bluuebunny/crossref_metadata_2025 huggingface dataset:
Purification and characterization of echinoderm casein kinase II. Regulation by protein kinase C
Authors: Sanghera J S , Charlton L A , Paddon H B , Pelech S L
Published: August 2015

Casein kinase II (CKII) is one of several protein kinases that become activated before germinal-vesicle breakdown in maturing sea-star oocytes. Echinoderm CKII was purified over 11,000-fold with a recovery of approximately 10% by sequential fractionation of the oocyte cytosol on tyrosine-agarose, heparin-agarose, casein-agarose and MonoQ. The purified enzyme contained 45, 38 and 28 kDa polypeptides, which corresponded to its alpha, alpha' and beta subunits respectively. The beta-subunit was autophosphorylated on one major tryptic peptide on serine residues, whereas the alpha'-subunit incorporated phosphate into at least two tryptic peptides primarily on threonine residues. Western-blotting analysis of sea-star oocyte extracts with two different anti-peptide antibodies that recognized conserved regions of the alpha-subunit indicated that the protein levels of the alpha- and alpha'-subunits of CKII were unchanged during oocyte maturation. The purified CKII was partly inactivated (by 25%) by preincubation with protein-serine/threonine phosphatase 2A, but protein-tyrosine phosphatases had no effect. The beta-subunit of CKII was phosphorylated on a serine residue(s) up to 0.54 mol of P/mol of beta-subunit by purified protein kinase C, and this correlated with a 1.5-fold enhancement of its phosphotransferase activity with phosvitin as a substrate. CKII was not a substrate for the maturation-activated myelin basic protein kinase p44mpk from sea-star oocytes, nor for cyclic-AMP-dependent protein kinase. These studies point to possible regulation of CKII by protein phosphorylation.
Site-specific serine phosphorylation of spinach leaf sucrose-phosphate synthase
Authors: Huber J L A , Huber S C
Published: August 2015

We recently reported [Huber, Huber & Nielsen (1989) Arch. Biochem. Biophys. 270, 681-690] that spinach (Spinacia oleracea L.) sucrose-phosphate synthase (SPS; EC 2.4.1.14) was phosphorylated in vivo when leaves were fed [32P]Pi. In vitro the enzyme was phosphorylated and inactivated by using [gamma-32P]ATP. We now report that SPS is phosphorylated both in vivo and in vitro on serine residues. The protein is phosphorylated at multiple sites both in vivo and in vitro as indicated by two-dimensional peptide maps of the immunopurified SPS protein. After being fed with radiolabel, leaves were illuminated or given mannose (which activates the enzyme), in the presence or absence of okadaic acid. Feeding okadaic acid to leaves decreased the SPS activation state in the dark and light and in leaves fed mannose. Across all the treatments, the activation state of SPS in situ was inversely related to the labelling of two phosphopeptides (designated phosphopeptides 5 and 7). These two phosphopeptides are phosphorylated when SPS is inactivated in vitro with [gamma-32P]ATP, and thus are designated as regulatory (inhibitory) sites [Huber & Huber (1991) Biochim. Biophys. Acta 1091, 393-400]. Okadaic acid increased the total 32P-labelling of SPS and in particular increased labelling of the two regulatory sites, which explains the decline in activation state. In the presence of okadaic acid, two cryptic phosphorylation sites became labelled in vivo that were not apparent in the absence of the inhibitor. Overall, the results suggest that light/dark regulation of SPS activity occurs as a result of regulatory serine phosphorylation. Multiple sites are phosphorylated in vivo, but two sites in particular appear to regulate activity and dephosphorylation of these sites in vivo is sensitive to okadaic acid.
Multisubunit erythroid complexes binding to the enhancer element of the chicken histone H5 gene
Authors: Penner C G , Davie J R
Published: August 2015

We identified the factor(s) that bind to the chicken erythroid-cell-specific histone H5 enhancer region which is located on the 3′ end of the gene. In DNAase I footprinting and u.v. cross-linking experiments with nuclear extracts from adult chicken immature erythrocytes, we determined that the trans-acting factor GATA-1 was the predominating protein interacting with the histone H5 enhancer. GATA-2 and GATA-3 were not detected. In contrast, gel-mobility-shift assays and competition experiments demonstrated that several specific complexes formed with the histone H5 enhancer region. Gel-mobility-shift assays with 23 bp oligonucleotides containing the GATA-binding site (AGATAA) of the histone H5 enhancer or of the beta-globin enhancer showed that the GATA sequence was sufficient for the formation of at least five complexes. Diagonal mobility-shift assays demonstrated that multisubunit complexes were forming with the GATA-1 protein. Our interpretation of the results is that GATA-1 interacts with a protein of approx. 105 kDa which, in turn, can associate with protein or protein complexes of approx. 26 kDa, 146 kDa and a protein(s) of molecular mass greater than 450 kDa. The different multisubunit complexes formed via the trans-acting factor GATA-1 may impart different transcriptional responses to the promoter and enhancer elements of the histone H5 and globin genes.
Regulation of protein synthesis in Swiss 3T3 fibroblasts. Rapid activation of the guanine-nucleotide-exchange factor by insulin and growth factors
Authors: Welsh G I , Proud C G
Published: August 2015

Insulin, whole serum, phorbol esters and epidermal growth factor each rapidly stimulate protein synthesis in serum-depleted Swiss 3T3 fibroblasts. The activation of protein synthesis by each of these agents is associated with stimulation of the activity of the guanine-nucleotide-exchange factor (GEF). This protein recycles the initiation factor eIF-2 by promoting exchange of GDP bound to eIF-2 for GTP. Activation of GEF is rapid, becoming maximal within 15 min. The degree of activation of GEF by these stimuli (to greater than 170% of control for insulin, serum or epidermal growth factor; 120% for phorbol dibutyrate) is more than enough to account for their effects on the overall rate of translation. Stimulation of protein synthesis and GEF activity occurs at low nanomolar insulin concentrations, indicating they are mediated through the insulin receptor. The best-characterized mechanism for regulating GEF activity is through changes in the phosphorylation of the smallest subunit of eIF-2 (eIF-2 alpha); however, none of the stimuli studied altered the level of phosphorylation of eIF-2 alpha in Swiss fibroblasts. It seems that direct regulation of GEF activity may be occurring here, and possible mechanisms for this are discussed.
Mechanism of action of purpuromycin
Authors: Landini P , Corti E , Goldstein B P , Denaro M
Published: August 2015

Purpuromycin, an antibiotic active against both fungi and bacteria, shows different modes of action against these two kinds of micro-organisms; in Candida albicans it inhibits RNA synthesis, whereas in Bacillus subtilis protein synthesis is primarily affected, with DNA and RNA synthesis blocked at higher concentrations of the drug. In bacterial cell-free protein-synthesis systems, purpuromycin did not inhibit synthesis from endogenous mRNA (elongation of peptides initiated within the intact cell) but inhibited MS2-phase RNA-dependent protein synthesis (which requires initiation) by 50% at 0.1 mg/l. Poly(U)-directed polyphenylalanine synthesis was 50% inhibited by 20 mg of purpuromycin/l when added to a complete system; however, when purpuromycin was preincubated with ribosomes dissociated into 30 S and 50 S subunits, the concentration for 50% inhibition fell to 0.1 mg/l. By contrast, in a C. albicans cell-free system poly(U)-directed polyphenylalanine synthesis was partially inhibited only at 200 mg/l. Purpuromycin also inhibited polynucleotide synthesis in vitro in reactions using Escherichia coli or wheat-germ RNA polymerases or E. coli DNA polymerase I. We suggest that in bacteria the primary target of purpuromycin is on ribosomes and that its action precedes the elongation step of protein synthesis. The effect on nucleic acid synthesis in both fungi and bacteria may be due to interaction of purpuromycin with DNA.
Secretion of a functional soluble form of neutral endopeptidase-24.11 from a baculovirus-infected insect cell line
Authors: Fossiez F , Lemay G , Labonté N , Parmentier-Lesage F , Boileau G , Crine P
Published: August 2015

Neutral endopeptidase (NEP; EC 3.4.24.11) is an integral membrane protein found at the plasma membrane of many cell types. A secreted form of NEP (sec-NEP) was recently obtained by transfection of COS-1 cells with a recombinant expression vector consisting of the cDNA encoding the signal peptide of pro-opiomelanocortin fused in-frame to the cDNA sequence of the complete ectodomain of rabbit NEP [Lemay, Waksman, Roques, Crine & Boileau (1989) J. Biol. Chem. 264, 15620-15623]. In order to produce large quantities of this enzyme for structural studies we have expressed this recombinant soluble form of NEP at high yields using a baculovirus/insect-cell system. A recombinant Autographa californica nuclear polyhedrosis-virus genome containing the sec-NEP sequence was used to infect host Spodoptera frugiperda Sf9 cells. Infected cells secreted an N-glycosylated soluble form of neutral endopeptidase which was enzymically active. The yield was about 80 nmol of enzyme/litre of culture. The soluble form of the recombinant enzyme purified by immunoaffinity showed the same catalytic properties as the wild-type enzyme extracted from the kidney brush-border membranes. Treatment of the recombinant enzyme with endo-beta-N-acetylglucosaminidase H showed, however, that invertebrate cells did not glycosylate the enzyme to the same extent as did mammalian cells. Our findings demonstrate that insect cells can be used as hosts for the production of the soluble form of neutral endopeptidase. We also conclude that neither a full complement of carbohydrate side chains nor the membrane anchor appear to be essential for the production and targeting to the cell surface of a fully functional enzyme in this expression system.
Release of choline by phospholipase D and a related phosphoric diester hydrolase in human erythrocytes. 1H spin-echo n.m.r. studies
Authors: Selle H , Chapman B E , Kuchel P W
Published: August 2015

A previously detected phosphatidylcholine-specific phospholipase D from lysates of human red blood cells has been further characterized by 1H spin-echo n.m.r. spectroscopy. A second choline-releasing enzymic activity was observed after addition of glycerophosphocholine. Both of these phosphoric diester hydrolase activities were activated to different extents by different concentrations of calcium ions. Differences between the two activities were also observed on inhibition by barium and phosphate ions. These distinct, choline-yielding, reactions which occur in the cytoplasm of red blood cells may be involved in the regulation of the levels of membrane phosphatidylcholine.
Molecular biology of the 2-haloacid halidohydrolase IVa from Pseudomonas cepacia MBA4
Authors: Murdiyatmo U , Asmara W , Tsang J S H , Baines A J , Bull A T , Hardman D J
Published: August 2015

The structural gene (hdl IVa) for the Pseudomonas cepacia MBA4 2-haloacid halidohydrolase IVa (Hdl IVa) was isolated on a 1.6 kb fragment of Ps. cepacia MBA4 chromosomal DNA. The recombinant halidohydrolase was expressed in Escherichia coli and Pseudomonas putida and the structural gene was subcloned on to the tac expression vector pBTac1. High-level expression from the tac promoter was seen to be temperature-dependent, a consequence of the nucleotide sequence adjacent to the fragment encoding the halidohydrolase. The nucleotide sequence of the fragment encoding the Hdl IVa was determined and analysed. Three ATG codons were identified in one of the open reading frames and the one corresponding to the start of the hdl IVa structural gene was determined by comparison of the predicted amino acid sequences with the experimentally determined N-terminal sequences of halidohydrolase IVa. The hdl IVa gene encoded a 231-amino acid-residue protein of M(r) 25,900. The sequence and predicted structural data are discussed and comparison is made with sequence data for other halidohydrolases.
Binding of human plasminogen to basement-membrane (type IV) collagen
Authors: Stack M S , Moser T L , Pizzo S V
Published: August 2015

Plasminogen, the zymogen form of the serine proteinase plasmin, has been implicated in numerous physiological and pathological processes involving extracellular-matrix remodelling. We have previously demonstrated that the activation of plasminogen catalysed by tissue plasminogen activator is dramatically stimulated in the presence of basement-membrane-specific type IV collagen [Stack, Gonzalez-Gronow & Pizzo (1990) Biochemistry 29, 4966-4970]. The present paper describes the binding of plasminogen to type IV collagen. Plasminogen binds to both the alpha 1(IV) and alpha 2(IV) chains of basement-membrane collagen, with binding to the alpha 2(IV) chain preferentially inhibited by 6-aminohexanoic acid. This binding is specific and saturable, with Kd,app. values of 11.5 and 12.7 nM for collagen and gelatin respectively. Although collagen also binds to immobilized plasminogen, this interaction is unaffected by 6-aminohexanoic acid. Limited elastase proteolysis of plasminogen generated distinct collagen-binding fragments, which were identified as the kringle 1-3 and kringle 4 domains. No binding of collagen to mini-plasminogen was observed. These studies demonstrate a specific interaction between plasminogen and type IV collagen and provide further evidence for regulation of plasminogen activation by protein components of the extracellular matrix.
The action of glycosylases on dopachrome (2-carboxy-2,3-dihydroindole-5,6-quinone) tautomerase
Authors: Aroca P , Martinez-Liarte J H , Solano F , García-Borrón J C , Lozano J A
Published: August 2015

It is shown that dopachrome (2-carboxy-2,3-dihydroindole-5,6-quinone) tautomerase (DCT) is a glycoprotein containing N-linked oligosaccharides. The enzymic activity can be stimulated by partial deglycosylation with a number of glycosylases such as neuraminidase, beta-mannosidase and beta-galactosidase. However, the stability of the enzyme after the hydrolytic treatment becomes lower. Thus total deglycosylation with peptide N-glycosidase F directly provokes an inactivation of DCT. The native enzyme also shows a strong affinity for concanavalin A-Sepharose. This affinity decreases after treatment with neuraminidase and/or beta-mannosidase. The DCT associated with coated vesicles seems to be mostly glycosylated, since the action of glycosylases on the enzyme obtained from these vesicles produced a similar stimulation to that with the melanosomal enzyme. Treatment of cultured melanocytes with tunicamycin elicited a decrease in the amount of active DCT inside the cells. All data suggest that the structure of the carbohydrate moiety of DCT should be very similar to, if not identical with, the structure proposed for tyrosinase by Ohkura, Yamashita, Mishima & Kobata (1984) Arch. Biochem. Biophys. 235, 63-77.
The esterolytic activity of epidermolytic toxins
Authors: Bailey C J , Redpath M B
Published: August 2015

The two epidermolytic toxins were shown to have intrinsic N-t-butyloxycarbonyl-L-glutamic acid alpha-phenyl esterase activity. The activity was dependent on free toxin pKa values of 6.6 and 6.8 for ETA and ETB respectively. ETB incorporated 0.97 mol of radiolabelled di-isopropyl phosphorofluoridate/mol of protein with loss of esterolytic and epidermolytic activities. The correspondence of epidermolytic and esterolytic activities in ETA and ETB during thermal inactivation and reaction with di-isopropyl phosphorofluoridate, together with the inactivity of the mutant protein ETA S195G, demonstrates that the two activities are dependent on a single active serine residue in each protein.
Kinetic barriers under steady-state conditions
Authors: Südi J
Published: August 2015

It is shown by analysis of a numerical example that kinetic barrier diagrams [Südi (1991) Biochem. J. 276; 265-268] are also useful for displaying the phenomenological resistance of an enzymic turnover to net chemical fluxes observed under steady-state conditions. Most importantly, a new additivity rule is revealed by net flux profiles, which refers to limiting (‘ideal’) conditions. The rule states that the net fluxes that one obtains under initial steady-state conditions are strictly identical with the overall flux in the corresponding equilibrium system. This finding amounts to defining the relation between unidirectional fluxes and net fluxes, and explains why the total ‘one-way flux resistance’ to the overall reaction at chemical equilibrium on the one hand, and the ‘net flux resistance’ to both initial steady-state reactions on the other hand, have to be equal. The conclusions are claimed to be generally valid for consecutive chemical reactions.
Evidence for an increase in positive surface charge and an increase in susceptibility to trypsin of Sarcophaga lectin (from the flesh fly, Sarcophaga peregrina) on its interaction with galactose, a hapten sugar of the lectin
Authors: Komano H , Kurama T , Nagasawa Y , Natori S
Published: August 2015

When Sarcophaga lectin (from the flesh fly, Sarcophaga peregrina), an insect humoral lectin, was eluted from a column of DEAE-cellulose in the presence of galactose (a hapten sugar of this lectin), it emerged at a lower salt concentration than when galactose was absent. In the presence of galactose the lectin was, in addition, more susceptible to trypsin digestion. The lectin was found to have an affinity for basic proteins such as histone H3 and sarcotoxin IA, but this property was lost in the presence of galactose. These results suggested that the lectin changes its conformation on interaction with galactose. This change is suggested to result in the exposure of some hidden lysine and/or arginine residues.
Uptake of injected 125I-ricin by rat liver in vivo. Subcellular distribution and characterization of the internalized ligand
Authors: Frénoy J P , Turpin E , Janicot M , Gehin-Fouque F , Desbuquois B
Published: August 2015

Subcellular-fractionation techniques were used to characterize the endocytic pathway followed by ricin in rat liver in vivo and tentatively identify the site(s) at which the ricin interchain disulphide bridge is split. After injection of 125I-ricin, hepatic uptake of radioactivity was maximum at 30 min (40% of injected dose). At 5 min, about 80% of the radioactivity in the homogenate was recovered in the microsomal (P) fraction, but later on the recovery of the radioactivity in the mitochondrial-lysosomal (ML) fractions progressively increased (50% at 30 min) at the expense of that in the P fraction. Subfractionation of the P and ML fractions on analytical sucrose-density gradients revealed a time-dependent translocation of the radioactivity from low- to high-density endocytic structures, with median relative densities at 5 and 60 min of about 1.15 and 1.16 (P fraction) and 1.19 and 1.22 (ML fraction) respectively. The late distribution of the radioactivity in the ML fraction was similar to that of the lysosomal marker acid phosphatase. Studies with co-injected lactose and mannan showed that ricin was internalized mainly via the mannose receptor. In the presence of mannan, the late recovery of radioactivity in the ML fraction was decreased, and the distribution of the radioactivity associated with the P fraction was shifted toward lower densities (median relative density 1.13), indicating a different pathway of endocytosis. Analysis of the radioactivity associated with the ML and S fractions by SDS/PAGE revealed a time-dependent increase in the amount of intact A- and B-chains and low-molecular-mass products. When ML fractions containing partially processed ricin were incubated at 37 degrees C at pH 5 or at pH 7.2 in the presence of ATP, only low-molecular-mass products were generated. We conclude that internalized ricin associates with endocytic structures whose size and density of equilibration increase with time, and that, although detectable in these structures, reduction of the ricin interchain disulphide bridge occurs to a large extent in the cytosol.
Site of palmitoylation of a phospholipase C-resistant glycosylphosphatidylinositol membrane anchor
Authors: Ferguson M A J
Published: August 2015

The site of palmitoylation of the phosphatidylinositol moiety of the glycosyl-phosphatidylinositol membrane anchor of Trypanosoma brucei procyclic acidic repetitive protein was studied by using periodate oxidation. Analysis of the products by g.c.-m.s. allowed the assignment of 40 and 60% of the palmitate to the 2-position and the 3-position respectively of the myo-inositol ring.
Fructose metabolism in the human erythrocyte. Phosphorylation to fructose 3-phosphate
Authors: Petersen A , Kappler F , Szwergold B S , Brown T R
Published: August 2015

In human erythrocytes, the first step in the metabolism of fructose is generally thought to be phosphorylation to fructose 6-phosphate catalysed by hexokinase. In variance with this assumption, we show here that fructose in these cells is metabolized primarily to fructose 3-phosphate by a specific 3-phosphokinase. This process has an overall estimated Km of 30 mM with respect to extracellular fructose and an apparent Vmax. of 0.6 mumol/h per ml. At a fixed concentration of fructose in the medium, the accumulation of fructose 3-phosphate was linearly dependent on the duration of incubation up to 5 h and was not affected by glucose. Once accumulated, fructose 3-phosphate appears to be degraded and/or relatively slowly metabolized, decreasing by only approximately 30% after a 12 h incubation in a fructose-free medium.
Induction and degradation of the uncoupling protein thermogenin in brown adipocytes in vitro and in vivo. Evidence for a rapidly degradable pool
Authors: Puigserver P , Herron D , Gianotti M , Palou A , Cannon B , Nedergaard J
Published: August 2015

The induction and degradation of the brown-fat-specific uncoupling protein thermogenin in brown fat cell cultures was investigated. Cultures were initiated with undifferentiated precursor cells from young mice and the amount of thermogenin was determined by immunoblotting. High levels of thermogenin could be induced by noradrenaline treatment in cells grown for more than 5 days in culture, and in such cell cultures continuously stimulated with noradrenaline, the thermogenin level continued to increase for at least a further 5 days. In cell cultures stimulated for only 24 h, the induced thermogenin was subsequently specifically and rapidly degraded, with a half-life of 20 h. As the half-life was prolonged by cycloheximide treatment, the degradation was apparently due to the induction of specific proteins after cessation of adrenergic stimulation. In cell cultures continuously stimulated with noradrenaline for 5 days, the induced thermogenin was degraded much more slowly after noradrenaline removal, with a half-life of 70 h. This half-life was unchanged by cycloheximide treatment, and the degradation after cycloheximide was in parallel with the degradation of protein in general, and was therefore non-specific. The prolongation of the half-life of thermogenin after the chronic treatment may be related to mitochondrial incorporation of thermogenin and consequent stabilization of the protein. The half-life of thermogenin in an in vivo situation of similar experimental design (the reacclimation of mice to warm after 5 days in the cold), was also long (about 7 days), and the loss was also non-specific, as it paralleled the loss of protein. Thus different molecular events are involved in thermogenin degradation when the protein is found in different functional pools.
Multiplicity of UDP-glucuronosyltransferases in fish. Purification and characterization of a phenol UDP-glucuronosyltransferase from the liver of a marine teleost, Pleuronectes platessa
Authors: Clarke D J , George S G , Burchell B
Published: August 2015

The aim of this work was to determine if a non-mammalian species had multiple UDP-glucuronosyltransferase (UDPGT) isoforms. At least six highly purified UDPGT isoenzymes were partially resolved by anion-exchange chromatography and UDP-hexanolamine-Sepharose 4B affinity chromatography from liver microsomes of a fish, the plaice. Q-Sepharose FF, chromatofocusing and affinity-chromatographic procedures were employed to separate and purify the phenol UDPGT isoform to apparent homogeneity. The purified enzyme conjugated 1-naphthol, but not bilirubin or steroids, and displayed a pI of 7.0 and a subunit molecular mass of 55 kDa. Bilirubin and testosterone UDPGT activities were more labile and, although purified over 200-fold, these preparations also contained the phenol UDPGT and had multiple polypeptides with molecular masses of 52-57 kDa. Antisera to rat bilirubin/phenol UDPGT and testosterone/phenol UDPGT isoforms cross-reacted strongly with the partially purified plaice UDPGT isoforms of molecular masses 52, 53 and 57 kDa and less strongly with phenol UDPGT 54 kDa and 56 kDa isoforms. Fish and mammalian UDPGTs therefore apparently possess a high degree of evolutionary conservation.
Histamine H1-receptor-mediated calcium influx in DDT1MF-2 cells
Authors: Dickenson J M , Hill S J
Published: August 2015

Undifferentiated monolayers of the hamster vas deferens smooth-muscle cell line, DDT1MF-2, were grown on glass coverslips and loaded with the Ca(2+)-sensitive fluorescent dye fura-2. Stimulation with histamine produced a rapid and maintained increase in intracellular free Ca2+ ([Ca2+]i), with an EC50 of 7.0 +/- 0.7 microM. The initial rise in [Ca2+]i can be attributed to Ca2+ release from intracellular stores, whereas the maintained or plateau phase is due to influx of extracellular Ca2+. The Ca2+ influx associated with the plateau phase required the continued presence of histamine on the receptor, since the H1-antagonist mepyramine (10 microM) attenuated the rise in [Ca2+]i observed when extracellular Ca2+ was re-applied after the cells had been stimulated with histamine, in experiments performed in nominally Ca(2+)-free buffer. Pretreatment with the inorganic Ca(2+)-channel blockers Ni2+ (1 mM) and Co2+ (1 mM) inhibited the influx component, whereas the organic voltage-operated Ca(2+)-channel antagonists nifedipine (10 microM) and PN-200-110 (10 microM) had no effect. These data suggest that histamine stimulates Ca2+ influx through an H1-receptor-activated Ca2+ channel. Experiments with Mn2+ indicated that the receptor-mediated Ca(2+)-influx pathway(s) is impermeable to Mn2+. Furthermore, the refilling of Ca2+ stores can occur independently of H1-receptor-mediated influx, since store refilling can be demonstrated even when the receptor-mediated Ca2+ entry is blocked by mepyramine. In conclusion, H1-receptor activation in the smooth-muscle cell line DDT1MF-2 stimulates both release of Ca2+ from intracellular stores [inositol 1,4,5-triphosphate (InsP3)-mediated] and Ca2+ influx through a receptor-activated Ca2+ channel. The subsequent refilling of the InsP3-sensitive intracellular Ca2+ store is independent of histamine H1-receptor stimulation (mepyramine-insensitive) and occurs without an observable rise in cytosolic free Ca2+.
Identification of the 52 kDa cytoskeletal-like protein of cytochalasin D-stimulated normal rat kidney (NRK/CD) cells as substrate-associated glycoprotein p52 [plasminogen-activator inhibitor type-1 (PAI-1)]. Expression of p52 (PAI-1) in NRK/CD cells is regulated at the level of mRNA abundance
Authors: Higgins P J , Ryan M P
Published: August 2015

Cell shape profoundly affects cellular metabolic activity, protein and nucleic acid synthesis, and cytoskeletal organization. To examine the influence of cell shape on protein expression, normal rat kidney (NRK) cells were exposed to the microfilament-disrupting drug cytochalasin D (CD), labelled with [35S]methionine, and newly synthesized cellular and cytoskeletal proteins examined by two-dimensional gel electrophoresis. CD produced dramatic changes in cell shape (from a flat to round phenotype) with concomitant 3-7-fold increases in the cellular content and cytoskeletal deposition of the microfilament-associated proteins actin, alpha-actinin, and tropomyosin isoform 1. Augmented actin protein content in NRK/CD cells was paralleled by a corresponding increase in actin mRNA abundance and was inhibited by prior addition of actinomycin D. A detergent-insoluble protein of 52 kDa was also detected at high levels in the cytoskeletal fraction of NRK/CD cells. Two-dimensional electrophoretic mapping of total cellular and cytoskeletal proteins revealed this 52 kDa protein to be the previously described glycoprotein p52 [Higgins & Ryan (1989) Biochem. J. 257, 173-182]. By using electrophoretic and immunochemical criteria, p52 was identified as plasminogen-activator inhibitor type-1 (PAI-1). Like actin, CD-induced p52(PAI-1) synthesis, cellular content, and partitioning to the detergent-insoluble cytoskeletal compartment reflected a corresponding increase in p52(PAI-1) mRNA. Such induction was similarly inhibited by actinomycin D. p52(PAI-1) expression in the NRK-cell system is thus responsive to CD-mediated shape changes and requires ongoing RNA synthesis for its induction. Differential extraction of detached cell bodies and the substrate-adherent ‘remnant’ fraction of NRK/CD cultures, furthermore, indicated that p52(PAI-1) was not an intrinsic internal cytoskeletal element but, rather, selectively localized to the extracellular residue. p52(PAI-1) retained its detergent-insoluble characteristics even in this isolated ‘remnant’ fraction, where it was also the predominant protein species resolved.
Maltose/proton co-transport in Saccharomyces cerevisiae. Comparative study with cells and plasma membrane vesicles
Authors: Van Leeuwen C C M , Weusthuis R A , Postma E , Van den Broek P J A , Van Dijken J P
Published: August 2015

Maltose/proton co-transport was studied in intact cells and in plasma membrane vesicles of the yeast Saccharomyces cerevisiae. In order to determine uphill transport in vesicles, plasma membranes were fused with proteoliposomes containing cytochrome c oxidase as a proton-motive force-generating system. Maltose accumulation, dependent on the electrical and pH gradients, was observed. The initial uptake velocity and accumulation ratio in vesicles proved to be dependent on the external pH. Moreover, kinetic analysis of maltose transport showed that Vmax. values greatly decreased with increasing pH, whereas the Km remained virtually constant. These observations were in good agreement with results obtained with intact cells, and suggest that proton binding to the carrier proceeds with an apparent pK of 5.7. The observation with intact cells that maltose is co-transported with protons in a one-to-one stoichiometry was ascertained in the vesicle system by measuring the balance between proton-motive force and the chemical maltose gradient. These results show that maltose transport in vesicles prepared by fusion of plasma membranes with cytochrome c oxidase proteoliposomes behaves in a similar way as in intact cells. It is therefore concluded that this vesicle model system offers a wide range of new possibilities for the study of maltose/proton co-transport in more detail.
Relationships between the degree of cross-linking of surface immunoglobulin and the associated inositol 1,4,5-trisphosphate and Ca2+ signals in human B cells
Authors: McConnell F M , Shears S B , Lane P J L , Scheibel M S , Clark E A
Published: August 2015

Cross-linking of surface immunoglobulin (Ig) receptors on human B cells leads to the activation of a tyrosine kinase. The activated tyrosine kinase subsequently phosphorylates a number of substrates, including phospholipase C-gamma. This enzyme breaks down phosphoinositol bisphosphate to form two intracellular messengers, diacylglycerol and inositol 1,4,5-trisphosphate, leading to the activation of protein kinase C and the release of intracellular Ca2+ respectively. We have used h.p.l.c. and flow cytometry to measure accurately the inositol phosphate turnover and Ca2+ release in anti-Ig-stimulated human B cells. In particular, we have examined the effect of dose of the cross-linking antibody on the two responses. The identity of putative messenger inositol phosphates has been verified by structural analysis, and the amounts of both inositol phosphates and Ca2+ present have been quantified. In the Ramos Burkitt lymphoma, which is very sensitive to stimulus through its Ig receptors, both inositol phosphate production and Ca2+ release were found to be related to the dose of anti-Ig antibody applied. This suggests that phospholipase C-mediated signal transduction in human B cells converts the degree of cross-linking of the immunoglobulin receptor quantitatively into intracellular signals.
Human homologues of the bacterial heat-shock protein DnaJ are preferentially expressed in neurons
Authors: Cheetham M E , Brion J P , Anderton B H
Published: August 2015

The bacterial heat-shock protein DnaJ has been implicated in protein folding and protein complex dissociation. The DnaJ protein interacts with the prokaryotic analogue of Hsp70, DnaK, and accelerates the rate of ATP hydrolysis by DnaK. Several yeast homologues of DnaJ, with different proposed subcellular localizations and functions, have recently been isolated and are the only eukaryotic forms of DnaJ so far described. We have isolated cDNAs corresponding to two alternatively spliced transcripts of a novel human gene, HSJ1, which show sequence similarity to the bacterial DnaJ protein and the yeast homologues. The cDNA clones were isolated from a human brain-frontal-cortex expression library screened with a polyclonal antiserum raised to paired-helical-filament (PHF) proteins isolated from extracts of the brains of patients suffering from Alzheimer's disease. The similarity between the predicted human protein sequences and the bacterial and yeast proteins is highest at the N-termini, this region also shows a limited similarity to viral T-antigens and is a possible common motif involved in the interaction with DnaK/Hsp70. Northern-blot analysis has shown that human brain contains higher levels of mRNA for the DnaJ homologue than other tissues examined, and hybridization studies with riboprobes in situ show a restricted pattern of expression of the mRNA within the brain, with neuronal layers giving the strongest signal. These findings suggest that the DnaJ-DnaK (Hsp70) interaction is general to eukaryotes and, indeed, to higher organisms.
Interaction of 7-n-alkoxycoumarins with cytochrome P-4502 and their partitioning into liposomal membranes. Assessment of methods for determination of membrane partition coefficients
Authors: Vermeir M , Boens N , Heirwegh K P M
Published: August 2015

A study was made of the binding of 7-ethoxy-, 7-n-propoxy- and 7-n-pentoxy-coumarin to cytochrome P-450(2) reconstituted into large unilamellar liposomes composed of a mixture of egg L-alpha-phosphatidylcholine, egg phosphatidylethanolamine and dipalmitoyl phosphatidic acid (2:1:0.06, by weight). The apparent spectral dissociation constants Ksapp. increased linearly with increasing proteoliposomal concentration. When both cytochrome P-450(2) and NADPH:cytochrome P-450 reductase were reconstituted into liposomes, the apparent Michaelis constants Kmapp. for O-dealkylation of 7-methoxy-, 7-ethoxy- and 7-n-propoxy-coumarin showed a similar dependence on the proteoliposomal concentration. The results were in accordance with models for kinetic or equilibrium processes in biphasic systems containing membrane-bound catalytic or acceptor sites, in which a linear solute partition in the bilayer membrane is postulated. The methyl, ethyl and n-propyl ether were readily dealkylated. However, the O-dealkylation rate of 7-n-butoxycoumarin was low and became very small for longer alkyl ethers. Both the effective dissociation constants and effective Michaelis constants decreased with elongation of the alkyl side chain of the coumarins. From plots of the apparent dissociation constants and apparent Michaelis constants against the lipid volume fraction of the proteoliposomes, the membrane partition coefficients for several homologues were calculated. When protein-free liposomes were added to 7-n-alkoxycoumarin solutions, the fluorescence intensity of the coumarins decreased and eventually became negligible in the presence of an excess of liposomal material. On the assumption that the overall fluorescence can be ascribed exclusively to the fraction of 7-n-alkoxycoumarin molecules present in the aqueous phase, partition coefficients for liposomal accumulation of the test compounds could be determined directly. For several coumarin ethers, comparable values were derived for the membrane partition coefficients from binding, kinetic and fluorescence intensity measurements. The change in free energy per methylene group of the 7-n-alkoxycoumarins for partitioning between n-octanol and buffer was significantly different from the value for liposome partitioning.
Oxytocin receptors on cultured astroglial cells. Kinetic and pharmacological characterization of oxytocin-binding sites on intact hypothalamic and hippocampic cells from foetal rat brain
Authors: Di Scala-Guenot D , Strosser M T
Published: August 2015

The ability of astroglial cells to exhibit oxytocin (OT)-binding sites has been investigated in embryonic hypothalamic and hippocampic astroglial cell cultures. The differential characteristics of binding of OT and [Arg8]vasopressin (AVP) agonists and antagonists to the OT-binding sites using the highly selective iodinated OT antagonist d(CH2)5-[Tyr(Me)2,Thr4,Tyr-NH2(9)]OVT ([125I]OTA) have been evaluated using intact cells maintained for 12 days in culture. The specific binding displayed features of reversibility. Computer analysis of the saturation studies using the LIGAND program indicated that, at 4 degrees C, the antagonist binds to a homogeneous population of sites with a Kd value of 0.02 nM and a low binding-site density of around 2 fmol/dish for hypothalamic cells and 6 fmol/dish for hippocampic cells. For hypothalamic cells, competition curves using unlabelled OT, AVP or V2 AVP agonist were characterized by a pseudo-Hill coefficient below unity (0.7), indicating possible heterogeneity among the binding sites. On the other hand, the dose-inhibition curves resulting from competition studies with hippocampic cells had a pseudo-Hill coefficient close to unity, except for OT. Computer analysis (LIGAND) indicated that the OT dose-inhibition curve was significantly better fitted to a two-site model, and this can be explained by two apparent forms of the receptor having high and low affinities for the displacing drug. The relative potencies of the peptides tested for binding to the high-affinity site were: AVP greater than OT greater than V1 AVP antagonist ([d(CH2)5-Tyr(Me)2]AVP) = V2 AVP agonist greater than AVP-Sar ([d(CH2)5-Sar7,Arg8]VP) in hypothalamic cultures, and OT = AVP greater than V1 AVP antagonist greater than V2 AVP agonist in hippocampic cultures. In addition, autoradiography allowed visualization of OT-binding sites, which are located on both soma and processes of astrocyte-like type of cells. In conclusion, these data provide evidence that glial cell cultures contain specific OT-binding sites which display pharmacological characteristics different from those already reported in neuronal cultures and in the adult rat brain.
Oxytocin receptors on cultured astroglial cells. Regulation by a guanine-nucleotide-binding protein and effect of Mg2+
Authors: Di Scala-Guenot D , Strosser M T
Published: August 2015

Specific binding sites for the radio-iodinated oxytocin (OT) antagonist d(CH2)5-[Tyr(Me)2,Thr4, Tyr-NH2(9)]OVT ([125I]OTA) have been characterized on cultured hypothalamic astroglial cell membranes. The rate of association of the ligand to OT-binding sites was identical in the presence and the absence of the non-hydrolysable GTP analogue guanosine 5′-[beta gamma-imido]triphosphate (Gpp[NH]p, 0.1 mM), whereas the monophasic dissociation reaction became biphasic in the presence of Gpp[NH]p. Scatchard analysis of equilibrium binding of [125I]OTA resulted in a linear plot with a single class of binding sites (Kd 0.06 nM) which were insensitive to the addition of Gpp[NH]p. Unlabelled OT and [Arg8]vasopressin (AVP) bound to high- (H) and low- (L) affinity states with a dissociation constant ratio (KL/KH) of 100 for both hormones. Binding with both high and low affinity required the presence of Mg2+ in the incubation buffer, and the addition of Gpp[NH]p decreased the KL/KH ratio to 10 and increased the percentage of low-affinity binding sites. On the other hand, neither omission of Mg2+ from the buffer nor the addition of Gpp[NH]p altered the binding of either OT or V1 AVP antagonists to OT receptors. In the presence of a G-protein inactivator (N-ethylmaleimide; 3 mM) during OT competition studies the affinities of the two OT-binding sites were unchanged, but 90% of the high-affinity binding sites were converted into the low-affinity state. These results obtained with cultured hypothalamic astroglial cells provide further evidence for a coupling of OT receptors with a guanine-nucleotide-binding protein, with a requirement for Mg2+.
Thrombospondin receptor expression in human neutrophils coincides with the release of a subpopulation of specific granules
Authors: Suchard S J , Burton M J , Stoehr S J
Published: August 2015

The extracellular matrix (ECM) protein thrombospondin (TSP) binds specifically to polymorphonuclear leucocyte (PMN) surface receptors and promotes cell adhesion and motility. TSP receptor expression increases 30-fold after activation with the synthetic chemotactic peptide, N-formylmethionyl-leucylphenylalanine (FMLP) or the Ca2+ ionophore A23187, in combination with cytochalasin B. The expression of TSP receptors was correlated with the exocytosis of both specific and azurophil granules. Newly expressed TSP receptors are not derived from easily mobilized specific granules since agents that trigger some specific granule release [phorbol myristate acetate (PMA), FMLP or ionophore A23187 alone] do not increase TSP receptor expression. In this study we used the anion-channel blocker, 4,4′-di-isothiocyanatostilbene-2,2′-disulphonic acid (DIDS) to investigate the source of these newly expressed receptors. When PMNs were exposed to cytochalasin B and FMLP or to cytochalasin B and ionophore A23187 in the presence of 30-100 microM-DIDS, TSP receptor expression increased coincidently with vitamin B12-binding protein release from specific granules. Under these same conditions, the release of the azurophil granule component, myeloperoxidase, was significantly inhibited. Using agonists that cause release of specific granules, or both specific granules and azurophil granules, we determined that DIDS blocked the release of PMA-mobilized specific granules and cytochalasin B plus FMLP- or cytochalasin B plus ionophore A23187-mobilized myeloperoxidase-containing azurophil granules but not specific granules mobilized by cytochalasin B plus FMLP or cytochalasin B plus ionophore A23187. These results suggested that PMNs contain at least two subpopulations of specific granules: one that is easily mobilized, lacks TSP receptors and is inhibitable by DIDS, and one that is difficult to mobilize, contains a large pool of TSP receptors and the release of which is enhanced in the presence of DIDS.
Depletion of the inositol 1,4,5-trisphosphate-sensitive intracellular Ca2+ store in vascular endothelial cells activates the agonist-sensitive Ca2+-influx pathway
Authors: Schilling W P , Cabello O A , Rajan L
Published: August 2015

Previous studies in non-excitable cells have suggested that depletion of internal Ca2+ stores activates Ca2+ influx from the extracellular space via a mechanism that does not require stimulation of phosphoinositide hydrolysis. To test this hypothesis in vascular endothelial cells, the effect of the Ca(2+)-ATPase/pump inhibitor 2,5-di-t-butylhydroquinone (BHQ) on cytosolic free Ca2+ concentration ([Ca2+]i) was examined. BHQ produced a dose-dependent increase in [Ca2+]i, which remained elevated over basal values for several minutes and was substantially inhibited in the absence of extracellular Ca2+. Application of bradykinin after BHQ demonstrated that the BHQ-sensitive compartment partially overlapped the bradykinin-sensitive store. Similar results were obtained with thapsigargin and cyclopiazonic acid, two other Ca(2+)-ATPase inhibitors. Although BHQ had no effect on phosphoinositide hydrolysis, both 45Ca2+ influx and efflux were stimulated by this agent. These results suggest that depletion of the agonist-sensitive Ca2+ store is sufficient for activation of Ca2+ influx. Several characteristics of the Ca(2+)-influx pathway activated by internal store depletion were compared with those of the agonist-activated pathway. Bradykinin-stimulated Ca2+ influx was increased at alkaline extracellular pH (pHo), and was inhibited by extracellular La3+, by depolarization of the membrane, and by the novel Ca(2+)-influx blocker 1-(beta-[3-(4-methoxyphenyl)propoxy]-4- methoxyphenethyl)-1H-imidazole hydrochloride (SKF 96365). Additionally, bradykinin stimulated influx of both 45Ca2+ and 133Ba2+, consistent with the hypothesis that the agonist-activated influx pathway is permeable to both of these bivalent cations. Likewise, activation of Ca2+ influx by BHQ, thapsigargin and cyclopiazonic acid was blocked by La3+, membrane depolarization and SKF 96365, but was unaffected by nitrendipine or BAY K 8644. Furthermore, Ca2+ influx stimulated by BHQ was increased at alkaline pHo and BHQ stimulated the influx of both 45Ca2+ and 133Ba2+ to the same extent. These results demonstrate that the agonist-activated Ca(2+)-influx pathway and the pathway activated by depletion of the agonist-sensitive internal Ca2+ store are indistinguishable.
A thermostable NADH oxidase from anaerobic extreme thermophiles
Authors: Maeda K , Truscott K , Liu X L , Scopes R K
Published: August 2015

A high-abundance NADH-oxidizing enzyme (NADH: acceptor oxidoreductase, EC 1.6.99.3) has been identified and isolated from a range of anaerobic extreme thermophiles, including strains of Clostridium thermohydrosulfuricum and Thermoanaerobium brockii. By use of a pseudo-affinity salt-promoted adsorbent, a nearly pure sample was obtained in one step; remaining impurities were separated by ion-exchange. The fully active purified enzyme contains FAD (two molecules per subunit of 75-78 kDa) and iron-sulphur, and is hexameric in its most active form. The reaction with oxygen is a one- or two-electron transfer to produce superoxide radical and H2O2; other acceptors include tetrazolium salts, dichlorophenol-indophenol, menadione and ferricyanide. The role of the enzyme is not clear; it was found not to be NAD:ferredoxin oxidoreductase, which is a major NADH-utilizing enzyme in these organisms.