bluuebunny/crossref_metadata_2025 huggingface dataset:
Characterization of phosphorylation sites in the cytoplasmic domain of the 300 kDa mannose-6-phosphate receptor
Authors: Rosorius O , Mieskes G , Issinger O G , Körner C , Schmidt B , von Figura K , Braulke T
Published: August 2015

The human 300 kDa mannose-6-phosphate receptor (MPR 300) is phosphorylated in vivo at serine residues of its cytoplasmic domain. Two-dimensional separation can resolve tryptic phosphopeptides into four major species. To identify the kinases involved in MPR 300 phosphorylation and the phosphorylation sites the entire coding sequence of the cytoplasmic tail was expressed in Escherichia coli. The isolated cytoplasmic domain was used as a substrate for four purified serine/threonine kinases [casein kinase II (CK II), protein kinase A (PKA), protein kinase C and Ca2+/calmodulin kinase]. All kinases phosphorylate the cytoplasmic tail exclusively on serine residues. Inhibition studies using synthetic peptides, partial sequencing of isolated tryptic phosphopeptides and co-migration with tryptic phosphopeptides from MPR 300 labelled in vivo showed that (i) PKA phosphorylates the cytoplasmic MPR 300 domain at Ser20 and at a non-identified site, neither of which are phosphorylated in vivo, and that (ii) the two sites phosphorylated by CK II in vivo and in vitro are Ser82 and Ser157. The results indicate that the human MPR 300 is a physiological substrate of either CK II or a related kinase which may play a role in the transport function of MPR 300 and/or interaction with other proteins.
Ligand-induced conformation change in folate-binding protein
Authors: Kaarsholm N C , Kolstrup A M , Danielsen S E , Holm J , Hansen S I
Published: August 2015

C.d. and fluorescence spectroscopy have been used to investigate the effect of ligand binding on the structure and stability of folate-binding protein (FBP) from cow's whey. The c.d. spectrum of unligated FBP predicts the following secondary structure: 22% helix, 25% antiparallel beta-strand, 5% parallel beta-strand, 17% turn and 31% random-coil structure. Folate binding to FBP results in significant changes in the c.d. spectrum. Analysis of the spectrum shows a 10% decrease in antiparallel beta-strand as a result of ligand binding. Folate binding also leads to strong quenching of FBP tryptophan fluorescence. The magnitude of the quench is proportional to ligand binding. The guanidinium chloride-induced unfolding of FBP is shown to be a multistate process. Detection by c.d. and fluorescence spectroscopy lead to non-identical transitions. Modelling studies are consistent with the existence of a stable folding intermediate. Ligand binding to FBP increases the apparent folding stability of the molecule. Simultaneous detection by c.d. and fluorescence indicate that the apparent increased folding stability is derived from ligand-induced aggregation of FBP.
The cerebrospinal-fluid soluble form of Alzheimer's amyloid β is complexed to SP-40,40 (apolipoprotein J), an inhibitor of the complement membrane-attack complex
Authors: Ghiso J , Matsubara E , Koudinov A , Choi-Miura N H , Tomita M , Wisniewski T , Frangione B
Published: August 2015

The amyloid fibrils deposited in Alzheimer's neuritic plaque cores and cerebral blood vessels are mainly composed of aggregated forms of a unique peptide, 39-42 amino acids long, named amyloid beta (A beta). A similar, although soluble, A beta (‘sA beta’) has been identified in cerebrospinal fluid, plasma and cell supernatants, indicating that it is normally produced by proteolytic processing of its precursor protein, amyloid precursor protein (APP). Using direct binding experiments we have isolated and characterized an 80 kDa circulating protein that specifically interacts with a synthetic peptide identical with A beta. The protein was unmistakably identified as SP-40,40 or ApoJ, a cytolytic inhibitor and lipid carrier, by means of amino acid sequence and immunoreactivity with specific antibodies. Immunoprecipitation with anti-SP-40,40 retrieved soluble A beta from cerebrospinal fluid, indicating that the interaction occurs in vivo.
Cyclic AMP-dependent protein kinase phosphorylates rabbit reticulocyte elongation factor-2 kinase and induces calcium-independent activity
Authors: Redpath N T , Proud C G
Published: August 2015

The catalytic subunit of cyclic AMP-dependent protein kinase (PKA) phosphorylated purified calcium/calmodulin-dependent eukaryotic elongation factor-2 (eEF-2) kinase, isolated from rabbit reticulocyte lysates. It maximally incorporated about 1 mol of phosphate/mol of eEF-2 kinase. The Km of eEF-2 kinase for PKA was calculated to be 7 microM. Phosphorylation of eEF-2 kinase by PKA induced calcium-independent activity which amounted to 40-50% of the total activity measured in the presence of calcium. Furthermore, the level of calcium-independent activity induced by phosphorylation by PKA was similar to that induced by the calcium-stimulated autophosphorylation of eEF-2 kinase. Phosphopeptide mapping of eEF-2 kinase labelled by autophosphorylation and by PKA revealed a number of common phosphopeptides. This suggests that PKA may phosphorylate the same site(s) which are phosphorylated autocatalytically and which are responsible for the induction of calcium-independent activity. The possible implications these findings have for the control of translation are discussed.
Evidence for a transient inhibitory effect of insulin on GLUT2 expression in the liver: studies in vivo and in vitro
Authors: Postic C , Burcelin R , Rencurel F , Pegorier J P , Loizeau M , Girard J , Leturque A
Published: August 2015

The glucose transporter GLUT2 is expressed predominantly in the liver. Previous studies have shown that glucose increases GLUT2 mRNA concentration in primary cultures of rat hepatocytes. Since insulin controls the glucose metabolism in the liver, it could be involved in the regulation of GLUT2 gene expression. In vivo, hyperinsulinaemia induced a transient inhibitory effect on liver GLUT2 gene expression, the maximal inhibition of GLUT2 mRNA concentration (93 +/- 6%) being observed after 6 h. When hyperglycaemia was associated with hyperinsulinaemia, the decrease in liver GLUT2 mRNA concentration was partially prevented. The respective effects of glucose and insulin were studied in vitro by primary culture of rat hepatocytes. Insulin alone exerted a transient inhibitory effect on GLUT2 mRNA concentration. When insulin and glucose (10-20 mM) were associated, the stimulatory effect of glucose on GLUT2 gene expression was predominant. In conclusion, the present study shows that GLUT2 mRNA concentration was conversely regulated by insulin and glucose, both in vitro and in vivo.
Structure of the murine rab3A gene: correlation of genomic organization with antibody epitopes
Authors: Baumert M , Fischer von Mollard G , Jahn R , Südhof T C
Published: August 2015

Rab3A is a neuronal low-molecular-mass GTP-binding protein that is modified post-translationally by two geranylgeranyl groups and specifically targeted to synaptic vesicles. We have now cloned and characterized the murine gene coding for rab3A. With a size of less than 8 kb including the promoter, the rab3A gene is relatively small. It contains five exons, the first of which is non-coding. The organization of the rab3A coding sequence into exons in the gene is different from that of ras proteins, the only other low-molecular-mass GTP-binding proteins with currently characterized gene structures. Nevertheless, the intron placement in the primary structure of rab3A may be indicative of a domain division of the protein, since each coding exon contains one of the four major conserved rab protein sequence motifs. The epitopes of monoclonal and polyclonal antibodies to rab3A were mapped with the hypothesis that antibody epitopes might represent distinct exposed protein domains and correlate with exon structures. Two monoclonal antibodies, named 42.1 and 42.2, were found to recognize epitopes with a different degree of conservation between different rab3 isoforms. These epitopes were mapped to relatively short amino acid sequences corresponding to exons 4 and 5 respectively, whereas a polyclonal antibody recognized a complex epitope that required the presence of intact rab3A. Comparison of the sequence of rab3A with that of ras, whose crystal structure has been determined, revealed that the epitopes for the monoclonal antibodies correspond to regions in ras that are highly exposed. Taken together, these results suggest that exons 4 and 5 at least represent distinct exposed protein domains that also form major natural epitopes in rab3A.
Requirement of the C-terminal proline residue for stability of the Ca2+-activated photoprotein aequorin
Authors: Watkins N J , Campbell A K
Published: August 2015

cDNA coding for the Ca(2+)-activated photoprotein aequorin from the jellyfish Aequorea victoria has been engineered to investigate the role of the C-terminal proline residue in bioluminescence. Recombinant aequorin proteins were synthesized by PCR followed by in vitro transcription/translation, and characterized by specific activity, stability, and affinity for coelenterazine. The C-terminal proline residue of aequorin was shown to be essential for the long-term stability of the bound coelenterazine. Aequorin minus proline had only 1% of the specific activity of the wild-type after 2 h, and was virtually inactive after 18 h. The instability of this variant was further demonstrated by re-activating with a coelenterazine analogue (epsilon-coelenterazine), where maximum reactivation was reached in 15 min, and the luminescent activity was almost completely abolished within 3 h. Replacement of the C-terminal proline residue with histidine or glutamic acid decreased the specific activity to 10 and 19% of that of the wild-type respectively. However these variants were also unstable, having t1/2 values of 2.4 h and 2.3 h respectively. Enhancement of the Ca(2+)-independent light emission when proline was replaced by histidine confirmed the stabilizing role of the C-terminal proline. No significant effect of removal of the C-terminal proline was detected on the affinity for coelenterazine.
Cytosolic phospholipase A2 from human monocytic cells: characterization of substrate specificity and Ca2+-dependent membrane association
Authors: Rehfeldt W , Resch K , Goppelt-Struebe M
Published: August 2015

A cytosolic phospholipase A2 (PLA2) has been purified to homogeneity from the human monocytic tumour cell line THP-1 by a combination of ion-exchange, heparin-agarose and hydrophobic-interaction chromatography and non-denaturing PAGE. The purified PLA2 exhibited a molecular mass of 100 kDa on SDS/PAGE, whereas non-denaturing PAGE showed a molecular mass of about 80 kDa. Investigation of the substrate specificity of PLA2 revealed no preference for arachidonic acid- over linoleic acid-containing phospholipids when labelled THP-1 membranes were used as substrate. Treatment of THP-1 cells for 1-72 h with the phorbol ester phorbol 12-myristate 13-acetate (PMA) led to a stable enzyme activation, which was also found after partial purification of PLA2 from PMA-stimulated THP-1 cells. The properties of the partially purified PLA2 from PMA-stimulated cells were compared with those of the PLA2 from unstimulated cells. Both enzymes showed an identical Ca(2+)-dependent enzyme activation (EC50 = 300 nM Ca2+). A significant difference was found in the Ca(2+)-dependent membrane association of the enzymes obtained from the two sources. PLA2 from PMA-stimulated cells showed membrane association of PLA2 activity at significantly lower Ca2+ concentrations (100-300 nM) compared with the PLA2 from unstimulated THP-1 cells (300-500 nM). The altered Ca(2+)-dependency was due to a stable modification of the enzyme. Changes in the ability of PLA2 to associate with cellular membranes may thus be regarded as an important molecular mechanism underlying the increase in fatty acid metabolism observed in activated cells.
Rapid activation of hepatic glutaminase in rats fed on a single high-protein meal
Authors: Ewart H S , Brosnan J T
Published: August 2015

We report that hepatic glutaminase is rapidly activated in rats fed on a single high-protein (60% casein) meal. Rats previously fed on a normal-protein (15% casein) diet for 3-4 days were given a high-protein meal for 2 h. The high-protein meal increased the rate of flux through glutaminase in intact liver mitochondria nearly 3-fold (20.6 +/- 1.7 nmol/min per mg of protein versus 7.5 +/- 2.9 nmol/min per mg of protein) at a P(i) concentration of 10 mM. The activation of flux through glutaminase by a high-protein meal involved an increased sensitivity of glutaminase to P(i), an activator of the enzyme. The Ka for P(i) was 1.0 mM and 24.1 mM in mitochondria from rats fed on the high-protein and normal-protein meals respectively. We measured the concentration of P(i) in the mitochondrial matrix and found that it did not differ in mitochondria from rats fed on the high-protein and normal-protein meals, suggesting that the effect of the high-protein meal on the P(i)-sensitivity of glutaminase was not due to a change in the distribution of P(i) across the mitochondrial inner membrane. Glutaminase activity was measured by using mitochondrial membranes from frozen-thawed mitochondria. Glutaminase activity and its dependence on P(i) were similar for preparations from rats fed on high-protein and normal-protein meals. These findings show that hepatic glutaminase is stimulated rapidly by a high-protein meal. This is part of the physiological hepatic response to increased protein intake which permits the liver to cope with the influx of glutamine occurring at this time.
Trichomonas vaginalis: characterization of ornithine decarboxylase
Authors: Yarlett N , Goldberg B , Moharrami M A , Bacchi C J
Published: August 2015

Ornithine decarboxylase (ODC), the lead enzyme in polyamine biosynthesis, was partially purified from Trichomonas vaginalis and its kinetic properties were studied. The enzyme appears to be of special significance in this anaerobic parasite, since the arginine dihydrolase pathway generates ATP as well as putrescine from arginine. ODC from T. vaginalis had a broad substrate specificity, decarboxylating ornithine (100%), lysine (1.0%) and arginine (0.1%). The enzyme had a pH optimum of 6.5, a temperature optimum of 37 degrees C and was pyridoxal 5′-phosphate-dependent. Attempts to separate ornithine- from lysine-decarboxylating activity by thermal-stability and pH-optima curves were not successful. Although Km values for ornithine and lysine were 109 and 91 microM respectively, and the Vmax values for these substrates were 1282 and 13 nmol/min per mg of protein respectively, the most important intracellular substrate is ornithine, since intracellular ornithine levels are 3.5 times those of lysine and extracellular putrescine levels are 7.5 times those of cadaverine. Ornithine was also an effective inhibitor of lysine-decarboxylating activity (Ki 150 microM), whereas lysine was relatively ineffective as inhibitor of ornithine-decarboxylating activity (Ki 14.5 mM). Crude ODC activity was localized (86%) in the 43,000 g supernatant and 3303-fold purification was obtained by (NH4)2SO4 salting and DEAE-Sephacel, agarose-gel and hydroxyapatite chromatography steps. The enzyme bound difluoro[3H]methylornithine ([3H]DFMO) with a ratio of drug bound to activity of 2500 fmol/unit, where 1 unit corresponds to 1 nmol of CO2 released from ornithine/min. The enzyme had a native M(r) of 210000 (gel filtration), with a subunit M(r) of 55,000 (by SDS/PAGE), suggesting that the trichomonad enzyme is a tetramer. From the subunit M(r) and binding ratio of DFMO, there is about 137 ng of ODC per mg of T. vaginalis protein (0.013%). The significant amount of ODC protein present supports the view that putrescine synthesis in T. vaginalis plays an important role in the metabolism of the parasite.
Purification, characterization and binding interactions of the Chinese-cobra (Naja naja atra) serum antitoxic protein CSAP
Authors: Shao J , Shen H , Havsteen B
Published: August 2015

The characterization of the single-chain protein in Chinese-cobra (Naja naja atra) blood serum, which yields strong specific protection against the venom of the same snake, is reported. The protein, CSAP (cobra serum antitoxic protein), was purified to electrophoretic homogeneity. Over the pH range 5-9 it formed stable complexes with the neuro- and the cardio-toxin of the snake. The molecular size of the CSAP was estimated to be 70.3 +/- 0.3 kDa. Tryptic hydrolysis of CSAP yielded several peptides that were able to bind to the toxin. The native CSAP maximally bound 8 +/- 1 toxin molecules/molecule. Six tryptic fragments, containing 5-39 residues, were sequenced. The longest of these displayed sequence similarity to rat serum albumin. The protective effect of the CSAP was demonstrated in vivo on mice and in vitro by measurement of the rate of haemolysis. Kinetic and thermodynamic parameters of the binding interactions of the neurotoxin and the CSAP were determined from the rates of displacement of 125I-labelled toxin from its complexes with the CSAP by unlabelled toxin by using a DEAE-cellulose filter assay for CSAP-toxin complexes. The toxin molecules rapidly dissociated from one type of site and slowly from a second. The binding capacity and concentration of the CSAP suffice to explain the protective effect of the latter against the toxin.
Binding of a substrate analogue can induce co-operative structure in the plasmin serine-proteinase domain
Authors: Teuten A J , Cooper A , Smith R A , Dobson C M
Published: August 2015

Human miniplasminogen and miniplasmin were studied by n.m.r. spectroscopy and differential scanning calorimetry (d.s.c.) in order to investigate the structural properties of the serine-proteinase domain. The d.s.c. thermograms of both miniplasminogen and non-inactivated miniplasmin at pH 4.0 can be closely fitted to two transitions, at 62 +/- 2 and 72 +/- 2 degrees C, corresponding to unfolding of the kringle 5 and proteinase domains respectively. No evidence was found, under these conditions, for non-co-operative unfolding of the proteinase domain. On inactivation of miniplasmin with an affinity label, a number of additional resonances arising from residues of the proteinase domain are observed in resolved regions of the n.m.r. spectrum. A combination of variable-temperature n.m.r. and d.s.c. has shown that part of the proteinase domain undergoes a major conformational transition on heating which is distinct from the unfolding of the remainder of the proteinase domain or the kringle 5 domain. This additional transition occurs at a temperature that depends on the nature of the affinity label present and is not observed in the absence of an inactivating agent. These results provide direct evidence for the existence of a region of the proteinase domain which, under these conditions, becomes structured only in the presence of a bound substrate.
Radical-induced chain oxidation of proteins and its inhibition by chain-breaking antioxidants
Authors: Neužil J , Gebicki J M , Stocker R
Published: August 2015

Exposure of proteins to oxygen-centred radicals results in dramatic changes in their structure, stability and function; properties that have been studied in many laboratories from a qualitative viewpoint. To allow a quantitative evaluation, we subjected aerated solutions of BSA to hydroxyl and superoxide anion radicals generated radiolytically under conditions where all radicals formed reacted with the protein (as judged by maximum damage to BSA). We observed that for each radical generated approx. 15 amino acids were consumed initially. Similar results were found with lysozyme and melittin. Such a massive consumption of BSA's amino acids was not observed when irradiations were carried out under anaerobic conditions. When bilirubin or Trolox (a water-soluble analogue of vitamin E) was added at a 2-fold molar excess over BSA, initial consumption of all measured amino acids, except tryptophan, decreased 4-fold. The total mass of amino acids initially protected (from consumption) exceeded the mass of antioxidants consumed by more than a factor of 20. Such protection of amino acids was not observed when the antioxidant-inactive acetyl Trolox was used. These results suggest that radical-mediated oxidation of proteins can proceed via a previously unrecognized chain reaction that may be inhibited by chain-breaking antioxidants.
DNA substrate specificity of pea DNA methylase
Authors: Houlston C E , Cummings M , Lindsay H , Pradhan S , Adams R L P
Published: August 2015

DNA methylase, present in low-salt extracts of nuclei prepared from Pisum sativum shoot tips, methylates model DNA substrates containing CNG trinucleotides or CI dinucleotides only. The binding to the hemimethylated trinucleotide substrates is very much stronger and more persistent than the binding to the unmethylated substrates or to the hemimethylated dinucleotide substrate. When the DNA concentration is limiting, the rate of methyl-group transfer with the hemimethylated CNG substrate is much greater than that with the unmethylated CNG. However, the Vmax. is similar for the two CNG substrates. On fractionation using Q-Sepharose, two peaks of activity are seen with different relative activities using the di- and trinucleotide substrates. The relative activity with these substrates changes during purification, during plant growth and on heating at 35 degrees C as well, indicating that more than one enzyme or more than one form of the enzyme may be present.
The role of the 5′-flanking region in the cell-specific transcription of the human von Willebrand factor gene
Authors: Ferreira V , Assouline Z , Schwachtgen J L , Bahnak B R , Meyer D , Kerbiriou-Nabias D
Published: August 2015

Transcriptional regulation of the human von Willebrand factor (vWF) gene was investigated in calf pulmonary artery endothelial (CPAE), HeLa, COS 7 and Hep G2 cells. Various lengths of flanking sequences extending up to 2123 bp 5′ of the transcription initiation site and containing 19 bp of the first exon, were linked to the bacterial chloramphenicol acetyltransferase (CAT) gene and these constructs were assayed in transient transfection assays. Sequences up to 89 bp upstream of the cap site showed transcriptional activity in all cell types. Sequences between -147 and -419 bp markedly reduced CAT activity in CPAE cells and abolished it in other cell lines. A domain from -592 to -810 bp generated low levels of expression only in CPAE cells. This transcriptional activity was repressed with constructs containing 1041 to 1240 bp upstream of the cap site. Endothelial cell-specific transcription was restored by a construct that contained 1286 bp upstream of the cap site. The additional 46 bp upstream of the negative regulatory domain were within the 5′ end of an inverse human Alu-family DNA repeat. RNAase-protection assays confirmed the correct transcriptional initiation. The sequence between -89 and -420 contained at least one negative regulatory element able to repress the CAT gene expression controlled by the heterologous thymidine kinase promoter in all cell types. A construct that included the sequence from -89 to -1286 bp increased the transcriptional activity directed by the thymidine kinase promoter only in CPAE cells. These results indicate that negative and positive elements in the 5′-flanking region interact to regulate vWF gene expression.
Correlation between secretion and phospholipase D activation in differentiated HL60 cells
Authors: Stutchfield J , Cockcroft S
Published: August 2015

Receptor-directed agonists including N-formylmethionyl-leucyl-phenylalanine (fMetLeuPhe), C5a, ATP and UTP all activate phospholipase D (PLD), which is accompanied by secretion in differentiated HL60 cells. Interference in the production of phosphatidase (PA) by the PLD pathway by diverting it towards the production of phosphatidylethanol (PEt) in the presence of ethanol leads to near-total inhibition of the secretion evoked by ATP and UTP and a partial inhibition of that evoked by fMetLeuPhe and C5a. In streptolysin-O-permeabilized cells, fMetLeuPhe is able to activate PLD, and this is dependent on the presence of a low concentration of guanosine 5′-[gamma-thio]-triphosphate (GTP[S]). Ca2+ (10 microM) and GTP[S] individually or in combination are also able to activate PLD and secretion. The stimulation of secretion in permeabilized cells stimulated by Ca2+ alone or fMetLeuPhe or GTP[S] is also abrogated when the production of PA is diverted to PEt by the presence of ethanol. Activation of PLD by GTP[S] or fMetLeuPhe is decreased if the cells are permeabilized first and GTP[S] or fMetLeuPhe is added subsequently. This corresponds well with the loss of the secretory response. We conclude that the ability of GTP[S] or fMetLeuPhe to stimulate secretion from permeabilized cells is dependent on a prior activation of the PLD signalling pathway. PA, generated as a consequence of PLD activation, acts as second messenger that can provide an initiating signal for secretion and is not required for exocytosis itself.
Biosynthesis of 3,4-didehydroretinol from retinol by human skin keratinocytes in culture
Authors: Rollman O , Wood E J , Olsson M J , Cunliffe W J
Published: August 2015

The uptake and metabolism of radiolabelled retinol was studied in cultivated human skin cells. Normal epidermal keratinocytes in primary culture were able to incorporate unbound [11,12-3H]all-trans-retinol from the growth medium and transform it into 3,4-didehydroretinol (dehydroretinol) in a dose- and time-dependent manner. A total of 23% of the radioactive label became cell-associated during a 48-h incubation period when added at 7 nM to differentiated keratinocytes submerged in serum-containing, high-calcium (1.56 mM) culture medium. At that time point, 25-30% of cell-bound radioactive retinol had been converted into dehydroretinol, with no labelled retinal, dehydroretinal, retinoic acid or dehydroretinoic acid being detected in cells or medium. Thus dehydroretinol, which occurs physiologically in mammalian skin tissue in vivo, was identified as the predominant neutral retinol metabolite in cultured keratinocytes using h.p.l.c. and anhydro-derivatization procedures. At least 94% of the product, along with its precursor, was present in the cells in esterified form, with no traces of the compound being secreted into the cell environment. The rate of formation of dehydroretinol from its precursor was significantly lower in keratinocytes grown in serum-free, low-calcium (0.09 mM) culture medium, and in medium pre-incubated with excess unlabelled substrate. Furthermore, the application of 13-cis-retinoic acid (isotretinoin), a therapeutic retinoid drug known to markedly reduce dehydroretinol levels in human skin, blocked the biosynthesis of this metabolite in cultured keratinocytes. The 3,4-dehydrogenation pathway observed in this study could not be shown to operate to any significant extent in cultures of human epidermal melanocytes or dermal fibroblasts, supporting the hypothesis that keratinocytes represent the principal cell type involved in dehydroretinol formation from retinol in human skin.
Nuclear import of the human androgen receptor
Authors: Jenster G , Trapman J , Brinkmann A O
Published: August 2015

Nuclear import of the human androgen receptor was investigated by immunocytochemical analysis of androgen receptor deletion and substitution mutants, which were transiently expressed in COS-1 cells. The signal responsible for nuclear import is encoded by amino-acid residues 608-625 and is functionally similar to the bipartite nucleoplasmin nuclear-localization signal. Although the subcellular distribution of androgen receptors mutated in the DNA-binding domain was unchanged compared with the wild-type androgen receptor, in the presence of ligand these mutations resulted in part of the receptor population forming clusters. Depending on the presence or absence of the bipartite nuclear localization signal, clusters were formed in the nucleus or in the cytoplasm, respectively. Expression of the wild-type androgen receptor in different cell lines revealed a cell-line-specific subcellular distribution of the unliganded receptor. The androgen receptor was predominantly nuclear when expressed in HeLa cells, whereas mainly cytoplasmic staining was observed when it was expressed in COS-1 cells. In the presence of hormone, the androgen receptor was located in the nucleus, independent of the cell line that was expressing the receptor. Anti-androgens and various steroid hormones induced the nuclear localization of the wild-type androgen receptor in a dose-dependent way, without activating transcription of an androgen-regulated reporter gene. This indicates that the inability of the tested compounds to activate transcription is not due to inhibited nuclear import.
Differences in expression of transcription factor AP-1 in human promyelocytic HL-60 cells during differentiation towards macrophages versus granulocytes
Authors: Mollinedo F , Gajate C , Tugores A , Flores I , Naranjo J R
Published: August 2015

Commitment of HL-60 cells to macrophage or granulocytic differentiation was achieved by incubation with 4 beta-phorbol 12-myristate 13-acetate (PMA) for 30-60 min or with dimethyl sulphoxide (DMSO) for 24 h respectively. The commitment stage towards PMA-induced macrophage differentiation was associated with increases in jun B and c-fos mRNA levels, as well as with an increase in the binding activity of transcription factor AP-1. Nevertheless, gel retardation analysis indicated that the AP-1 activity detected in untreated cells was drastically reduced during the commitment stage of DMSO-induced HL-60 differentiation towards granulocytes. When HL-60 cells were treated with sodium butyrate, which induced monocytic differentiation, a remarkable increase in AP-1 binding activity was detected. Treatment of HL-60 cells with 1 alpha,25-dihydroxyvitamin D3, another monocytic differentiation agent, induced a weak, but appreciable, increase in AP-1 activity. Furthermore, addition of sodium butyrate or 1 alpha,25-dihydroxyvitamin D3 to HL-60 cells induced the expression of c-fos, c-jun, jun B and jun D proto-oncogenes. In contrast, when HL-60 cells were treated with retinoic acid, a granulocytic differentiation inducer, no enhanced AP-1 binding activity was observed, and only a weak increase in jun D mRNA level was detected. These data indicate that formation of AP-1 is not required for the induction of HL-60 differentiation towards granulocytes, whereas induction of monocytic differentiation is correlated with an increase in AP-1 activity. The differential expression of AP-1 activity may be critical in the differentiation of HL-60 cells towards monocytic or granulocytic lineages.
Endothelin (ETA) receptor number and calcium signalling are up-regulated by protein kinase C-β 1 overexpression
Authors: Pachter J A , Mayer-Ezell R , Cleven R M , Fawzi A B
Published: August 2015

To evaluate the role of protein kinase C (PKC) in regulation of cellular responsiveness to mitogens, we used rat 6 (R6) fibroblasts that stably overexpress the beta 1 isoenzyme of protein kinase C (PKC-beta 1). The potent vasoconstrictor and mitogen endothelin-1 (ET-1; 100 nM) was substantially more effective in stimulating InsP3 accumulation in PKC-beta 1-overexpressing fibroblasts (PKC3 cells) than in control fibroblasts lacking the PKC-beta 1 cDNA insert. PKC3 cells were found to express a 7-fold greater number of endothelin receptors than did control cells, whereas both cell lines showed equivalent Kd values. These receptors were of the ETA subtype, as defined by a 1000-fold greater affinity for ET-1 than for ET-3. Changes in intracellular free Ca2+ levels ([Ca2+]i) in response to ET-1 measured with the fluorescent Ca2+ indicator fura-2 showed that ET-1 was more potent and efficacious in stimulating [Ca2+]i in PKC3 cells than in control fibroblasts. The ET-1-induced Ca2+ rise was completely blocked by the selective ETA antagonist BQ123, but only slightly diminished by extracellular application of 2 mM EGTA. In contrast with the effects of PKC-beta 1 overexpression on responsiveness to ET-1, alpha-thrombin, which was previously found to have a weaker effect on InsP3 accumulation in PKC-beta 1-overexpressing cells, was also a less effective stimulator of [Ca2+]i in PKC3 cells than in control cells. These results demonstrate that, although the Ca2+ response to alpha-thrombin is diminished by PKC-beta 1 overexpression, ETA receptor number and cellular responsiveness to ET-1 are increased in PKC-beta 1-overexpressing cells.
Investigations into the post-translational modification and mechanism of isopenicillin N:acyl-CoA acyltransferase using electrospray mass spectrometry
Authors: Aplin R T , Baldwin J E , Roach P L , Robinson C V , Schofield C J
Published: August 2015

Electrospray mass spectrometry (e.s.m.s.) was used to confirm the position of the post-translational cleavage of the isopenicillin N:acyl-CoA acyltransferase preprotein to give the alpha- and beta-subunits. The e.s.m.s. studies suggested partial modification of the alpha-subunit in vivo by exogenously added substituted acetic acids. E.s.m.s. has also allowed the observation in vitro of the transfer of the acyl group from several acyl-CoAs to the beta-subunit. N.m.r. data for the CoA species have been deposited as Supplementary Publication SUP 500173 (2 pages) at the British Library Document Supply Centre (DSC), Boston Spa, Wetherby, West Yorkshire LS23 7BQ, from whom copies can be obtained on the terms indicated in Biochem. J. (1993) 289, 9.
Identification of a putative tyrosine-O-sulphate (TyrS) receptor possibly functioning in the biosynthetic transport of tyrosine-sulphated proteins in Madin-Darby canine kidney cells
Authors: Liu J , Han J R , Liu C C , Suiko M , Liu M C
Published: August 2015

By employing an affinity-gel fractionation technique coupled to Western-blot analysis, we have identified a 175 kDa tyrosine-O-sulphate (TyrS)-binding protein present in Madin-Darby canine kidney (MDCK) cells. The binding of this TyrS-binding protein to TyrS covalently bonded to Sepharose gel was found to be pH-dependent, being strong from pH 8.0 down to pH 6.5 and increasingly weak at pH 6.0 and below. Results obtained from Triton X-114 temperature-induced phase separation and sodium carbonate buffer (pH 11) extraction experiments indicated that the TyrS-binding protein is an integral membrane protein. This 175 kDa TyrS-binding protein was found to be present in association with a major tyrosine-sulphated protein, the apically secreted 80 kDa glycoprotein (gp 80), in cell lysate prepared from MDCK cells maintained under normal growth conditions. When the cell lysate used was prepared from MDCK cells pretreated with 20 mM sodium chlorate, a metabolic sulphation inhibitor, the complex formed between the two proteins could no longer be detected, indicating that the binding of the TyrS-binding protein is through the TyrS residue(s) of gp 80. Both cell-surface biotinylation and cell-surface trypsinization studies demonstrated the predominantly, if not exclusively, intracellular location of the TyrS-binding protein. Furthermore, radioactive pulse-chase experiments revealed that the newly synthesized radiolabelled fibronectin and gp 80 were present in complexes with the TyrS-binding protein in MDCK cells pulse-labelled with [35S]methionine or [35S]sulphate. Exogenous [35S]methionine-labelled gp 80 added to the medium, on the other hand, was not found to be present in association with the TyrS-binding protein in MDCK cells over a 2-h time course. These results strongly suggested the identity of the 175 kDa TyrS-binding protein as a putative ‘TyrS receptor’, possibly functioning in the biosynthetic transport of tyrosine-sulphated proteins in MDCK cells.
The C-terminal region of α-crystallin: involvement in protection against heat-induced denaturation
Authors: Takemoto L , Emmons T , Horwitz J
Published: August 2015

Recent studies have demonstrated that the alpha-crystallins can protect other proteins against heat-induced denaturation and aggregation. To determine the possible involvement of the C-terminal region in this activity, the alpha-crystallins were subjected to limited tryptic digestion, and the amount of cleavage from the N-terminal and C-terminal regions of the alpha-A and alpha-B crystallin chains was assessed using antisera specific for these regions. Limited tryptic digestion resulted in cleavage only from the C-terminal region of alpha-A crystallin. This trypsin-treated alpha-A crystallin preparation showed a decreased ability to protect proteins from heat-induced aggregation using an in vitro assay. Together, these results demonstrate that the C-terminal region of alpha-A crystallin is important for its ability to protect against heat-induced aggregation, which is consistent with the hypothesis that post-translational changes that are known to occur at the C-terminal region may have significant effects on the ability of alpha-A crystallin to protect against protein denaturation in vivo.
Interaction of CCAAT/enhancer-binding protein α and β with the rat caeruloplasmin gene promoter
Authors: Bingle C D , Fleming R E , Gitlin J D
Published: August 2015

To determine the mechanisms of expression of the rat caeruloplasmin gene, the promoter region was analysed by DNAase I footprinting. Using nuclear extract from rat liver, a prominent site of protein-DNA interaction was detected from -93 to -48 upstream of the caeruloplasmin gene transcription start and sequence analysis of this region revealed three potential CCAAT/enhancer-binding protein (C/EBP) consensus elements. Mobility-shift analysis using an oligonucleotide encoding this region identified specific binding of proteins from rat liver nuclear extract, and some of these complexes were supershifted using antisera to the C/EBP alpha and beta family members. Mobility-shift studies using a polypeptide encoding the DNA-binding domain of C/EBP alpha also revealed a specific interaction with this region of the caeruloplasmin promoter, and DNAase I footprinting using this polypeptide protected the identical region from -93 to -48. Co-transfection of expression plasmids encoding C/EBP alpha or a related leucine-zipper factor D-binding protein (DBP) revealed a C/EBP-specific increase in reporter gene activity in HepG2 cells transfected with caeruloplasmin-chloramphenicol acetyltransferase containing the -93 to -48 region. A similar result was obtained when these constructs were co-transfected into mouse L cells which were shown not to express the endogenous caeruloplasmin gene. Taken together, these data indicate a role for C/EBP alpha and beta in mediating transcription from the caeruloplasmin gene promoter and suggest that this region of the promoter is not responsible for tissue-specific expression.
Localization of the FK506-binding protein, FKBP 13, to the lumen of the endoplasmic reticulum
Authors: Nigam S K , Jin Y J , Jin M J , Bush K T , Bierer B E , Burakoff S J
Published: August 2015

The function of the immunophilins, FKBP 12 and FKBP 13, which are binding proteins for the immunosuppressant drug FK506 and rapamycin, remains poorly defined, although it has been suggested that immunophilins and immunophilin-like proteins may play a role in protein sorting/folding and intracellular calcium ion regulation. As a first step towards understanding the function of FKBP 13, we studied its subcellular localization by immunoblotting of well-defined subcellular fractions from a canine pancreatic homogenate and immunocytochemical analysis of an overexpressed cloned cDNA for FKBP 13. Whereas FKBP 12 fractionated entirely into the cytosol, virtually all FKBP 13 was found in the rough microsomal fraction which consisted of highly purified rough endoplasmic reticulum (ER), along with several well-characterized ER markers [the immunoglobulin heavy-chain binding protein (BiP), grp 94 and ribophorin I]. Moreover, FKBP 13 co-banded with the ER markers on isopycnic sucrose gradients. By immunofluorescence, the overexpressed cDNA for FKBP 13 in Hela cells gave an ER-staining pattern highly similar to that of known ER proteins. Addition of the ligand FK506 did not appear to alter the distribution of FKBP 13. Separation of the ER luminal contents and membrane revealed FKBP 13 to be a luminal ER protein. Since the lumen of the ER is where the folding of membrane and secreted proteins occurs, as well as a major site of intracellular calcium storage, it seems possible that FKBP 13 may be involved in one of these functions.
Cloning and analysis of the esterase genes conferring insecticide resistance in the peach-potato aphid, Myzus persicae (Sulzer)
Authors: Field L M , Williamson M S , Moores G D , Devonshire A L
Published: August 2015

Full-length cDNA clones encoding the esterases (E4 and FE4) that confer insecticide resistance in the peach-potato aphid [Myzus persicae (Sulzer)] were isolated and characterized. The E4 cDNA contained an open reading frame of 1656 nucleotides, coding for a protein of 552 amino acids. The FE4 cDNA shared 99% identity with E4 over this region, the most important difference being a single nucleotide substitution resulting in the FE4 mRNA having an extra 36 nucleotides at the 3′ end. The derived amino acid sequences for the N-terminus of E4 and FE4 were identical, with the first 23 residues being characteristic of a signal peptide and the next 40 residues being an exact match to the N-terminal sequence determined by Edman degradation of both purified proteins. The predicted molecular masses of 58.8 and 60.2 kDa for the E4 and FE4 polypeptides were consistent with those previously observed by in vitro translation of mRNA. Five potential N-linked glycosylation sites were present in both polypeptides, in accordance with earlier evidence that the native esterases are glycoproteins. Comparison of the aphid esterase protein sequences with other serine hydrolases provided evidence that their activity involves a charge-relay system with a catalytic triad the same as that found in acetylcholinesterase. Restriction mapping and sequencing of cloned genomic DNA showed that the E4 gene is spread over 4.3 kb with six introns and that the previously reported differences between the 3′ ends of the E4 and FE4 genes result from single nucleotide substitutions and not gross differences in the DNA sequences.
Subcloning and characterization of the binding domain of fragment B of diphtheria toxin
Authors: Esbensen Q Y , Falnes P O , Olsnes S , Madshus I H
Published: August 2015

The binding domain (R domain) of diphtheria toxin as defined from the recently published crystal structure [Choe, Bennett, Fujii, Curmi, Kantardjieff, Collier and Eisenberg (1992) Nature (London) 357, 216-222] was subcloned. The 17 kDa peptide containing amino acids 378-535 from fragment B of diphtheria toxin preceded by the tripeptide Met-His-Gly bound specifically and with high affinity to diphtheria-toxin receptors. It efficiently inhibited the toxicity of full-length toxin. The binding domain entered the detergent phase of Triton X-114 at pH values below 6, indicating that it exposed hydrophobic regions at acidic pH.
Vinculin is a major platelet protein that undergoes Ca2+-dependent tyrosine phosphorylation
Authors: Vostal J G , Shulman N R
Published: August 2015

When intracellular Ca2+ pools are released during platelet stimulation by thrombin, elevation of platelet cytosolic Ca2+ concentration induces tyrosine phosphorylation of a 130 kDa protein, and refilling the pools mediates dephosphorylation of this protein [Vostal, Jackson and Shulman (1991) J. Biol. Chem. 266, 16911-16916]. In the present work the 130 kDa protein was identified as vinculin by the following criteria. (1) It is detected on protein immunoblots of thrombin-activated platelets by both monoclonal anti-phosphotyrosine and anti-vinculin antibodies. (2) Removal of N-linked sugars with peptide-N-glycosidase or reduction did not change the molecular mass of vinculin or of the 130 kDa protein on SDS/PAGE. (3) The 130 kDa tyrosine-phosphorylated protein associates with Triton-soluble fraction of platelets as does vinculin. (4) The 130 kDa protein immunoprecipitated by anti-vinculin monoclonal antibody reacts with anti-phosphotyrosine antibody; when immunoprecipitated by anti-phosphotyrosine antibody it reacts with anti-vinculin antibody. (5) The 130 kDa tyrosine-phosphorylated protein and vinculin focus isoelectrically at pI 5.4-5.8. Our finding that vinculin is a major platelet protein that undergoes Ca(2+)-dependent tyrosine phosphorylation during platelet activation may provide clues to the function of this protein.
Endopeptidase 3.4.24.11 converts N-1-(R,S)carboxy-3-phenylpropyl-Ala-Ala-Phe-p-carboxyanilide into a potent inhibitor of angiotensin-converting enzyme
Authors: Williams C H , Yamamoto T , Walsh D M , Allsop D
Published: August 2015

It was reported recently that N-1-(R,S)carboxy-3-phenylpropyl-Ala-Ala-Phe-p-carboxyanilide (CPP-A-A-F-pAB), an inhibitor of endopeptidase 3.4.24.15 (E-24.15), also inhibits angiotensin-converting enzyme (ACE) from rabbit lung. We have found that this compound is without effect on ACE purified from pig kidney, at a concentration some 1000-fold greater than the Ki reported for inhibition of the enzyme from lung. However, preincubation of CPP-A-A-F-pAB with neutral endopeptidase 3.4.24.11 (E-24.11) does result in potent inhibitory effects on ACE. We have shown this to be due to formation of a fragment, CPP-A-A, the structure of which is closely related to ACE inhibitors such as enalaprilat. CPP-A-A was found to be a potent inhibitor of pig ACE. Under the conditions used it had an IC50 value of 1.6 x 10(-8) M, compared with the value obtained for captopril of 7.5 x 10(-10) M. These results have important implications for studies of E-24.15 when using CPP-A-A-F-pAB in vivo or in crude tissue extracts where E-24.11 might also be present.
Distinct β-subunits are present in hybrid insulin-like-growth-factor-1 receptors in the central nervous system
Authors: Moss A M , Livingston J N
Published: August 2015

Previous work suggests the existence of different isoforms of the insulin-like-growth-factor-1 (IGF-1) receptor in various tissues. In the present study we provide support for the concept that heterogeneous IGF-1 receptors exist in the brain and that part of the heterogeneity is derived from IGF-1 receptor hybrids formed from different beta-subunits. IGF-1 receptors were extracted from adult-rat forebrain synaptosomes and partially purified by wheat-germ agglutinin (WGA) chromatography. Hormone-binding studies in this preparation demonstrate the presence of receptors for IGF-1 and insulin. An antibody, a-RIR, specific for the rat insulin receptor was used to remove insulin receptors from the WGA extract. Studies with the immunodepleted material demonstrated two proteins of 92 and 99 kDa that are phosphorylated on tyrosine during incubation with low concentrations of IGF-1. Both proteins bound with high affinity and specificity to IGF-1 immobilized on agarose, and each underwent phosphorylation when the agarose beads were incubated with [gamma-32P]ATP and MnCl2. Two-dimensional phosphopeptide maps after exhaustive trypsin treatment of the two proteins showed significant differences in their structure as well as differences from the phosphopeptide map for the beta-subunit of the insulin receptor. The relationship of the two proteins to the IGF-1 receptor was further probed by an antibody (a-HF) raised against a specific sequence in the beta-subunit of the human IGF-1 receptor, and a polyclonal antibody raised against the liver insulin receptor (L1) which cross-reacts with the IGF-1 receptor. Both antibodies immunoprecipitated the two phosphorylated proteins. However, reduction of the receptors to form receptor dimers or monomers showed that a-HF precipitated only the 99 kDa protein, whereas L1 precipitated primarily the 92 kDa protein. In conclusion, the brain IGF-1 receptor apparently has two structurally different beta-subunits, one of 92 kDa and a second of 99 kDa. Interestingly, at least a portion of the IGF-1 receptor population has both isoforms in the same receptor.