bluuebunny/crossref_metadata_2025 huggingface dataset:
Development of thyroxine type II deiodinase activity in brains of Zucker rats
Authors: Marie V , Dupuy F , Bazin R
Published: August 2015

The present study was undertaken to determine whether the capacity for 3,5,3′,5′-tetraiodothyronine (T4) conversion into 3,5,3′-tri-iodothyronine (T3), as measured by the activity of thyroxine type II 5′-monodeiodinase (T4-5′D), was altered in the brains of young Zucker fa/fa rats during the period of intense maturation of the central nervous system (i.e. from 10 to 20 days of life). From 7 to 14 days of age, no difference in brain T4-5′D activity could be detected between lean and pre-obese rats; serum free T4 was not affected by the fa gene. During the suckling to weaning transition, T4-5′D reached a plateau in brains of lean rats, while it increased by 50% in brains of pre-obese rats; concurrently, serum free T4 increased in Fa/fa rats, whereas it did not change in fa/fa rats. The increased capacity for conversion of T4 into T3 observed in brains of pre-obese compared with lean rats could not be ascribed to a variation in the amount of T4-5′D, since Vmax. did not differ between the two genotypes; however, it could be totally accounted for by an increased affinity of the enzyme for T4. This change may represent an adaptive response to low serum free T4 in order to maintain the cerebral T3 concentration in pre-obese rats. These results show that the alteration in T4 metabolism in brains of fa/fa rats is not an early event and thus cannot interfere with maturation of the central nervous system. However, the decreased serum free T4 which was observed in pre-obese rats after suckling might play a secondary role in development of this genetic obesity.
Guanosine 5′-[γ-thio]triphosphate induces membrane localization of cytosol-independent phospholipase D activity in a cell-free system from U937 promonocytic leucocytes
Authors: Kusner D J , Dubyak G R
Published: August 2015

Activation of phospholipase D (PLD) in phagocytic leucocytes requires protein components present in both the plasma membrane and the cytosol, but the catalytic and regulatory factors are not fully defined. We have characterized the effect of guanosine 5′-[gamma-thio]triphosphate (GTP[S]) on the subcellular requirements for reconstitution of PLD activity, using a cell-free system from U937 human promonocytic leucocytes. Incubation of permeabilized cells with 100 microM GTP[S] resulted in a membrane-localized PLD activity which was independent of added cytosol. The PLD activity of membranes from GTP[S]-treated cells was 7-fold greater than the basal activity of control membranes, and could be further augmented by the addition of ATP. This was the first demonstration of a stable agonist-regulated PLD activity in membranes from phagocytic leucocytes which was quantitatively comparable with that seen in a fully reconstituted system. Cytosol from GTP[S]-treated cells had a decreased capacity to support PLD activation, consistent with GTP[S]-induced depletion of a factor essential for reconstitution of PLD activity. Incubation of isolated membrane and cytosol with GTP[S] also resulted in a cytosol-independent PLD activity in the re-isolated membranes. The effect of GTP[S] could be mimicked by guanosine 5′-[beta gamma-imido]triphosphate, but not by aluminium fluoride, consistent with the involvement of a low-molecular-mass GTP-binding protein(s). Incubation of isolated subcellular fractions with GTP[S], followed by removal of unbound nucleotide, suggested that at least one of the GTP-binding proteins involved in the membrane localization of PLD activity was itself present in the membrane fraction. These data were consistent with a model in which activation of GTP-binding protein(s) resulted in the stable assembly of an active PLD signalling complex at the membrane surface.
l-carnosine (β-alanyl-l-histidine) and carcinine (β-alanylhistamine) act as natural antioxidants with hydroxyl-radical-scavenging and lipid-peroxidase activities
Authors: Babizhayev M A , Seguin M C , Gueyne J , Evstigneeva R P , Ageyeva E A , Zheltukhina G A
Published: August 2015

Carnosine (beta-alanyl-L-histidine) and carcinine (beta-alanylhistamine) are natural imidazole-containing compounds found in the non-protein fraction of mammalian tissues. Carcinine was synthesized by an original procedure and characterized. Both carnosine and carcinine (10-25 mM) are capable of inhibiting the catalysis of linoleic acid and phosphatidylcholine liposomal peroxidation (LPO) by the O2(-.)-dependent iron-ascorbate and lipid-peroxyl-radical-generating linoleic acid 13-monohydroperoxide (LOOH)-activated haemoglobin systems, as measured by thiobarbituric-acid-reactive substance. Carcinine and carnosine are good scavengers of OH. radicals, as detected by iron-dependent radical damage to the sugar deoxyribose. This suggests that carnosine and carcinine are able to scavenge free radicals or donate hydrogen ions. The iodometric, conjugated diene and t.l.c. assessments of lipid hydroperoxides (13-monohydroperoxide linoleic acid and phosphatidylcholine hydroperoxide) showed their efficient reduction and deactivation by carnosine and carcinine (10-25 mM) in the liberated and bound-to-artificial-bilayer states. This suggests that the peroxidase activity exceeded that susceptible to direct reduction with glutathione peroxidase. Imidazole, solutions of beta-alanine, or their mixtures with peptide moieties did not show antioxidant potential. Free L-histidine and especially histamine stimulated iron (II) salt-dependent LPO. Due to the combination of weak metal chelating (abolished by EDTA), OH. and lipid peroxyl radicals scavenging, reducing activities to liberated fatty acid and phospholipid hydroperoxides, carnosine and carcinine appear to be physiological antioxidants able to efficiently protect the lipid phase of biological membranes and aqueous environments.
Inactive membrane protein kinase Cs: a possible target for receptor signalling
Authors: Chakravarthy B R , Whitfield J F , Durkin J P
Published: August 2015

The activation of the multifunctional cell signalling enzymes, the protein kinase Cs (PKCs), is generally thought to result from the translocation of inactive cytosolic enzymes to activation sites in cell membranes. However, recent studies suggest that PKCs may also be stimulated in cells by processes independent of translocation. One possible mechanism is the modulation of the activity of PKCs already resident in membranes. A PKC assay that measures enzyme activity directly in isolated native membranes has revealed the presence of an activatable pool of PKCs resident in native membranes of various cells and tissues. In 3T3-L1 cells, some or all of this pool of membrane PKCs was stimulated within 10 min of exposing the cells to 10 ng/ml epidermal growth factor or 100 ng/ml fibroblast growth factor. Similar increases in PKC activity were observed in native membranes isolated from CTLL-2, WEHI-231 and S49 lymphoma cells that had been exposed to interleukin-2. These growth factors all stimulated membrane PKC activity without detectably translocating cytosolic enzymes to the membranes. In intact WEHI cells, low concentrations (5-10 microM) of a diacylglycerol, 1-oleoyl-2-acetyl-sn-glycerol (OAG), or low concentrations (2-10 nM) of phorbol 12-myristate 13-acetate sufficed to activate PKCs already resident in membranes, but much higher concentrations (50-100 microM and 50-100 nM respectively) were needed to detectably stimulate the translocation of cytosolic PKCs. A phosphatidylcholine-specific phospholipase C also selectively stimulated membrane PKCs in WEHI cells at concentrations that were much less than those needed to induce the translocation of cytosolic enzymes. Furthermore, interleukin-2 and low concentrations of OAG both stimulated the phosphorylation of the 85 kDa PKC-selective substrate protein in intact WEHI cells in which translocation of PKCs was not evident. These results suggest that the membranes of some cells maintain a pool of activatable PKCs that respond to lower levels of extracellular stimuli than cytosolic PKCs, and that can be stimulated by signals which produce diacylglycerols through the hydrolysis of phospholipids other than polyphosphoinositides.
Purification and characterization of the short-chain alkylsulphatase of coryneform B1a
Authors: Matts P J , White G F , Payne W J
Published: August 2015

Using a combination of streptomycin sulphate precipitation, and DEAE-cellulose and butyl-agarose chromatography, an alkylsulphatase active towards short-chain alkyl sulphates has been purified approx. 70-fold from extracts of coryneform B1a grown on butyl-1-sulphate. The enzyme protein is dimeric with a subunit molecular mass of 77.6 kDa, has an isoelectric point of pI 7.2, and converts butyl-1-sulphate stoichiometrically into butan-1-ol and inorganic sulphate. Stoichiometric incorporation of 18O from H2(18)O into sulphate during the reaction showed that enzymic hydrolysis occurred at the O-S bond of the C-O-S ester linkage. The enzyme was active on C3-C7 linear primary alkyl sulphates but not on higher (C8,9) or lower (C1,2) homologues, although the latter pair were competitive inhibitors. The specificity constant (kcat./Km) was highest for pentyl sulphate (Km 1.89 +/- 0.38 mM; kcat. 6.86 +/- 0.52 s-1) and decreased for higher and lower homologues. No activity was detected towards C3-C9 racemic alkyl-2-sulphates, D- or L-enantiomers of butyl-2-sulphate, the symmetrical secondary alkyl sulphates pentyl-3-sulphate, heptyl-4-sulphate, nonyl-5-sulphate, C1-C8 alkane sulphonates, choline sulphate, or butyric acid-4-sulphate; none of these compounds (except the symmetrical esters and butyric acid-4-sulphate, which were not tested) was demonstrably inhibitory. The enzyme was compared with other alkylsulphatases in terms of substrate specificity and mode of action.
Receptor occupancy regulates Ca2+ entry and intracellular Ca2+ redistribution in activated human platelets
Authors: Brüne B , von Appen F , Ullrich V
Published: August 2015

Fura-2-loaded human platelets were used to study Ca2+ release from intracellular compartments, as well as Ca2+ influx from the extracellular space. We investigated the response towards the endoperoxide/thromboxane-receptor agonist. U46619, and the inhibitor of the endoplasmic-reticulum Ca(2+)-ATPase, thapsigargin. U46619 dose-dependently depleted intracellular Ca2+ stores, followed by active sequestration of released Ca2+. Ca2+ influx induced by U46619 largely relies on receptor occupancy. Removing the thromboxane analogue from its receptor by using the endoperoxide/thromboxane-receptor antagonist BM 13177 largely blunted U46619-mediated Ca2+ influx. The Ca(2+)-ATPase inhibitor thapsigargin evoked a gradual rise in intracellular Ca2+, which was potentiated by a preceding activation of platelets with the receptor agonist U46619. This agonist-sensitizing effect also depends on receptor occupancy. Removing U46619 from its receptor by addition of the endoperoxide/thromboxane-receptor antagonist BM13177 suppressed the sensitizing effect completely. Furthermore, interrupting downstream receptor signalling events by raising intracellular levels of cyclic nucleotides (cyclic AMP, cyclic GMP) again suppressed the U46619-sensitizing effect on thapsigargin-induced Ca2+ release. This study indicates that the process of Ca2+ release followed by resequestration in response to a platelet agonist by its own is not sufficient to produce the sensitizing effect. Rather, a continuously occupied receptor triggering sustained downstream signalling events seems to be required for sensitization. The presence of a receptor agonist may induce an increased cycling of Ca2+ between the agonist-responsive and the thapsigargin-dischargeable compartment, leading to faster and more intense accumulation of Ca2+ in the cytosolic compartment after inhibition of the Ca(2+) ATPase. Suggestively, receptor occupancy increases the Ca(2+)-releasing potency of thapsigargin by coupling the thapsigargin-sensitive Ca(2+)-storing compartments with an agonist-responsive compartment that exhibits a high leakage rate in stimulated platelets.
Autocrine regulation of milk secretion by a protein in milk
Authors: Wilde C J , Addey C V P , Boddy L M , Peaker M
Published: August 2015

Frequency or completeness of milk removal from the lactating mammary gland regulates the rate of milk secretion by a mechanism which is local, chemical and inhibitory in nature. Screening of goat's milk proteins in rabbit mammary explant cultures identified a single whey protein of M(r) 7600 able to inhibit synthesis of milk constituents. The active whey protein, which we term FIL (Feedback inhibitor of Lactation), also decreased milk secretion temporarily when introduced into a mammary gland of lactating goats. FIL was synthesized by primary cultures of goat mammary epithelial cells, and was secreted vectorially together with other milk proteins. N-terminal amino acid sequencing indicated that it is a hitherto unknown protein. The evidence indicates that local regulation of milk secretion by milk removal is through autocrine feedback inhibition by this milk protein.
Physical and biological characterization of a growth-inhibitory activity purified from the neuroepithelioma cell line A673
Authors: Stam K , Stewart A A , Qu G Y , Iwata K K , Fenyö D , Chait B T , Marshak D R , Haley J D
Published: August 2015

Epithelial- and haematopoietic-cell growth-inhibitory activities have been identified in the conditioned medium of the human peripheral neuroepithelioma cell line A673. An A673-cell-derived growth-inhibitory activity was previously fractionated into two distinct components which inhibited the proliferation of human carcinoma and leukaemia cells in culture. One inhibitory activity was shown to comprise interleukin-1 alpha (IL-1 alpha). Here, we have purified to homogeneity a distinct activity which inhibited the growth of the epithelial cells in vitro. Using a combination of protein-sequence analysis and mass spectrometry, we demonstrated that biological activity can be assigned to a dimeric protein with a molecular mass of 25,576 (+/- 4) Da and an N-terminal sequence identical with that of transforming growth factor-beta 1 (TGF-beta 1). Further characterization of the growth inhibitor with TGF-beta-isoform-specific antibodies showed that > 90% of the bioactivity consists of TGF-beta 1 and not TGF-beta 2 or TGF-beta 3. Although A673 cells were growth-inhibited by exogenous TGF-beta 1, we showed that TGF-beta 1 in A673-cell-conditioned media was present in the latent, biologically inactive, form which did not act as an autocrine growth modulator of A673 cells in vitro.
Time-dependent inhibition of peptidylprolyl cis-trans-isomerases by FK506 is probably due to cis-trans isomerization of the inhibitor's imide bond
Authors: Zarnt T , Lang K , Burtscher H , Fischer G
Published: August 2015

Free in solution, the immunosuppressive compounds cyclosporin A (CsA), FK506, ascomycin and rapamycin are present in many solvents in various slowly interconverting conformations. Together with their cellular receptor proteins, cyclophilin (CyP) and FK506-binding protein (FKBP), however, these inhibitors have been shown to have a homogeneous conformation. The existence of a slow cis-trans interconversion of an imidic bond in the inhibitor molecule during the course of the formation of the CsA-CyP18cy complex (where CyP18cy is human 18 kDa cytosolic CyP) prompted us to investigate the reaction of the peptidomacrolides FK506, ascomycin and rapamycin with two specific binding-proteins in more detail. Since formation of the FK506-FKBP complex results in the inhibition of the peptidylprolyl cis-trans-isomerase activity of the binding protein, we used the enzyme's decrease in enzymic activity to monitor binding of the inhibitors to their enzyme targets. For FK506, the kinetics of inhibition of human 12 kDa cytosolic FKBP (FKBP12cy) were clearly dependent on time. Subsequent to a rapid inactivation reaction, not resolved in its kinetics due to manual mixing, a slow dominant first-order inactivation process with a relaxation time of 1163 s at 10 degrees C was observed. Concomitantly the Ki value of the slow phase dropped 2.6-fold within the first 60 min of incubation. Using the FKBP12cy homologue 25 kDa membrane FKBP (FKBP25mem), a bacterial peptidylprolyl cis-trans-isomerase, the rate and amplitudes of the inhibition reactions were very similar to FKBP12cy. On the other hand, the kinetics and amplitudes of the inhibition of FKBP12cy varied significantly if rapamycin was used as an inhibitor instead of FK 506. Owing to reduced conformation transition in rapamycin upon binding to FKBP12cy, the slow phase during inhibition was significantly decreased in amplitude. A likely reason for this became apparent when the activation-enthalpy and the pH-dependence of the rate constants of the slow phase were determined. We conclude that the cis to trans interconversion of the pipecolinyl bond of the three peptidomacrolides may be responsible for the slow process. There was no indication of a suicide catalysis of this process by FKBPs.
Activation of precursors for matrix metalloproteinases 1 (interstitial collagenase) and 3 (stromelysin) by rat mast-cell proteinases I and II
Authors: Suzuki K , Lees M , Newlands G F J , Nagase H , Woolley D E
Published: August 2015

Histological studies have previously demonstrated an association between mast-cell activation/degranulation and areas of connective-tissue lysis in vivo; in addition, mast-cell extracts have been shown to activate latent forms of collagenase and stromelysin. In the present study we have examined the potential roles of rat mast-cell proteinase (RMCP) I and RMCP II as activators of the precursors of matrix metalloproteinase (MMP)-1 (interstitial collagenase), MMP-2 (gelatinase A) and MMP-3 (stromelysin 1). Both RMCPs I and II activated proMMP-3 by converting the 57 kDa precursor into a 45 kDa polypeptide. The N-terminal amino acid of 45 kDa MMP-3 activated by RMCP II was identified as Phe83. By contrast, only RMCP II activated the 52 kDa proMMP-1 by converting it into a 41 kDa protein and generating the new N-termini, namely Gln80 and Val82. The collagenolytic activity which resulted from this cleavage was only 35% of the full activity, but this could not be augmented by subsequent treatment with MMP-3, the latter being a crucial enzyme for the generation of the fully active MMP-1 with Phe81 at the N-terminus, in conjunction with other serine proteinases. Thus RMCP II activates proMMP-1 via a mechanism different from that reported for the stepwise processing by combinations of other trypsin-like enzymes and MMP-3. ProMMP-2 (pro-gelatinase A) was not activated by either RMCP I or RMCP II, despite processing to smaller products.
Non-detergent sulphobetaines: a new class of mild solubilization agents for protein purification
Authors: Vuillard L , Braun-Breton C , Rabilloud T
Published: August 2015

The action of non-detergent sulphobetaines (NDSBs) as new mild agents for protein purification is described. The solubilization effects of non-detergent sulphobetaines are shown in different examples; all obtained under non-denaturing conditions: (1) microsomal proteins extraction; (2) recovery after dialysis of nuclear proteins; (3) reduction of precipitation in isoelectric focusing experiments under non-denaturing conditions; and (4) purification of a membrane-bound serine protease from Plasmodium falciparum involved in erythrocyte invasion by malaria merozoites. The absence of a significant denaturation effect induced by NDSBs is demonstrated by tests on beta-galactosidase and alkaline phosphatase. A simple NDSB synthesis and some possible explanations of the action of NDSBs are also presented.
Non-enzymic glycation of fibrous collagen: reaction products of glucose and ribose
Authors: Bailey A J , Sims T J , Avery N C , Halligan E P
Published: August 2015

Non-enzymic glycation of collagen involves a series of complex reactions ultimately leading to the formation of intermolecular cross-links resulting in changes in its physical properties. During analysis for the fluorescent cross-link pentosidine we identified the presence of an additional component (Cmpd K) in both glucose and ribose incubations. Cmpd K was formed more quickly than pentosidine in glucose incubations and more slowly than pentosidine in ribose incubations. Cmpd K represented 45% of the total fluorescence compared with 15% for pentosidine in glucose incubations and 25% of the total fluorescence compared with 30% for pentosidine in the ribose incubations. Cmpd K is not an artefact of in vitro incubations, as it was shown to be present in dermal tissue from diabetic patients. Subsequent high-resolution h.p.l.c. analysis of glucose-incubated collagen revealed Cmpd K comprise two components (K1 and K2). Further, a similar analysis of Cmpd K from the ribose incubations revealed two different components (K3 and K4). These differences indicate alternative mechanisms for the reactions of glucose and ribose with collagen. The amounts of these fluorescent components and the pentosidine cross-link determined for both glucose and ribose glycation were found to be far too low (about one pentosidine molecules per 200 collagen molecules after 6 months incubation with glucose) to account for the extensive cross-linking responsible for the changes in physical properties, suggesting that a further additional series of cross-links are formed. We have analysed the non-fluorescent high-molecular-mass components and identified a new component that increases with time of in vitro incubation and is present in the skin of diabetic patients. This component is present in sufficient quantities (estimated at one cross-link per two collagen molecules) to account for the changes in physical properties occurring in vitro.
Identification of candidate residues for interaction of protein S with C4b binding protein and activated protein C
Authors: Greengard J S , Fernandez J A , Radtke K P , Griffin J H
Published: August 2015

Protein S is a plasma factor essential for prevention of thrombosis, partly due to its activity as a cofactor for the plasma anticoagulant protease-activated protein C. To expand knowledge about structure-function relationships in homologous protein S molecules, studies of protein S from different species have been performed. Protein S anti-coagulant activity in human, monkey, bovine, and porcine plasma has been inactivated by purified human C4b binding protein (C4BP) with dose-dependence, suggesting that each protein S can bind human C4BP and that only the free form of each is anti-coagulantly active. Purified porcine protein S has a 10-fold higher Kd for human C4BP than has human protein S. Protein S residues 420-434 provide an essential binding site for the negative regulator C4BP. cDNA sequences show that protein S residues 420-434 are highly conserved in all four species with the notable exception of Lys-429-Ile in porcine protein S. Differences between porcine and human protein S, e.g. Lys-429-Ile, Lys-43-Ala, Ser-197-Leu, Ser 199-Phe, Glu-463-Gly, Lys-571-Glu, Asn-602-Ile, Gln-607-Pro, may contribute to the decreased affinity of porcine protein S for human C4BP. Moreover, the species specificity of cofactor activities of various species of protein S is determined for human versus bovine-activated protein C, and these results, combined with sequence comparisons, agree with previous evidence that the thrombin-sensitive region and the first epidermal growth factor domain of protein S, i.e. residues 47-116, are responsible for recognition of activated protein C.
Kinetics of the cooperative binding of glucose to dimeric yeast hexokinase P-I
Authors: Hoggett J G , Kellett G L
Published: August 2015

Kinetic studies of the cooperative binding of glucose to yeast hexokinase P-I at pH 6.5 have been carried out using the fluorescence temperature-jump technique. Three relaxation effects were observed: a fast low-amplitude effect which could only be resolved at low glucose concentrations (tau 1(-1) = 500-800 s-1), an intermediate effect (tau 2) which showed a linear dependence of reciprocal relaxation time on concentration, and a slow effect (tau 3) which showed a curved dependence on glucose concentration, increasing from approximately 28 s-1 at low concentrations to 250 s-1 at high levels. The findings are interpreted in terms of the concerted Monod-Wyman-Changeux mechanism, the two faster relaxations being assigned to binding to the R and T states, and the slow relaxation to isomerization between the states. Quantitative fitting of the kinetic data to the mechanism has been carried out using independent estimates of the equilibrium parameters of the model; these have been derived from equilibrium dialysis data and by determining the enhancement of the intrinsic ATPase activity of the enzyme by the non-phosphorylatable sugar lyxose, which switches the conformation of the enzyme to the active R state.
Thapsigargin inhibits Ca2+ entry into human neutrophil granulocytes
Authors: Geiszt M , Káldi K , Szeberényi J B , Ligeti E
Published: August 2015

The mechanism of Ca2+ entry after ligand binding to receptors on the surface of non-excitable cells is a current focus of interest. Considerable attention has been given to Ca2+ influx induced by emptying of intracellular pools. Thapsigargin, an inhibitor of microsomal Ca(2+)-ATPase, is an important tool in inducing store-regulated Ca2+ influx. In the present paper we show that, at concentrations above 500 nM, thapsigargin also has an opposite effect: it inhibits store-regulated Ca2+ influx into Fura-2-loaded human neutrophil granulocytes. As thapsigargin has been frequently applied at concentrations up to 2 microM, its inhibitory action on plasma-membrane Ca2+ fluxes deserves consideration.
Effects of aluminium on the hepatic inositol polyphosphate phosphatase
Authors: Ali N , Craxton A , Sumner M , Shears S B
Published: August 2015

There is speculation that some of the toxic effects of Al3+ may originate from it perturbing inositol phosphate/Ca2+ signalling. For example, in permeabilized L1210 mouse lymphoma cells, 10-50 microM Al3+ activated Ins(1,3,4,5)P4-dependent Ca2+ mobilization and Ins(1,3,4,5)P4 3-phosphatase activity [Loomis-Husselbee, Cullen, Irvine and Dawson (1991) Biochem. J. 277, 883-885]. Ins(1,3,4,5)P4 3-phosphatase activity is performed by a multiple inositol polyphosphate phosphatase (MIPP) that also attacks Ins(1,3,4,5,6)P5 and InsP6 [Craxton, Ali and Shears (1995) Biochem. J. 305, 491-498]: 5-50 microM Al3+ increased MIPP activity towards both Ins(1,3,4,5)P4 (by 30%) and Ins(1,3,4,5,6)P5 (by up to 500%), without affecting metabolism of InsP6. Higher concentrations of Al3+ inhibited metabolism of all three substrates, and in the case of InsP6, Al3+ altered the pattern of accumulating products. When 1-50 microM Al3+ was present, InsP6 became a less effective inhibitor of Ins(1,3,4,5)P4 3-phosphatase activity; this effect did not depend on the presence of cellular membranes, contrary to a previous proposal. The latter phenomenon largely explains how, in a cell-free system where Ins(1,3,4,5)P4 3-phosphatase is inhibited by endogenous InsP6, the addition of Al3+ can apparently increase the enzyme activity. However, there was no effect of either 10 or 25 microM Al3+ (in either the presence or absence of apotransferrin) on inositol phosphate profiles in either Jurkat E6-1 lymphoma cells or AR4-2J pancreatoma cells.
A novel isoenzyme of aldehyde dehydrogenase specifically involved in the biosynthesis of 9-cis and all-trans retinoic acid
Authors: Labrecque J , Dumas F , Lacroix A , Bhat P V
Published: August 2015

The pleiotropic effects of retinoids are mediated by two families of nuclear receptors: RAR (retinoic acid receptors) and RXR (retinoid X receptors). 9-cis-Retinoic acid is a specific ligand for RXR receptors, whereas either 9-cis- or all-trans-retinoic acid activates the RAR receptor family. The existence of RXRs suggests a new role for isomerization in the biology of retinoic acid. We report here the identification of an aldehyde dehydrogenase in the rat kidney that catalysed the oxidation of 9-cis- and all-trans-retinal to corresponding retinoic acids with high efficiency, 9-cis-retinal being 2-fold more active than all-trans-retinal. Based on several criteria, such as amino acid sequence, pH optimum, and inhibition by chloral hydrate, this enzyme was found to be a novel isoenzyme of aldehyde dehydrogenase. 9-cis-Retinol, the precursor for the biosynthesis of 9-cis-retinal was identified in the rat kidney. The occurrence of endogenous 9-cis-retinol and the existence of specific dehydrogenase which participates in the catalysis of 9-cis-retinal suggest that all-trans-retinoi(d) isomerization to 9-cis-retinoi(d) occurs at the retinol level, analogous to all-trans-retinol isomerization to 11-cis-retinol in the visual cycle.
Binding of histone H1e-c variants to CpG-rich DNA correlates with the inhibitory effect on enzymic DNA methylation
Authors: Santoro R , D'Erme M , Mastrantonio S , Reale A , Marenzi S , Saluz H P , Strom R , Caiafa P
Published: August 2015

Within the H1 histone family, only some fractions enriched in the H1e-c variants are effective in causing a marked inhibition, in vitro, of enzymic DNA methylation and, in gel retardation and Southwestern blot experiments, in binding double-stranded (ds) CpG-rich oligonucleotides. Both the 6-CpG ds-oligonucleotide and the DNA purified from chromatin fractions enriched in ‘CpG islands’ are good competitors for the binding of H1e-c to 6-meCpG ds-oligonucleotide. Because of their ability to bind any DNA sequence and to suppress the enzymic methylation in any sequence containing CpG dinucleotides, these particular H1 variants could play some role in maintaining linker DNA at low methylation levels and even in preserving the unmethylated state of the CpG-rich islands which characterize the promoter regions of housekeeping genes.
Identification of putative ligand-binding sites of the integrin α4β1 (VLA-4, CD49d/CD29)
Authors: Kamata T , Puzon W , Takada Y
Published: August 2015

Integrin alpha 4 beta 1 recognizes both fibronectin (CS-1 sequence) and vascular cell adhesion molecule-1 (VCAM-1). To localize the ligand-binding sites of alpha 4, we located the epitopes for function-blocking anti-alpha 4 monoclonal antibodies (mAbs), including those that recognize previously described (but not yet physically localized) functional epitopes (A, B1, B2 and C) using interspecies alpha 4 chimeras expressed in mammalian cells. Epitopes B1 and B2 were associated with ligand binding, and epitopes A and B2 with homotypic cellular aggregation. mAbs P4C2 (epitope B2), 20E4 and PS/2 were mapped within residues 108-182; mAbs HP2/1 (epitope B1), SG/73 and R1-2 within residues 195-268; mAbs HP1/3 (epitope A) and P4G9 within residues 1-52; and B5G10 (epitope C) within residues 269-548. The data suggest that residues 108-268, which do not include bivalent-cation-binding motifs, are related to VCAM-1 and CS-1 binding, and more N-terminal portions of alpha 4 (residues 1 and 52 and 108-182) to homotypic aggregation. Since mAbs PS/2 and HP2/1 block alpha 4 beta 7 binding to mucosal addressin cell adhesion molecule-1 (MAdCAM-1), the MAdCAM-1-binding site is close to, or overlapping with, VCAM-1- and CS-1-binding sites. The role of Asp-130 of beta 1 in the binding to VCAM-1 and CS-1 peptide was examined. Chinese hamster ovary (CHO) cells expressing beta 1 (D130A) (Asp-130 to Ala mutant of beta 1) and alpha 4 showed much less binding to both ligands than CHO cells expressing wild-type beta 1 and alpha 4 [a dominant negative effects of beta 1 (D130A)], suggesting that Asp-130 of beta 1 is critical for binding to both ligands and that the two ligand share common binding mechanisms [corrected].
Absolute and empirical determination of the enzymatic activity and kinetic investigation of the action of hyaluronidase on hyaluronan using viscosimetry
Authors: Vercruysse K P , Lauwers A R , Demeester J M
Published: August 2015

We describe an investigation of the action of hyaluronidase on hyaluronan using viscosimetry. A new viscosimetric approach was developed for determining the activity of the enzyme in katal units. This approach requires knowledge of several parameters (e.g. Mark-Houwink constants) which were determined by combining viscosimetric measurement and gel-permeation chromatography analysis. Using all the necessary parameters we determined the kinetic parameters of the enzyme and found that 250 i.u. correspond to 1 nkat. An empirical viscometric was used to estimate the activity of the enzyme, and the Km was determined using the kinetic dilution method. The estimates produced by the absolute and empirical approaches were in good agreement. We demonstrate that the empirical estimation of the reaction rate is related to the rate of reaction expressed in absolute units and thus provides a good estimate of enzyme activity. Furthermore, we have found an empirical relationship which enables investigation of the kinetics of the enzyme in a simple and sensitive way by viscosimetry.
Structure of heparan sulphate from human brain, with special regard to Alzheimer's disease
Authors: Lindahl B , Eriksson L , Lindahl U
Published: August 2015

Heparan sulphate (HS) was isolated after proteolytic digestion of cerebral cortex, obtained at autopsy, of patients with Alzheimer's disease (AD) and of control subjects. Deaminative cleavage in combination with selective radiolabelling procedures showed that the N-acetylated regions in the intact polysaccharides ranged from isolated residues to approximately 10 consecutive N-acetylated disaccharide units, without any apparent difference between AD and control HS. The yield of disaccharide deamination products was slightly higher with AD than with control HS, suggesting a differential distribution of N-sulphate groups. Separation of the disaccharides by anion-exchange h.p.l.c. yielded four mono-O-sulphated and one di-O-sulphated disaccharide; these components occurred in strikingly similar proportions in all cerebral HS preparations (except polysaccharide from neonatal brain) irrespective of the age of the individual and the histopathology of the cortex specimen. No significant difference was noted between HS obtained from control and from AD tissue. By contrast, the composition of HS isolated from brain differed significantly from that of HS preparations derived from other human organs.
The proteolytic processing site of the precursor of lysyl oxidase
Authors: Cronshaw A D , Fothergill-Gilmore L A , Hulmes D J S
Published: August 2015

The precise cleavage site of the N-terminal propeptide region of the precursor of lysyl oxidase has not yet been established, due to N-terminal blocking of the mature protein. Using a combination of peptide fragmentation, amino acid sequencing, time-of-flight m.s. and partial chemical unblocking procedures, it is shown that the mature form of lysyl oxidase begins at residue Asp-169 of the precursor protein (numbered according to the human sequence). The cleavage site is 28 residues to the C-terminal side of the site previously suggested on the basis of apparant molecular mass by SDS/PAGE, with the consequence that the two putative, N-linked glycosylation sites and the position of the Arg/Gln sequence polymorphism are now all in the precursor region.
Enzymic characterization of Bacillus subtilis GTP cyclohydrolase I. Evidence for a chemical dephosphorylation of dihydroneopterin triphosphate
Authors: De Saizieu A , Vankan P , van Loon A P GM
Published: August 2015

GTP cyclohydrolase I catalyses the first committing step in the biosynthesis of the pterin moiety of folic acid: conversion of GTP to dihydroneopterin triphosphate. GTP cyclohydrolase I of Bacillus subtilis was purified to homogeneity and shown to have a homo-octameric structure. The enzyme had an apparent Km for GTP of 4 microM and, in the absence of cations, a Vmax. of 80 nmol/min per mg of protein. K+ ions moderately increased its Vmax., whereas UTP and Ca2+ and Mg2+ ions drastically increased its Km for GTP. Dihydrofolate and other products of the folate and tetrahydrobiopterin pathways did not inhibit GTP cyclohydrolase I. In addition to their effect on the enzyme activity, Ca2+ and Mg2+ ions catalysed the chemical dephosphorylation of dihydroneopterin triphosphate to non-cyclic dihydroneopterin monophosphate, the substrate for the phosphomonoesterase reaction in folate biosynthesis. This dephosphorylation was specific and did not require the action of a phosphatase. We suggest a physiological role for Ca2+ ions and UTP in regulation of folate biosynthesis at the levels of GTP cyclohydrolase I and dephosphorylation of dihydroneopterin triphosphate.
Promotion of binding of von Willebrand factor to platelet glycoprotein Ib by dimers of ristocetin
Authors: Hoylaerts M F , Nuyts K , Peerlinck K , Deckmyn H , Vermylen J
Published: August 2015

In the absence of high shear forces, the in vitro binding of human von Willebrand factor (vWF) to its platelet receptor glycoprotein Ib (GPIb) can be promoted by two well-characterized mediators, botrocetin and ristocetin. Using purified vWF and GPIb, we have investigated the mechanisms by which ristocetin mediates this binding. Specific binding of vWF monomers to GPIb occurred with a 1:1 stoichiometry, but high-affinity binding required the participation of two ristocetin dimers. Binding was strongly dependent on pH and inhibited by low poly-L-lysine concentrations, indicating ristocetin-dependent charge neutralization during the interaction. With increasing ristocetin concentrations, vWF binding depended progressively less on the involvement of its A1 loop, which is compatible with a model in which the two ristocetin dimers bridge the vWF-GPIb complex on secondary sites. In agreement with this model, the ristocetin-dimer-promoted stabilization of vWF on GPIb was abolished by low concentrations of poly(Pro-Gly-Pro), which is known to complex ristocetin dimers. Mechanistic analysis of the inhibition of vWF binding by the recombinant vWF fragment Leu504-Ser728 (VCL), which covers the entire A1 loop, revealed an affinity of VCL for GPIb comparable with that of the botrocetin-vWF complex for GPIb, and identified a specific but 20-fold lower affinity of VCL in the presence of ristocetin. The proline-rich peptides flanking the vWF A1 loop, Cys474-Val489 and Leu694-Asp709, inhibited vWF binding semispecifically by competitively interfering with the formation of the GPIb-vWF complex rather than by complexation of free ristocetin dimers. In conclusion, ristocetin-promoted binding of vWF to its GPIb receptor results from charge neutralization and interactions involving proline residues in the vicinity of the natural interaction sites present on both GPIb and the A1 domain of vWF.
Mechanism of ligand binding to α1-acid glycoprotein (orosomucoid): correlated thermodynamic factors and molecular parameters of polarity
Authors: Urien S , Giroud Y , Tsai R S , Carrupt P A , Brée F , Testa B , Tillement J P
Published: August 2015

Eight ligands were used in this study, four basic, three neutral and one acidic. Their binding to serum alpha 1-acid glycoprotein (orosomucoid) was measured at several temperatures, and the data were analysed together by a general model with three unknowns, number of binding sites, delta H0 and delta S0. The partition coefficients of the ligands were measured in octanol/water and heptane/water systems (log Poct. and log Phep.), and their molecular volumes were calculated by molecular modelling techniques. These structural properties allow determination of polarity parameters (delta log Poct.-hep., lambda oct. and lambda hep.) which encode in different proportions the various polar interactions between the solute and the aqueous and organic phases, i.e. hydrogen-bonding capacity and dipolarity/polarizability. This study shows that good correlations exist between delta H0 or delta S0 and polarity parameters, such that the enthalpic contribution to binding increases with increasing polarity of the ligands, mainly hydrogen-bond-donor acidity, whereas their entropic contribution to binding decreases.
Calcium mobilization and protein kinase C activation are required for cholecystokinin stimulation of pancreatic cholesterol esterase secretion
Authors: Brodt-Eppley J , Hui D Y
Published: August 2015

The bile salt-stimulated cholesterol esterase is a digestive enzyme synthesized by the acinar cells of the pancreas. Previous results have shown that cholesterol esterase biosynthesis and secretion in the AR42J pancreatoma cells could be increased 3-5-fold by intestinal hormones such as cholecystokinin (CCK). The purpose of the current study is to explore the signalling mechanism by which CCK stimulation of AR42J cells results in increased biosynthesis and secretion of the cholesterol esterase. The results showed that the CCK-induced cholesterol esterase secretion could be mimicked by addition of the Ca2+ ionophore A23187 or by transient incubation of AR42J cells with the protein kinase C activator phorbol 12-myristate 13-acetate (PMA). Cholesterol esterase stimulation by CCK, A23187 and PMA could be abolished by the calcium chelator BAPTA or by specific protein kinase C inhibitors such as chelerythrine. Additionally, prolonged incubation of AR42J cells with PMA to reduce the protein kinase C level, also reduced CCK-stimulated cholesterol esterase secretion to a level similar to that observed in control cells. Taken together, these data suggested that CCK activation of cholesterol esterase secretion may be mediated by a Ca(2+)-dependent protein kinase C pathway, requiring increases in calcium mobilization and activation of protein kinase C.
The membrane immunoglobulin receptor utilizes a Shc/Grb2/hSOS complex for activation of the mitogen-activated protein kinase cascade in a B-cell line
Authors: Kumar G , Wang S , Gupta S , Nel A
Published: August 2015

Ligation of membrane immunoglobulin M (mIgM) receptor in the Ramos B-cell line induced tyrosine phosphorylation of several intracellular substrates, including the adaptor protein. Shc. Phosphorylated Shc could be seen to associate with Grb2 in a complex which included hSOS. Inasmuch as hSOS is involved in p21ras activation, we also demonstrated that mIgM ligation activated a Ras-dependent kinase cascade in which sequential activation of Raf-1 and MEK-1 culminates in the activation of p42 mitogen-activated protein (MAP) kinase (ERK-2). The tumour promoter and protein kinase C agonist, phorbol 12-myristate 13-acetate (PMA), also activated Raf-1, MEK-1, and MAP kinase in Ramos cells, but did not induce tyrosine phosphorylation of Shc or Shc/Grb2 association. Okadaic acid, another tumour promoter and serine/threonine phosphatase inhibitor, activated p42 MAP kinase without activating Raf-1 or MEK-1, suggesting the existence of a serine/threonine phosphatase which directly regulates MAP kinase activity.
Expression of active recombinant pallidipin, a novel platelet aggregation inhibitor, in the periplasm of Escherichia coli
Authors: Haendler B , Becker A , Noeske-Jungblut C , Krätzschmar J , Donner P , Schleuning W D
Published: August 2015

The platelet aggregation inhibitor pallidipin is a protein present in the saliva of the blood-sucking triatomine bug Triatoma pallidipennis. Expression of recombinant pallidipin in the periplasm of Escherichia coli was achieved by placing its coding sequence downstream of the alkaline phosphatase (APase) or trc promoter in frame with bacterial leader peptide DNA sequences derived from APase or from the periplasmic form of cyclophilin (Cph). In each case the DNA sequence of mature pallidipin was merged to the leader peptide coding part, either directly, or while introducing additional amino acids, in order to assess their influence on the activity of the leader peptidase and on the biological activity of the recombinant protein. All tested constructs gave rise to abundant periplasmic expression of pallidipin, which was then purified by a combination of cation- and anion-exchange chromatography followed by size-exclusion gel chromatography. Recombinant pallidipin had the expected molecular mass (approximately 19 kDa) and was correctly processed, as demonstrated by SDS/PAGE and N-terminal amino acid sequencing. The highest expression levels were obtained with the three APase-derived expression plasmids. Platelet aggregation tests revealed that E. coli-derived pallidipin was fully active, with an IC50 of 33-89 nM, comparable with that of the native protein, except when an additional N-terminal lysyl-isoleucyl dipeptide was present, which resulted in an IC50 more than ten times higher.
Determination of the structure of an N-substituted protoporphyrin isolated from the livers of griseofulvin-fed mice
Authors: Bellingham R M A , Gibbs A H , de Matteis F , Lian L Y , Roberts G C K
Published: August 2015

Feeding mice with griseofulvin, a widely used anti-fungal agent which induces protoporphyria as a side-effect, leads to the formation in the liver of two green pigments which have been shown to be porphyrin adducts. In this work, the major porphyrin adduct isolated from the livers of griseofulvin-fed mice has been characterized structurally using one- and two-dimensional NMR spectroscopy. The adduct was shown to be an N-alkylated protoporphyrin IX in which the whole of griseofulvin (less a hydrogen atom) is attached to a pyrrole ring nitrogen of the porphyrin. It was shown that the drug-to-porphyrin linkage is an an -O-CH2-Npyrrole = linkage, to either the 4- or 6-position of ring a of griseofulvin. In an attempt to identify which pyrrole nitrogen is involved in this linkage, the 1H spectra of the free base and zinc complex of the adduct were compared with the corresponding spectra of the four regioisomers of N-methylprotoporphyrin. These comparisons indicated that the adduct isolated from the livers of griseofulvin-fed mice is either the NC or the ND regioisomer, although a clear distinction between these two could not be made on the available evidence. The mechanism of formation of the adduct and its relation to griseofulvin-induced protoporphyria are discussed.
Secondary structure and biophysical activity of synthetic analogues of the pulmonary surfactant polypeptide SP-C
Authors: Johansson J , Nilsson G , Strömberg R , Robertson B , Jörnvall H , Curstedt T
Published: August 2015

Native pulmonary-surfactant-associated lipopolypeptide SP-C, its chemically depalmitoylated form and several synthetic analogues lacking the palmitoylcysteine residues were analysed for secondary structure in phospholipid micelles and for biophysical activity in 1,2-dipalmitoyl-sn-glycero-3- phosphocholine/phosphatidylglycerol/palmitic acid (68:22:9, by wt.). Compared with the native molecule, with the entire poly-valyl part in a known alpha-helical conformation, depalmitoylated SP-C was found to be still mainly alpha-helical, but with an approx. 20% decrease in the helical content. A synthetic hybrid polypeptide where the entire poly-valyl alpha-helical part of native SP-C had been replaced with the amino acid sequence of a transmembrane helix of bacteriorhodopsin is also predominantly alpha-helical. In contrast, synthetic SP-C analogues lacking only the palmitoyl groups, by replacement of the palmitoylcysteine residues with cysteine, phenylalanine or serine, or lacking the positively charged amino acids by replacement with alanine, are considerably less alpha-helical than both native and depalmitoylated SP-C. The data indicate that the SP-C palmitoyl groups are important for maintenance of the alpha-helical conformation in parts of the polypeptide, and that the poly-valyl alpha-helical conformation is not fully formed in synthetic SP-C polypeptides. Furthermore, the helical structure of both native and depalmitoylated SP-C in dodecylphosphocholine micelles is very resistant to thermal denaturation, exhibiting ordered structure at 90 degrees C. The alpha-helical content grossly parallels the peptide-induced acceleration of the spreading of phospholipids at an air/water interface and the increase of surface pressure. The data suggest that the alpha-helical conformation itself, rather than just the covalent structure, is of prime importance for the biological function of synthetic pulmonary-surfactant peptides.