bluuebunny/crossref_metadata_2025 huggingface dataset:
Ryanodine receptor/calcium release channel conformations as reflected in the different effects of propranolol on its ryanodine binding and channel activity
Authors: ZCHUT Sigalit , FENG Wei , SHOSHAN-BARMATZ Varda
Published: August 2015

1. Propranolol, a β-blocker, inhibited or stimulated ryanodine binding to both the membrane-bound and purified ryanodine receptor (RyR) depending on the assay conditions. At high NaCl concentrations, propranolol increased the number of ryanodine-binding sites (Bmax) with no effect on the binding affinity. In the presence of 0.2 M NaCl, ryanodine binding was inhibited by propranolol. Half-maximal inhibition was obtained at 1.2 mM and complete inhibition at 2 mM propranolol. The inhibitory effect of propranolol obtained at low NaCl concentration was not restored by increasing the NaCl concentration to 1 M. 2. Modulators of the RyR that are known to alter its conformational states, such as adenine nucleotides, Ca2+ concentration and pH, modified the effect of propranolol on ryanodine binding. In the presence of propranolol and at low NaCl concentrations, ryanodine binding was inhibited and showed no Ca2+-, pH- or time-dependence. 3. Propranolol immediately and completely blocked the channel opening of RyR reconstituted into a planar lipid bilayer. Propranolol-modified non-active channel was reactivated to a subconductive state (about 40% of the control conductance) by ATP. 4. Competition experiments between lidocaine (a stimulatory drug) or tetracaine (an inhibitory drug) and propranolol at 0.2 or 1.0 M NaCl, respectively, suggest the existence of different interaction sites for local anaesthetics and propranolol. 5. These results suggest that propranolol interacts directly with the RyR and modifies its ryanodine binding and single-channel activities. Propranolol effects are altered by the RyR conformational state, suggesting its possible use as a conformational probe for RyR.
The purification and some properties of the Mg2+-activated cytosolic aldehyde dehydrogenase of Saccharomyces cerevisiae
Authors: DICKINSON Francis M.
Published: August 2015

A purification procedure has been developed for the cytosolic aldehyde dehydrogenase of Saccharomyces cerevisiae that yields homogeneous enzyme. The enzyme seems to be a tetramer of identical 58 kDa subunits. The enzyme reaction is strongly stimulated by Mg2+ at low NADP+ concentrations but there is no absolute requirement for bivalent cations. The kinetics of the reaction have been studied in the presence and absence of MgCl2. NADP+ binding studies of the quenching of protein fluorescence in the presence and absence of MgCl2 show that the effect of Mg2+ is to increase the affinity of the enzyme for NADP+ by approx. 100-fold. NADP+ binding causes a slow conformational change in the enzyme and converts the enzyme from the inactive or low-activity form in which it is isolated into the fully active form. This conformational change seems to explain the marked lag-phases seen in enzyme assays. The enzyme is strongly inhibited by disulfiram and pyridoxal 5-phosphate.
Expression in Escherichia coli, purification and characterization of heparinase I from Flavobacterium heparinum
Authors: ERNST Steffen , VENKATARAMAN Ganesh , WINKLER Stefan , GODAVARTI Ranga , LANGER Robert , COONEY Charles L. , SASISEKHARAN SASISEKHARAN
Published: August 2015

The use of heparin for extracorporeal therapies has been problematical due to haemorrhagic complications; as a consequence, heparinase I from Flavobacterium heparinum is used for the determination of plasma heparin and for elimination of heparin from circulation. Here we report the expression of recombinant heparinase I in Escherichia coli, purification to homogeneity and characterization of the purified enzyme. Heparinase I was expressed with an N-terminal histidine tag. The enzyme was insoluble and inactive, but could be refolded, and was purified to homogeneity by nickel-chelate chromatography. The cumulative yield was 43%, and the recovery of purified heparinase I was 14.4 mg/l of culture. The N-terminal sequence and the molecular mass as analysed by matrix-assisted laser desorption MS were consistent with predictions from the heparinase I gene structure. The reverse-phase HPLC profile of the tryptic digest, the Michaelis–Menten constant Km (47 μg/ml) and the specific activity (117 units/mg) of purified recombinant heparinase I were similar to those of the native enzyme. Degradation of heparin by heparinase I results in a characteristic product distribution, which is different from those obtained by degradation with heparinase II or III from F. heparinum. We developed a rapid anion-exchange HPLC method to separate the products of enzymic heparin degradation, using POROS perfusion chromatography media. Separation of characteristic di-, tetra- and hexa-saccharide products is performed in 10 min. These methods for the expression, purification and analysis of recombinant heparinase I may facilitate further development of heparinase I-based medical therapies as well as further investigation of the structures of heparin and heparan sulphate and their role in the extracellular matrix.
Kinetic mechanism of octopus hepatopancreatic glutathione transferase in reverse micelles
Authors: TANG Shiao-Shek , CHANG Gu-Gang
Published: August 2015

Octopus glutathione transferase (GST) was enzymically active in aerosol-OT [sodium bis-(2-ethylhexyl)sulphosuccinate]/iso-octane reverse micelles albeit with lowered catalytic constant (kcat). The enzyme reaction rate was found to be dependent on the [H2O]/[surfactant] ratio (ωo) of the system with maximum rate observed at ωo 13.88, which corresponded to vesicles with a core volume of 64 nm3. According to the physical examinations, a vesicle of this size is barely large enough to accommodate a monomeric enzyme subunit. Dissociation of the enzyme in reverse micelles was confirmed by cross-linking of the associated subunits with glutaraldehyde and separation of the monomers and dimers with electrophoresis in the presence of SDS. The kinetic properties of the enzyme were investigated by steady-state kinetic analysis. Both GSH and 1-chloro-2,4-dinitrobenzene (CDNB) showed substrate inhibition and the Michaelis constant for CDNB was increased by 36-fold to 11.05 mM in reverse micelles. Results on the initial-velocity and product-inhibition studies indicate that the octopus GST conforms to a steady-state sequential random Bi Bi mechanism. The results from a log kcat versus pH plot suggest that amino acid residues with pKa values of 6.56±0.07 and 8.81±0.17 should be deprotonated to give optimum catalytic function. In contrast, the amino acid residue with a pKa value of 9.69±0.16 in aqueous solution had to be protonated for the reaction to proceed. We propose that the pKa1 (6.56) is that for the enzyme-bound GSH, which has a pKa value lowered by 1.40–1.54 pH units compared with that of free GSH in reverse micelles. The most probable candidate for the observed pKa2 (8.81) is Tyr7 of GST. The pKa of Tyr7 is 0.88 pH unit lower than that in aqueous solution and is about 2 pH units below the normal tyrosine. This tyrosyl residue may act as a base catalyst facilitating the dissociation of enzyme-bound GSH. The possible interaction of GST with plasma membrane in vivo is discussed.
Pre-steady-state kinetic study of the effects of K+ on the partial reactions of the catalytic cycle of the plasma membrane Ca2+-ATPase
Authors: HERSCHER Claudio J. , REGA Alcides F. , ADAMO Hugo P.
Published: August 2015

The effects of 100 mM K+ on the partial reactions that take place during ATP hydrolysis by the calcium ion-dependent ATPase from plasma membrane (PM-Ca2+-ATPase) were studied at 37 °C on fragmented intact membranes from pig red cells by means of a rapid chemical quenching technique. At 10 μM [γ-32P]ATP plus non-limiting concentrations of Ca2+ and Mg2+, K+ increased the kapp of formation by 140% to 84±11 s-1 and the steady-state level of phosphoenzyme (EP) by 25% to 3.4±0.17 pmol/mg of protein. If added together with [γ-32P]ATP at the beginning of phosphorylation, K+ was much less effective than if added earlier, indicating that it did not act on the phosphorylation reaction. Measurements of the E2 → E1 transition by phosphorylation showed that in medium with Ca2+ and Mg2+, K+ increased the kapp of the transition by 55% to 14±3 s-1 and the apparent concentration of E1 by 45%, suggesting that this may be the cause of the increased rate of phosphorylation observed in enzyme preincubated with K+. The presence of K+ did not change the slow decay of EP without Mg2+ but activated the decay of EP made with Mg2+, increasing its kapp by 60% to 91±12 s-1. In contrast with observations made during phosphorylation, if added at the beginning of dephosphorylation K+ was fully effective in favouring decomposition of EP made in medium containing no K+. In the presence of either 3 mM ATP or 3 mM ATP plus calmodulin, which activate hydrolysis of CaE2P, the effect of K+ on dephosphorylation was conserved. Because the sites for K+ are intracellular and the concentration of K+ in normal red cells is above 100 mM, the effects described here must be taken into account to describe the catalytic cycle of the PM-Ca2+-ATPase under physiological conditions.
Processing of N3, a mammalian proteasome β-type subunit
Authors: THOMSON Stuart , RIVETT A. Jennifer
Published: August 2015

Proteasome subunits are encoded by members of the same gene family and can be divided into two groups based on their similarity to the alpha and beta subunits of the simpler proteasome isolated from Thermoplasma acidophilum. RN3 is the beta-type subunit, N3, of rat proteasomes which has been implicated in the peptidylglutamyl-peptide hydrolase activity of the proteinase complex. We have expressed recombinant RN3 protein in Escherichia coli in order to raise subunit-specific polyclonal antibodies. Identification of the position of RN3 on two-dimensional PAGE gels of purified rat liver proteasomes showed a single protein spot of molecular mass 24 kDa and of pI value of about 5. This protein has a free N-terminus, having undergone post-translational processing. After immunoprecipitation from [35S]methionine-labelled human embryo lung L-132 cells using anti-RN3 antibodies, two radiolabelled spots were observed on two-dimensional PAGE gels, one corresponding to the mature N3, the other of molecular mass 28.5 kDa and pI value around 5, which was probably the unprocessed form of N3. However, the latter protein had a higher molecular mass (31 kDa) than was predicted from the sequence of previously cloned cDNA. Therefore rapid amplification of cDNA ends (‘RACE’) was carried out to determine the full sequence. The lack of detectable RN3 precursor in purified rat liver proteasomes suggests that the processing probably accompanies assembly of the complex. The half-life of the processing was determined to be 31 min in growing L-132 cells. The unprocessed form of N3 was not observed after immunoprecipitation of 35S-labelled complexes with anti-proteasome antibodies. There was no evidence to suggest that unprocessed N3 is found in precursor complexes which have been implicated in the assembly of some other unprocessed beta-type subunits. Interestingly also, the site of cleavage of N3 (ITR↓TQN) differs significantly from those of other processed animal beta-type proteasome subunits [(H/T)G↓TT(T/L)], many of which resemble more closely the cleavage site of the Thermoplasma acidophilum beta subunit.
Rac GTPase interacts specifically with phosphatidylinositol 3-kinase
Authors: BOKOCH Gary M. , VLAHOS Chris J. , WANG Yan , KNAUS Ulla G. , TRAYNOR-KAPLAN Alexis E.
Published: August 2015

The Rac GTP-binding proteins are members of the Rho family and regulate growth factor-stimulated actin assembly in a variety of cells. The formation of phosphorylated inositol lipids has been implicated in control of the processes initiating and regulating such actin polymerization. Associations of Rho family GTP-binding proteins with enzymes involved in lipid metabolism have been described. Here we demonstrate a direct and specific interaction of Rac proteins with phosphatidylinositol (PI) 3-kinase. This interaction is dependent upon Rac being in a GTP-bound state and requires an intact Rac effector domain. In contrast, direct binding of RhoA to PI 3-kinase could not be detected. Rac–GTP also bound to PI 3-kinase in Swiss 3T3 fibroblast and human neutrophil lysates, and increased PI 3-kinase activity became associated with Rac–GTP in platelet-derived growth factor-stimulated cells. Interaction of Rac–GTP with PI 3-kinase in vitro stimulated the activity of the enzyme by 2–9-fold. A specific interaction of active Rac with PI 3-kinase might be important in regulation of the actin cytoskeleton.
New insights into the pathogenesis of copper toxicosis in Wilson's disease: evidence for copper incorporation and defective canalicular transport of caeruloplasmin
Authors: CHOWRIMOOTOO George F. E. , AHMED Hafez A. , SEYMOUR Carol A.
Published: August 2015

Previous studies have suggested that copper is incompletely incorporated into caeruloplasmin, the major plasma form of copper-transporting protein, in the genetic copper toxic condition, Wilson's disease. In this paper we have investigated the role of copper and caeruloplasmin in the abnormal biliary copper transport that characterizes Wilson's disease. Using SDS/PAGE and Western blotting, we have demonstrated the presence of holocaeruloplasmin in liver samples from patients with Wilson's disease (abnormal biliary copper excretion) and in control patients (normal biliary copper excretion). The presence of holocaeruloplasmin was also confirmed by measurement of caeruloplasmin oxidase activity using staining with o'Dianisidine. In contrast with the findings in liver tissue, holocaeruloplasmin was absent from bile from patients with Wilson's disease, but as expected it was present in the bile from control subjects. We have also identified and partially characterized a 189–200 kDa protein from purified human biliary canalicular membranes which binds copper and possesses caeruloplasmin-like activity when probed with a specific human anti-caeruloplasmin antibody. In conclusion, we have demonstrated that copper incorporation in caeruloplasmin is normal in patients with Wilson's disease contrary to previous reports. Secondly, we have shown that the defect in Wilson's disease appears to lie in the biliary canalicular excretion of holocaeruloplasmin resulting in its retention within the hepatocyte causing copper toxicosis. Finally we have identified and partially characterized a caeruloplasmin-binding protein from biliary canalicular membranes which may provide a link to the gene defect in Wilson's disease.
1H-NMR characterization of l-tryptophan binding to TRAP, the trp RNA-binding attenuation protein of Bacillus subtilis
Authors: RAMESH Vasudevan , BROWN Tom
Published: August 2015

A 1H-NMR study of the binding of L-tryptophan to the trp RNA-binding attenuation protein of Bacillus subtilis (TRAP), an ondecamer (91.6 kDa), has been implemented. The assignment of the aromatic indole ring proton resonances of the bound tryptophan ligand has been successfully carried out by two-dimensional chemical exchange experiments. The observation of only a single set of chemical shifts of the bound ligand demonstrates that the tryptophan binding site is identical in all the 11 subunits of the protein. Further, the large change in ligand chemical shifts suggests that the conformation of tryptophan ligand undergoes a significant rearrangement after complex formation with TRAP. This is further substantiated by the extensive ligand-induced chemical shift changes observed to the protein resonances and identification of several strong ligand–protein intermolecular nuclear Overhauser effects. A correlation of these preliminary NMR data with the X-ray crystal structure of the TRAP–tryptophan complex also suggests, tentatively, that the observed changes to the NMR spectra of the protein might correspond to changes associated with residues surrounding the tryptophan binding pocket owing to complex formation.
Biosynthesis of the MUC2 mucin: evidence for a slow assembly of fully glycosylated units
Authors: SHEEHAN John K. , THORNTON David J. , HOWARD Marj , CARLSTEDT Ingemar , CORFIELD Anthony P. , PARASKEVA Christos
Published: August 2015

The human colonic cell line PC/AA was grown to near confluency over 24 days and labelled with [14C]proline and [3H]glucose over the last 48 h in culture. The cell layer was extracted with 6 M guanidinium chloride and the mature fully glycosylated mucins were isolated at a density of 1.45 g/ml by using density-gradient centrifugation in CsCl/4 M guanidinium chloride. These mucins were identified as MUC2 with an anti-peptide antibody. The macromolecules were fragmented by reduction into two distinct populations of MUC2 subunits as assessed by agarose electrophoresis. The MUC2 mucin was polydisperse in length, ranging from 500 nm to many microns and its molecular-mass distribution, assessed by rate-zonal centrifugation, ranged from 5×106 to 40×106 Da. However, the metabolically labelled MUC2 mucins, though found throughout the whole distribution, were of much smaller average size. Since the entire distribution is not uniformly radiolabelled over 48 h, the formation of the largest species must be preceded by glycosylation and occur slowly, over several days, via the assembly of fully glycosylated units which are likely to be at least dimers [Asker, Baeckstrom, Axelsson, Carlstedt, and Hansson (1995) Biochem. J. 308, 873–880].
Collagen fibril formation
Authors: KADLER Karl E. , HOLMES David F. , TROTTER John A. , CHAPMAN John A.
Published: August 2015

Collagen is most abundant in animal tissues as very long fibrils with a characteristic axial periodic structure. The fibrils provide the major biomechanical scaffold for cell attachment and anchorage of macromolecules, allowing the shape and form of tissues to be defined and maintained. How the fibrils are formed from their monomeric precursors is the primary concern of this review. Collagen fibril formation is basically a self-assembly process (i.e. one which is to a large extent determined by the intrinsic properties of the collagen molecules themselves) but it is also sensitive to cell-mediated regulation, particularly in young or healing tissues. Recent attention has been focused on ‘early fibrils’ or ‘fibril segments’ of ~10 μm in length which appear to be intermediates in the formation of mature fibrils that can grow to be hundreds of micrometres in length. Data from several laboratories indicate that these early fibrils can be unipolar (with all molecules pointing in the same direction) or bipolar (in which the orientation of collagen molecules reverses at a single location along the fibril). The occurrence of such early fibrils has major implications for tissue morphogenesis and repair. In this article we review the current understanding of the origin of unipolar and bipolar fibrils, and how mature fibrils are assembled from early fibrils. We include preliminary evidence from invertebrates which suggests that the principles for bipolar fibril assembly were established at least 500 million years ago.
cDNA cloning and tissue-specific expression of the phosphatidylcholine transfer protein gene
Authors: B. H. GEIJTENBEEK Teunis , SMITH Alexander J. , BORST Piet , WIRTZ Karel W. A.
Published: August 2015

We have isolated a cDNA containing the complete coding sequence of bovine liver phosphatidylcholine transfer protein (PC-TP). The deduced amino acid sequence consists of 213 amino acid residues and is, except for a lysine instead of an arginine at position 167, identical to the sequence determined by Edman degradation [Akeroyd, Moonen, Westerman, Puyk and Wirtz (1981) Eur. J. Biochem. 114, 385–391]. A cDNA encoding amino acid residues 41–214 of mouse lung PC-TP was also isolated. The predicted amino acid sequence was 90% similar (81% identical) to the corresponding sequence of bovine liver PC-TP, demonstrating that PC-TP is conserved among mammalian species. By Southern blot analysis, evidence was obtained for the presence of a single bovine PC-TP-encoding gene. The expression of the PC-TP gene was determined during mouse embryonic development and in adult mouse tissues using an RNase protection assay. PC-TP RNA was present in embryos at all stages of development as early as the embryonic stem cell, suggesting a role for PC-TP in cell growth and differentiation. Towards the end of embryonic development, just before term, high levels of PC-TP RNA were found in the liver. This level was even higher 7 days post-term. In addition to adult liver, high levels of PC-TP RNA were also found in kidney and testis. The prominent presence of PC-TP in developing and adult liver is compatible with its proposed role in bile formation.
Thrombopoietin potentiates activation of human platelets in association with JAK2 and TYK2 phosphorylation
Authors: RODRÍGUEZ-LIÑARES Belén , WATSON Steve P.
Published: August 2015

Thrombopoietin (TPO), also known as the c-mpl ligand, stimulates rapid tyrosine phosphorylation of multiple proteins in human platelets including the Janus family kinases JAK2 and TYK2. On its own, TPO has no effect on platelet aggregation and dense-granule secretion but induces a general potentiation of these responses by other stimuli. The most dramatic effect is observed against threshold concentrations of agonists for aggregation. Shape change or weak reversible aggregation induced by low concentrations of thrombin, collagen and the thromboxane mimetic, U46619, are converted into irrreversible aggregation in the presence of TPO. A similar result is obtained in the presence of the ADP scavenger apyrase and cyclo-oxygenase inhibitor indomethacin. TPO also induces potentiation of dense-granule secretion measured through release of 5-hydroxy[3H]tryptamine. This effect is most striking against low concentrations of stimuli and is independent of aggregation as it is observed in the presence of chelation of extracellular Ca2+ with EGTA. TPO potentiates activation of phospholipase C and elevation of intracellular Ca2+, providing a molecular explanation for potentiation of functional responses. TPO may have an important physiological role in priming platelet activation in thrombocytopenia, an action that may help to compensate for the reduced platelet density.
Isolation and characterization of vascular smooth muscle inositol 1,4,5-trisphosphate receptor
Authors: ISLAM Md. Omedul , YOSHIDA Yutaka , KOGA Takaki , KOJIMA Masayasu , KANGAWA Kenji , IMAI Shoichi
Published: August 2015

myo-Inositol 1,4,5-trisphosphate (InsP3) receptor of porcine aorta was purified to near homogeneity and its biochemical properties were compared with those of cerebellar InsP3 receptor of the same animal species. The aortic InsP3 receptor consisted of equal amounts of two polypeptides with slightly differing molecular masses of around 240 kDa and was found to possess a single population of InsP3-binding site (Kd of 1.2 nM). The InsP3 receptor purified from porcine cerebellum was also comprised of two polypeptides. However, the molecular mass was slightly but definitely larger, being 250 kDa, and the amounts of the two polypeptides were not equal. The aortic InsP3 receptor cross-reacted with polyclonal antibody specific to type 1 InsP3 receptor as did the cerebellar InsP3 receptor. The aortic InsP3 receptor bound to calmodulin–Sepharose in a Ca2+-dependent manner, while the cerebellar InsP3 receptor did not. Reverse transcriptase-PCR analysis revealed two splicing variants of the type 1 InsP3 receptor in porcine aortic smooth muscle distinct from those of the type 1 InsP3 receptor of porcine cerebellum. The possible relevance of this difference to difference in calmodulin-binding property was discussed.
Casein kinase 2 inhibits the renaturation of complementary DNA strands mediated by p53 protein
Authors: FILHOL Odile , BAUDIER Jacques , CHAMBAZ Edmond M. , COCHET Claude
Published: August 2015

Considerable effort is currently being devoted to understand the functions of protein p53, a major regulator of cell proliferation. The protein p53 has been reported to catalyse the annealing of complementary DNA or RNA strands. We report that this activity is inhibited in the presence of the serine/threonine protein kinase CK2. It is shown that this inhibition can be explained by the occurrence of a high-affinity molecular association between p53 and CK2. The molecular complex involves an interaction between the C-terminal domain of p53 and the β subunit of the oligomeric kinase. Accordingly, the isolated α subunit of the kinase was without effect. In addition, after phosphorylation by CK2, phosphorylated p53 lost its DNA annealing activity. Because the C-terminal domain of p53 is both involved in the association with CK2 and phosphorylated by it, our results suggest that either protein–protein interaction or phosphorylation of this domain might control the base pairing of complementary sequences promoted by p53 in processes related to DNA replication and repair.
Binding of a protein to an AU-rich domain of tumour necrosis factor α mRNA as a 35 kDa complex and its regulation in primary rat astrocytes
Authors: KIM Youn-uck , RUS Horea G. , FISHER Stephen N. , PITHA Paula M. , SHIN Moon L.
Published: August 2015

Newcastle disease virus (NDV) induces tumour necrosis factor α (TNFα) gene transcription and increases the mRNA stability. NDV stabilizes TNFα mRNA by preventing poly(A) shortening in a protein kinase C-dependent manner. TNFα3´-untranslated region (UTR) contains an AU-rich domain (ARD) with seven AUUUA pentamers, a motif implicated in poly(A) removal and mRNA degradation. In this report, protein binding to TNFα ARD and the effects of NDV and kinases on ARD-binding activity were investigated in primary rat astrocytes. Both nuclear and cytoplasmic extracts contained proteins binding to centrally located 27 nt AUUUAUUAUUUAUUUAUUAUUUAUUUA, within TNFαARD. Portions of ARD with a single AUUUA did not show ARD-binding activity. The ARD–protein complexes migrated as two bands on electrophoretic mobility-shift assay. The slower moving complexes appeared either as a broader band or doublets. The UV cross-linked ARD–protein complexes, however, migrated as a single 35 kDa band on SDS/PAGE. In cytoplasmic extracts treated with alkaline phosphatase there was a decrease in the faster moving complex and an increase in the slower moving complex, whereas NDV infection produced the reverse effect. In addition, the faster moving complex was decreased when cytoplasmic extracts from NDV-infected cells were treated with protein phosphatase 1 or 2A. Neither NDV infection nor phosphatase treatment affected the mobility pattern of nuclear extracts. The data indicate that a protein of molecular mass less than 35 kDa binds to a segment of TNFαARD containing primarily UUAUUUAUU motifs, and the ARD-binding activity in cytoplasmic compartment is post-transcriptionally modified.
Cyclic GMP regulates activation of phosphoinositidase C by bradykinin in sensory neurons
Authors: HARVEY Justine S. , BURGESS Gillian M.
Published: August 2015

Prior exposure of cultured neonatal rat dorsal root ganglion (DRG) neurons to bradykinin resulted in marked attenuation of bradykinin-induced activation of phosphoinositidase C (PIC). The (logconcentration)–response curve for bradykinin-induced [3H]inositol trisphosphate ([3H]IP3) formation was shifted to the right and the maximum response was reduced. Bradykinin increases cyclic GMP (cGMP) in DRG neurons [Burgess, Mullaney, McNeill, Coote, Minhas and Wood (1989) J. Neurochem. 53, 1212–1218] and treatment of the neurons with dibutyryl cGMP (dbcGMP) had a similar, inhibitory, effect on bradykinin-induced [3H]IP3 formation. NG-Nitro-L-arginine (LNNA) blocked bradykinin-induced formation of cGMP. It prevented the functional uncoupling induced by pretreatment with bradykinin, but not the inhibitory effect of dbcGMP on [3H]IP3 formation. The ability of LNNA to prevent desensitization was reversed by excess L-arginine, indicating that its actions were mediated through inhibition of nitric oxide synthase. In addition to functional desensitization, exposure to bradykinin reduced the number of cell-surface receptors detected with [3H]bradykinin, without affecting its KD value for the remaining sites. In contrast to bradykinin, pretreatment with dbcGMP had no effect on either the KD or Bmax for [3H]bradykinin binding. This implies that the inhibitory effect of dbcGMP was downstream from the binding of bradykinin to its receptor and upstream of IP3 formation. The lack of effect of dbcGMP on [3H]bradykinin binding suggests that the decrease in receptor number induced by bradykinin was mediated by a different mechanism and was not a key factor in the rapid phase of desensitization in these cells.
Molecular cloning, expression and regulatory activity of Gα11- and βγ-subunit-stimulated phospholipase C-β from avian erythrocytes
Authors: WALDO Gary L. , PATERSON Andrew , BOYER José L. , NICHOLAS Robert A. , HARDEN T. Kendall
Published: August 2015

A turkey erythrocyte phospholipase C (PLC) has been instrumental in delineating the role of G-proteins in receptor-regulated inositol lipid signalling. This isoenzyme is uniquely regulated both by α-subunits of the Gq family and by G-protein βγ-subunits. A 4819 bp cDNA encoding this PLC has been cloned from a turkey erythrocyte cDNA library. The open reading frame of this cDNA encodes a 1211-amino-acid protein (calculated molecular mass 139050 Da) that contains amino acid sequences of 16 peptides sequenced from the turkey erythrocyte PLC. The predicted sequence of the turkey PLC shows considerable similarity with the sequences of previously cloned members of the PLC-β family, with the highest identity (71%) shared with PLC-β2 and lesser identities observed with PLC-β1 (49%), PLC-β3 (46%) and PLC-β4 (37%). The largest differences in sequence between the turkey PLC-β and other PLC-β isoenzymes occur in the C-terminal domain and in the region between the X- and Y-domains. The turkey isoenzyme and PLC-β2, which differ in their regulation by G-protein α-subunits, are only 44% similar across the approx. 400 amino acid residues of the C-terminal domain that has been implicated in αq activation of these proteins. Recombinant turkey PLC-β was purified to homogeneity following expression from a recombinant baculovirus in Sf9 insect cells. The immunoreactivity and mobility on SDS/PAGE of the recombinant enzyme were the same as observed with native turkey erythrocyte PLC-β. Moreover, the catalytic activities of the recombinant enzyme were indistinguishable from those of native turkey erythrocyte PLC-β in assays carried out in the presence of cholate and Ca2+, or in assays of activity after reconstitution with Gα11 or G-protein βγ-subunits. The turkey PLC-β was more sensitive to activation by Gα11 than was PLC-β2, and was more sensitive to activation by βγ-subunits than either PLC-β2 or PLC-β1.
Phospholipase C in mouse oocytes: characterization of β and γ isoforms and their possible involvement in sperm-induced Ca2+ spiking
Authors: DUPONT Genevieve , McGUINNESS Orla M. , JOHNSON Martin H. , BERRIDGE Michael J. , BORGESE Franck
Published: August 2015

This study involved an investigation of the role of phospholipase C (PLC) in generating repetitive Ca2+ spikes at fertilization. Using a PCR-based strategy we have demonstrated that mouse oocytes have mRNA coding for PLCβ1, PLCβ3 and PLCγ isoenzymes. Furthermore, immunodetection of PLCγ1 using monoclonal antibodies reveals that PLCγ1 protein is present in mature mouse oocytes, ruling out the possibility that mRNA was being transcribed but not expressed. We were unsuccessful at detecting the presence of PLCβ protein, but the presence of this isoform can be inferred from functional studies. The PLC inhibitor, U73122, exerted an inhibitory effect on oocytes activated by spermatozoa or acetylcholine at concentrations of 10 and 30 μM respectively, while its inactive analogue had no effect. The soluble tyrosine kinase inhibitors, genistein (100 μM), herbimycin (10 μM) and geldanamycin (0.6 μM) which could affect signalling through PLCγ hindered but never completely inhibited Ca2+ spiking in response to fertilization. We conclude that the activation of PLC to generate InsP3 may play a critical role in fertilization.
Cloning, sequencing and expression of rat liver pyruvate carboxylase
Authors: JITRAPAKDEE Sarawut , BOOKER Grant W. , CASSADY A. Ian , WALLACE John C.
Published: August 2015

Overlapping clones encoding rat liver pyruvate carboxylase (PC) have been isolated by screening a liver cDNA library and by performing rapid amplification of cDNA ends polymerase chain reaction on total liver RNA. The sequence of rat PC cDNA contains an open reading frame of 3537 nucleotides encoding a polypeptide of 1178 amino acids with a calculated Mr of 129848. This is flanked by a 5′ untranslated region of 66 bp and a 3′ untranslated region of 421 bp including the poly(A) tail. The inferred protein sequence is 96.6% identical with mouse and 96.3% identical with human PCs, 68.4% identical with mosquito PC and 53.5% identical with yeast PC isoenzymes PC1 and PC2. On the basis of partial proteolysis and sequence homology with PC from other organisms (yeast, mosquito, mouse and human) and with other biotin enzymes, three functional domains, namely the biotin carboxylation domain, the transcarboxylation domain and the biotinyl domain, have been identified. Comparison with the known structure of the biotin carboxylase subunit of Escherichia coli acetyl-CoA carboxylase [Waldrop, Rayment and Holden (1994) Biochemistry 33, 10249–10256] highlights the functional importance of 11 highly conserved residues. Northern analysis revealed that PC mRNA is highly expressed in rat liver, kidney, adipose tissue and brain, moderately expressed in heart, adrenal gland and lactating mammary gland, and expressed at a low level in spleen and skeletal muscle.
SDS-resistant aggregation of membrane proteins: application to the purification of the vesicular monoamine transporter
Authors: SAGNÉ Corinne , ISAMBERT Marie-Françoise , HENRY Jean-Pierre , GASNIER Bruno
Published: August 2015

The vesicular monoamine transporter, which catalyses a H+/monoamine antiport in monoaminergic vesicle membrane, is a very hydrophobic intrinsic membrane protein. After solubilization, this protein was found to have a high tendency to aggregate, as shown by SDS/PAGE, especially when samples were boiled in the classical Laemmli buffer before electrophoresis. This behaviour was analysed in some detail. The aggregation was promoted by high temperatures, organic solvents and acidic pH, suggesting that it resulted from the unfolding of structure remaining in SDS. The aggregates were very stable and could be dissociated only by suspension in anhydrous trifluoroacetic acid. This SDS-resistant aggregation behaviour was shared by very few intrinsic proteins of the chromaffin granule membrane. Consequently, a purification procedure was based on this property. A detergent extract of chromaffin granule membranes enriched in monoamine transporter was heated and the aggregates were isolated by size-exclusion HPLC in SDS. The aggregates, containing the transporter, were dissociated in the presence of trifluoroacetic acid and analysed on the same HPLC column. This strategy might be of general interest for the purification of membrane proteins that exhibit SDS-resistant aggregation.
Biosynthesis of anandamide and related acylethanolamides in mouse J774 macrophages and N18 neuroblastoma cells
Authors: Di MARZO Vincenzo , De PETROCELLIS Luciano , SEPE Nunzio , BUONO Anna
Published: August 2015

Anandamide (arachidonoylethanolamide, AnNH) has been recently proposed as the endogenous ligand at the brain cannabinoid receptor CB1. Two alternative pathways have been suggested for the biosynthesis of this putative mediator in the central nervous system. Here we present data (1) substantiating further the mechanism by which AnNH is produced by phospholipase D (PLD)-catalysed hydrolysis of N-arachidonoylphosphatidylethanolamine in mouse neuroblastoma N18TG2 cells, and (2) suggesting for the first time that AnNH is biosynthesized via the same mechanism in a non-neuronal cell line, mouse J774 macrophages, together with other acylethanolamides and is possibly involved in the control of the immune/inflammatory response. Lipids from both neuroblastoma cells and J774 macrophages were shown to contain a family of N-acylphosphatidylethanolamines (N-aPEs), including the possible precursor of AnNH, N-arachidonoyl-PE. Treatment with exogenous PLD, but not with exogenous phospholipase A2 and ethanolamine, resulted in the production of a series of acylethanolamides (AEs), including AnNH, from both cell types. The formation of AEs was accompanied by a decrease in the levels of the corresponding N–aPEs. Enzymically active homogenates from either neuroblastoma cells or J774 macrophages were shown to convert synthetic N-[3H]arachidonoyl-PE into [3H]AnNH, thus suggesting that in both cells an enzyme is present which is capable of catalysing the hydrolysis of N–aPE(s) to the corresponding AE(s). Finally, as previously shown in central neurons, on stimulation with ionomycin, J774 macrophages also produced a mixture of AEs including AnNH and palmitoylethanolamide, which has been proposed as the preferential endogenous ligand at the peripheral cannabinoid receptor CB2 and, consequently, as a possible down-modulator of mast cells. On the basis of this as well as previous findings it is now possible to hypothesize for AnNH and palmitoylethanolamide, co-synthesized by macrophages, a role as peripheral mediators with multiple actions on blood cell function.
Chemical cross-linking of pleckstrin in human platelets: evidence for oligomerization of the protein and its dissociation by protein kinase C
Authors: McDERMOTT Alison M. , HASLAM Richard J.
Published: August 2015

The major substrate of protein kinase C (PKC) in platelets is the 40 kDa protein, pleckstrin. Addition of the homobifunctional reagent, bis(sulphosuccinimidyl)suberate (BS3), to platelet lysate, cytosol fraction or to electropermeabilized platelets resulted in cross-linking of pleckstrin to give higher-molecular-mass complexes of 68 kDa, 90 kDa and 100–120 kDa respectively, which were visualized by immunoblotting with an anti-pleckstrin antibody. Higher levels of cross-linking were observed in permeabilized platelets than in platelet lysates. The yields of the cross-linked complexes were much reduced after dilution of platelet lysate or lysis of electropermeabilized platelets and, in the case of the 90 kDa and 100–120 kDa species, after activation of PKC by phorbol 12-myristate 13-acetate. Similar experiments with purified pleckstrin indicated that the 90 kDa and 100–120 kDa species consist, at least in part, of pleckstrin dimers and higher oligomers. After incubation of purified pleckstrin (0.45 mg/ml) for 1 h with 2 mM BS3, about 25% of the protein was present in cross-linked species. The results indicate that pleckstrin undergoes a reversible self-association that can be prevented by phosphorylation of the protein, and also interacts with an unidentified platelet protein of about 28 kDa.
Evidence that filaggrin is a component of cornified cell envelopes in human plantar epidermis
Authors: SIMON Michel , HAFTEK Marek , SEBBAG Mireille , MONTÉZIN Martine , GIRBAL-NEUHAUSER Elisabeth , SCHMITT Daniel , SERRE Guy
Published: August 2015

Cornified cell envelope (CE) is generated during the late stages of epidermal differentiation and is made up of proteins covalently linked together by transglutaminases. To determine whether filaggrin is a component of this structure in humans, we analysed highly purified CE from plantar stratum corneum. An immunoelectron microscopy analysis showed specific binding of four different anti-(pro)filaggrin monoclonal antibodies to the surface of the CE, proved previously to be free of non-covalently linked proteins. Moreover, the anti-filaggrin activity of one of the antibodies was absorbed by preincubation with the plantar CE, as determined by ELISA. Convincingly, fragments of CE produced by proteolytic digestion of the structures were stained by this antibody on immunoblots. These data provide direct evidence that filaggrin is a component of CE purified from human plantar stratum corneum. Cross-linking between CE and the filaggrin-containing fibrous matrix may enhance the structural cohesion of the corneocytes and thus the resistance of the stratum corneum.
Cloning and biochemical characterization of the cyclophilin homologues from the free-living nematode Caenorhabditis elegans
Authors: PAGE Antony P. , MacNIVEN Kenneth , HENGARTNER Michael O.
Published: August 2015

Cyclosporin A (CsA) is the most widely used immunosuppressive agent, whose properties are exerted via an interaction with cyclophilin, resulting in down-regulation of signal-transduction events in the T-cell. Cyclophilin is identical with peptidylprolyl cis–trans isomerase (PPI; EC 5.2.1.8), an enzyme which catalyses the isomerization between the two proline conformations in proteins, thereby acting as a catalyst in protein-folding events. Several reports indicate that CsA has potent anti-parasitic activity, effective against both protozoan and helminth species. In order to understand the various biological roles that cyclophilins play we have initiated a study of these proteins in the genetically tractable nematode Caenorhabditis elegans. Here we describe the cloning and characterization of 11 cyclophilin genes (cyp-1 to -11) derived from this nematode; this is currently the greatest number of isoforms described in a single species. Southern blotting and physical mapping indicated that these genes are dispersed throughout the nematode genome. A high degree of conservation exists between several isoforms, which also share characteristics with the ubiquitous isoforms previously described. The remaining isoforms are divergent, having altered CsA-binding domains and additional non-cyclophilin domains, which may impart compartmental specificity. Ten of these isoforms have been expressed in Escherichia coli, and the resultant fusion proteins have been examined biochemically for PPI activity, which they all possess. Isomerase activity is highest in the conserved and lowest in divergent isoforms, perhaps indicating a more specific substrate for the latter. Analysis of the C. elegans cyp genes will provide answers as to the roles played by cyclophilins in protein folding and signal transduction.
Proteoglycan synthesis in human and murine haematopoietic progenitor cell lines: isolation and characterization of a heparan sulphate proteoglycan as a major proteoglycan from the human haematopoietic cell line TF-1
Authors: STÖCKER Georg , DRZENIEK Zofia , JUST Ursula , OSTERTAG Wolfram , SIEBERTZ Barbara , GREILING Helmut , HAUBECK Hans-Dieter
Published: August 2015

Proteoglycans of bone-marrow stromal cells and their extracellular matrix are important components of the microenvironment of haematopoietic tissues. Proteoglycans might also be involved in the interaction of haematopoietic stem and stromal cells. Recently, several studies have been reported on the proteoglycan synthesis of stromal cells, but little is known about the proteoglycan synthesis of haematopoietic stem or progenitor cells. Here we report on the isolation and characterization of proteoglycans from two haematopoietic progenitor cell lines, the murine FDCP-Mix A4 and the human TF-1 cell line. Proteoglycans were isolated from metabolically labelled cells and purified by several chromatographic steps, including anion-exchange and size-exclusion chromatography. Biochemical characterization was performed by electrophoresis or gel-filtration chromatography before and after digestion with glycosaminoglycan-specific enzymes or HNO2 treatment. Whereas FDCP-Mix A4 cells synthesize a homogeneous chondroitin 4-sulphate proteoglycan, isolation and characterization of proteoglycans from the human cell line TF-1 revealed, that TF-1 cells synthesize, in addition to a chondroitin sulphate proteoglycan, a heparan sulphate proteoglycan as major proteoglycan. For this heparan sulphate proteoglycan a core protein size of approx. 59 kDa was determined. Immunochemical analysis of this heparan sulphate proteoglycan revealed that it is not related to the syndecan family nor to glypican.
Affinity purification of 5-methylthioribose kinase and 5-methylthioadenosine/S-adenosylhomocysteine nucleosidase from Klebsiella pneumoniae
Authors: CORNELL Kenneth A. , WINTER R. W. , TOWER Paula A. , RISCOE Michael K.
Published: August 2015

Two enzymes in the methionine salvage pathway, 5-methylthioribose kinase (MTR kinase) and 5´-methylthioadenosine/S-adenosylhomocysteine nucleosidase (MTA/SAH nucleosidase) were purified from Klebsiellapneumoniae. Chromatography using a novel 5´-(p-aminophenyl)thioadenosine/5-(p-aminophenyl)thioribose affinity matrix allowed the binding and selective elution of each of the enzymes in pure form. The molecular mass, substrate kinetics and N-terminal amino acid sequences were characterized for each of the enzymes. Purified MTR kinase exhibits an apparent molecular mass of 46–50 kDa by SDS/PAGE and S200HR chromatography, and has a Km for MTR of 12.2 μM. Homogeneous MTA/SAH nucleosidase displays a molecular mass of 26.5 kDa by SDS/PAGE, and a Km for MTA of 8.7 μM. Comparisons of the N-terminal sequences obtained for each of the enzymes with protein-sequence databases failed to reveal any significant sequence similarities to known proteins. However, the amino acid sequence obtained for the nucleosidase did share a high degree of sequence similarity with the putative translation product of an open reading frame in Escherichia coli, thus providing a tentative identification of this gene as encoding an MTA/SAH nucleosidase.
Functional characterization of a recombinant sodium-dependent nucleoside transporter with selectivity for pyrimidine nucleosides (cNT1rat) by transient expression in cultured mammalian cells
Authors: FANG Xiao , PARKINSON Fiona E. , MOWLES Delores A. , YOUNG James D. , CASS Carol E.
Published: August 2015

We have demonstrated that monkey kidney (COS-1) cells have a single type of nucleoside transport process, which, because it was equilibrative, sodium-independent and could be inhibited by nitrobenzylthioinosine (NBMPR), was identified as the ‘equilibrative sensitive’ or ‘es’ transporter. Using NBMPR or dilazep to inhibit the endogenous nucleoside transport activity, we have transiently expressed a cDNA that encodes an inhibitor-insensitive, concentrative nucleoside transporter protein (cNT1rat) of rat intestine in COS-1 cells. The production of recombinant cNT1rat was examined by immunoblotting using an epitope-tagged construct and by analysis of inward fluxes of 3H-labelled nucleosides. Recombinant cNT1rat was sodium-dependent and selective for pyrimidine nucleosides, with approximate Km values of 21 μM, 12.5 μM and 15 μM for uridine, thymidine and adenosine, respectively. Although adenosine exhibited high affinity for the recombinant transporter, its Vmax value was low. A variety of anti-viral and anti-cancer nucleoside drugs inhibited cNT1rat-mediated uptake of uridine by transfected COS-1 cells although to different extents (Floxidine > Idoxuridine > Zidovudine > Zalcitabine > Cytarabine > Gemcitabine), suggesting that the concentrative pyrimidine-selective nucleoside transporters, of which cNT1rat is a representative, may play a role in cellular uptake of these drugs. The cNT1rat/COS-1 expression system is a useful tool for analysis of cNT1rat-mediated transport processes.