ‘futile cycling’ reactions.
Human small intestinal angiotensin-converting enzyme: intracellular transport, secretion and glycosylation
Authors: Naim H Y
Published: August 2015

Human intestinal angiotensin-converting enzyme (ACE) exists in the brush-border membrane as a monomeric protein of apparent molecular mass 184 kDa. It is associated with the membrane via a hydrophobic segment and has a transmembrane orientation [Naim (1992) Biochem. J. 286, 451-457]. In addition to the membrane-bound form (ACEm), hydrophilic forms of ACE (ACEsec) can be identified in biosynthetically labelled intestinal cells. Thus the culture medium of biosynthetically labelled human biopsy samples contains an ACE molecule which has an apparent molecular mass similar to that of its membrane-bound counterpart. The secreted ACEsec forms follow a precursor/product relationship with the mature ACE molecule. The effect of the monomeric structure of ACE in its intracellular transport and secretion was investigated by pulse-chase experiments on human biopsy samples labelled with [35S]methionine. The results reveal 2-3-fold slower transport of ACE from the endoplasmic reticulum (ER) to the Golgi as compared with the homodimeric proteins dipeptidylpeptidase IV and aminopeptidase N. Further, the transport kinetics of ACE are comparable with those of human sucrase-isomaltase and human maltase-glucoamylase, two brush-border disaccharidases that do not form homodimers in the ER of human small-intestinal cells. These findings strongly suggest that homodimerization of brush-border proteins may influence the rate of transport of these proteins from the ER to the Golgi. The effect of glycosylation on the transport and secretion of ACE was investigated by utilizing several inhibitors of glycan processing. Here, secretion of ACE molecules continued to take place, albeit to a considerably lesser extent. In fact, approx. 2-fold less ACE molecules were secreted in the presence of inhibitors of ER glucosidases I and II and cis-Golgi mannosidase-I, suggesting that carbohydrate processing is important in the attainment of a transport-competent conformation.
Phorbol 12-myristate 13-acetate-stimulated phosphorylation of erythrocyte membrane skeletal proteins is blocked by calpain inhibitors: possible role of protein kinase M
Authors: Al Z , Cohen C M
Published: August 2015

Human erythrocytes contain cytosolic protein kinase C (PKC) which, when activated by phorbol 12-myristate 13-acetate (PMA), induces the phosphorylation of the membrane skeletal proteins band 4.1, band 4.9 and adducin. We found that brief treatments of erythrocytes with PMA resulted in a decrease in cytosolic PKC content and in the transient appearance in the cytosol of a Ca(2+)- and phospholipid-independent 55 kDa fragment of PKC, called PKM. Prolonged treatment with PMA resulted in the complete and irreversible loss of erythrocyte PKC. To investigate the possible role of calpain in this process, the calpain inhibitors leupeptin and E-64 were sealed inside erythrocytes by reversible haemolysis. Both inhibitors prolonged the lifetime of PKC in PMA-treated cells, and leupeptin was shown to block the PMA-stimulated appearance of PKM in the cytosol. Significantly, leupeptin also completely blocked PMA-stimulated phosphorylation of membrane and cytosolic substrates. This effect was mimicked by other calpain inhibitors (MDL-28170 and calpain inhibitor I), but did not occur when other protease inhibitors such as phenylmethanesulphonyl fluoride, pepstatin A or chymostatin were used. In addition, the phosphorylation of exogenous histone sealed inside erythrocytes was also blocked by leupeptin. Immunoblotting showed that leupeptin did not prevent the PMA-induced translocation of PKC to the erythrocyte membrane. Thus inhibition of PKC phosphorylation of membrane skeletal proteins by calpain inhibitors was not due to inhibition of PKC translocation to the membrane. Our results suggest that PMA treatment of erythrocytes results in the translocation of PKC to the plasma membrane, followed by calpain-mediated cleavage of PKC to PKM. This cleavage, or some other leupeptin-inhibitable process, is a necessary step for the phosphorylation of membrane skeletal substrates, suggesting that the short-lived PKM may be responsible for membrane skeletal phosphorylation. Our results suggest a potential mechanism whereby erythrocyte PKC may be subject to continual down-regulation during the lifespan of the erythrocyte due to repeated activation events, possibly related to transient Ca2+ influx. Such down-regulation may play an important role in erythrocyte survival or pathophysiology.
Bradykinin-dependent activation of adenylate cyclase activity and cyclic AMP accumulation in tracheal smooth muscle occurs via protein kinase C-dependent and -independent pathways
Authors: Stevens P A , Pyne S , Grady M , Pyne N J
Published: August 2015

Treatment of cultured tracheal smooth-muscle cells (TSM) with phorbol 12-myristate 13-acetate (PMA) (100 nM) or bradykinin (100 nM) elicited enhanced basal and guanosine 5′-[beta gamma-imido]-triphosphate-stimulated adenylate cyclase activities in subsequently isolated membranes. Combined stimulation of cells was non-additive, indicating that both agents activate adenylate cyclase via similar routes. Both PMA (100 nM) and bradykinin (100 nM) allowed the alpha subunit of Gs to act as a more favourable substrate for its cholera-toxin-catalysed ADP-ribosylation in vitro. PMA was without effect on intracellular cyclic AMP in control cells. However, constitutive activation of Gs by treatment in vivo with cholera toxin (0.5 ng/ml, 18 h) sensitized the cells to PMA stimulation, resulting in a concentration-dependent increase in intracellular cyclic AMP accumulation (EC50 = 7.3 +/- 2.5 nM, n = 5). Bradykinin also elicited a concentration-dependent increase in intracellular cyclic AMP (EC50 = 63.3 +/- 14.5 nM, n = 3). Constitutive activation of Gs resulted in an increased maximal response (10-fold) and potency (EC50 = 6.17 +/- 1.6 nM, n = 3) to bradykinin. This response was not affected by the B2-receptor antagonist, NPC567 [which selectively blocks bradykinin-stimulated phospholipase C (PLC), with minor activity against phospholipase D (PLD) activity]. Des-Arg9-bradykinin (a B1-receptor agonist) was without activity. These results suggest that the receptor sub-type capable of activating PLD may also be stimulatory for cyclic AMP accumulation. Furthermore, pre-treatment of the cells with butan-l-ol (0.3%, v/v), which traps phosphatidate derived from PLD reactions, blocked the bradykinin-stimulated increase in intracellular cyclic AMP. These studies suggest that there may be a causal link between PLD-derived phosphatidate and the positive modulation of adenylate cyclase activity. In support of this, the concentration-dependence for bradykinin-stimulated adenylate cyclase activity was identical with that of bradykinin-stimulated phospholipase D activity (EC50 = 5 nM). Bradykinin, but not PMA, was also capable of eliciting the inhibition of cyclic AMP phosphodiesterase activity in TSM cells (EC50 > 100 nM) via an unidentified mechanism. These studies indicate that cross-regulation between the cyclic AMP pathway and phospholipid-derived second messengers in TSM cells does not occur as a consequence of PLC-catalysed PtdIns(4,5)P2 hydrolysis, but may involve, in part, PLD-catalysed phosphatidylcholine hydrolysis.
Platelet-derived growth factor (PDGF)-AB-mediated phosphorylation of PDGF β receptors
Authors: Coats S R , Love H D , Pledger W J
Published: August 2015

Platelet-derived growth factor (PDGF) stimulates the proliferation of Balb/c-3T3 fibroblasts through binding and subsequent activation of PDGF receptors. Activation of the PDGF receptors has been proposed to involve receptor dimerization. PDGF-AB has been shown to bind PDGF alpha and beta receptor subunits to form PDGF alpha beta and alpha alpha receptor dimers. In this paper we demonstrate that, following the down-regulation of PDGF alpha receptors, the binding of PDGF-AB to beta receptors occurred at 37 degrees C but not at 4 degrees C. PDGF-AB stimulated the phosphorylation of PDGF beta receptor monomers in cells depleted of PDGF alpha receptors by prior exposure to PDGF-AA.
Chromosomal localization of human genes for arylamine N-acetyltransferase
Authors: Hickman D , Risch A , Buckle V , Spurr N K , Jeremiah S J , McCarthy A , Sim E
Published: August 2015

Arylamine N-acetyltransferase is encoded at two loci, AAC-1 and AAC-2, on human chromosome 8. The products of the two loci are able to catalyse N-acetylation of arylamine carcinogens, such as benzidine and other xenobiotics. AAC-2 is polymorphic and individuals carrying the slow-acetylator phenotype are more susceptible to benzidine-induced bladder cancer. We have identified yeast artificial chromosome clones encoding AAC-1 and AAC-2 and have used the cloned DNAs as fluorescent probes for in situ hybridization. The hybridization patterns allow assignment of AAC-1 and AAC-2 to chromosome 8p21.3-23.1, a region in which deletions have been associated with bladder cancer [Knowles, Shaw and Proctor (1993) Oncogene 8, 1357-1364].
Rate of calcium entry determines the rapid changes in protein kinase C activity in angiotensin II-stimulated adrenal glomerulosa cells
Authors: Kojima I , Kawamura N , Shibata H
Published: August 2015

The present study was conducted to monitor precisely the activity of protein kinase C (PKC) in adrenal glomerulosa cells stimulated by angiotensin II (ANG II). PKC activity in cells was monitored by measuring phosphorylation of a synthetic KRTLRR peptide, a specific substrate for PKC, immediately after the permeabilization of the cells with digitonin [Heasley and Johnson J. Biol. Chem. (1989) 264, 8646-8652]. Addition of 1 nM ANG II induced a gradual increase in KRTLRR peptide phosphorylation, which reached a peak at 30 min, and phosphorylation was sustained thereafter. When the action of ANG II was terminated by adding [Sar1,Ala8]ANG II, a competitive antagonist, both Ca2+ entry and KRTLRR phosphorylation ceased rapidly, whereas diacylglyercol (DAG) content was not changed significantly within 10 min. Similarly, when blockade of Ca2+ entry was achieved by decreasing extracellular Ca2+ to 1 microM or by adding 1 microM nitrendipine, KRTLRR peptide phosphorylation was decreased within 5 min. In addition, restoration of Ca2+ entry was accompanied by an immediate increase in KRTLRR peptide phosphorylation. Under the same condition, DAG content did not change significantly. We then examined the role of the PKC pathway in ANG II-induced aldosterone production. Ro 31-8220 inhibited ANG II-induced KRTLRR phosphorylation without affecting the activity of calmodulin-dependent protein kinase II. In the presence of Ro 31-8220, ANG II-mediated aldosterone production was decreased to approx. 50%. Likewise, intracellular administration of PKC19-36, a sequence corresponding to residues 19-36 of the regulatory domain of PKC known to inhibit PKC activity, attenuated ANG II-mediated activation of PKC and aldosterone output. These results indicate a critical role of Ca2+ entry in the regulation of PKC activity by ANG II.
Bradykinin-induced growth inhibition of normal rat kidney (NRK) cells is paralleled by a decrease in epidermal-growth-factor receptor expression
Authors: Van Zoelen E J J , Peters P H J , Afink G B , Van Genesen S , De Roos A D G , Van Rotterdam W , Theuvenet A P R
Published: August 2015

Normal rat kidney fibroblasts, grown to density arrest in the presence of epidermal growth factor (EGF), can be induced to undergo phenotypic transformation by treatment with transforming growth factor beta or retinoic acid. Here we show that bradykinin blocks this growth-stimulus-induced loss of density-dependent growth arrest by a specific receptor-mediated mechanism. The effects of bradykinin are specific, and are not mimicked by other phosphoinositide-mobilizing agents such as prostaglandin F2 alpha. Northern-blot analysis and receptor-binding studies demonstrate that bradykinin also inhibits the retinoic acid-induced increase in EGF receptor levels in these cells. These studies provide additional evidence that EGF receptor levels modulate EGF-induced expression of the transformed phenotype in these cells.
Contribution of interactions with the core domain of hirudin to the stability of its complex with thrombin
Authors: Betz A , Hopkins P C R , Le Bonniec B F , Stone S R
Published: August 2015

The importance of interactions with residues 15-21 in the core domain of hirudin for the formation of the complex with thrombin has been investigated by site-directed mutagenesis. Contacts made by Leu-15 were found to be particularly important; replacement of this residue by alanine led to a decrease in the binding energy (delta delta Gbo) of 15 kJ.mol-1. Comparison with effects obtained in previous mutagenesis studies indicate that interactions with the side chain of Leu-15 contribute more to the stability of the complex than those of any other hirudin residues. Interactions with the side chains of Glu-17, Asn-20 and Val-21 also contributed significantly to binding energy; the delta delta Gbo value for these mutations was between 3 and 6 kJ.mol-1. Examination of the crystal structure of the thrombin-hirudin complex suggested the possibility that ionic interactions that would increase binding energy could be engineered by mutating Ser-19, Asn-20 and Gln-49 to acidic residues. The stability of the thrombin-hirudin complex was not, however, increased by these substitutions. The results obtained are discussed in terms of the crystal structure of the thrombin-hirudin complex.
Annexin II contains two types of Ca2+-binding sites
Authors: Jost M , Weber K , Gerke V
Published: August 2015

The annexins are a multigene family of Ca(2+)-dependent phospholipid-binding proteins which contain novel types of Ca2+ sites. Using site-directed mutagenesis, we generated mutant proteins that show defects in the Ca(2+)-binding sites in a particular member of this family, the src tyrosine kinase substrate annexin II. Analysis of the relative Ca(2+)-binding affinities of annexin II mutants in a combined Ca2+/phospholipid-binding assay revealed two distinct types of Ca(2+)-binding sites. Three so-called type II sites are found in annexin repeats 2, 3 and 4 respectively. Two so-called type III sites are located in the first repeat and involve the glutamic acid residues at positions 52 and 95. Both types of sites were recently identified by X-ray crystallography in annexins V and I [Huber, Schneider, Mayr, Römisch and Paques (1990) FEBS Lett. 275, 15-21; Weng, Luecke, Song, Kang, Kim and Huber (1993) Protein Sci. 2, 448-458], indicating that similar principles govern Ca2+ binding to annexins in crystals and in solution. The two types of Ca(2+)-binding sites differ not only in their architecture but also in their affinity for the bivalent cation. The Ca2+ concentration needed for half-maximal phosphatidylserine binding is 5-10 microM for an annexin II derivative with intact type II but defective type III sites (TM annexin II) whereas a mutant protein containing defective type II but unaltered type III sites (CM annexin II) requires 200-300 microM Ca2+ for the same activity. Annexin II mutants with defects in the type II and/or type III sites also show different subcellular distributions. When expressed transiently in HeLa cells, TM annexin II acquires the typical location in the cortical cytoskeleton observed for the wild-type molecule. In contrast, CM annexin II remains essentially cytosolic, as does a mutant protein containing defects in both type II and type III Ca(2+)-binding sites (TCM annexin II). This indicates that the intracellular association of annexin II with the submembraneous cytoskeleton depends only on the occupation of type II Ca(2+)-binding sites.
Presence and differential expression of SGLT1, GLUT1, GLUT2, GLUT3 and GLUT5 hexose-transporter mRNAs in Caco-2 cell clones in relation to cell growth and glucose consumption
Authors: Mahraoui L , Rodolosse A , Barbat A , Dussaulx E , Zweibaum A , Rousset M , Brot-Laroche E
Published: August 2015

Seven clones from the Caco-2 cell line, three isolated from passage 29 (PD7, PD10, PF11) and four from passage 198 (TB10, TC7, TF3, TG6), all of them selected on the basis of differences in the levels of expression of sucrase-isomaltase and rates of glucose consumption, were analysed for the expression of hexose-transporter mRNAs (SGLT1, GLUT1-GLUT5) in relation to the phases of cell growth and the associated variations of the rates of glucose consumption. All clones showed a similar pattern of evolution of the rates of glucose consumption, which decreased from the exponential to the late-stationary phase, but differed, in a 1-40-fold range, in the values observed at late postconfluency. According to these values, clones could be divided into high- (PD10, PF11) and low-glucose-consuming cells (PD7, TB10, TC7, TF3 and TG6). GLUT1 and GLUT3 mRNAs were expressed in all clones and showed a similar pattern of evolution: their level decreased, from the exponential to the stationary phase, in close correlation with the decrease in rates of glucose consumption, with only high-glucose-consuming clones maintaining high levels in the stationary phase. In contrast, SGLT1, GLUT2 and GLUT5 mRNAs were only expressed, like sucrase-isomaltase mRNA, in the low-glucose-consuming clones, and their level increased from the exponential to the stationary phase, in parallel with the differentiation of the cells. GLUT4 was undetectable in all the clones. Glucose deprivation generally resulted in a discrete decrease in the levels of all transporter mRNAs in all clones, one exception being GLUT2, which in the high-glucose-consuming clones is only detectable when the cells are grown in low glucose. These clones should be ideal tools with which to study in vitro, at the single-cell level, how these transporters concur to the utilization and transport of hexoses and how their exclusive or co-ordinated expression is regulated.
The potential dolichol recognition sequence of β-1,4-mannosyltransferase is not required for enzymic activity using phytanyl-pyrophosphoryl-α-N,N'- diacetylchitobioside as acceptor
Authors: Revers L , Wilson I B H , Webberley M C , Flitsch S L
Published: August 2015

The ALG1 gene of Saccharomyces cerevisiae encodes beta-1,4-mannosyltransferase, an essential membrane-associated enzyme involved in the assembly of dolichyl-linked oligosaccharide precursors for N-glycosylation [Albright and Robbins (1990) J. Biol. Chem. 265, 7042-7049], which catalyses the transfer of a mannose residue from GDP-mannose to dolichyl-pyrophosphoryl-alpha-N,N'- diacetylchitobioside; it also possesses a putative transmembrane domain, bearing an 11-amino-acid consensus sequence, which has been proposed to mediate dolichol recognition. Here we report the construction and bacterial expression of a mutant beta-1,4-mannosyltransferase derived from ALG1, which carries a 34-amino-acid deletion resulting in the absence of the entire N-terminal transmembrane domain. This truncated enzyme has an apparent Km value of 17 microM for phytanyl-pyrophosphoryl-alpha-N,N'-diacetylchitobioside, a known acceptor for beta-1,4-mannosyltransferase [Flitsch, Pinches, Taylor and Turner (1992) J. Chem. Soc., Perkin Trans. 1, 2087-2093]. The intact enzyme, expressed in the same system, has an apparent Km value of 25 microM. These figures are in good agreement with previously reported values for wild-type beta-1,4-mannosyl-transferase incubated with the natural dolichyl-linked substrate. Gel-filtration chromatography (before and after beta-mannosidase digestion) of the products of both forms of the enzyme verifies the formation of Man beta 1->4GlcNAc beta 1->4GlcNAc. We therefore conclude that the putative dolichol recognition sequence is not necessary for recognition of the phytanyl analogue of its natural dolichol substrate and suggest it probably also is not needed for its natural substrate.
Experimental approach to the kinetic study of unstable site-directed irreversible inhibitors: kinetic origin of the apparent positive co-operativity arising from inactivation of trypsin by p-amidinophenylmethanesulphonyl fluoride
Authors: Espín J C , Tudela J
Published: August 2015

Experimental characterization of enzyme inactivation by unstable irreversible inhibitors has only previously been carried out by using discontinuous methods involving preincubation, removal of samples and further residual activity assays. A continuous method for the kinetic study of these inhibitors in the presence of an auxiliary substrate was recently proposed in a theoretical study. This method was based on approximate expressions for the evolution of the product concentration, which contained series expansions with five or more exponential terms, seriously complicating their use in practice. In the present paper, a new experimental method has been developed for the kinetic study of unstable and site-directed irreversible inhibitors, considering two different ranges of inhibitor concentration. Thus at low inhibitor concentrations, the system evolves from an initial to a final steady state, the rates of which are described by exact analytical equations. At high inhibitor concentrations, however, the product accumulation can be described by an exact uniexponential equation. This simple and efficient method has been applied to the kinetic study of trypsin inactivation by p-amidinophenylmethanesulphonyl fluoride, near the optimum pH of the enzyme. The dependence of the final steady-state rate on the substrate concentration shows apparent positive co-operativity which has not previously been reported. The kinetic origin of this type of co-operativity is predicted by one of the exact analytical equations derived here. The instability of new protein and non-protein irreversible inhibitors has to be carefully characterized to prevent true unstable irreversible inhibitors being wrongly described as allosteric reversible inhibitors.