bluuebunny/crossref_metadata_2025 huggingface dataset:
The zinc chelator 1,10-phenanthroline enhances the stimulatory effects of protein kinase C activators and staurosporine, but not sphingosine and H2O2, on phospholipase D activity in NIH 3T3 fibroblasts
Authors: Kiss Z
Published: August 2015

Protein kinase C (PKC), an enzyme which is believed to mediate the stimulatory effects of the PKC activator phorbol 12-myristate 13-acetate (PMA) on phospholipase D (PLD) activity, has a zinc-dependent structure required for phorbol ester binding. Accordingly, zinc or zinc chelators would be expected to promote or inhibit, respectively, the stimulatory effects of PMA on PLD-mediated phospholipid hydrolysis. Instead, treatment of [14C]choline- and [14C]ethanolamine-labelled NIH 3T3 fibroblasts with the high-affinity zinc chelator 1,10-phenanthroline (0.2-1 mM) for 20-30 min was found to enhance the stimulatory effects of PMA on PLD-mediated hydrolysis of phosphatidylcholine and phosphatidylethanolamine. In [14C]palmitic acid-labelled fibroblasts, in the presence of ethanol, phenanthroline also enhanced the stimulatory effect of PMA on the synthesis of phosphatidylethanol, a marker of PLD activity. Addition of zinc (250 microM) to phenanthroline-treated fibroblasts reversed the stimulatory effects of the chelator. The potentiating effects of phenanthroline were also partially reversed by cadmium, whereas iron, lead, copper, magnesium and calcium were without effects. Of the other activators of PLD tested, phenanthroline also enhanced the stimulatory effects of platelet-derived growth factor and staurosporine, but not that of sphingosine and H2O2, on the hydrolysis of both phospholipids. These results suggest that regulation of PLD by PKC activators and staurosporine involves a common intermediate step, which is inhibited by a chelatable cellular pool of zinc.
The role of His66 and His72 in the reaction mechanism of bovine liver low-Mr phosphotyrosine protein phosphatase
Authors: Chiarugi P , Cirri P , Camici G , Manao G , Fiaschi T , Raugei G , Cappugi G , Ramponi G
Published: August 2015

Site-directed mutagenesis of a synthetic gene coding for low-M(r) phosphotyrosine protein phosphatase from bovine liver has been carried out. The two histidine residues in the enzyme have been mutated to glutamine; both single and double mutants were produced. The mutated and non-mutated sequences have been expressed in Escherichia coli as fusion proteins, in which the low-M(r) phosphotyrosine protein phosphatase was linked to the C-terminal end of the maltose-binding protein. The fusion enzymes were easily purified by single-step affinity chromatography. The mutants were studied for their kinetic properties. Both single mutants showed decreased kcat. values (30 and 7% residual activities for His66 and His72 respectively), and alterations of the Ki values relative to four-competitive inhibitors were observed. The kinetic mechanism of p-nitrophenyl phosphate hydrolysis in the presence of both single mutants was determined and compared with that of the non-mutated enzyme. The rate-determining step of the catalytic process of the His66→Gln mutant was the same as that found for non-mutated enzyme, whereas for the His72→Gln mutant, both the kinetic constant of the step that causes the formation of a phosphoenzyme covalent intermediate, and the kinetic constant of the step that causes the dephosphorylation of the enzyme covalent intermediate, determined the kcat. value. This observation was confirmed by phosphoenzyme covalent intermediate trapping experiments. The participation of both histidine residues (His66 and His72) at the active site is strongly suggested by the results of diethyl pyrocarbonate inactivation of both single mutants, each containing a single histidine residue. Both mutants are completely inactivated by diethyl pyrocarbonate treatment; the competitive inhibitor Pi protects both mutants from inactivation. The His66/His72 double mutant was completely inactive.
Covalent structure of bovine brain calreticulin
Authors: Matsuoka K , Seta K , Yamakawa Y , Okuyama T , Shinoda T , Isobe T
Published: August 2015

The covalent structure of bovine brain calreticulin, a major Ca(2+)-binding protein in the lumen of the endoplasmic reticulum, was determined by analysis of the purified protein. The protein consisted of 400 amino acids, with an N-linked oligosaccharide attached to the polypeptide chain. The polypeptide sequence determined was compatible with the sequence of calreticulin deduced from cDNA of different sources, with a number of differences presumably due to species-specific amino acid substitutions. The protein retained the C-terminal tetrapeptide, KDEL, involved in retention of proteins resident in the endoplasmic reticulum, whereas the N-terminal signal peptide predicted from the cDNA sequence had been removed in the purified protein. The bovine brain protein contained a high-mannose type of oligosaccharide attached to Asn162, which is typical of resident endoplasmic reticulum proteins. The carbohydrate moiety was heterogeneous and had the composition GlcNAc2Man4-9, of which GlcNAc2Man5 was the most abundant in the bovine brain preparation. Glycosylation of calreticulin, however, appeared to be a species-specific modification, as Asn162 is replaced by Asp in the sequences already determined for a number of species. Analysis of the purified protein also identified an intramolecular disulphide bridge between Cys120 and Cys146.
Protein kinase C-dependent prostaglandin production mediates angiotensin II-induced atrial-natriuretic peptide release
Authors: Church D J , Braconi S , van der Bent V , Vallotton M B , Lang U
Published: August 2015

The respective roles of protein kinase C (PKC) and endogenous prostaglandin formation in angiotensin II (Ang II)-induced myocardial secretion of atrial natriuretic peptide (ANP) was studied in cultured, spontaneously beating, neonatal-rat cardiomyocytes. Incubation of cardiomyocytes with 0.1 microM Ang II led to a rapid but transient increase in particulate-bound PKC activity, a response accompanied by marked increases in cellular 6-oxo-prostaglandin F1 alpha (6-oxo-PGF1 alpha) generation and ANP secretion. A role for PKC in Ang II-induced 6-oxo-PGF1 alpha formation and ANP secretion was apparent, insofar as both responses were suppressed in the presence of the PKC inhibitors staurosporine (1 microM) and CGP 41251 (1 microM), as well as in cells in which PKC had been previously down-regulated by pretreatment with phorbol diester. Furthermore, Ang II-induced 6-oxo-PGF1 alpha production was found to be strongly correlated with Ang II-induced ANP release (r = 0.87, P < 0.001, n = 6), indicating a role for prostacyclin (PGI2) in Ang II-induced ANP secretion in these cells. This hypothesis was confirmed by finding that both Ang II-induced 6-oxo-PGF1 alpha production and ANP release were abolished in the presence of the respective phospholipase A2 and cyclo-oxygenase inhibitors quinacrine (10 microM) and indomethacin (10 microM), whereas exogenously applied PGI2 (1 microM) and prostaglandin E2 (0.1 microM) mimicked Ang II-induced ANP secretion in this system. Taken together, these results suggest that Ang II induces ANP secretion in spontaneously beating rat cardiomyocytes via a PKC-dependent autocrine pathway involving a cyclo-oxygenase product and a yet-to-be-identified myocardial prostanoid receptor.
Stimulation of low-density lipoprotein uptake in HepG2 cells by epidermal growth factor via a tyrosine kinase-dependent, but protein kinase C-independent, mechanism
Authors: Graham A , Russell L J
Published: August 2015

Epidermal growth factor (EGF), a potent mitogenic polypeptide, stimulated the uptake and degradation of [3H]sucrose-labelled low-density lipoprotein (LDL) by HepG2 cells. The increase in LDL uptake was prevented by the presence of the tyrosine kinase inhibitor genistein. Activation of protein kinase C with phorbol 12-myristate 13-acetate (PMA) also stimulated the uptake of [3H]LDL by HepG2 cells. When EGF and PMA were added together, PMA increased the response to EGF in an additive manner. The protein kinase C inhibitor Ro-31-8220 prevented the increase in LDL uptake caused by PMA, but did not affect EGF stimulation of LDL uptake. Similarly, down-regulation of protein kinase C activity by chronic treatment with PMA also did not affect the EGF stimulation of LDL uptake. These results suggest that the EGF stimulation of LDL uptake and degradation by HepG2 cells is mediated by a tyrosine kinase-dependent, but protein kinase C-independent, mechanism.
Pancreatic plasma membranes: promiscuous partners in membrane fusion
Authors: Lee E G , Marciniak S J , MacLean C M , Edwardson J M
Published: August 2015

We have developed a system in which the fusion of pancreatic plasma membranes with zymogen granules can be studied in vitro. We show here that pancreatic plasma membranes fuse not only with pancreatic zymogen granules but also with parotid secretory granules. In contrast, parotid membranes fuse only with parotid granules and not with pancreatic granules. The extent of fusion is insensitive to Ca2+ for all combinations of plasma membranes and granules. Guanosine 5′-[gamma-thio]triphosphate (GTP[S]), on the other hand, stimulates fusion of pancreatic membranes with both pancreatic granules and parotid granules, but inhibits fusion between parotid membranes and parotid granules.
Triacylglycerol metabolism by lymphocytes and the effect of triacylglycerols on lymphocyte proliferation
Authors: Calder P C , Yaqoob P , Newsholme E A
Published: August 2015

This study investigates the ability of lymphocytes to utilize fatty acids originating from triacylglycerols and the effect of triacylglycerols upon mitogen-stimulated lymphocyte proliferation. Lymphocytes isolated from rat lymph nodes, spleen, thymus and lymphatic duct had a lipoprotein lipase activity of approx. 10 units/mg of protein, indicating that the fatty acids of circulating triacylglycerols are accessible to these cells. In culture lymph node lymphocytes hydrolysed triacylglycerols added to the medium as emulsions. Both non-esterified fatty acids and free glycerol appeared in the cell culture medium, but their concentrations indicated that a high proportion of each (65-90% of fatty acids and 60-80% of glycerol) was taken up by the cells. The incorporation and fate of triacylglycerol-fatty acids was studied by culturing the cells in the presence of tri[3H]oleoylglycerol or tri[14C]inoleoylglycerol. Both fatty acids were incorporated into lymphocyte lipids in a time-dependent manner; linoleic acid was incorporated at a significantly greater rate than oleic acid. The majority of oleic acid (greater than 70%) was incorporated into cellular triacylglycerol, while less than 10% was incorporated into phospholipids. In contrast, linoleic acid incorporation into cellular triacylglycerol never exceeded 25%, while up to 45% was incorporated into phospholipids. Triacylglycerols containing polyunsaturated fatty acids inhibited concanavalin A-stimulated lymphocyte proliferation in a concentration- and time-dependent manner; triacylglycerols containing saturated fatty acids or oleic acid were not inhibitory. Such direct effects of certain triacylglycerols on lymphocyte function may explain why some clinical trials of polyunsaturated fatty acid-rich diets have been successful in improving the condition of patients suffering from inflammatory diseases.
Characterization of the antimicrobial peptide derived from sapecin B, an antibacterial protein of Sarcophaga peregrina (flesh fly)
Authors: Yamada K , Natori S
Published: August 2015

Sapecin B, an antibacterial protein of Sarcophaga peregrina, was divided into four peptides. A hendecapeptide derived from its helix region was found to have comparable antibacterial activity with that of the complete protein. This peptide had a much wider spectrum of antimicrobial activity than that of sapecin B, exhibiting activity on not only Staphylococcus aureus (Gram-positive) and Escherichia coli (Gram-negative), but also some yeasts, including Candida albicans. The peptide was shown to bind to liposomes containing acidic phospholipids and cause release of entrapped glucose, suggesting that its primary site of action is the bacterial membrane. Its antimicrobial activity could be increased by substituting various amino acid residues for hydrophobic and/or basic ones.
Cytodifferentiation in Tetrahymena vorax is linked to glycosyl-phosphatidylinositol-anchored protein assembly
Authors: Yang X , Ryals P E
Published: August 2015

The role of glycosyl-PtdIns (GPI)-anchored proteins in the cytodifferentiation of Tetrahymena vorax was examined. Labelling of cells with [3H]myristate or [3H]palmitate followed by electrophoresis showed an array of proteins carrying covalently bound lipids. Electrophoresis of protein from cells labelled with the GPI-anchor components [3H]Ins and [14C]ethanolamine revealed three polypeptides on fluorograms which have apparent molecular masses of approx. 28, 50 and 82 kDa. Labelled lipid associated with these polypeptides was susceptible to release by in vitro exposure to Bacillus thuringiensis PtdIns-specific phospholipase C (PI-PLC). Using labelled fatty acids, cells induced to differentiate showed altered GPI-anchored protein-labelling patterns in comparison with undifferentiated control cells, with a heavily labelled 32 kDa band appearing upon differentiation. Pre-incubation of cells in 10 mM D-mannosamine, an inhibitor of GPI incorporation into protein, resulted in a reduction of the incorporation of label into the three GPI-anchored proteins, nearly complete inhibition of differentiation and a reduction in the rate of digestive vacuole formation. A 50% inhibition of differentiation was obtained using 500 microM mannosamine. The inhibitory impact of D-mannosamine on differentiation could be competitively and completely reversed by the inclusion of D-mannose, but not D-glucose. Neither glucosamine nor tunicamycin inhibited differentiation. Incubation of cells in PI-PLC (5 units/ml) plus the differentiation inducer resulted in an acceleration of differentiation and generally higher percentages of differentiated cells versus controls.
Periplasmic expression of human interferon-α 2c in Escherichia coli results in a correctly folded molecule
Authors: Voss T , Falkner E , Ahorn H , Krystek E , Maurer-Fogy I , Bodo G , Hauptmann R
Published: August 2015

Human interferon-alpha 2c (IFN-alpha 2c) was produced in Escherichia coli under the control of the alkaline phosphatase promoter using a periplasmic expression system. Compared with other leader sequences, the heat-stable enterotoxin II leader of E. coli (STII) resulted in the highest rate of correct processing as judged by Western-blot analysis. The fermentation was designed as a batch-fed process in order to obtain a high yield of biomass. The processing rate of IFN-alpha 2c could be increased from 25% to more than 50% by shifting the fermentation pH from 7.0 to 6.7. IFN-alpha 2c extracted from the periplasm was purified by a new four-step chromatographic procedure. Whereas cytoplasmically produced IFN-alpha 2c does not have its full native structure, IFN-alpha 2c extracted from the periplasm was found to be correctly folded, as shown by c.d. spectroscopy. Peptide-map analysis in combination with m.s. revealed the correct formation of disulphide bridges. N-terminal sequence analysis showed complete removal of the leader sequence, creating the authentic N-terminus starting with cysteine.
Temporal regulation of the IgE-dependent 1,2-diacylglycerol production by tyrosine kinase activation in a rat (RBL 2H3) mast-cell line
Authors: Lin P , Fung S J , Li S , Chen T , Repetto B , Huang K S , Gilfillan A M
Published: August 2015

We explored the possible role of tyrosine kinases in the IgE-dependent regulation of 1,2-diacylglycerol (DAG) production in RBL 2H3 cells. When triggered via their high-affinity IgE receptors (Fc epsilon RI), there was a rapid phosphorylation of tyrosine residues on a number of proteins. The phosphorylation of these proteins and ultimately histamine release were inhibited in a concentration-dependent manner by the tyrosine kinase inhibitor, tyrphostin. In cells labelled with [3H]myristic acid, we observed a characteristic biphasic increase in [3H]DAG production. In the presence of tyrosine kinase inhibitor, the initial increase in DAG was still observed, but the secondary increase, which was dependent on phosphatidylcholine-specific phospholipase D (PC-PLD) activation, was completely abolished. Tyrphostin significantly inhibited IgE-dependent activation of PC-PLD, suggesting that PC-PLD activation was regulated by tyrosine phosphorylation. Furthermore, when proteins from RBL 2H3 cells were immunoprecipitated with an anti-phosphotyrosine antibody, PC-PLD activity was recovered from the immunoprecipitated fraction. These results demonstrate that the secondary, but not the initial, phase of 1,2-DAG production in response to Fc epsilon RI aggregation is regulated by the initial activation of tyrosine kinases and that PC-PLD may be regulated directly by this mechanism.
A new genomic polymorphism of Rh-polypeptide genes
Authors: Umenishi F , Kajii E , Ikemoto S
Published: August 2015

To determine which Rh-polypeptide genes are related to which Rh (rhesus) antigens, genomic DNAs prepared from donors of ten kinds of different Rh phenotypes were analysed by Southern hybridization method using two probes for the 5′ moiety and 3′ moiety of an Rh-polypeptide cDNA. A polymorphism for the Rh blood-group system was observed with BamHI, HindIII, PstI and EcoRI restriction endonucleases between genomic DNAs from the RhD-positive and RhD-negative individuals as has previously been reported. Further study showed an additional genomic polymorphism. The new polymorphism was detected between the RhC/c phenotypes unrelated to the RhD/d phenotypes with RsaI restriction endonuclease using the probe for the 5′ moiety of Rh polypeptide cDNA. A 1.9 kb RasI restriction fragment was missing in the genomes from individuals with the RhC phenotypes. However, this fragment was present in the genomes of Rhc phenotypes and in its half dose in the genomes of RhCc phenotypes. These results clearly demonstrate a new polymorphism between the RhC/c phenotypes regardless of the RhD-positive and RhD-negative phenotypes, and it is predicted that the Rh polypeptide genes encoding RhD, RhC/c and RhE/e antigens are different.
A C-terminal peptide of bovine rhodopsin binds to the transducin α-subunit and facilitates its activation
Authors: Phillips W J , Cerione R A
Published: August 2015

In order to investigate the possible roles of the intracellular domains of rhodopsin in the functional coupling of the photoreceptor to transducin, different peptides that correspond to parts of the known sequence of rhodopsin were synthesized. Since we have found tht the binding of rhodopsin to the alpha subunit of transducin (alpha T) increases the susceptibility of alpha T to phosphorylation by protein kinase C-beta 1, we used this phosphorylation reaction as an initial screen for peptides that mimic the actions of rhodopsin. The results of this screen indicated that a peptide from the C-terminal tail of rhodopsin (amino acids 325-338; KNPLGDDEASTTVS-amide; designated as peptide 3) was capable of interacting with the alpha T subunit. Evidence that peptide 3 binds to alpha T at a site that overlaps the rhodopsin-binding domain was obtained from experiments showing that peptide 3 inhibited the rhodopsin-stimulated GTPase activity of alpha T and that this inhibition was overcome at high levels of rhodopsin. A potentially important outcome of the peptide 3/alpha T interaction is the facilitation of the activation of the alpha T subunit. This was first demonstrated in fluorescence experiments where the binding of peptide 3 was shown to strongly promote the enhancement of the tryptophane emission of alpha T that is elicited by the addition of NaF. Specifically, the EC50 for NaF was shifted from approximately 4 mM in the absence of peptide 3 to below 0.5 mM in the presence of peptide 3. Further verification that peptide 3 facilitated the ability of NaF to activate the alpha T subunit was obtained from experiments measuring the alpha T GDP/NaF-stimulated hydrolysis of cyclic GMP by the cyclic GMP phosphodiesterase.
A slowly ADP-ribosylated pertussis-toxin-sensitive GTP-binding regulatory protein is required for vasopressin-stimulated Ca2+ inflow in hepatocytes
Authors: Berven L A , Hughes B P , Barritt G J
Published: August 2015

The roles of heterotrimeric GTP-binding regulatory proteins (G-proteins) and inositol polyphosphates in the mechanism by which vasopressin stimulates Ca2+ inflow in hepatocytes were investigated by using single cells loaded with fura2 by microinjection. Vasopressin-stimulated Ca2+ inflow was mimicked by microinjection of guanosine 5′-[gamma-thio]triphosphate (GTP[S]) or guanosine 5′-[beta gamma-imido]triphosphate to the cells, but not adenosine 5′-[gamma-thio]triphosphate (ATP[S]) or guanosine 5′-[beta-thio]diphosphate (GDP[S]). Extracellular Gd3+ (5 microM) inhibited both vasopressin- and GTP[S]-stimulated Ca2+ inflow. GDP[S], but not GMP, administered to hepatocytes by microinjection, completely inhibited vasopressin-stimulated Ca2+ inflow and partially inhibited vasopressin-induced release of Ca2+ from intracellular stores. The microinjection of pertussis toxin had no effect either on the release of Ca2+ from intracellular stores or on Ca2+ inflow induced by vasopressin, but completely inhibited changes in these processes induced by epidermal growth factor (EGF). Hepatocytes isolated from rats treated with pertussis toxin for 24 h exhibited no vasopressin- or GTP[S]-stimulated Ca2+ inflow, whereas the vasopressin-stimulated release of Ca2+ from intracellular stores was similar to that observed for control cells. Heparin or ATP[S] inhibited, or delayed the onset of, both vasopressin-induced release of Ca2+ from intracellular stores and vasopressin-stimulated Ca2+ inflow. Vasopressin-induced oscillations in intracellular [Ca2+] were observed in some heparin-treated cells. It is concluded that the stimulation by vasopressin of Ca2+ inflow to hepatocytes requires inositol 1,4,5-trisphosphate (InsP3) and, by implication, the pertussis-toxin-insensitive G-protein required for the activation of phospholipase C beta [Taylor, Chae, Rhee and Exton (1991) Nature (London) 350, 516-518], and another G-protein which is slowly ADP-ribosylated by pertussis toxin and acts between InsP3 and the putative plasma-membrane Ca2+ channel. EGF-stimulated Ca2+ inflow involves at least one G-protein which is rapidly ADP-ribosylated and is most likely required for InsP3 formation.
Isomerization of all-trans-retinoic acid to 9-cis-retinoic acid
Authors: Urbach J , Rando R R
Published: August 2015

The discovery of the biological activity of 9-cis-retinoic acid raises questions as to its mode of biosynthesis. A simple mechanism involves the direct isomerization of all-trans-retinoic acid to 9-cis-retinoic acid. It is shown here that bovine liver membranes, but not supernatant fractions, can isomerize all-trans-retinoic acid into 9-cis-retinoic acid and 13-cis-retinoic acid. The concentration of 9-cis-retinoic acid generated approaches its equilibrium concentration, which is determined here to be approximately 15%. However, the isomerization process could not be shown to be saturable, and is first-order in all-trans-retinoic acid in the concentration range measured (8.3 nM to 3 microM). Isomerization reactions measured using bovine liver microsomes appear to be mediated by thiol groups, as they can be blocked by group-specific thiol-blocking reagents such as N-ethylmaleimide. It is interesting to note that the non-stereospecific behaviour observed here mimics what is observed when all-trans-retinoic acid is applied to cells. Finally, significant formation of 9-cis-retinoids was not found when the reaction was carried out with liver microsomes and either all-trans-retinol or all-trans-retinal.
Tyrosine phosphorylation of P-selectin in intact platelets and in a disulphide-linked complex with immunoprecipitated pp60c-src
Authors: Modderman P W , von dem Borne A E G K , Sonnenberg A
Published: August 2015

P-selectin is a 140 kDa membrane glycoprotein found in secretory granules of platelets and endothelial cells where it is rapidly translocated to the plasma membrane upon cell activation. It then functions as a receptor for various types of leucocytes. Metabolic labelling of resting platelets with 32Pi showed that P-selectin is primarily phosphorylated on serine residues, although some tyrosine phosphorylation was observed as well. However, tyrosine phosphorylation of P-selectin was greatly stimulated by treatment with the permeating phosphatase inhibitor, pervanadate. When P-selectin immunoprecipitates were incubated with [gamma-32P]ATP (in vitro kinase assay), a fraction of P-selectin was phosphorylated on its tyrosine residues by a co-precipitated kinase. P-selectin phosphorylated in vitro co-migrated with 140 kDa surface-labelled 125I-P-selectin during SDS/PAGE under reducing conditions. Under non-reducing conditions, however, phosphorylated P-selectin was disulphide-linked to unknown protein(s) in a 205 kDa complex. In vitro kinase assays of the most abundant platelet tyrosine kinase, pp60c-src, demonstrated the presence of similar 140 and 205 kDa phosphorylated proteins in SDS/PAGE under reducing and non-reducing conditions respectively. Extraction and reprecipitation studies with proteins phosphorylated in vitro indicated that P-selectin and pp60c-src form a 205 kDa 1:1 disulphide-linked complex. In the complex, pp60c-src autophosphorylation is inhibited and P-selectin is phosphorylated on tyrosine residues. As protein disulphides in the cytoplasm of intact cells are extremely rare, our results suggest that P-selectin and pp60c-src, which co-localize in platelet dense granules, may be non-covalently associated and spontaneously form disulphide bridges during lysis. In addition, the observed tyrosine phosphorylation of P-selectin in intact platelets suggests that its function might be regulated by phosphorylation by pp60c-src.
Threonine synthesis from homoserine as a selectable marker in mammalian cells
Authors: Rees W D , Grant S D , Hay S M , Saqib K M
Published: August 2015

The plasmid pSVthrBC expresses the Escherichia coli thrB (homoserine kinase) and thrC (threonine synthase) genes in mouse cells and enables them to synthesize threonine from homoserine. After transfection with pSVthrBC and culture in medium containing homoserine, only cells that have incorporated pSVthrBC survive. Homoserine at concentrations greater than 1 mM is toxic to mammalian cells. Mouse cells selected from medium containing 5 mM homoserine had incorporated 20-100 copies of the plasmid per cell and had homoserine kinase activities of 0.001-0.012 nmol/min per mg of protein per copy. Cells selected from medium containing 10 mM homoserine had incorporated one or two copies of the plasmid per cell and had homoserine kinase activities of 0.06-0.39 nmol/min per mg of protein per copy. By using high concentrations of homoserine, it is possible to use pSVthrBC to select and isolate cell lines that have one or two copies of the plasmid incorporated into an active region of chromatin. CHO and HeLa cells have also been successfully transfected with pSVthrBC. COS-7 cells are naturally resistant to homoserine as they are able to metabolize homoserine.
Synthesis of dehydrogenation polymers of ferulic acid with high specificity by a purified cell-wall peroxidase from French bean (Phaseolus vulgaris L.)
Authors: Zimmerlin A , Wojtaszek P , Bolwell G P
Published: August 2015

A cationic (pI 8.3) wall-bound peroxidase has been purified to homogeneity from suspension-cultured cells of French bean (Phaseolus vulgaris L.). The enzyme was a glycoprotein and its M(r) was 46,000 as determined by SDS/Page and h.p.l.c. gel filtration. It was localized biochemically to microsomes and the cell wall, and the latter subcellular distribution was confirmed by immunogold techniques. The native enzyme showed absorption maxima at 403, 500 and 640 nm, with an RZ (A405/A280) of 3.3. The peroxidase oxidized guaïacol and natural phenolic acids. By desorption-chemical-ionization mass spectrometry the enzyme was found to oxidize the model compound, ferulic acid, into dehydrodiferulic acid. Kinetics studies indicated an apparent Km of 113.3 +/- 22.9 microM and a Vmax of 144 mumol.min-1.nmol-1 of protein at an H2O2 concentration of 100 microM. In comparison with a second French-bean peroxidase (FBP) and horseradish peroxidase, as a model, it acted with a 6-10-fold higher specificity in this capacity. It is a member of the peroxidase superfamily of bacterial, fungal and plant haem proteins by virtue of its highly conserved amino acid sequence within the proximal and distal haem-binding sites. This is good evidence that this particular FBP may function in constructing covalent cross-linkages in the wall during development and response to pathogens.
Conformation-dependent platelet adhesion to collagen involving integrin alpha 2 beta 1-mediated and other mechanisms: multiple alpha 2 beta 1-recognition sites in collagen type I.
Authors: Morton L F , Peachey A R , Zijenah L S , Goodall A H , Humphries M J , Barnes M J
Published: August 2015

Platelet adhesion has been measured to type-I monomeric collagen, collagen fibres, alpha 1(I) and alpha 2(I) chains and the chain fragments alpha 1(I)CB3, alpha 1(I)CB6, alpha 1(I)CB7 and alpha 1(I)CB8, and alpha 2(I)CB3,5 and alpha 2(I)CB4. Little if any adhesion occurred to any denatured species at 37 degrees C, demonstrating the importance of the collagen helix. However, on coating at 4 degrees C to promote helix formation, and assaying at room temperature to avoid denaturation, adhesion was observed to both alpha-chain types and all fragments, the exact level of which depended on the identity of the species in question. Adhesion was strongly Mg(2+)-dependent. Antibodies against the integrin alpha 2 beta 1 partially inhibited adhesion to alpha-chains and all fragments except alpha 1(I)CB6, indicating a wide distribution of alpha 2 beta 1-binding sites in the collagen molecule. ‘Activation-dependent’ adhesion to monomeric collagen, totally secondary to alpha 2 beta 1-mediated adhesion, involved at least two mechanisms, one mediated by integrin alpha IIb beta 3 and insensitive to prostaglandin E1, the other inhibitable by prostaglandin E1 but independent of integrin alpha IIb beta 3. alpha IIb beta 3-mediated adhesion to fragments was, at least in part, independent of the alpha 2 beta 1-mediated adhesion. Adhesion to fibres was largely bivalent-cation-independent with only minor involvement of integrin alpha 2 beta 1. Some alpha IIb beta 3-mediated adhesion occurred but was independent of any alpha 2 beta 1-initiated adhesion. Total ‘activation-dependent’ adhesion to fibres was less than to monomeric collagen. Affinity chromatography revealed bivalent-cation-independent binding to fibres of three main platelet surface proteins, 90, 150 and 190 kDa in size.
Permissive stimulation of Ca(2+)-induced phospholipase A2 by an adenosine receptor agonist in a pertussis toxin-sensitive manner in FRTL-5 thyroid cells: a new ‘cross-talk’ mechanism in Ca2+ signalling.
Authors: Shimegi S , Okajima F , Kondo Y
Published: August 2015

We have described the pertussis toxin (PTX)-sensitive potentiation of P2-purinergic agonist-induced phospholipase C activation, Ca2+ mobilization and arachidonic acid release by an adenosine receptor agonist, N6-(L-2-phenylisopropyl)adenosine (PIA), which alone cannot influence any of these cellular activities [Okajima, Sato, Nazarea, Sho and Kondo (1989) J. Biol. Chem. 264, 13029-13037]. In the present study we have found that arachidonic acid release was associated with lysophosphatidylcholine production, and conclude that arachidonic acid is produced by phospholipase A2 in FRTL-5 thyroid cells. This led us to assume that PIA augments P2-purinergic arachidonic acid release by increasing [Ca2+]i which, in turn, activates Ca(2+)-sensitive phospholipase A2. The arachidonic acid-releasing response to PIA was, however, always considerably higher (3.1-fold increase) than the Ca2+ response (1.3-fold increase) to the adenosine derivative. In addition, arachidonic acid release induced by the [Ca2+]i increase caused by thapsigargin, an endoplasmic-reticulum Ca(2+)-ATPase inhibitor, or calcium ionophores was also potentiated by PIA without any effect on [Ca2+]i and phospholipase C activity. This action of PIA was also PTX-sensitive, but not affected by the forskolin- or cholera toxin-induced increase in the cellular cyclic AMP (cAMP), suggesting that a PTX-sensitive G-protein(s) and not cAMP mediates the PIA-induced potentiation of Ca(2+)-generated phospholipase A2 activation. Although acute phorbol ester activation of protein kinase C induced arachidonic acid release, P2-purinergic and alpha 1-adrenergic stimulation of arachidonic acid release was markedly increased by the protein kinase C down-regulation caused by the phorbol ester. This suggests a suppressive role for protein kinase C in the agonist-induced activation of arachidonic acid release. We conclude that PIA (and perhaps any of the G1-activating agonists) augments an agonist (maybe any of the Ca(2+)-mobilizing agents)-induced arachidonic acid release by activation of Ca(2+)-dependent phospholipase A2 in addition to enhancement of agonist-induced phospholipase C followed by an increase in [Ca2+]i.
Identification, purification and partial characterization of a carboxypeptidase from the matrix of rat liver mitochondria: a novel metalloenzyme
Authors: Figueiredo E , Duque-Magalhães M C
Published: August 2015

A novel carboxypeptidase has been purified to apparent homogeneity from the matrix fraction of rat liver mitochondria by using a procedure mainly based on immobilized-metal-affinity chromatography (IMAC). This carboxypeptidase has been named mCP-III, since it represents the third major peak of carboxypeptidase activity after the IMAC step of purification. mCP-III hydrolyses a number of N-blocked dipeptides, with preference for Cbz-Phe-Ala, and shows no degrading activity towards 125I-casein. The optimal pH of its activity is 7.6, the apparent Km for Cbz-Phe-Ala is 0.12 mM and the specific activity is 145.5 mumol/min per mg of protein. The enzyme is a typical metalloproteinase, is inhibited by 1,10-phenanthroline and carboxypeptidase inhibitor and re-activated by added Zn2+ and Co2+. The molecular mass estimated by molecular-sieve h.p.l.c. was approx. 115 kDa with two protein bands of 61 and 50 kDa shown by SDS/PAGE analysis, indicating that the enzyme is active as a dimer. This is the first clearly identified carboxypeptidase within mitochondria.
Increase in retinyl palmitate concentration in eyes and livers and the concentration of interphotoreceptor retinoid-binding protein in eyes of vitiligo mutant mice
Authors: Smith S B , Duncan T , Kutty G , Kutty R K , Wiggert B
Published: August 2015

Retinyl esters play an important role in the visual cycle because they are involved in regeneration of 11-cis-retinal for use in rhodopsin formation. In the present study, retinyl ester concentrations were significantly elevated in eyes and livers of mice homozygous for the vitiligo mutation (mivit/mivit). Vitiligo mice demonstrate a slowly progressing retinal degeneration characterized by gradual loss of photoreceptor cells and rhodopsin as well as uneven pigmentation of the retinal pigment epithelium (RPE). Analysis of retinoids by h.p.l.c. indicated that the retinyl palmitate level was increased fivefold in eyes of affected mice at 10 weeks postnatally and was threefold higher at 22 weeks of age. Accumulation of retinyl palmitate occurred in the RPE rather than the neural retina. Furthermore, the concentration of all-trans-retinol was elevated in the RPE of vitiligo mice. Levels of interphotoreceptor retinoid binding protein (IRBP) were increased in vitiligo mice between ages 4 and 14 weeks, but returned to normal by 16 weeks. Increased IRBP levels were not due to increased protein synthesis because IRBP mRNA levels did not differ significantly between control and affected animals. To examine possible systemic involvement in vitiligo mice, retinoids were evaluated in liver and plasma. Mean hepatic total vitamin A levels in affected mice were approximately 1.7 times higher than controls. Analysis of esterified and non-esterified retinoids in liver showed that the concentration of retinyl palmitate was elevated. Plasma retinol levels were normal. This study provides the first evidence of altered systemic retinoid metabolism in vitiligo mice, which occurs, significantly, under normal dietary conditions.
Fusion between Newcastle disease virus and erythrocyte ghosts using octadecyl Rhodamine B fluorescence assay produces dequenching curves that fit the sum of two exponentials
Authors: Cobaleda C , García-Sastre A , Villar E
Published: August 2015

The kinetics of fusion between Newcastle disease virus and erythrocyte ghosts has been investigated with the octadecyl Rhodamine B chloride assay [Hoekstra, De Boer, Klappe, and Wilschut (1984) Biochemistry 23, 5675-5681], and the data from the dequenching curves were fitted by non-linear regression to currently used kinetic models. We used direct computer-assisted fitting of the dequenching curves to the mathematical equations. Discrimination between models was performed by statistical analysis of different fits. The experimental data fit the exponential model previously published [Nir, Klappe, and Hoekstra (1986) Biochemistry 25, 2155-2161] but we describe for the first time that the best fit was achieved for the sum of two exponential terms: A1[1-exp(-k1t)]+A2[1-exp(-k2t)]. The first exponential term represents a fast reaction and the second a slow dequenching reaction. These findings reveal the existence of two independent, but simultaneous, processes during the fusion assay. In order to challenge the model and to understand the meaning of both equation, fusion experiments were carried out under different conditions well known to affect viral fusion (changes in pH, temperature and ghost concentration, and the presence of disulphide-reducing agents or inhibitors of viral neuraminidase activity), and the same computer fitting scheme was followed. The first exponential equation represents the viral protein-dependent fusion process itself, because it is affected by the assay conditions. The second exponential equation accounts for a nonspecific reaction, because it is completely independent of the assay conditions and hence of the viral proteins. An interpretation of this second process is discussed in terms of probe transfer between vesicles.
A semi-preparative enzymic synthesis of malonyl-CoA from [14C]acetate and 14CO2: labelling in the 1, 2 or 3 position
Authors: Roughan G
Published: August 2015

A semi-preparative enzymic synthesis of [1-14C]malonyl-CoA from [1-14C]acetate and bicarbonate, and of [3-14C]malonyl-CoA from Na2(14)CO3 and acetate, was achieved by using chloroplasts rapidly isolated from 7-8-day-old pea shoots. Around 70% of the [1-14C]acetate was converted into malonyl-CoA in 2-3 h, and the specific radioactivity of [3-14C]malonyl-CoA synthesized in the system was 25-30 Ci/mol. Reactions were monitored and labelled products were purified by h.p.l.c.
Inhibition of prolyl 4-hydroxylase by oxalyl amino acid derivatives in vitro, in isolated microsomes and in embryonic chicken tissues
Authors: Baader E , Tschank G , Baringhaus K H , Burghard H , Günzler V
Published: August 2015

The potency of oxalyl amino acid derivatives as inhibitors of prolyl 4-hydroxylase was studied in vitro, in isolated microsomes and in chicken embryonic-tissue culture. These compounds represent structural analogues of 2-oxoglutarate in which the -CH2- moiety at C-3 is replaced by -NH-, with or without further structural modifications. The most efficient inhibitor of purified prolyl 4-hydroxylase was oxalylglycine. Its mode of inhibition was competitive with respect to 2-oxoglutarate. The Ki value varied between 1.9 and 7.8 microM, depending on the variable substrate used. Oxalylalanine inhibited purified enzyme with a Ki of 40 microM. Other oxalyl amino acid derivatives showed little inhibitory activity. In microsomes isolated from embryonic chicken bone, oxalylglycine and oxalylalanine inhibited prolyl hydroxylation with IC50 values of 23 and 120 microM respectively. Dimethyloxalylglycine was not an inhibitor of purified prolyl 4-hydroxylase and only weakly active in the microsomal system, but efficiently suppressed hydroxyproline synthesis in embryonic chicken calvaria and lung. The data suggest that dimethyloxalyl amino acids are converted into active inhibitors in intact cells, most likely in the cytoplasmic compartment.
Role of dimerization in the control of the functioning of the human haemoglobin mutant haemoglobin Howick (β37 Trp→Gly)
Authors: Brittain T
Published: August 2015

Haemoglobin Howick shows a high oxygen affinity (p50 = 1 mmHg) and a low co-operativity (n = 1.3). Equilibrium studies show the protein to be essentially totally dimeric in the oxygenated form. A wide range of rapid kinetic experiments indicate that the deoxygenated form of the protein exists in a tetramer<-->dimer equilibrium with an associated equilibrium constant of 3 microM. These kinetic data also indicate that the oxygenated form of the protein exists in a tetramer<-->dimer equilibrium with an associated equilibrium constant of 35 mM, and furthermore clearly identifies a large increase in the rate of the tetramer-to-dimer dissociation process as the origin of the vastly increased dissociation equilibrium constants. Simulations of the protein-concentration-dependence of the oxygen-binding properties of haemoglobin Howick, based on the measured equilibrium parameters, closely fits the experimental data. The change in dimerization constant for the deoxygenated form of the protein corresponds remarkably well to the free-energy change predicted for the simple transfer of the amino acid side chain at position beta 37 from a hydrophobic to a hydrophilic environment during the dimerization process.
Expression of liver carnitine palmitoyltransferase I and II genes during development in the rat
Authors: Thumelin S , Esser V , Charvy D , Kolodziej M , Zammit V A , McGarry D , Girard J , Pegorier J P
Published: August 2015

The enzyme activity and the expression (protein and mRNA concentrations) of genes encoding for hepatic carnitine palmitoyl-transferases (CPT) I and II were studied during neonatal development, in response to nutritional state at weaning and during the fed-starved transition in adult rats. The activity, the protein concentration and the level of mRNA encoding CPT I are low in foetal-rat liver and increase 5-fold during the first day of extra-uterine life. The activity and gene expression of CPT I are high during the entire suckling period, in the liver of 30-day-old rats weaned at 20 days on to a high-fat diet and in the liver of 48 h-starved adult rats. The activity and CPT I gene expression are markedly decreased in the liver of rats weaned on to a high-carbohydrate diet. By contrast, the activity, the protein concentration and the level of mRNA encoding CPT II are already high in the liver of term foetuses and remain at this level throughout the suckling period, irrespective of the nutritional state of the animals either at weaning or in the adult.
Controlling the speed of hirudin folding
Authors: Chang J Y
Published: August 2015

The folding of hirudin undergoes an initial stage of non-specific packing, followed by consolidation (re-organization) of partially packed intermediates to attain the native structure [Chatrenet and Chang (1993) J. Biol. Chem. 268, 20988-20996]. Non-specific packing leads to the formation of scrambled hirudins as folding intermediates. A systematic study was carried out to search for conditions which would selectively control and enhance the processes of packing and consolidation. It is demonstrated here that under optimized conditions, including the use of cystine/cysteine and protein disulphide isomerase, the folding of hirudin in vitro can be achieved quantitatively within 30 s.
The role of Glu-60 in the specificity of the recombinant ribonuclease from Bacillus amyloliquefaciens (barnase) towards dinucleotides, poly(A) and RNA
Authors: Bastyns K , Froeyer M , Volckaert G , Engelborghs Y
Published: August 2015

A computer model of the complex between G2′p5′G and barnase, the recombinant ribonuclease of Bacillus amyloliquefaciens, was constructed, based on the known structure of the complex RNAase T1.G2′p5′G. This model suggests that the conserved residue Glu-60 plays an important role in the specificity of barnase for guanosine. A barnase mutant was therefore made in which Glu-60 was replaced by Gln. This mutation increases the Km for the dinucleotides GpC and GpA, by a factor of 10, but does not change the kcat. For ApA, the kcat/Km decreases by a similar factor, but the individual parameters could not be determined. The mutation, however, has no influence on the kcat and the Km of barnase action towards RNA and poly(A). This demonstrates that the interactions between the substrate and the residue at position 60 must be different in the case of ApA and poly(A). For RNA, this conclusion is also likely, but not absolutely certain, because barnase/RNA might be a Briggs-Haldane type enzyme/substrate pair. Therefore, if the effect of the mutation were limited to an increase of the dissociation rate constant of the substrate (k-1), this would not be evident in Km or kcat/Km. In view of the clear cut situation with poly(A), the pH profile for and the effect of salt concentration on the kinetic parameters of the mutant barnase were studied for this substrate. The influence of salt on the Km can be interpreted via the linked function concept and shows a cooperative dissociation of 7-10 counterions upon poly(A) binding. The binding of the substrate is strongly reduced at high pH, and the pKa involved decreases strongly at high salt concentrations. Poly(A) and RNA show a pH dependency of their absorbance spectrum, indicating a pH-dependent change of base stacking, which may influence the catalytic parameters.
Regulation of expression of the human fructose transporter (GLUT5) by cyclic AMP
Authors: Mahraoui L , Takeda J , Mesonero J , Chantret I , Dussaulx E , Bell G I , Brot-Laroche E
Published: August 2015

The effect of cyclic AMP on the expression of the fructose transporter, GLUT5, was studied in Caco-2 cells, a human colon cancer cell line that differentiates spontaneously in culture into cells with the properties of small intestine enterocytes. Treatment of differentiated Caco-2 cells with 50 microM forskolin, which stimulates adenylate cyclase and raises intracellular cyclic AMP levels, increased fructose uptake 2-fold and raised GLUT5 protein and mRNA levels 5- and 7-fold respectively. The increased GLUT5 mRNA levels in forskolin-treated cells are a result of stabilization of GLUT5 mRNA in these cells and increased transcription. The effect of cyclic AMP on GLUT5 transcription was assessed by measuring the activity of human GLUT5 promoter-reporter gene constructs in forskolin-treated differentiated Caco-2 cells. The results showed that forskolin stimulated the activity of the GLUT5-reporter gene constructs and this stimulatory effect was mediated by cis-acting regulatory sequences.