bluuebunny/crossref_metadata_2025 huggingface dataset:
Characterization and purification of proteins which bind high-density lipoprotein. A putative cell-surface receptor
Authors: Bond H M , Morrone G , Venuta S , Howell K E
Published: August 2015

High-density lipoprotein (HDL) is shown by ligand blotting to bind membrane-associated polypeptides with sizes of 60, 100 and 210 kDa. Binding was concentration-dependent and competed by excess unlabelled HDL. All the major apolipoproteins of HDL, apoA-I, apoA-II and apoA-IV, bound independently. The 100 kDa and 210 kDa HDL-binding activities were purified from membranes of Hep3B tumour cells by ion-exchange chromatography and gel filtration. The binding activities at 100 kDa and 210 kDa co-purified. After treatment with disulphide-reducing reagent, the 210 kDa band was no longer present and an increase was observed in the amount and binding ability of the 100 kDa polypeptide. The 100 kDa binding protein labelled at the cell surface with 125I could be immunoprecipitated after cross-linking to cell-surface-bound HDL. It is proposed that this HDL-binding activity, a putative cell-surface receptor for HDL, exists totally or in part as a high-molecular-mass complex composed of 100 kDa subunits.
Effects of phorbol esters and secretagogues on nitrobenzylthioinosine binding to nucleoside transporters and nucleoside uptake in cultured chromaffin cells
Authors: Delicado E G , Sen R P , Miras-Portugal M T
Published: August 2015

Secretagogues inhibited adenosine uptake in chromaffin cells without causing apparent changes in the uptake affinity. The inhibition caused by carbachol, nicotine and acetylcholine reached 50%. This inhibition was reproduced by the action of protein kinase C activators such as phorbol 12-myristate 13-acetate (PMA; 100 nM), phorbol 12,13-dibutyrate (PDBu; 100 nM), dicaproin (10 micrograms/ml) and tricaprylin (10 micrograms/ml), with inhibitions of Vmax. of 18, 20, 37 and 47% respectively. No changes in the affinity of uptake were observed with these effectors. Down-regulation of protein kinase C by phorbol esters decreased the inhibitory effects of carbachol on adenosine uptake. Binding studies with nitrobenzylthioinosine (NBTI) showed a similar decrease in the number of transporters when chromaffin cells were treated with the same effectors used for the uptake studies. The high-affinity dissociation constants showed minor changes with respect to the control. The ratio between maximal uptake capacity and the transporter number per cell was not significantly modified by the action of secretagogues or direct effectors of protein kinase C. The number of high-affinity binding sites for NBTI was decreased in cellular homogenates by the direct action of protein kinase C activators, with staurosporine able to reverse this action. Protein kinase C from bovine brain in the presence of ATP and effectors, decreased the number of high-affinity NBTI-binding sites in purified chromaffin cell plasma membranes. These data suggest the possibility of a molecular modification at the transporter level.
Spectroscopic study of the Schiff bases of dodecylamine with pyridoxal 5′-phosphate and 5′-deoxypyridoxal. A model for the Schiff bases of pyridoxal 5′-phosphate in biological systems
Authors: Vázquez M A , Muñoz F , Donoso J , García Blanco F
Published: August 2015

We recorded the absorption spectra of the Schiff bases of pyridoxal 5′-phosphate (PLP) and 5′-deoxypyridoxal (DPL) with dodecylamine (DOD) at different pH values. By applying deconvolution techniques to the spectra and analysing their different components we found that the above-mentioned Schiff bases in aqueous solutions of pH 7 adopted a conformation in which the pyridine ring is embedded in a very hydrophobic medium from which water is virtually completely excluded. This conformation in the same as that adopted by PLP when it acts as coenzyme for some enzymes such as glycogen phosphorylase. The experimental results obtained also show such a conformation to be highly favoured but sensitive to the protonation of the pyridine nitrogen, which makes the aromatic ring more readily accessible to the solvent.
Purification and partial characterization of rat factor D
Authors: Baker B C , Campbell C J , Grinham C J , Turcatti G
Published: August 2015

Rat factor D has been purified to homogeneity (10,559-fold) from serum by chromatography on CM-Sepharose Fast Flow, phenyl-Sepharose CL-4B and Mono S and has been shown to resemble its human and mouse counterparts both in substrate specificity and in its susceptibility to inhibition by the organophosphorous inhibitor di-isopropylfluorophosphate. The rat enzyme, however, is heavily glycosylated and binds to wheat-germ lectin-Sepharose 6MB and 5-hydroxytryptamine-agarose, but not to concanavalin A-Sepharose 4B. All of the carbohydrate chains are N-linked. Enzymic removal of this carbohydrate decreased the Mr by approx. 15,000. The deglycosylated rat enzyme had the same mobility as native human factor D on SDS/PAGE, corresponding to an Mr of 24,500. N-Terminal sequence analysis of the first 30 amino acids of rat factor D highlighted the sequence similarity with human factor D (greater than 76%) and, in particular, with mouse adipsin (greater than 93%).
Transforming-growth-factor-β activation elements in the distal promoter regions of the rat α1 type I collagen gene
Authors: Ritzenthaler J D , Goldstein R H , Fine A , Lichtler A , Rowe D W , Smith B D
Published: August 2015

We have located a cis-acting element (alpha 1-TAE) within the promoter sequences of the rat collagen alpha 1(I) gene (COL1A1) 1600 bases upstream of the transcription start site which mediates transcriptional activation by transforming growth factor beta (TGF-beta). The functional significance of this region was established by (1) deletion analysis of the alpha 1(I) promoter cloned upstream of the bacterial chloramphenicol acetyltransferase (CAT) gene and (2) by co-transfection of promoter constructs with double-stranded oligonucleotides. DNA-mobility-shift assays with radiolabelled alpha 1-TAE demonstrated increased nuclear binding activity after TGF-beta stimulation. Oligonucleotides encoding the alpha 1-TAE, additional upstream regions within the alpha 1(I) promoter, as well as consensus nuclear-factor-1 (NF-1) sequences, competed with the alpha 1-TAE sequence. The two collagen type I genes are stimulated by TGF-beta through different regions of their promoters.
Postnatal changes in sialylation of glycoproteins in rat liver
Authors: Oda-Tamai S , Kato S , Akamatsu N
Published: August 2015

Glycoproteins containing N-linked oligosaccharides were prepared from plasma and liver microsomes of rats aged 0-5 weeks, and galactose and sialic acid content were determined. The sialic acid/galactose ratios in plasma membrane N-glycans remained at about 1 throughout the postnatal period, suggesting that most of the galactose residues are sialylated. In the same way, it was suggested that most of the galactose residues of microsomal N-glycans were sialylated at 0, 4 and 5 weeks of age, but that the degree of sialylation was lower at the other ages, with a minimum at 2 weeks. When the activities of sialyltransferase and galactosyltransferase in liver Golgi membranes were determined, age-dependent changes were found, not only in the specific activities of the enzymes, but also in the Golgi membrane content per g of liver. The activity of galactosyltransferase per g of liver increased immediately after birth, whereas that of sialyltransferase remained at a low level for 2 weeks and then increased to a constant level at 4 weeks. It is probable that this delayed increase in the activity of sialyltransferase results in the decreased sialylation of microsomal N-glycans at 1, 2 and 3 weeks. Sialyltransferase was solubilized from the liver microsomes of rats aged 2, 3 and 4 weeks and characterized. Phosphocellulose column chromatography separated the activity into two subfractions, designated transferase I and transferase II in the order of elution. The increase in total sialyltransferase activity during this period was caused mainly by an increase in transferase I. Rechromatography of each transferase from 3-week-old rats after neuraminidase treatment showed that transferase I but not transferase II contained sialic acid residue(s) and that desialylated transferase I was eluted in a similar way as transferase II. Although the apparent Km value for CMP-N-acetylneuraminic acid and the heat stability of transferase I were different from those of transferase II, the difference was abolished by treating transferase I with neuraminidase, suggesting that transferase II may be a desialylated form of transferase I. These changes in the sialylation of membrane glycoproteins, including sialyltransferase, may be related to the control of liver growth during postnatal development.
The cysteine-rich domain of human proteins, neuronal chimaerin, protein kinase C and diacylglycerol kinase binds zinc. Evidence for the involvement of a zinc-dependent structure in phorbol ester binding
Authors: Ahmed S , Kozma R , Lee J , Monfries C , Harden N , Lim L
Published: August 2015

Diacylglycerol (DG) and its analogue phorbol 12-myristate 13-acetate (PMA) activate the ubiquitous phospholipid/Ca2(+)-dependent protein kinase, protein kinase C (PKC), and cause it to become tightly associated with membranes. DG is produced transiently as it is rapidly metabolized by DG kinase (DGK) to phosphatidic acid. Phorbol esters such as PMA are not metabolized and induced a prolonged membrane association of PKC. Until recently, PKC was the only known phorbol ester receptor. We have shown that a novel brain-specific cDNA, neuronal chimaerin (NC), expressed in Escherichia coli, binds phorbol ester with high affinity, stereospecificity and a phospholipid requirement [Ahmed, Kozma, Monfries, Hall, Lim, Smith & Lim (1990) Biochem. J. 272, 767-773]. The proteins NC, PKC and DGK possess a cysteine-rich domain with the motif HX11/12CX2CXnCX2CX4HX2CX6/7C (where n varies between 12 and 14). The partial motif, CX2CX13CX2C, is present in a number of transcription factors including the steroid hormone receptors and the yeast protein, GAL4, in which zinc plays a structural role of co-ordinating cysteine residues and is essential for DNA binding (protein-nucleic acid interactions). The cysteine-rich domain of NC and PKC is required for phospholipid-dependent phorbol is required for phospholipid-dependent phorbol ester binding, suggesting an involvement of this domain in protein-lipid interactions. We have expressed recombinant NC, PKC and DGK glutathione S-transferase and TrpE fusion proteins in E. coli to investigate the relationship between the cysteine-rich motif, HX11/12CX2CX10-14CX2CX4HX2CX6/7C, zinc and phorbol ester binding. The cysteine-rich domain of NC, PKC and DGK bound 65Zn2+ but only NC and PKC bound [3H]phorbol 12,13-dibutyrate. When NC and PKC were subjected to treatments known to remove metal ions from GAL4 and the human glucocorticoid receptor, phorbol ester binding was inhibited. These data provide evidence for the role of a zinc-dependent structure in phorbol ester binding.
High-affinity folate binding in human choroid plexus. Characterization of radioligand binding, immunoreactivity, molecular heterogeneity and hydrophobic domain of the binding protein
Authors: Holm J , Hansen S I , Høier-Madsen M , Bostad L
Published: August 2015

High-affinity [3H]folate binding in solubilized human choroid plexus homogenate displayed characteristics, e.g. apparent positive co-operativity, which are typical of specific folate binding. The highest folate-binding activity per g of protein was associated with the 27000 g membrane pellet where the membrane-marker enzyme gamma-glutamyltransferase had its main localization. Ultrogel AcA 44 chromatography revealed two major folate-binding proteins (molecular masses greater than 110 kDa and approx. 100 kDa) and one minor one (molecular mass approx. 25 kDa) and approx. 100 kDa) and one minor one (molecular mass approx. 25 kDa) in the Triton X-100-solubilized membrane pellet. After exposure of the membrane pellet to phosphatidylinositol-specific phospholipase C there was only one large 25 kDa peak of folate binding. This could suggest that the folate-binding protein is anchored to the membrane by a glycosylphosphatidylinositol moiety, which can be inserted into Triton X-100 micelles and thus can give rise to forms of large molecular size on gel filtration. This notion was supported by the identical molecular masses of the greater than 110 kDa and 25 kDa folate-binding peaks determined by SDS/PAGE and immunoblotting. The folate-binding protein in choroid plexus cross-reacted with rabbit antibodies against the 25 kDa human milk folate-binding protein, and paraffin-embedded sections of choroid plexus showed immunostaining after exposure to rabbit anti-(human milk folate-binding protein) serum (1:8000 dilution).
Regulation of arachidonic acid release in vascular endothelium. Ca2+-dependent and -independent pathways
Authors: Buckley B J , Barchowsky A , Dolor R J , Whorton A R
Published: August 2015

Ca2+ metabolism and its relationship to arachidonic acid release were studied in cultured pig aortic endothelial cells. When cells were treated with bradykinin, a rapid rise in intracellular Ca2+ concentration ([Ca2+]i) occurred. Arachidonic acid release from cells prelabelled with [3H]arachidonic acid and subjected to flow-through conditions closely followed the changes in [Ca2+]i. Attenuation of the Ca2+ response by chelating extracellular and intracellular Ca2+ or by desensitization of receptors led to comparable attenuation of arachidonate release. Activation of protein kinase C inhibited Ca2+ mobilization in response to bradykinin and stimulated arachidonic acid release. Inhibition of protein kinase C had no effect on bradykinin-stimulated arachidonic acid release, suggesting that protein kinase C does not mediate the bradykinin response. The role of GTP-binding regulatory proteins (G-proteins) in mediating the bradykinin response was also investigated. Bradykinin-stimulated arachidonic acid release was not diminished by preincubation with pertussis toxin. Treatment with the G-protein activator AlF4- resulted in the release of a large pool of arachidonic acid and the formation of lysophospholipids. Combined treatment with AlF4- and bradykinin resulted in a greater than additive effect on arachidonic acid release. In contrast with bradykinin, AlF(4-)-stimulated arachidonic acid release was not dependent on the presence of extracellular Ca2+ or the mobilization of intracellular Ca2+. These results demonstrate Ca(2+)-dependent (bradykinin) and Ca(2+)-independent (AlF4-) pathways of phospholipase A2 activation.
Relative sensitivity to inhibition by cimetidine and clonidine differentiates between the two types of Na+-H+ exchangers in cultured cells
Authors: Ramamoorthy S , Tiruppathi C , Nair C N , Mahesh V B , Leibach F H , Ganapathy V
Published: August 2015

Available evidence indicates that there are two types of Na(+)-H+ exchangers, type A (housekeeping type) and type B (epithelial or apical type), in mammalian cells. We have recently reported, using isolated membrane vesicles, that these two types can be differentiated by their relative sensitivities to inhibition by clonidine and cimetidine [Kulanthaivel, Leibach, Mahesh, Cragoe & Ganapathy (1990) J. Biol. Chem. 264, 1249-1252]. The present study was undertaken to determine whether this approach is also effective in cultured cells. The JAR human placental choriocarcinoma cell line and the opossum kidney (OK) cell line, when grown as confluent monolayer cultures on an impermeable plastic support, express Na(+)-H+ exchanger activity which is measurable by determining Na+ uptake into the cells from the culture medium. The JAR cell Na(+)-H+ exchanger was found to be about 100 times more sensitive to inhibition by dimethylamiloride than the OK cell Na(+)-H+ exchanger. Inhibition studies with clonidine and cimetidine were able to differentiate between these two exchangers very clearly. Cimetidine was 18 times more potent than clonidine in inhibiting the JAR cell Na(+)-H+ exchanger. In contrast, clonidine was at least 8 times more potent than cimetidine in inhibiting the Na(+)-H+ exchanger of the OK cell. The results show that the JAR cell expresses the type A Na(+)-H+ exchanger, whereas the OK cell expresses the type B Na(+)-H+ exchanger. This approach also proved to be very effective in correctly identifying the type of Na(+)-H+ exchanger in a third cell line (HeLa). It is concluded that the relative susceptibility to inhibition by clonidine and cimetidine offers an easy and efficient means of differentiating between the two types of Na(+)-H+ exchangers in cultured cells.
Allosteric regulation of phosphonoacetaldehyde hydrolase by n-butylphosphonic acid
Authors: Dumora C , Lacoste A M , Cassaigne A , Mazat J P
Published: August 2015

The effect of n-butylphosphonic acid on the activity of phosphonoacetaldehyde hydrolase from Pseudomonas aeruginosa was investigated: at low concentrations this compound appeared as an activator of the enzyme activity, whereas at higher concentrations it exhibited inhibitory properties. The experimental results were modelled according to an allosteric model involving two different classes of sites for n-butylphosphonic acid.
Masses of inositol phosphates in resting and tetanically stimulated vertebrate skeletal muscles
Authors: Mayr G W , Thieleczek R
Published: August 2015

The masses of inositol phosphates have been determined in isolated skeletal muscles from Xenopus laevis (sartorius, tibialis anterior and iliofibularis) and rat (gastrocnemius and soleus) which were quick-frozen in the resting state and at different stages of an isometric (Xenopus) or isotonic (rat) tetanus. The isomeric spectrum of inositol phosphates detected was similar to that in other tissues and cell types. The total sarcoplasmic concentrations of the isomers Ins-(1,4,5,6)P4/Ins(3,4,5,6)P4 (0.2-0.9 microM), Ins(1,3,4,6)P4 (not detectable), Ins(1,3,4,5,6)P5 (about 1 microM) and InsP6 (3.2-4.6 microM) were lower than in other cell types. Variations in these concentrations were due to the muscle type rather than to the donor species. The putative second messenger Ins(1,4,5)P3, as well as its dephosphorylation product Ins(1,4)P2, were present at surprisingly high total myoplasmic resting concentrations, ranging from 1.2 to 2.5 microM and 3.5 to 6.9 microM respectively. Upon tetanic stimulation these two inositol phosphates in particular exhibited significantly increased total sarcoplasmic concentrations, up to 4.2 microM and 11.3 microM respectively, with a time scale of seconds. From the initial rate of increase in the total sarcoplasmic concentrations of Ins(1,4,5)P3 and its rapidly formed metabolic products, a minimal phosphoinositidase C (PIC) activity in tetanically activated Xenopus skeletal muscle of about 1.7-2.6 microM/s can be estimated. This PIC activity observed in vivo seems to be far too low to account for a functional role for Ins(1,4,5)P3 as a chemical transmitter in the fast excitation-contraction coupling (ECC) process in skeletal muscle. The presence of Ins(1,3,4,5)P4 in all muscle types is indicative of a Ca(2+)-activated Ins(1,4,5)P3 3-kinase activity. The rapid transient increases in Ins(1,3,4)P3 and Ins(1,3)P2 in isometrically contracting Xenopus muscles suggest that corresponding Ins(1,3,4,5)P4 phosphatases are operating in skeletal muscle as well. In all muscles investigated except rat soleus, the fructose 1,6-bisphosphate [Fru(1,6)P2] concentration increased substantially during a tetanus, up to about 2 mM. This increase is correlated with a simultaneous decrease in phosphocreatine, whereas the energy charge of the muscles was essentially unaffected by the applied tetani. The time course of the rise in Fru(1,6)P2 was used to model changes in the free concentrations of high-affinity aldolase-binding inositol phosphates during the course of a tetanus. These calculations demonstrate that the free concentration of Ins(1,4,5)P3 and other aldolase-bound inositol phosphates can increase much faster and to a larger extent than the corresponding total concentrations as a result of their competitive displacement from aldolase-binding sites by the rapidly rising concentration of Fru(1,6)P2.
Tissue-specific ceruloplasmin gene expression in the mammary gland
Authors: Jaeger J L , Shimizu N , Gitlin J D
Published: August 2015

Using a ceruloplasmin cDNA clone in RNA blot analysis, a single 3.7 kb ceruloplasmin-specific transcript was detected in rat mammary gland tissue from pregnant and lactating animals. Ceruloplasmin gene expression in the mammary gland was tissue-specific, with no evidence of expression in brain, heart or other extrahepatic tissues. Ceruloplasmin mRNA was also detected in mammary gland tissue from male, virgin female and non-pregnant/multiparous animals, and the abundance of ceruloplasmin-specific transcripts in virgin female rats was independent of their stage of oestrus. In virgin female mammary gland the content of ceruloplasmin mRNA was 20% of that in hepatic tissue from these animals and approx. 2-3-fold greater than that found in mammary gland tissue of pregnant or lactating animals. Development studies revealed ceruloplasmin gene expression in male and female mammary gland by only 2 weeks of age, prior to the onset of puberty. Biosynthetic studies indicated that the ceruloplasmin mRNA in mammary gland tissue was translated into a 132 kDa protein qualitatively similar to that synthesized in liver. By in situ hybridization, ceruloplasmin gene expression was localized to the epithelium lining the mammary gland alveolar ducts, without evidence of expression in the surrounding mesenchyme. Ceruloplasmin gene expression was also detected in a human breast adenocarcinoma cell line and in biopsy tissue from women with invasive ductal carcinoma. Taken together, these data indicate that the mammary gland is a prominent site of extrahepatic ceruloplasmin gene expression and add to the evidence that ceruloplasmin biosynthesis is associated with growth and differentiation in non-hepatic tissues.
Phospholipase C-β1 is regulated by a pertussis toxin-insensitive G-protein
Authors: Martin T F J , Lewis J E , Kowalchyk J A
Published: August 2015

Regulation of phospholipase C (PLC) by receptors is mediated either through protein tyrosine phosphorylation or by activation of GTP-binding proteins (Gp). For the latter, pertussis toxin (PT)-sensitive and -insensitive pathways have been described, indicating PLC regulation by at least two types of G-proteins. The identity of PLC isoenzymes which are regulated by either type of Gp remains to be determined. Thyrotropin-releasing hormone stimulates a PLC in GH3 cells via a PT-insensitive Gp. Reconstitution methods for the assay of the GH3-cell Gp were developed. Previously, the membrane PLC was found to be reversibly extracted from membranes by high salt and to be activated by guanosine 5′-[gamma-thio]triphosphate (GTP[S]) only when membrane-associated, suggesting that Gp was retained in salt-extracted membranes. In the present work, Gp was cholate-solubilized from PLC-deficient membranes and incorporated into phospholipid vesicles, which were found to confer GTP[S]- and AlF4(-)-stimulated activity on a solubilized membrane PLC. The reconstitution provided a direct assay for the GH3-cell Gp which was shown to be distinct from Gi, Go and Gs proteins by immunodepletion studies. Incorporation of G-protein beta-gamma subunits into phospholipid vesicles with Gp inhibited GTP[S]-stimulated activity in the reconstitution. The results indicated that Gp is a heterotrimeric G-protein with the properties expected for the PT-insensitive GH3-cell Gp protein. PLC-beta 1 was fully purified and shown to be regulated by Gp in the reconstitution. In contrast, PT-sensitive G-proteins failed to affect the activity of PLC-beta 1. The results indicate (1) that a PT-insensitive Gp regulates PLC-beta 1 and (2) that PT-sensitive and -insensitive pathways of PLC regulation employ different PLC isoenzymes as well as different G-proteins.
Thyroid hormone concentrative uptake in rat erythrocytes. Involvement of the tryptophan transport system T in countertransport of tri-iodothyronine and aromatic amino acids
Authors: Zhou Y , Samson M , Francon J , Blondeau J P
Published: August 2015

The kinetic properties of transport system T, which is specific for uptake of aromatic amino acids, were studied in rat erythrocytes in the presence of leucine in order to block the neutral amino acid transport system L. Since the triiodothyronine (T3) transport system and system T are closely related, the trans effect of T3 and tryptophan on [3H]tryptophan transport and the trans effects of aromatic amino acids on [125I]T3 transport were studied. Equilibrium-exchange, zero-trans and infinite-trans studies of [3H]tryptophan transport indicated that system T in rat erythrocytes is a simple carrier with exchanging properties resulting in trans-acceleration of influx and trans-inhibition of efflux when tryptophan was present at the trans side of the membrane. In erythrocytes preloaded with unlabelled tryptophan, countertransport resulted in a 7-fold accumulation of labelled substrate inside the cells. T3 on the trans side of the membrane inhibited both influx and efflux of tryptophan, with Ki values similar to the Km values of the T3 transport system. Extracellular tryptophan trans-inhibited [125I]T3 efflux in a manner similar to [3H]tryptophan efflux. Preloading erythrocytes with tryptophan resulted in trans-acceleration of T3 uptake and a transient 5-fold accumulation of free T3 into erythrocytes. Phenylalanine and tyrosine (but not the D-isomer of tryptophan or non-aromatic amino acids) also produced trans-acceleration for T3 uptake and T3 countertransport. These results are compatible with a kinetic model assuming a common simple carrier of T3 and tryptophan transport and point to a countertransport pathway driving the uphill uptake of T3 by hetero-exchange with intracellular aromatic amino acids.
Retinoic acid stimulates expression of thrombomodulin, a cell surface anticoagulant glycoprotein, on human endothelial cells. Differences between up-regulation of thrombomodulin by retinoic acid and cyclic AMP
Authors: Horie S , Kizaki K , Ishii H , Kazama M
Published: August 2015

Thrombomodulin (TM) is a surface protein on endothelial cells, and represents one of the most valuable regulatory factors in the anticoagulant system. In this paper, we demonstrate that retinoic acid (RA) causes an increase in TM antigen on human umbilical vein endothelial cells (HUVECs) in vitro. The effect of RA on the surface TM level of HUVECs was dose-dependent in the range from 0.01 to 10 microM-RA. Antigen levels began to increase 3 h after addition of 10 microM-RA, and plateaued at a maximum level of approx. 2.5 times that of the untreated control at 24 h. TM levels remained at a maximum for a further 12 h, and then gradually decreased. The effects of RA on cell surface TM activity and antigen levels were parallel in all experiments. TM expression was also increased by treatment with 10 microM-retinal or 10 microM-retinol for 24 h, though the increases were approx. 70% and 30% respectively of that produced by 10 microM-RA. Pretreatment of HUVECs with cycloheximide inhibited the effect of RA. When HUVECs were incubated with both 10 microM-RA and 5 mM-8-bromo cyclic AMP (or 1 mM-3-isobutyl-1-methylxanthine, a phosphodiesterase inhibitor), the increase in TM antigen was greater than that observed with either compound alone. Northern blot analysis showed that treatment of HUVECs with 8-bromo cyclic AMP, RA or RA plus 8-bromo cyclic AMP increased TM mRNA levels by 2.2-, 4.5- and 5.5-fold respectively compared with the untreated control. Furthermore, no significant difference in cellular cyclic AMP levels was observed between RA-treated and control cells. These results indicate that the expression of TM is not only controlled by the intracellular cyclic AMP level but is also affected by RA, and suggest that RA-induced up-regulation of TM on HUVECs is independent of cyclic AMP regulation.
Direct detection of a globin-derived radical in leghaemoglobin treated with peroxides
Authors: Davies M J , Puppo A
Published: August 2015

The root nodules of leguminous plants contain an oxygen-carrying protein which is somewhat similar to myoglobin. Reaction of the Fe3+ form of this protein (metleghaemoglobin; MetLb) with H2O2 is known to generate a ferryl [iron(IV)-oxo] species. This intermediate, which is analogous to Compound II of peroxidases and ferryl myoglobin, is one oxidizing equivalent above the initial level. In the present study it is shown that the second oxidizing equivalent from the peroxide is rapidly transferred into the surrounding protein, generating a protein radical which has been detected by e.p.r. spectroscopy; this reaction is analogous to that observed with metmyoglobin. An identical protein-derived species is observed with all three forms of MetLb tested (a, c1, c3) and with a number of other hydroperoxides and two-electron oxidants. This latter result, the observation that the concentration of this species is not affected by certain hydroxyl-radical scavengers, and the loss of the radical when the oxy or deoxy forms are used, demonstrate that this species is formed by electron transfer within the protein rather than by the generation and subsequent reaction of hydroxyl radicals (and related species from the other hydroperoxides). The e.p.r. signal of this species, which decays rapidly with a half-life of approx. 40 s, is consistent with the formation of a sterically constrained tyrosine-derived phenoxyl radical; protein-iodination experiments lend support to this assignment. Reaction between the radical and a number of other compounds has been observed, demonstrating that it is at least partially exposed on the surface of the protein. Analysis of the protein structure suggest that the radical may be centred on a tyrosine residue present at position 132 in the protein; this residue is close to the haem prosthetic group, which would facilitate rapid electron transfer.
Mechanistic and active-site studies on d(–)-mandelate dehydrogenase from Rhodotorula graminis
Authors: Baker D P , Kleanthous C , Keen J N , Weinhold E , Fewson C A
Published: August 2015

D(–)-Mandelate dehydrogenase, the first enzyme of the mandelate pathway in the yeast Rhodotorula graminis, catalyses the NAD(+)-dependent oxidation of D(–)-mandelate to phenylglyoxylate. D(–)-2-(Bromoethanoyloxy)-2-phenylethanoic acid [‘D(–)-bromoacetylmandelic acid’], an analogue of the natural substrate, was synthesized as a probe for reactive and accessible nucleophilic groups within the active site of the enzyme. D(–)-Mandelate dehydrogenase was inactivated by D(–)-bromoacetylmandelate in a psuedo-first-order process. D(–)-Mandelate protected against inactivation, suggesting that the residue that reacts with the inhibitor is located at or near the active site. Complete inactivation of the enzyme resulted in the incorporation of approx. 1 mol of label/mol of enzyme subunit. D(–)-Mandelate dehydrogenase that had been inactivated with 14C-labelled D(–)-bromoacetylmandelate was digested with trypsin; there was substantial incorporation of 14C into two tryptic-digest peptides, and this was lowered in the presence of substrate. One of the tryptic peptides had the sequence Val-Xaa-Leu-Glu-Ile-Gly-Lys, with the residue at the second position being the site of radiolabel incorporation. The complete sequence of the second peptide was not determined, but it was probably an N-terminally extended version of the first peptide. High-voltage electrophoresis of the products of hydrolysis of modified protein showed that the major peak of radioactivity co-migrated with N tau-carboxymethylhistidine, indicating that a histidine residue at the active site of the enzyme is the most likely nucleophile with which D(–)-bromoacetylmandelate reacts. D(–)-Mandelate dehydrogenase was incubated with phenylglyoxylate and either (4S)-[4-3H]NADH or (4R)-[4-3H]NADH and then the resulting D(–)-mandelate and NAD+ were isolated. The enzyme transferred the pro-R-hydrogen atom from NADH during the reduction of phenylglyoxylate. The results are discussed with particular reference to the possibility that this enzyme evolved by the recruitment of a 2-hydroxy acid dehydrogenase from another metabolic pathway.
Immunosuppressive activity of corticotrophin-releasing factor. Inhibition of interleukin-1 and interleukin-6 production by human mononuclear cells
Authors: Hagan P , Poole S , Bristow A F
Published: August 2015

Corticotrophin-releasing factor (CRF) from the hypothalamus stimulates corticotrophin (ACTH) secretion. Concentrations of CRF in the peripheral circulation are normally low, and increase during pregnancy due to CRF secretion by the placenta [Cunnah, Jessop, Besser & Rees (1987) J. Endocrinol. 113, 123-131], although CRF in maternal blood does not appear to stimulate the hypothalamo-pituitary-adrenal (HPA) axis [Potter, Behan, Fischer, Linton, Lowry & Vale (1991) Nature (London) 349, 423-426]. We have examined the possibility that the placental. CRF might contribute to the suppression of the maternal immune system, which is necessary to prevent rejection of the foetus, by studying endotoxin-evoked cytokine production by monocytes as a model for activation of the immune response. CRF inhibited endotoxin-evoked cytokine production from human mononuclear cells (MNCs), the fraction of peripheral blood containing monocytes. The effects of CRF were reversed by a specific CRF receptor antagonist, and were additive with glucocorticoid inhibition of cytokine secretion. Anti-interleukin-1 (IL-1) antisera inhibited endotoxin-evoked IL-6 production; however, the CRF effect was not additive, suggesting that CRF inhibition of IL-6 production may be secondary to CRF inhibition of IL-1. These results suggest a role for CRF as an immunosuppressant during pregnancy.
Monomerization of tetrameric bovine caudate nucleus acetylcholinesterase. Implications for hydrophobic assembly and membrane anchor attachment site
Authors: Heider H , Brodbeck U
Published: August 2015

Tetrameric detergent-soluble bovine caudate nucleus acetylcholinesterase (AChE) was reduced and alkylated under conditions in which at least 95% of initial activity is retained. This treatment alone did not result in monomerization of AChE, nor did it create a hydrophilic enzyme. However, in the presence of SDS the enzyme became monomerized. Incubation of AChE with trypsin in the presence of the reversible inhibitor edrophonium rendered the enzyme hydrophilic and led to catalytically active monomers being produced. SDS/PAGE of this preparation in non-reducing conditions revealed only a small decrease in the subunit molecular mass. N-Terminal sequencing of the enzyme, before and after trypsin treatment, yielded identical N-termini showing that the enzyme was monomerized subsequent to C-terminal tryptic cleavage. From our results, we conclude that the most C-terminal cysteine residue is involved in inter-subunit disulphide bonding as well as in the attachment of AChE to the membrane anchor. Furthermore, the C-terminal region in the primary structure provides an area for hydrophobic contacts between the different subunits and also between the subunits and the membrane anchor.
Mastoparan promotes exocytosis and increases intracellular cyclic AMP in human platelets. Evidence for the existence of a Ge-like mechanism of secretion
Authors: Wheeler-Jones C P D , Saermark T , Kakkar V V , Authi K S
Published: August 2015

Recent studies have shown that mastoparan, an amphiphilic peptide derived from wasp venom, accelerates guanine nucleotide exchange and GTPase activity of purified GTP-binding proteins. In the present study we have examined the functional consequences of exposure of intact human platelets to mastoparan. Mastoparan promoted rapid (less than or equal to 1 min) dose-dependent increases in 5-hydroxy[14C]tryptamine and beta-thromboglobulin release from dense-granule and alpha-granule populations respectively. The exocytotic response did not result from a lytic effect of mastoparan and occurred in the complete absence of platelet shape change and aggregation. Liberation of [3H]arachidonate and increases in cytosolic [Ca2+] (detected with fura 2) were not observed in platelets stimulated with mastoparan. Similarly, in platelets preloaded with [3H]inositol during reversible electroporation, mastoparan did not cause the accumulation of [3H]inositol phosphates. Mastoparan-induced secretion was unaffected by preincubation with either the protein kinase C inhibitor staurosporine (10 nM-10 microM) or prostacyclin (PGI2; 100 ng/ml) and was not accompanied by phosphorylation of the 45 kDa protein kinase C substrate or the 20 kDa protein normally associated with platelet activation. The G-protein inhibitor guanosine 5′-[beta-thio]diphosphate (GDP[S]; 1 mM) attenuated the secretion induced by mastoparan in both intact and saponin-permeabilized platelets. Encapsulation of GDP[S] during reversible permeabilization inhibited mastoparan-induced secretion, providing evidence for an intracellular action of GDP[S]. In all these studies thrombin (0.05-0.2 unit/ml) elicited characteristic responses, and thrombin-induced secretion was inhibited by staurosporine, PGI2 and GDP[S]. Mastoparan also increased intra-platelet cyclic AMP in a dose-dependent manner. Mastoparan and PGI2 increased 32P incorporation into a protein of approx. 24 kDa, whereas phosphorylation of a 50 kDa substrate was only seen in PGI2-stimulated platelets. These results indicate that mastoparan promotes secretion by a mechanism which does not involve stimulation of phospholipase C and suggest that the secretory event may result either from a direct fusogenic action of mastoparan and/or from stimulation of the putative exocytosis-linked G-protein, Ge.
‘Macromolecular crowding’ is a primary factor in the organization of the cytoskeleton
Authors: Cuneo P , Magri E , Verzola A , Grazi E
Published: August 2015

We propose that, in the cell, the reversible conversion of actin filaments into actin bundles is controlled by the concentration of the macromolecules [we have employed poly(ethylene glycol) 6000 to mimic the macromolecules of the cell] as well as by the nature of the ancillary cytoskeletal proteins that decorate actin filaments. The proposal is based on the following evidence. (1) Under our experimental conditions the transition from filaments into bundles occurs at increasing concentrations of poly(ethylene glycol), with the following sequence: caldesmon-actin, 3%; filamin-actin, 4-5%; caldesmon-tropomyosin-actin, 5-7%; actin, 6-7%; tropomyosin-actin, 9-10%. (2) Under conditions of low osmoelastic stress [3% poly(ethylene glycol)], preformed caldesmon-actin bundles are dissociated by the addition of either tropomyosin or tropomyosin-decorated actin. The dissociation of the bundles promoted by the addition of tropomyosin-decorated actin is faster than that promoted by the addition of tropomyosin.
Cloning and expression of a chick liver glutathione S-transferase CL 3 subunit with the use of a baculovirus expression system
Authors: Chang L H , Fan J Y , Liu L F , Tsai S P , Tam M F
Published: August 2015

Glutathione S-transferase CL 3 subunits purified from 1-day-old-chick livers were digested with Achromobacter proteinase I and the resulting fragments were isolated for amino acid sequence analysis. An oligonucleotide probe was constructed accordingly for cDNA library screening. A cDNA clone of 1342 bases, pGCL301, encoding a protein of 26209 Da was isolated and sequenced. Including conservative substitutions, this protein has 75-79% sequence similarity to other Alpha family glutathione S-transferases. The coding sequence of pGCL301 was inserted into a baculovirus vector for infection of Spodoptera frugiperda (SF9) cells. The expressed protein has a high relative activity with ethacrynic acid (47% of the specific activity with 1-chloro-2,4-dinitrobenzene). The enzyme has a subunit molecular mass of 25.2 +/- 1.2 kDa (by SDS/PAGE), a pI of 9.45 and an absorption coefficient A1%1cm of 13.0 +/- 0.5 at 280 nm.
Activation of prothrombin in the presence of human umbilical-vein endothelial cells
Authors: Schoen P , Reutelingsperger C , Lindhout T
Published: August 2015

Addition of Factor Xa, Factor Va and prothrombin to immobilized cultured human umbilical-vein endothelial cells resulted after a time delay in thrombin formation. The prothrombin-converting (prothrombinase) activity, however, was not associated with the cell surface. Rather, perturbation by thrombin, either formed in situ or exogenously added, induced a procoagulant phospholipid surface in the fluid phase, which, in the presence of Factor Xa and Factor Va, enabled the assembly of prothrombinase.
Lipid A stimulates phospholipase D activity in rat mesangial cells via a G-protein
Authors: Harrris W E , Bursten S L
Published: August 2015

Stimulation of mesangial cells (MC) with the bacterial endotoxin Lipid A activated two enzymes involved in lipid metabolism. First, a phospholipase D hydrolyses phosphatidylethanolamine (PE) to phosphatidic acid (PA), followed by dephosphorylation of PA to 1,2-diacylglycerol (DAG) by PA phosphohydrolase. MC or microsomes from these cells were pre-labelled with [3H]glycerol. A 30-60 s stimulation with 10-100 ng of Lipid A/ml caused a decrease in [3H]glycerol in PE and increased radioactive glycerol in PA. The enzyme responsible for this hydrolysis preferred PE containing unsaturated acyl side chains. DAG was formed from PA within the first 1 min after Lipid A stimulation. Microsomes incubated with 25 mM-NaF to inhibit phospholipase C and to stimulate GTP-binding proteins also caused PE to be converted into PA. The [3H]glycerol and acyl mass of phosphatidylcholine, phosphatidylserine and phosphatidylinositol did not change with either Lipid A or NaF. Addition of guanosine 5′-[gamma-thio]triphosphate to MC microsomes caused the rapid decrease in proportion of PE and increase in PA, followed by an increase in DAG unsaturated acyl mass. These data suggest the concurrent G-protein-dependent activation by Lipid A of a PE-directed phospholipase D and a PA phosphohydrolase.
Purification, characterization and function of bacterioferritin from the cyanobacterium Synechocystis P.C.C. 6803
Authors: Laulhère J P , Labouré A M , Van Wuytswinkel O , Gagnon J , Briat J F
Published: August 2015

Storage and buffering of iron is achieved by a class of proteins, the ferritins, widely distributed throughout the living kingdoms. All ferritins have in common their three-dimensional structure and their ability to store large amounts of iron in their central cavity. However, eukaryotic ferritins from plants and animals and bacterioferritins have no sequence similarity, and besides non-haem iron bacterioferritins contain haem residues whereas eukaryotic ferritins do not. In this paper we report the first purification and characterization of a bacterioferritin from a cyanobacterium. It has a molecular mass of 400 kDa and is built up from 19 kDa subunits. Its N-terminal sequence shows 73% identity with that of the Escherichia coli bacterioferritin subunit. It contains 2300 atoms of iron and 1500 molecules of phosphate per ferritin molecule and 0.25 haem residue per subunit; the alpha-peak of the cytochrome has its maximum at 559 nm. In contrast with what is known for eukaryotic ferritins, we found that bacterioferritin from Synechocystis is not inducible by iron under the conditions that we have tested and that it has a constant concentration whatever the iron status of the cells, even at very low iron concentration. Bacterioferritin from Synechocystis P.C.C. 6803 is fully assembled in vivo and it is shown by labelling with 59Fe that it is able to load iron in vitro as well as in vivo. Bacterioferritin from Synechocystis is shown to have an iron-buffering function while the bulk of cellular iron is found associated with a pool of low-molecular-mass electronegative molecules. The role of Synechocystis bacterioferritin in iron metabolism is discussed.
Glutamyl-tRNA reductase activity in Bacillus subtilis is dependent on the hemA gene product
Authors: Schröder I , Hederstedt L , Kannangara C G , Gough S P
Published: August 2015

The Bacillus subtilis hemAXCDBL operon encodes enzymes for the synthesis of 5-aminolaevuline acid via the C5 pathway (hemA and hemL) and uroporphyrinogen III (hemB, hemC and hemD). B. subtilis HemA protein (molecular mass 50 kDa) was overexpressed in hemA mutant of both Escherichia coli and B. subtilis. A mutant B. subtilis HemA protein with a Cys to Tyr change at position 105 was also overexpressed. Both wild-type and mutant HemA proteins migrated as oligomers (molecular mass greater than or equal to 230 kDa) on gel-filtration columns. All column fractions containing wild-type HemA protein had glutamyl-tRNA reductase activity. No glutamyl-tRNA reductase activity was found with the mutant HemA protein. It is concluded that the B. subtilis hemA gene product is identical to, or part of, the glutamyl-tRNA reductase of the C5 pathway.
Analysis of human hepatic microsomal glucose-6-phosphatase in clinical conditions where the T2 pyrophosphate/phosphate transport protein is absent
Authors: Nordlie R C , Scott H M , Waddell I D , Hume R , Burchell A
Published: August 2015

The availability of a rare set of human hepatic microsomes in which T2, a pyrophosphate/phosphate transport protein of the glucose-6-phosphatase system, has been shown immunologically to be completely absent, has permitted further characterization of multicomponent glucose-6-phosphatase (EC 3.1.3.9). Pyrophosphatase activity in intact microsomes was found to be totally absent, but was normal in disrupted microsomes. However, Pi did not accumulate within the lumen of the microsomes when glucose 6-phosphate was the substrate. This was not as predicted if there is only one transport protein in the endoplasmic reticulum capable of transporting Pi, produced by glucose-6-phosphatase, out of the lumen. The results suggest that the pyrophosphate/phosphate transport system of human hepatic endoplasmic reticulum must be more complex than previously thought, as it must comprise at least two protein components.
Levels of G-proteins in liver and brain of lean and obese (ob/ob) mice
Authors: McFarlane-Anderson N , Bailly J , Bégin-Heick N
Published: August 2015

G-protein levels were assessed in liver and brain membranes of lean and obese mice. ADP-ribosylation and immunodetection studies revealed a decrease in the abundance of Gs and Gi alpha-subunits in the liver membranes of obese mice compared with lean mice. In contrast, in brain membranes, the abundance of these proteins was not significantly different between lean and obese mice. Studies at the mRNA level in both liver and brain revealed no difference in gene expression between lean and obese mice. Protein and mRNA studies both showed that Gs, Gi alpha 1, Gi alpha 2, Go alpha and G beta subunits are present in brain membranes, and Gi alpha 3 is barely detectable. In liver, Ga alpha, Gi alpha 2 and G beta subunits are the major constituents, whereas Gi alpha 1, Gi alpha 3 and Go alpha are barely detectable. It is possible that the differences observed at the protein level are due to different rates of translation of the mRNA. Different rates of release of the alpha-subunits from the membrane and/or different rates of degradation would also explain these results.