bluuebunny/crossref_metadata_2025 huggingface dataset:
Ca2+ concentration during binding determines the manner in which annexin V binds to membranes
Authors: Trotter P J , Orchard M A , Walker J H
Published: August 2015

Annexins are a family of calcium-binding proteins that have been implicated in a wide range of intracellular processes. We have previously reported that stimulation of platelets with thrombin can induce the association of intracellular annexin V with membranes in two distinct ways. First, in such a way that it can be eluted from the membrane with EGTA and secondly in a manner such that it is tightly bound to the membrane and requires the non-ionic detergent Triton X-100 for its solubilization. We report that exposure of platelets to the calcium ionophore A23187 mimics the relocation induced by stimulation with thrombin. In separate experiments we demonstrate that a calcium ion concentration [Ca2+] of 0.8 microM is sufficient for maximum binding of the EGTA-resistant form to membranes. In contrast a higher [Ca2+] was required to induce maximal binding of the annexin V which could be extracted with EGTA. We demonstrate that following temperature-induced phase separation in Triton X-114, the membrane-associated annexin V partitions predominantly into the aqueous phase. We also show that the isoelectric point of annexin V does not change following membrane association. These observations suggest that a covalent modification, of annexin V itself, is not responsible for its association with the membrane. Millimolar [Ca2+] is required for maximal binding of purified annexin V to phospholipid vesicles. We show that binding to phospholipids can be reversed entirely by subsequent treatment with EGTA. This suggests that the EGTA-resistant form of annexin V is binding to a membrane component other than phosphatidylserine. Annexin V has previously been shown to bind to protein kinase C. Relocation of annexin V to membranes paralleled that of protein kinase C in thrombin-stimulated cells but not in cells treated with A23187, suggesting that these proteins are not functionally linked in platelet activation. Using bifunctional cross-linking reagents we have identified an 85 kDa complex containing annexin V. This may represent an association between annexin V and an annexin V-binding protein with a molecular mass of approximately 50 kDa.
Signal-transduction-pathway-specific desensitization of expression of orphan nuclear receptor TIS1
Authors: Lim R W , Yang W L , Yu H
Published: August 2015

Induction of many primary-response genes by a variety of stimuli occurs in a transient manner. The precise mechanism responsible for these transient kinetics is not completely understood. We report here that the orphan nuclear receptor, TIS1, which is a potent sequence-specific transcription factor, was transiently induced by the adrenergic agonist isoprenaline in the C2C12 skeletal-muscle cell line. Moreover, we showed that the rapid decline in mRNA level after peak induction was due in part to a specific desensitization of the isoprenaline-mediated induction pathway. Desensitization of the induction response presumably occurred at the level of the receptor, as agents that either bypass the adrenergic receptor or activate alternative signalling pathways were able to induce TIS1 expression in the desensitized cells. However, stimulation by agents that directly activate intracellular enzymes also resulted in the signal-transduction-pathway-specific desensitization of TIS1 inducibility. Our results suggest that the pathway-specific nature of the desensitization process may be important for directing an integrated response to multiple physiological stimuli.
Replacement of Lys by Glu in a transmembrane segment strongly impairs the function of the uracil permease from Saccharomyces cerevisiae
Authors: Urban-Grimal D , Pinson B , Chevallier J , Haguenauer-Tsapis R
Published: August 2015

The co-transport of uracil and protons through the plasma membrane of the yeast Saccharomyces cerevisiae is mediated by a specific permease encoded by the FUR4 gene. The uracil permease is a multi-spanning membrane protein that follows the secretory pathway to the plasma membrane. Recent experimental data led to the proposal of a two-dimensional model of its topology. A spontaneous mutant corresponding to the substitution of Lys-272 by glutamic acid was obtained. The influence of this mutation was studied by comparing the wild-type and mutant permeases produced in a strain carrying a chromosomal deletion of the FUR4 gene. The mutant permease is correctly targeted to the plasma membrane and its stability is similar to that of the wild-type permease. The uptake parameters for the mutant permease were impaired and showed an approximately 65-fold increase of apparent K(m) and a decrease in apparent Vmax. Equilibrium binding measurements with enriched plasma membrane preparations showed an approximately 70-fold increase in apparent Kd in the mutant, whereas its Bmax. was similar to that of the wild type. Lys-272 is fully conserved in the uracil permease family and is predicted to lie in the fourth transmembrane segment of the protein. It seems to be essential for both efficient uracil binding and translocation.
Mutations in the B-domain of insulin-like growth factor-I influence the oxidative folding to yield products with modified biological properties
Authors: Milner S J , Francis G L , Wallace J C , Magee B A , Ballard F J
Published: August 2015

The oxidative folding of human insulin-like growth factor (IGF)-I yields two major disulphide folding isomers. In the present study, B-domain analogues of IGF-I were used to investigate the effect of mutations on the folding reaction and to investigate the functional implications of misfolding. The analogues used were substitutions of the native Glu3 by Gly or Arg, or the native Glu9 by Lys. IGF-I and these analogues were also prepared attached to a hydrophobic 13-amino-acid N-terminal extension, Met-Phe-Pro-Ala-Met-Pro-Leu-Ser-Ser-Leu-Phe-Val-Asn, referred to as ‘Long-IGF-I’ analogues. Each IGF was fully reduced and refolded to yield native and misfolded isomers, which were subsequently purified for biological characterization. Analysis of the folding reaction at equilibrium revealed a distribution of folding isomers characteristic for each peptide. The yield of the native disulphide folding isomer was increased for the Glu3 substitutions, but not for the Glu9 substitution. The main alternative folding isomer was present in the IGF-I analogues in reduced proportions. Except for [Gly3]IGF-I the N-terminal extension increased the yield of the native isomer which was maximal for the analogue Long-[Arg3]IGF-I. A folding intermediate for the latter analogue was isolated and partially characterized. The biological assays showed that all the main alternative isomers bound poorly to IGF-binding proteins (IGFBPs) secreted by L6 myoblasts. Moreover, these isomers bound to the type 1 IGF receptor with 0.5-25% the affinity of the native isomer. In a rat L6 myoblast protein-synthesis assay, the observed biological activity of the native and main alternative isomers was explained by their modified IGFBP- and receptor-binding properties. We propose that the N-terminal extension imparts a steric constraint at a crucial point in folding, thus allowing native disulphide bonds to form efficiently.
The human MUC2 mucin apoprotein appears to dimerize before O-glycosylation and shares epitopes with the ‘insoluble’ mucin of rat small intestine
Authors: Asker N , Baeckström D , Axelsson M A B , Carlstedt I , Hansson G C
Published: August 2015

Rabbit antiserum against a synthetic peptide corresponding to a tandemly repeated amino acid sequence in the human intestinal mucin apoprotein MUC2 was used in immunoprecipitation to study the biosynthesis of MUC2 in the colon-carcinoma cell line LS 174T. Under non-reducing conditions, two bands were precipitated, the smaller with an apparent size of about 700 kDa on SDS/PAGE. When analysed by two-dimensional electrophoresis after reduction, the larger band migrated to the same position as the smaller band and was interpreted as a putative disulphide-bond-stabilized dimer. Pulse-chase experiments showed only the monomer after 5 min and the appearance of the putative dimer after 30 min. The MUC2 apoprotein was also precipitated by antisera against the HF-deglycosylated peptides of the two highly glycosylated domains of the ‘insoluble’ mucin complex of rat small intestine [Carlstedt, Herrmann, Karlsson, Sheehan, Fransson and Hansson (1993) J. Biol. Chem. 268, [18771-18781]. Endoprotease Lys-C cleavage of the immunopurified apoprotein gave a large fragment of about 250 kDa that was detected by both the antiserum against the MUC2 tandem repeat and one of the glycopeptide antisera. This supports the view that the ‘insoluble’ mucin of rat small intestine is encoded by the Muc2 gene, as recently indicated by a partial cDNA sequence [Hansson, Baeckström, Carlstedt and Klinga-Levan (1994) Biochem. Biophys. Res. Commun. 198, 181-190] and that parts of the apoprotein are conserved between the species. A lectin from the snail Helix pomatia that detects terminal alpha-GalNAc residues did not bind to the monomer or putative dimer, suggesting that O-glycosylation starts after dimerization. The results indicate that the biosynthetic pathway of the MUC2 mucin may be similar to that of the von Willebrand factor with which MUC2 shares sequence similarities at its C- and N-termini.
Isolation and some properties of glycated d-glyceraldehyde-3-phosphate dehydrogenase from rabbit muscle
Authors: He R Q , Yang M D , Zheng X , Zhou J X
Published: August 2015

Glycated D-glyceraldehyde-3-phosphate dehydrogenases (GAPDH) from rabbit muscle and human erythrocytes have been investigated. The specific activity of the non-glycated GAPDH from rabbit muscle is approx. 180 units. (One unit is defined as the specific activity required to convert 1 microM of substrate/min per mg of enzyme.) The activity of the glycated enzyme, consisting of two sugars per tetramer, is lower than that of the non-glycated GAPDH. Non-enzymic transamination of the N-termini of glycated GAPDH (gGAPDH) indicates that they are not blocked by glycation. The rate of modification of thiols (Cys-149) with 5,5′-dithiobis-(2-nitrobenzoic acid) was greater for the glycated than the non-glycated enzymes. The rate of modification of amino groups of Lys residues of gGAPDH with o-phthalaldehyde was greater for the non-glycated enzyme. In 0.18 M guanidine-HC1 solution, the emission intensity at 410 nm of a fluorescent NAD+ derivative introduced into the active site decreased to 80%, whereas that of gGAPDH decreased to 50%. This suggests that the glycated sites are near the active site; glycation of the enzyme leads to a change of the microenvironment of Cys-149, alters the conformation of the active site and decreases the activity.
Secretion of active human lecithin-cholesterol acyltransferase by insect cells infected with a recombinant baculovirus
Authors: Chawla D , Owen J S
Published: August 2015

Difficulties in purifying the plasma enzyme lecithin-cholesterol acyltransferase (LCAT) have hampered detailed studies of its (patho)physiological role in lipoprotein metabolism and of structure-function relationships. Potentially, baculovirus-driven expression systems offer a powerful means to produce significant amounts of LCAT. Accordingly, full-length LCAT cDNA was cloned into pVL 1392, a high-level expression derivative of Autographa californica nuclear polyhedrosis virus (AcNPV), and the resultant plasmid was co-transfected into Trichoplusia ni insect cells (High 5 line) with a linearized viral DNA using lipofectin. Such viral DNA had a lethal mutation and grew only when recombined with a pVL1392-type rescue plasmid; cells infected with recombinant Autographa californica LCAT virus changed from a fibroblast-like morphology to rounded, but lacked the polyhedrin occlusion bodies characteristic of wild-type AcNPV infections. Enzymically active recombinant LCAT (rLCAT), sensitive to sulphydryl reagents, was secreted in the late phase of infection (36-48 h) but was absent with wild-type infections. The secreted protein had an apparent molecular mass of 53 kDa by SDS/PAGE, lower than that of native plasma LCAT; it was susceptible to endoglycosidase H digestion and was bound by concanavalin A, suggesting that precursor high-mannose N-glycan chains had not undergone full maturation to complex types. Pretreatment of the cells with tunicamycin to inhibit the first step of N-glycosylation led to intracellular accumulation of immature rLCAT (approximately 46-48 kDa) and a marked reduction in enzyme secreted. We conclude that the baculovirus gene-expression system will permit production of biologically active normal and mutant forms of LCAT protein.
Regulation of GLUT5 gene expression in rat intestinal mucosa: regional distribution, circadian rhythm, perinatal development and effect of diabetes
Authors: Castelló A , Gumá A , Sevilla L , Furriols M , Testar X , Palacín M , Zorzano A
Published: August 2015

1. GLUT5 gene expression was studied in small intestine under a variety of conditions characterized by altered intestinal absorption of monosaccharides. 2. RNA-blotting studies showed that GLUT5 mRNA was abundantly expressed in rat and rabbit intestine and kidney, but it was not detected in heart or brown adipose tissue. GLUT5 mRNA levels were higher in the upper segments of the small intestine (duodenum and proximal jejunum) than in the lower segments (distal jejunum and ileum). 3. The intestinal expression of GLUT5 mRNA in rat proximal jejunum showed circadian rhythm. A 12-fold increase in GLUT5 mRNA levels was detected at the end of the light cycle and at the beginning of the dark cycle when compared with the early light period. In keeping with this, GLUT5 protein content in brush-border membranes was also increased at the beginning of the dark cycle compared with that in the light period. 4. In streptozotocin-induced diabetes an 80% increase in GLUT5 mRNA levels in mucosa from the proximal jejunum was detected under conditions in which enhanced intestinal absorption of monosaccharides has been reported. 5. The intestinal expression of GLUT5 mRNA showed regulation during perinatal development. Levels of GLUT5 mRNA were low during fetal life, increased progressively during the postnatal period and reached levels comparable with the adult state after weaning. Weaning on to a high-fat diet partially prevented the induction of GLUT5 gene expression. 6. Our results indicate that GLUT5 gene expression is tightly regulated in small intestine. Regulation involves maximal expression in the upper part of the small intestine, circadian rhythm, developmental regulation dependent on the fat and carbohydrate content in the diet at weaning and enhanced expression in streptozotocin-induced diabetes. Furthermore, changes observed in intestinal GLUT5 expression correlate with reported alterations in intestinal absorption of fructose. This suggests a regulatory role for GLUT5 in fructose uptake by absorptive enterocytes.
Reactions of glutamate semialdehyde aminotransferase (glutamate-1-semialdehyde 2,1 aminomutase) with vinyl and acetylenic substrate analogues analysed by rapid scanning spectrophotometry
Authors: Tyacke R J , Contestabile R , Grimm B , Harwood J L , John R A
Published: August 2015

The reactions occurring when glutamate-1-semialdehyde amino-transferase (glutamate-1-semialdehyde 2,1 aminomutase, EC 5.4.3.8) was treated with two potential mechanism-based inactivators, namely 4-aminohex-5-enoate and 4-aminohex-5-ynoate, have been investigated by monitoring rapid transient changes in the absorption spectrum of the enzyme's prosthetic group, pyridoxal 5′-phosphate. In both cases a short-lived chromophore absorbing maximally at about 500 nm was formed in a few milliseconds. In the case of the vinyl analogue (4-aminohex-5-enoate) this chromophore, considered to be a quinonoid intermediate, converted rapidly into the pyridoxamine phosphate form of the co-enzyme in a single turnover which was accompanied by negligible inactivation. However, slow inactivation of the enzyme by this compound was observed when the enzyme was made to undergo multiple turnovers by including the efficient aldehyde substrate, succinic semialdehyde. The acetylenic compound, aminohexynoate, produced more complex spectral changes with the consecutive formation of compounds absorbing maximally at 496 nm, 450 nm, 564 nm and 330 nm. The enzyme was 90% inactivated by aminohexynoate within 10 s and thereafter lost no further activity unless aldehyde substrate was added. Mechanisms and kinetic constants consistent with the observations are proposed for each compound. The observation that the acetylenic compound is a much more potent inactivator than its vinyl analogue is attributed to the occurrence of a conjugated allene as intermediate.
Study of the roles of Arg-104 and Arg-225 in the 2-kinase domain of 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase by site-directed mutagenesis
Authors: Rider M H , Crepin K M , De Cloedt M , Bertrand L , Vertommen D , Hue L
Published: August 2015

The roles of Arg-104 and Arg-225 located in the 2-kinase domain of the bifunctional enzyme 6-phosphofructo-2-kinase (PFK-2)/fructose-2,6-bisphosphatase (FBPase-2) have been studied by site-directed mutagenesis. In recombinant rat liver PFK-2/FBPase-2, mutation of Arg-225 to Ser increased the Km of PFK-2 for fructose-6-phosphate (Fru-6-P) 7-fold at pH 6 and decreased PFK-2 activity at suboptimal substrate concentrations between pH 6 and 9.5. The mutation had no effect on the Vmax of PFK-2 or on the Km of PFK-2 for MgATP. The mutation also increased the Vmax. of FBPase-2 4-fold without changing the Km for Fru-2,6-P2 or IC50 of Fru-6-P. These findings are in agreement with a previous study [Rider and Hue (1992) Eur. J. Biochem. 207, 967-972] on the protection by Fru-6-P of the labelling of Arg-225 by phenylglyoxal, and suggest that Arg-225 participates in Fru-6-P binding. In recombinant rat muscle PFK-2/FBPase-2, mutation of Arg-104 to Ser increased the Km for Fru-6-P 60-fold, increased the IC50 of citrate, increased the Vmax. 1.5-3-fold at pH 8.5 and altered the pH profile of PFK-2 activity. It did not affect the Km of PFK-2 for MgATP. The mutation also decreased the Vmax. of FBPase-2 3-fold, increased the Km for Fru-2,6-P2 70-fold and increased the IC50 of Fru-6-P at least 300-fold. Although the dimeric structure was maintained in the mutant, its PFK-2 activity was more sensitive towards inactivation by guanidinium chloride than the wild-type enzyme activity. The findings indicate that Arg-104 is involved in Fru-6-P binding in the PFK-2 domain and that it might also bind citrate. Structural changes resulting from the mutation might be responsible for the changes in kinetic properties of FBPase-2.
Identification and characterization of a novel human neutrophil protein related to the S100 family
Authors: Guignard F , Mauel J , Markert M
Published: August 2015

A rabbit polyclonal antibody raised against myeloid-related protein 8 (MRP-8), a protein of the S100 family, recognized another S100 protein (MRP-14) as well as a protein of 6.5 kDa (p6) in the cytosol of resting neutrophils. p6 was found to be a novel member of the S100 family. It consisted of two isoforms with pI values of 6.2 (the minor form, p6a) and 6.3 (the major form, p6b) and constituted 5% of the total cytosolic proteins. Both isoforms were also demonstrated in the cytosol of monocytes, but not in lymphocytes, as previously shown for MRP-8 and MRP-14. Only the major isoform bound radioactive Ca2+, as also observed for MRP-8, whereas the different variants of MRP-14 were all labelled. On neutrophil activation with opsonized zymosan, a stimulant known to require extracellular Ca2+, 58% of p6a and 42% of p6b was translocated to the membrane. With phorbol 12-myristate 13-acetate, a Ca(2+)-independent stimulant, no translocation was detected. This translocation pattern was similar to that observed with MRP-8 and MRP-14. In addition, p6, MRP-8 and MRP-14 were specifically associated with the cytoskeletal fraction of the membrane. The Ca(2+)-dependent translocation of the novel S100 protein in parallel with MRP-8 and MRP-14 suggests a role for these proteins in regulating the Ca2+ signal to the membrane cytoskeleton and thus in regulating neutrophil activation.
A novel procedure for the rapid isolation of surfactant protein A with retention of its alveolar-macrophage-stimulating properties
Authors: van Iwaarden J F , Teding van Berkhout F , Whitsett J A , Oosting R S , van Golde L M G
Published: August 2015

Previous studies have shown that surfactant protein A (SP-A) derived from alveolar-proteinosis patients activates rat alveolar macrophages. However, it is not known if normal rat, dog and human SP-A can also stimulate alveolar macrophages. As alveolar-proteinosis SP-A has a slightly different structure from ordinary SP-A, it would be possible that the ascribed alveolar-macrophage-stimulating properties of SP-A are restricted to alveolar-proteinosis SP-A. To clarify this issue, we isolated SP-A from normal rat and dog pulmonary surfactants, using the same isolation technique commonly used for the isolation of alveolar-proteinosis SP-A, i.e. by butanol precipitation. In contrast with human alveolar-proteinosis SP-A, rat and dog SP-A obtained thus could not activate rat alveolar macrophages to produce oxygen radicals or enhance the phagocytosis of fluorescein isothiocyanate-labelled herpes simplex virus. However, rat, dog and normal human SP-A isolated by a novel method, involving extraction from pulmonary surfactant by using n-octyl beta-D-glucopyranoside and subsequent purification by cation-exchange chromatography, were able to elicit an oxidative burst in rat as well as normal human alveolar macrophages. In addition, dog and rat SP-A obtained thus stimulated the phagocytosis of herpes simplex virus by rat alveolar macrophages. These findings indicate that normal human, rat and dog SP-A have the same alveolar-macrophage-stimulating properties as human alveolar proteinosis SP-A. Dog and rat SP-A isolated by this novel method had the same Ca(2+)-dependent self-aggregation and lipid-aggregation properties as SP-A isolated by butanol precipitation. The new and milder isolation procedure yielded SP-A of high purity, as judged by SDS/PAGE and ELISA.
Identification of a band-3 binding site near the N-terminus of erythrocyte membrane protein 4.2
Authors: Rybicki A C , Musto S , Schwartz R S
Published: August 2015

Protein 4.2 (P4.2) is a major component of the erythrocyte plasma membrane accounting for approx. 5% of total membrane protein. The major membrane binding site for P4.2 is contained within the cytoplasmic domain of band 3 (cdb3), although the precise location of the cdb3 binding site is not known. To identify the cdb3 binding site, we used synthetic P4.2 peptides (15-mers) that spanned the entire 721-amino-acid large isoform of P4.2, and determined the binding of these peptides to cdb3 in an in vitro binding assay. One peptide, P8 (L61FVRRGQPFTIILYF), bound strongly to cdb3 and four others bound less strongly (P22, L271LNKRRGSVPILRQW; P27, G346EGQRGRIWIFQTST; P41, L556WRKKLHLTLSANLE; P48, I661HRERSYRFRSVWPE). These peptides have in common a cluster of two or three basic amino acid residues (arginine or lysine), in a region without nearby acidic residues. Cdb3 bound saturably to P8 with a Kd of 0.16 microM and a capacity of 0.56 mol of cdb3 monomer/mol of P8. Use of overlapping synthetic peptides further defined the cdb3 site as being contained within V63RRGQPFTIILYF. Replacement of R64R with R64G, G64R or G64G almost completely abolished cdb3 binding, suggesting that R64R is essential for cdb3 binding. P8 competitively inhibited binding of purified human erythrocyte P4.2 to cdb3. In blot overlay assays, cdb3 bound to a 23 kDa N-terminal P4.2 tryptic peptide containing V63RRGQPFTIILYF but not to other P4.2 tryptic peptides lacking this site. The V63RRGQPFTIILYF site is highly conserved in mouse and human erythrocyte P4.2 as well as between P4.2 and transglutaminase proteins, which are evolutionarily related to P4.2.
The modulation of phosphatidylinositol biosynthesis in hamster hearts by methyl lidocaine
Authors: Lee E , Tardi P G , Man R Y K , Choy P C
Published: August 2015

Methyl lidocaine is an experimental anti-arrhythmic drug which has been shown to enhance the biosynthesis of phosphatidyl-inositol (PI) in the hamster heart. In this study, the effect of methyl lidocaine on enzymes involved in the biosynthesis of PI in the heart was examined. When the hamster heart was perfused with labelled methyl lidocaine, the majority of the compound was not metabolized after perfusion. The direct action of methyl lidocaine on an enzyme was studied by the presence of the drug in enzyme assays, whereas its indirect action was studied by assaying the enzyme activity in the heart after methyl lidocaine perfusion. CTP:phosphatidic acid cytidylyl-transferase, a rate-limiting enzyme in PI biosynthesis, was stimulated by methyl lidocaine in a direct manner. Kinetic studies revealed that methyl lidocaine caused a change in the affinity between the enzyme and phosphatidic acid and resulted in the enhancement of the reaction. Alternatively, acyl-CoA:lysophosphatidic acid acyltransferase, another key enzyme for PI biosynthesis, was not activated by the presence of methyl lidocaine. However, the enzyme activity was stimulated in hearts perfused with methyl lidocaine. The enhancement of the acyl-transferase by methyl lidocaine perfusion was found to be mediated via the adenylate cyclase cascade with the elevation of the cyclic AMP level. The stimulation of protein kinase A activity by cyclic AMP resulted in the phosphorylation and activation of the acyltransferase. Interestingly, the activity of protein kinase C was not stimulated by methyl lidocaine perfusion. We conclude that the enhancement of PI biosynthesis by methyl lidocaine in the hamster heart resulted from the direct activation of the cytidylyltransferase, as well as the phosphorylation and subsequent activation of the acyltransferase.
Cloning of the cDNA encoding mast cell tryptase of Mongolian gerbil, Meriones unguiculatus, and its preferential expression in the intestinal mucosa
Authors: Murakumo Y , Ide H , Itoh H , Tomita M , Kobayashi T , Maruyama H , Horii Y , Nawa Y
Published: August 2015

By using the combination of reverse-transcription PCR and rapid amplification of cDNA ends methods, a cDNA encoding mast cell tryptase was successfully cloned from the small intestine of Mongolian gerbil, Meriones unguiculatus, infected with Nippostrongylus brasiliensis. The cDNA was 1219 bp long including 810 bp of an open reading frame. Based on the deduced amino acid sequences of known mast cell tryptases of other species, the gerbil mast cell tryptase (gMCT) was highly similar to mouse mast cell protease (mMCP)-7, and seems to be translated as a prepro-enzyme with 25 amino acids of signal and activation peptides and 245 amino acids of mature enzyme. The gMCT mRNA was preferentially transcribed in the intestinal mucosa and to a far lesser extent in the connective tissue such as skin and tongue. Moreover, kinetic study after infection revealed that the amount of gMCT mRNA in the small intestine correlated well with the degree of intestinal mastocytosis. Throughout the course of infection, enzyme-histochemically detectable tryptase activity was limited to mucosal mast cells. Since mucosal mast cells of other rodents, including mice and rats, do not express tryptases, this is the first report of rodent mast cell tryptase expressed in the intestinal mucosa.
The design of peptide-based substrates for the cdc2 protein kinase
Authors: Srinivasan J , Koszelak M , Mendelow M , Kwon Y G , Lawrence D S
Published: August 2015

The substrate sequence specificity of the cdc2 protein kinase from Pisaster ochraceus has been evaluated. The peptide, Ac-Ser-Pro-Gly-Arg-Arg-Arg-Arg-Lys-amide, serves as an efficient cdc2 kinase substrate with a Km of 1.50 +/- 0.04 microM and a Vmax. of 12.00 +/- 0.18 mumol/min per mg. The amino acid sequence of this peptide is not based on any sequence in a known protein substrate of the cyclin-dependent kinase, but rather was designed from structural attributes that appear to be important in the majority of cdc2 substrates. The cyclin-dependent enzyme is remarkably indiscriminate in its ability to recognize and phosphorylate peptides that contain an assortment of structurally diverse residues at the P-2, P-1 and P+2 positions. However, peptides that contain a free N-terminal serine or lack an arginine at the P+4 position are relatively poor substrates. These aspects of the substrate specificity of the cdc2 protein kinase are compared and contrasted with the previously reported substrate specificity of a cdc2-like protein kinase from bovine brain [Beaudette, Lew and Wang (1993) J. Biol. Chem. 268, 20825-20830].
A study of novel lectins and their involvement in the activation of the prophenoloxidase system in Blaberus discoidalis
Authors: Chen C , Durrant H J , Newton R P , Ratcliffe N A
Published: August 2015

Endogenous and exogenous lectins have been found to activate the prophenoloxidase (proPO) system of the cockroach, Blaberus discoidalis, to the same extent as laminarin, a previously known microbial activator of proPO. The lectins can also further enhance this laminarin activation of the proPO system. Non-lectin proteins did not display any activation properties. The time course of proPO activation was studied after reconstitution of the reaction system using purified lectins, a trypsin-like enzyme, a trypsin inhibitor and partially purified lectin-binding proteins from the cockroach haemolymph. Lectin activation of the proPO system is probably not mediated by the lectin sugar-binding sites, as specific inhibitory sugars failed to abrogate the enhanced effect. The results suggest that alternative binding site(s) on the lectins may be involved in the proPO activation process. Evidence also suggests that several different lectins are involved in the regulation of the proPO system through separate receptors or binding molecules on the haemocytes, and that they exert their effects early in the sequence of events leading to conversion of proPO into its active form, possibly via regulation of serine proteases and protease inhibitors.
Effect of transforming growth factor-β1 and basic fibroblast growth factor on the expression of cell surface proteoglycans in human lung fibroblasts. Enhanced glycanation and fibronectin-binding of CD44 proteoglycan, and down-regulation of glypican
Authors: Romarís M , Bassols A , David G
Published: August 2015

We have tested the effects of transforming growth factor-beta 1 (TGF-beta 1), basic fibroblast growth factor (bFGF) and TGF-beta 1 + bFGF on the expression of the cell surface proteoglycans (CD44, syndecans and glypican) in cultures of human lung fibroblasts (HLF). Cell surface proteoglycan expression was monitored by quantitative immunoprecipitation from metabolically labelled cells. Western and Northern blotting and evaluation of the glycanation of the proteoglycans. Stimulation of the cells with TGF-beta 1 increased the length of the chondroitin sulphate (CS) chains on CD44 (approximately 1.6-fold). bFGF, administered solely, also increased the length of the CS chains on CD44 (approximately 1.4-fold), whereas the combination of TGF-beta 1 + bFGF nearly doubled both the length and the number of the CS chains on CD44. None of these treatments lead to changes in CD44 message or core-protein expression. This enhanced glycanation of CD44 after the TGF-beta 1, bFGF and combined treatments correlated with a 2-fold increase in the affinity of the proteoglycan for fibronectin but had no influence on the binding to type I collagen. TGF-beta 1, alone or in combination with bFGF, also stimulated the CS content of syndecan-1, but none of the other syndecans was significantly affected by any of the factors or combinations tested. The expression of glypican however was significantly decreased (nearly halved) by the combination of TGF-beta 1 + bFGF, less so by TGF-beta 1 and not at all by bFGF. This decrease occurred both at the level of the message and of the core protein. These data demonstrate specific and differential effects of TGF-beta 1 and bFGF on the structure, expression and interactions of the cell surface proteoglycans of HLF.
Active site labelling of inositol 1,4,5-trisphosphate 3-kinase A by phenylglyoxal
Authors: Communi D , Lecocq R , Vanweyenberg V , Erneux C
Published: August 2015

Chemical modification by phenylglyoxal, an arginine-specific reagent, of both native and recombinant rat brain inositol 1,4,5-trisphosphate [Ins(1,4,5)P3] 3-kinase A was accompanied by irreversible inhibition of enzyme activity. This effect was prevented in the presence of the substrate ATP but not Ins(1,4,5)P3. The modification reaction obeyed pseudo-first-order rate kinetics. Complete inhibition of activity corresponded to incorporation of 1.2 mol of phenylglyoxal per mol of protein. A single [14C]phenylglyoxal-modified peptide was isolated following alpha-chymotrypsin digestion of the radiolabelled Ins(1,4,5)P3 3-kinase and reverse-phase HPLC. ATP prevented the incorporation of radioactivity to this peptide. The peptide sequence (i.e. QWREGISSSTTL) corresponded to amino acids 315 to 326 of rat brain Ins(1,4,5)P3 3-kinase A. An estimate of the radioactivity of the different phenylthiohydantoin amino acid derivative showed the modified amino acid to be Arg-317. The data directly identify a reactive arginine residue as part of the ATP-binding site. Arg-317 is located within a sequence segment which is conserved among the catalytic domain of Ins(1,4,5)P3 3-kinase isoenzymes A and B in human and rat species.
Mechanisms of platelet activation by a stimulatory antibody: cross-linking of a novel platelet receptor for monoclonal antibody F11 with the FcγRII receptor
Authors: Naik U P , Ehrlich Y H , Kornecki E
Published: August 2015

The mechanisms by which a stimulatory monoclonal antibody (mAb), called mAb F11, induces granular secretion and aggregation in human platelets have been characterized. Fab fragments of mAb F11, as well as an mAb directed against the platelet Fc gamma RII receptor (mAb IV.3) were found to inhibit mAb F11-induced platelet secretion and aggregation, indicating that the mAb F11 IgG molecule interacts with the Fc gamma RII receptor through its Fc domain and with its own antigen through its Fab domain. The mAb F11 recognized two platelet proteins of 32 and 35 kDa on the platelet membrane surface, as identified by Western blot analysis. We purified both proteins from human platelet membranes using DEAE-Sepharose chromatography followed by mAb F11 affinity chromatography. When added to platelet-rich plasma, the purified proteins dose-dependently inhibited mAb F11-induced platelet aggregation. The purified protein preparation also competitively inhibited the binding of 125I-labelled mAb F11 to intact platelets. The N-terminal 26 amino acid sequences of both the 32 and 35 kDa proteins were identical and contained a single unblocked serine in the N-terminal position. When digested with N-glycanase, the 32 and 35 kDa proteins were converted into a single approximately 29 kDa protein, indicating that these two proteins are derived from the same core protein but differ in their degree of glycosylation. Internal amino acid sequence analysis of the F11 antigen provided information concerning 68 amino acids and suggested two consensus phosphorylation sites for protein kinase C (PKC). The phosphorylation by PKC of the isolated F11 antigen was observed following stimulation by phorbol 12-myristate 13-acetate. Databank analysis of the N-terminal and internal amino acid sequences of the F11 antigen indicated that the N-terminal sequence exhibited the highest degree of similarity to the variable region of the alpha-chain of human T-cell receptors (TCR). In contrast, the F11 internal sequences did not exhibit any similarity to the TCR. Our results demonstrate that the F11 antigen is a novel platelet membrane surface glycoprotein which becomes cross-linked with the Fc gamma RII receptor when platelets are activated by the stimulatory mAb F11. These mechanisms may be relevant to the production of immune thrombocytopenia by platelet-activating antibodies.
1α,24(S)-dihydroxyvitamin D2: a biologically active product of 1α-hydroxyvitamin D2 made in the human hepatoma, Hep3B
Authors: Strugnell S , Byford V , Makin H L J , Moriarty R M , Gilardi R , LeVan L W , Knutson J C , Bishop C W , Jones G
Published: August 2015

A major metabolite of the vitamin D analogue 1 alpha-hydroxyvitamin D2 in human liver cells in culture has been identified as 1 alpha,24(S)-dihydroxyvitamin D2 [1 alpha,24(S)-(OH)2D2]. 1 alpha-Hydroxyvitamin D3 incubated with the same cells gives rise to predominantly 25- and 27-hydroxylated products. Our identification of 1 alpha,24(S)-dihydroxyvitamin D2 is based on comparisons of the liver cell metabolite with chemically synthesized 1 alpha,24(S)-(OH)2D2 and 1 alpha,24(R)-(OH)2D2 by using HPLC, GC and GC-MS techniques. The stereochemical orientation of the 24-hydroxyl group was inferred after X-ray-crystallographic analysis of the 24(R)-OH epimer. 1 alpha,24(S)-Dihydroxyvitamin D2 binds strongly to the vitamin D receptor and is biologically active in growth hormone and chloramphenicol acetyltransferase reporter gene expression systems in vitro, but binds poorly to rat vitamin D-binding globulin, DBP. We suggest that this metabolite, 1 alpha,24(S)-(OH)2D2, possesses the spectrum of biological properties to be useful as a drug in the treatment of psoriasis, metabolic bone disease and cancer.
Structural analysis of inositol phospholipids from Trypanosoma cruzi epimastigote forms
Authors: Bertello L E , Gonçalvez M F , Colli W , de Lederkremer R M
Published: August 2015

Inositol phospholipids (IPL) from epimastigote forms of Trypanosoma cruzi have been investigated by metabolic labelling with [3H]palmitic acid and by GLC-MS analysis of the lipids obtained from non-labelled parasites. The IPL fraction was separated into phosphatidylinositol (PI) and inositol-phosphoceramide subfractions, the latter accounting for 80-85% of the total IPL. The neutral lipids released from the IPLs by PI-specific phospholipase C (PI-PLC) from Bacillus thuringiensis were analysed by silica-gel and reverse-phase TLC for the radioactive lipids and by GLC-MS for the non-radioactive samples. Ceramides containing dihydrosphingosine and sphingosine with C16:0 and C18:0 fatty acids were identified. The main component in the [3H]palmitic acid-labelled ceramides was palmitoyldihydrospingosine, while in the non-labelled sample the ceramides contained mainly sphingosine. This could reflect partial uptake of phospholipid from the medium. The PI contain both alkylacyl- and diacyl-glycerol lipids, with the ether lipid being more abundant. The latter was identified as 1-O-hexadecylglycerol esterified by C18:2 and C18:1 fatty acids. Interestingly, the same lipid had been identified in the anchor of the 1G7 glycoprotein of T. cruzi metacyclic forms.
Elimination of glycosylation heterogeneity affecting heparin affinity of recombinant human antithrombin III by expression of a β-like variant in baculovirus-infected insect cells
Authors: Ersdal-Badju E , Lu A , Peng X , Picard V , Zendehrouh P , Turk B , Björk I , Olson S T , Bock S C
Published: August 2015

In order to promote homogeneity of recombinant antithrombin III interactions with heparin, an asparagine-135 to alanine substitution mutant was expressed in baculovirus-infected insect cells. The N135A variant does not bear an N-linked oligosaccharide on residue 135 and is therefore similar to the beta isoform of plasma antithrombin. Purified bv.hat3.N135A is homogeneous with respect to molecular mass, charge and elution from immobilized heparin. Second-order rate constants for thrombin and factor Xa inhibition determined in the absence and presence of heparin are in good agreement with values established for plasma antithrombin and these enzymes. Based on far- and near-UV CD, bv.hat3.N135A has a high degree of conformational similarity to plasma antithrombin. Near-UV CD, absorption difference and fluorescence spectroscopy studies indicate that it also undergoes an identical or very similar conformational change upon heparin binding. The Kds of bv.hat3.N135A for high-affinity heparin and pentasaccharide were determined and are in good agreement with those of the plasma beta-antithrombin isoform. The demonstrated similarity of bv.hat3.N135A and plasma antithrombin interactions with target proteinases and heparins suggest that it will be a useful base molecule for investigating the structural basis of antithrombin III heparin cofactor activity.
Time-course studies by neutron solution scattering and biochemical assays of the aggregation of human low-density lipoprotein during Cu2+-induced oxidation
Authors: Meyer D F , Mayans M O , Groot P H E , Suckling K E , Bruckdorfer K R , Perkins S J
Published: August 2015

The oxidative modification of low-density lipoproteins (LDL) is recognized to be a key event in the development of atherosclerotic plaques on artery walls. The characteristics of LDL oxidized by cells of the artery wall can be imitated by the addition of Cu2+ ions to initiate lipid peroxidation in LDL. Neutron scattering of LDL in 2H2O buffers enables the time course of changes in the gross structure of LDL during oxidation to be continuously monitored under conditions close to physiological. Oxidation of LDL [2 mg of apolipoprotein B (apoB) protein/ml] was studied in the presence of 6.4, 25.6 and 51.2 mumol of Cu2+/g of apoB by incubation at 37 degrees C for up to 70 h. Neutron Guinier analyses showed that the radius of gyration RG (indicative of size) and the forward-scattered intensity at zero angle I(0) (indicative of M(r)) continuously increased during oxidation, indicating that LDL had aggregated. Both the rate of aggregation and the change in RG and I(0) values after 10 and 50 h increased with Cu2+ concentration. Distance-distribution functions P(r) showed that, within 4 h, the maximum dimension of LDL increased from 23 to 55 nm. The P(r) curves of oxidatively modified LDL exhibited two peaks at 10-12 nm and 26 nm. The 10-12 nm peak corresponds to native LDL, and the 26 nm peak is assigned to the initial formation of LDL dimers and trimers and their progression to form higher oligomers. The growth of the 26 nm peak depended on Cu2+ concentration. Particle-size-distribution functions Dv(r) suggested that the polydisperse spherical structure of LDL ceased to exist after 30 h, at which point the LDL samples underwent a phase separation. Related, but not identical, changes in the I(Q) and P(r) curves were observed when native LDL was self-aggregated by brief vortexing. Parallel assessment of LDL protein modification by SDS/PAGE showed increased aggregation and degradation of apoB with increased Cu2+ concentrations, and that the main apoB protein band had diminished after 2-8 h, depending on the amount of Cu2+ added. The uptake and degradation of oxidized 125I-labelled LDL by mouse peritoneal macrophages occurred maximally within the first 10 h, and increased in proportion to the Cu2+ concentration. ApoB protein broke down within the first 10 h of oxidation, and this is the period when scavenger receptors on macrophages can recognize and internalize oxidized LDL. Within 10 h, the protein-lipid interactions responsible for the spherical LDL structure became destabilized by protein fragmentation.(ABSTRACT TRUNCATED AT 400 WORDS)
Molecular organization of the interferon γ-binding domain in heparan sulphate
Authors: Lortat-Jacob H , Turnbull J E , Grimaud J A
Published: August 2015

Interferon (IFN)-gamma, in common with a number of cytokines or growth factors, strongly interacts with heparan sulphate (HS). It has been shown previously that one of the C-terminal basic clusters of amino acids (a regulatory element of IFN-gamma activity) is involved in this interaction. The structural organization of the HS domain that binds to human IFN-gamma has been investigated here. IFN-gamma-affinity chromatography of HS oligosaccharides released by either enzymic or chemical cleavage showed that the binding site is not found in a domain that is resistant to either heparinase or heparitinase or exclusively N-sulphated or N-acetylated. This led us to take a ‘footprinting’ approach in which HS was depolymerized in the presence of IFN-gamma and the cytokine-protected sequences were separated from the digested fragments. Using this strategy we consistently isolated an IFN-gamma-protected domain (IPD; approx. 10 kDa) which displayed the same affinity as full-length HS for the cytokine. Treatment of IPD with either heparinase or heparitinase strongly reduced its affinity, confirming that the high-affinity binding site encompassed a mixture of HS structural domains. Patterns of depolymerization with either enzymic or chemical agents were consistent with IPD being composed of an extended internal domain (approx. 7 kDa) which is predominantly N-acetylated and GlcA-rich, flanked by small N-sulphated oligosaccharides (mainly hexa- to octasaccharides). This is the first description of an HS protein-binding sequence with this type of molecular organization. Furthermore, using a cross-linking strategy, we demonstrated that one HS molecule bound to an IFN-gamma dimer. Together these results lead us to propose a novel model for the interaction of HS with a protein, in which two sulphated terminal sequences of the binding domain interact directly with the two IFN-gamma C-termini and bridge the two cytokine monomers through an internal N-acetyl-rich sequence.
Recombinant soluble human tissue factor secreted by Saccharomyces cerevisiae and refolded from Escherichia coli inclusion bodies: glycosylation of mutants, activity and physical characterization
Authors: Stone M J , Ruf W , Miles D J , Edgington T S , Wright P E
Published: August 2015

Tissue factor (TF) is the cell-surface transmembrane receptor that initiates both the extrinsic and intrinsic blood coagulation cascades. The abilities of TF to associate with Factor VIIa and Factor X in a ternary complex and to enable proteolytic activation of Factor X by Factor VIIa reside in the extracellular domain of TF. We describe the expression of the surface domain of TF (truncated TF, tTF) in both Saccharomyces cerevisiae and Escherichia coli and the biochemical and physical characterization of the recombinant proteins. Wild-type tTF and several glycosylation-site mutants were secreted efficiently by S. cerevisiae under the control of the yeast prepro-alpha-signal sequence; the T13A,N137D double mutant was the most homogeneous variant expressed in milligram quantities. Wild-type tTF was expressed in a non-native state in E. coli inclusion bodies as a fusion protein with a poly(His) leader. The fusion protein could be fully renatured and the leader removed by proteolysis with thrombin; the correct molecular mass (24,729 Da) of the purified protein was confirmed by electrospray mass spectrometry. Recombinant tTFs from yeast, E. coli and Chinese hamster ovary cells were identical in their abilities to bind Factor VIIa, to enhance the catalytic activity of Factor VIIa and to enhance the proteolytic activation of Factor X by Factor VIIa. Furthermore, CD, fluorescence emission and NMR spectra of the yeast and E. coli proteins indicated that these proteins are essentially identical structurally.