bluuebunny/crossref_metadata_2025 huggingface dataset:
Low-density-lipoprotein receptors in human fibroblasts are not degraded in lysosomes
Authors: Casciola L A F , Grant K I , Gevers W , Coetzee G A , van der Westhuyzen D R
Published: August 2015

The rate of degradation of low-density-lipoprotein (LDL) receptors was measured in cultured human skin fibroblasts by [35S]methionine pulse-chase experiments. The half-life of LDL receptors was unaltered by inclusion of LDL in the medium (t1/2 11 h). Neither lysosomotropic inhibitors (chloroquine or NH4Cl) nor leupeptin inhibited the rate of receptor degradation in the absence of ligand. In cells incubated at 18 degrees C to inhibit the delivery of internalized ligands from endocytic vesicles to lysosomes, receptor degradation continued, but at the expected rate of about six times lower than that at 37 degrees C. Mutant LDL receptors defective in internalization were degraded at the same rate as normal receptors, suggesting that receptor internalization and recycling are not required for basal turnover. We conclude that the rate-limiting steps for, and probably the whole pathway of, degradation of normal LDL receptors does not take place in lysosomes.
δ-Aminolaevulinate synthase in human HepG2 hepatoma cells. Repression by haemin and induction by chemicals
Authors: Iwasa F , Sassa S , Kappas A
Published: August 2015

delta-Aminolaevulinate (ALA) synthase, the rate-limiting enzyme in haem biosynthesis in the normal liver, was examined in human HepG2 hepatoma cells. Haemin, up to 100 microM, had no effect on ALA synthase activity in vitro; it did, however, exhibit a dose-dependent inhibitory action when added to cells growing in culture (half-maximal inhibition at 1 microM). The half-life of ALA synthase activity after haemin treatment was 2 h, which was similar to that found after treatment with cycloheximide. Cells treated with actinomycin D showed a longer half-life of the enzyme activity, i.e. 4 h, compared with haemin or cycloheximide treatment. Treatment of cells with succinylacetone markedly inhibited the activity of ALA dehydratase and 59Fe incorporation into haem, but in increased ALA synthase activity. Both the haemin-induced repression and the succinylacetone-mediated de-repression of ALA synthase activity were reversible within 4 h after replacing the medium with fresh medium without the chemical. In addition to succinylacetone, dimethyl sulphoxide and 3-methylcholanthrene induced the enzyme. Induction of ALA synthase by these chemicals was also suppressed by treatment of cells with haemin. These findings indicate that the level of ALA synthase in HepG2 cells is maintained by both synthesis and degradation of the enzyme, and that the synthesis of the enzyme is regulated by the concentration of regulatory free haem in the cell.
Regulation of production of δ-aminolaevulinate synthase in tissues of chick embryos. Effects of porphyrogenic agents and of haem precursors
Authors: Drew P D , Ades I Z
Published: August 2015

Studies conducted by several groups have established that porphyrogenic agents which caused elevations in chick-embryo liver delta-aminolaevulinate (ALA) synthase activity also increased the concentrations of the enzyme's RNA, and that haemin inhibited these elevations. We have determined in this study, using immune-blot analyses, that administration in ovo of allylisopropylacetamide (AIA) in combination with diethyl 1,4-dihydro-2,4,6-trimethyl,3,5-pyridinedicarboxylate (DDC) increased the mass of ALA synthase in intestine and kidney of chick embryos. Furthermore, the molecular mass of the subunit of the enzyme in those tissues appeared identical with that of liver ALA synthase. Using a synthetic oligonucleotide complementary to ALA synthase mRNA, we determined by solution hybridization and Northern-blot analyses that AIA and DDC also increased the concentrations of ALA synthase mRNA in intestine and kidney and that testosterone elevated the concentration of the RNA in kidney. In analyses of RNA obtained from chick-embryo liver, intestine, kidney, heart, brain and lung, the probe bound primarily in each case to a single 2.3 kb RNA. Finally, the haem precursors ALA and FeCl3, when injected together into the fluid surrounding embryos, inhibited both the elevations in ALA synthase mass and RNA concentration brought about by porphyrogenic agents in liver, kidney and intestine. Thus the results indicated that: (1) certain porphyrogenic agents increased ALA synthase mass and RNA in chick-embryo intestine and kidney, in addition to liver; (2) ALA and FeCl3 inhibited the elevations; and (3) the sizes of ALA synthase's subunit as well as the enzyme's mRNA appeared identical, in each case, in all tissues examined.
Regulation of GH3 pituitary tumour-cell adenylate cyclase activity by activators of protein kinase C
Authors: Quilliam L A , Dobson P R M , Brown B L
Published: August 2015

The influence of protein kinase C (PKC) activation on cyclic AMP production in GH3 cells has been studied. The stimulation of cyclic AMP accumulation induced by forskolin and cholera toxin was potentiated by 4 beta-phorbol 12,13-dibutyrate (PDBu). Moreover, PDBu, which causes attenuation of the maximal response to vasoactive intestinal polypeptide (VIP), also induced a small right shift in the dose-response curve for VIP-induced cyclic AMP accumulation. PDBu-stimulated cyclic AMP accumulation was unaffected by pretreatment of cells with pertussis toxin or the inhibitory muscarinic agonist, oxotremorine. PDBu stimulation of adenylate cyclase activity required the presence of a cytosolic factor which appeared to translocate to the plasma membrane in response to the phorbol ester. The diacylglycerol-generating agents thyroliberin, bombesin and bacterial phospholipase C each stimulated cyclic AMP accumulation, but, unlike PDBu, did not attenuate the stimulation induced by VIP. These results suggest that PKC affects at least two components of the adenylate cyclase complex. Stimulation of cyclic AMP accumulation is probably due to modification of the catalytic subunit, whereas attenuation of VIP-stimulated cyclic AMP accumulation appears to be due to the phosphorylation of a different site, which may be the VIP receptor.
Characterization of the gene encoding mouse serum amyloid P component. Comparison with genes encoding other pentraxins
Authors: Whitehead A S , Rits M
Published: August 2015

A CBA/J-strain mouse serum amyloid P component (SAP) genomic clone was isolated and analysed. The clone contains the entire SAP gene and specifies a primary transcript of 1065 nucleotide residues. This comprises a first exon of 206 nucleotide residues containing the mRNA 5′-untranslated region and sequence encoding the pre-SAP leader peptide and the first two amino acid residues of mature SAP separated by a single 110-base intron from a 749-nucleotide-residue second exon containing sequence encoding the bulk of the mature SAP and specifying the mRNA 3′-untranslated region. The overall organization is similar to that of the human SAP gene, and the coding region and intron sequences are highly conserved. The SAP RNA cap site was defined by primer extension analysis of polyadenylated acute-phase liver RNA. The 5′-region of the mouse SAP gene contains modified CAAT and TATA promoter elements preceded by a putative hepatocyte-nuclear-factor-1-recognition site; these structures are in a region that is highly homologous to the corresponding region of the human SAP gene. Comparisons of the mouse SAP gene structure and derived amino acid sequence with those of other mammalian pentraxins were made.
Target size analysis by radiation inactivation of carnitine palmitoyltransferase activity and malonyl-CoA binding in outer membranes from rat liver mitochondria
Authors: Zammit V A , Corstorphine C G , Kolodziej M P
Published: August 2015

The functional molecular sizes of the protein(s) mediating the carnitine palmitoyltransferase I (CPT I) activity and the [14C]malonyl-CoA binding in purified outer-membrane preparations from rat liver mitochondria were determined by radiation-inactivation analysis. In all preparations tested the dose-dependent decay in [14C]malonyl-CoA binding was less steep than that for CPT I activity, suggesting that the protein involved in malonyl-CoA binding may be smaller than that catalysing the CPT I activity. The respective sizes computed from simultaneous analysis for molecular-size standards exposed under identical conditions were 60,000 and 83,000 DA for malonyl-CoA binding and CPT I activity respectively. In irradiated membranes the sensitivity of CPT activity to malonyl-CoA inhibition was increased, as judged by malonyl-CoA inhibition curves for the activity in control and in irradiated membranes that had received 20 Mrad radiation and in which CPT activity had decayed by 60%. Possible correlations between these data and other recent observations on the CPT system are discussed.
Biosynthesis of paf-acether. Paf-acether but not leukotriene C4 production is impaired in cultured macrophages
Authors: Dulioust A , Vivier E , Meslier N , Roubin R , Haye-Legrand I , Benveniste J
Published: August 2015

After adherence for 24 or 48 h mouse peritoneal macrophages, upon a zymosan challenge, synthesized 114 +/- 55 and 82 +/- 31 pmol of paf-acether (paf)/mg of protein respectively, as compared with 513 +/- 195 pmol of paf/mg of protein in 2 h-adherent macrophages (means +/- S.D., n = 10). By contrast, 24 h- and 48 h-adherent macrophages exposed to zymosan produced more leukotriene C4 (2.7 +/- 1.1 and 1.4 +/- 0.2 nmol/mg of protein respectively, n = 5) than did 2 h-adherent macrophages (0.5 +/- 0.2 nmol/mg of protein, n = 5). Paf production was not altered when 2 h- and 24 h-adherent cells were cultured and/or stimulated in the presence of 5 microM-indomethacin, 10 microM-nordihydroguaiaretic acid or 100 microM-BW755C as compared with untreated cells. These results indirectly exclude the regulation of paf production by arachidonic acid metabolites. We investigated the efficiency of the enzymic steps which govern paf synthesis. We showed that the anabolic process was not impaired since (1) the amounts of alkylacylglycerophosphocholine and lyso-paf were similar in 2 h-, 24 h- and 48 h-adherent macrophages; (2) adding synthetic lyso-paf or acetyl-CoA to intact cells did not increase paf production in zymosan-stimulated 24 h- and 48 h-adherent macrophages; (3) the basal level of acetyltransferase was comparable in 2 h-, 24 h- and 48 h-adherent macrophages and in all cases was increased by 2-3 times upon zymosan challenge. We also showed that impaired paf production in 24 h- and 48 h-cultured macrophages was not due to the nature of the stimulus used to induce its synthesis.
A kinetic investigation of the acyl-CoA oxidase reaction with the use of a novel spectrophotometric assay. Inhibition by acetyl-CoA, CoA and FMN
Authors: Hovik R , Osmundsen H
Published: August 2015

A direct-reading spectrophotometric assay for acyl-CoA oxidase activity is described. The assay is based on the strong absorption at 300 nm of deca-2-trans,4-cis-dienoyl-CoA, the product of oxidation of dec-4-cis-enoyl-CoA. By use of this assay, acetyl-CoA, CoA and FMN were found to be inhibitors of acyl-CoA oxidase, but with distinctly different kinetic characteristics.
Purification of TEM-1 β-lactamase by immunoaffinity chromatography
Authors: Bibi E
Published: August 2015

A monoclonal antibody prepared against TEM-1 beta-lactamase was found to compete with penicillins and cephalosporins for binding to the enzyme. The purified antibody preparation was linked to Sepharose 4B and used for immunoaffinity-chromatography purification of TEM-1 beta-lactamase. Elution with either benzylpenicillin or cloxacillin yielded a highly purified, concentrated and active enzyme preparation.
The quaternary structure in solution of human complement subcomponent C1r2C1s2
Authors: Perkins S J , Nealis A S
Published: August 2015

C1r2C1s2 is a subcomponent of first component C1 of the complement cascade. Previously two distinct models for its structure have been described, in which C1r2C1s2 is either a linear rod-like assembly of the globular domains found in each of C1s and C1r, or these domains are arranged to form an asymmetric X-shaped structure. These two models were evaluated by using hydrodynamic simulations and neutron scattering. The data on C1s, C1s2 and C1r are readily represented by straight hydrodynamic cylinders, but not C1r2 or C1r2C1s2. Tests of the X-structure for C1r2 and C1r2C1s2 successfully predicted the experimental sedimentation coefficients, thus supporting this model. Neutron scattering analyses on C1s and C1r2 are consistent with a linear structure for C1s, but not for C1r2. An X-shaped structure for C1r2 was found to give a good account of the neutron data at large scattering angles. The total length of the C1s and C1r monomers was determined as 17-20 nm, which is compatible with electron microscopy. On the basis of the known sequences of C1r and C1s, this length is accounted for by a linear arrangement of a serine-proteinase domain (length 4 nm), two short consensus repeat domains (2 x 4 nm), and a globular entity containing the I, II and III domains (4-7 nm).
Desialylation of transferrin by liver endothelium is selective for its triantennary chain
Authors: Irie S , Tavassoli M
Published: August 2015

Liver endothelium can remove and transport the glycoprotein transferrin (TF). During this process the molecules are desialylated; however, in contrast with other such glycoproteins, for example caeruloplasmin, only half of transported TF is desialylated. To explore which component of TF is desialylated, we double-labelled fully sialylated TF with [3H]sialic acid residues and a 125I-protein moiety. This was then ‘chased’ through purified liver endothelium in pulse-chase experiments. Endothelium-conditioned TF was fractionated on an RCA120 affinity column into sialylated and desialylated components. Each component was then re-fractionated on a concanavalin A affinity column, which separates the glycoprotein according to the branching pattern of its glycan chain. The desialylated fraction was eluted only as a triantennary component, whereas the non-desialylated fraction consisted only of bi- and tetra-antennary chains. The significance of this selective desialylation of triantennary chain of TF in the subsequent metabolism of its iron content and its possible role in the pathogenesis of alcohol-induced hepatic siderosis are discussed.
Defective phosphorylation of a calmodulin-binding protein in cystic-fibrosis submandibular glands
Authors: Shori D K , Dormer R L , Goodchild M C , McPherson M A
Published: August 2015

Calmodulin-binding proteins in fractions purified from human submandibular glands by calmodulin-Sepharose were phosphorylated with [gamma-32P]ATP, in the absence of exogenous protein kinase. The major proteins phosphorylated had molecular masses of 45, 51 and 61 kDa. Phosphorylation was increased by activators of protein kinase C and inhibited by H-7. Phosphorylation of the 61 kDa band was markedly decreased in cystic-fibrosis submandibular glands.
Phospholipid-protein interactions of the plasma-membrane Ca2+-transporting ATPase. Evidence for a tissue-dependent functional difference
Authors: Missiaen L , Raeymaekers L , Wuytack F , Vrolix M , de Smedt H , Casteels R
Published: August 2015

The aim of the present work was to investigate the stimulation of the plasma-membrane Ca2+-transporting ATPase by negatively charged phospholipids. The Ca2+-transporting ATPase was purified from pig stomach smooth muscle and from pig erythrocytes, and was reactivated with phosphatidylcholine (PC) in the presence and absence of negatively charged phospholipids. The substitution of phosphatidylinositol (PI), phosphatidylinositol 4-phosphate (PIP), phosphatidylinositol 4,5-bisphosphate (PIP2), phosphatidic acid (PA) or phosphatidylserine (PS) for PC induced profound changes in the Vmax, the K0.5 and the Hill coefficient of the Ca2+-activation curves for both ATPases. Low concentrations of each of the negatively charged phospholipids increased the Vmax., but high ratios of PIP, PIP2 or PA to PC decreased this parameter. PI, PA and PS increased the Vmax. of the erythrocyte enzyme to a larger extent than that of the smooth-muscle enzyme. This difference was less pronounced for PIP and absent for PIP2. PI (greater than 20% PC substituted), PIP, PIP2, PA and PS all increased the affinity of the two Ca2+-transporting ATPases for Ca2+ in the following order of potency: PIP2 greater than PIP greater than PI approximately PS approximately PA. PI, PA and PS increased the Ca2+ affinity of the smooth-muscle enzyme more than that of the erythrocyte enzyme; this difference was less pronounced for PIP and absent for PIP2. Even in the presence of calmodulin, all of the negatively charged phospholipids were still able to increase the Vmax. of the erythrocyte enzyme, whereas only PIP and PIP2 increased the affinity for Ca2+. The effect of PI at low concentrations (less than 20%) on the erythrocyte enzyme was peculiar in that it caused a decrease in the Ca2+ affinity instead of an increase. This effect was not observed for the smooth-muscle enzyme. All of the negatively charged phospholipids slightly increased the Hill coefficient for Ca2+ of both ATPases, and this effect was additive to that of calmodulin. The stimulation of the erythrocyte enzyme exhibited positive co-operativity towards PI and PIP, whereas that of the smooth-muscle enzyme did not. It is concluded (1) that there is a correlation between the number of negative charges on the phospholipids (PIP2 greater than PIP greater than PA approximately PI approximately PS) and the magnitude of their effect on the Vmax. and the K0.5 for Ca2+, and (2) that the action of the lipids on the smooth-muscle enzyme differs from that on the erythrocyte enzyme, indicating that these two Ca2+-transporting ATPases are not the same.
Molybdenum nitrogenase of Azotobacter chroococcum. Tight binding of MgADP to the MoFe protein
Authors: Miller R W , Eady R R
Published: August 2015

The dye-oxidized or dithionite-reduced forms of the MoFe protein of molybdenum nitrogenase of Azotobacter chroococcum were shown to bind 2 mol of MgADP/mol of protein, as determined by column equilibrium techniques. The gel-filtration elution profile of unbound Mg[14C]ADP was not symmetrical, consistent with a low rate of dissociation from the protein. Symmetrical elution profiles were observed for the oxidized Fe protein of nitrogenase, which bound 2 mol of MgADP/mol of protein. The low rate of dissociation of MgADP from MoFe protein was shown by non-equilibrium column techniques, where 1 mol of MgADP/mol of MoFe protein remained tightly bound during chromatography. Very weak binding of MgATP (less than 0.01 mol of MgATP/mol of MoFe protein) to dye-oxidized but not to dithionite-reduced MoFe protein was observed. These results are discussed in terms of their relevance to the catalytic cycle of nitrogenase catalysis.
Synthesis of hyaluronate in cultured bovine articular cartilage
Authors: Ng C K , Handley C J , Mason R M , Robinson H C
Published: August 2015

The synthesis and distribution of hyaluronate and proteoglycan were studied in bovine articular cartilage in short-term explant culture with [3H]acetate and H2(35)SO4 as precursors. The incorporation of [3H]acetate into hyaluronate and sulphated glycosaminoglycans was linear with time, except that hyaluronate synthesis showed a marked lag at the beginning of the incubation. [3H]Hyaluronate represented 4-7% of the total [3H]glycosaminoglycans synthesized over a 6 h period. However, the distributions of [3H]hyaluronate and 3H-labelled sulphated glycosaminoglycans were different: about 50% of the newly synthesized [3H]hyaluronate appeared in the medium, compared with less than 5% of the 3H-labelled sulphated proteoglycans. A pulse-chase experiment revealed that the release of newly synthesized [3H]hyaluronate from cartilage was rapid. No difference was observed in the distribution of [3H]hyaluronate between medium and tissue by cartilage from either the superficial layer or the deep layer of articular cartilage. When articular cartilage was incubated with 0.4 mM-cycloheximide, proteoglycan synthesis was markedly inhibited, whereas the synthesis of hyaluronate was only partially inhibited and resulted in more of the newly synthesized hyaluronate being released into the medium. Analysis of the hydrodynamic size of [3H]hyaluronate isolated from cartilage on Sephacryl-1000 revealed one population that was eluted as a broad peak (Kav. less than 0.7), compared with two populations (Kav. greater than 0.5 and less than 0.5) appearing in the medium of cultures. These data suggest that hyaluronate is synthesized in excess of proteoglycan synthesis and that the hyaluronate that is not complexed with proteoglycans is rapidly lost from the tissue.
Classification of prostaglandin receptors based on coupling to signal transduction systems
Authors: Muallem S , Merritt B S , Green J , Kleeman C R , Yamaguchi D T
Published: August 2015

A wide spectrum of prostaglandins (PG) stimulate both the production of cyclic AMP and an increase in free cytosolic Ca2+ concentration [(Ca2+]i) in the osteogenic osteosarcoma cell line, UMR-106-01, which has characteristics compatible with osteoblasts. Using PG-stimulated determinations of the second messengers cyclic AMP and [Ca2+]i, a method for classification of PG receptors is presented. UMR-106-01 cells demonstrate three subclasses of PG receptors. One receptor interacts with PGF2 alpha, PGD2, and thromboxane B2 (TxB2) to increase [Ca2+]i. A second receptor binds PGE2, PGE1, PGI2, PGA2 and 6-oxo-PGF1 alpha to increase [Ca2+]i by stimulation of a second separate phospholipase C pool. A third receptor accepts PGE2, PGE1, PGA2, PGI2 and to a lesser extent PGF2 alpha, PGD2 and TxB2 to increase cyclic AMP. Such a classification system may be applicable to other cells responding to multiple PGs by inducing changes in cellular second messengers.
Electroneutral, HCO3--independent, pH gradient-dependent uphill transport of Cl- by ileal brush-border membrane vesicles. Possible role in the pathogenesis of chloridorrhea
Authors: Vasseur M , Caüzac M , Alvarado F
Published: August 2015

By applying a rapid filtration technique to isolated brush border membrane vesicles from guinea pig ileum, 36Cl uptake was quantified in the presence and absence of electrical, pH and alkali-metal ion gradients. A mixture of 20 mM-Hepes and 40 mM-citric acid, adjusted to the desired pH with Tris base, was found to be the most suitable buffer. Malate and Mes could be used to replace the citrate, but succinate, acetate and maleate proved to be unsuitable. In the absence of a pH gradient (pHout:pHin = 7.5:7.5), Cl- uptake increased slightly when an inside-positive membrane potential was applied, but uphill transport was never observed. A pH gradient (pHout:pHin = 5.0:7.5) induced both a 400% increase in the initial Cl- influx rate and a long-lasting (20 to 300 s) overshoot, indicating that a proton gradient can furnish the driving force for uphill Cl- transport. Under pH gradient conditions, initial Cl- entry rates had the following characteristics. (1) They were unaffected by cis-Na+ and/or -K+, indicating the absence of Cl-/K+, Cl-/Na+ or Cl-/K+/Na+ symport activity. (2) Inhibition by 20-100 mM-trans-Na+ and/or -K+ occurred, independent of the existence of an ion gradient. (3) Cl- entry was practically unaffected by short-circuiting the membrane potential with equilibrated potassium and valinomycin. (4) Carbonyl cyanide m-chlorophenylhydrazone was strongly inhibitory and so, to a lesser extent, was 4-acetamido-4′-isothiocyanostilbene-2,2′-disulphonic acid [(SITS)], independent of the sign and size of the membrane potential. (5) Cl- entry was negligibly increased (less than 30%) by either trans-Cl- or -HCO3-, indicating the absence of an obligatory Cl-/anion antiport activity. In contrast, the height of the overshoot at 60 s was increased by trans-Cl-, indicating time-dependent inhibition of 36Cl efflux. That competitive inhibition of 36Cl fluxes by anions is involved here is supported by initial influx rate experiments demonstrating: (1) the saturability of Cl- influx, which was found to exhibit Michaelis-Menten kinetics; and (2) competitive inhibition of influx by cis-Cl- and -Br-. Quantitatively, the conclusion is warranted that over 85% of the total initial Cl- uptake energized by a pH gradient involves an electroneutral Cl-/H+ symporter or its physicochemical equivalent, a Cl-/OH- antiporter, exhibiting little Cl- uniport and either Cl-/Cl- or Cl-/HCO3- antiport activities.(ABSTRACT TRUNCATED AT 400 WORDS)
The amino acid sequence of a flavodoxin from the eukaryotic red alga Chondrus crispus
Authors: Wakabayashi S , Kimura T , Fukuyama K , Matsubara H , Rogers L J
Published: August 2015

The amino acid sequence of the constitutive flavodoxin from the red alga Chondrus crispus was determined from the analyses of peptide fragments derived by enzymic digestions of the carboxymethylated protein. This is the first sequence reported for a flavodoxin from a eukaryote. The protein is composed of 173 amino acid residues and is a member of the longer-chain group of flavodoxins. The extent of sequence homology to the three other flavodoxins in the group for which sequences are available is in the range 36-39%, with the most strongly conserved regions being those implicated in binding of the FMN, the redox-active prosthetic group. Nevertheless, Chondrus crispus flavodoxin stands apart in a number of respects, in particular the possession of an unusually high content of proline, with these residues distributed more or less regularly along the peptide chain.
Cytosolic ferritin and lipid-associated ferritin are metabolically different in guinea-pig livers
Authors: Cham B E , Roeser H P , Nikles A C
Published: August 2015

A distinct pool of liver ferritin has been described in man, guinea pigs and rats [Cham, Roeser, Nikles & Ridgway (1986) Clin. Chim. Acta 158, 71-79]. This ferritin accounts for approx. 30% of total intracellular ferritin. It differs from previously described cytosolic and ‘microsomal-fraction’ ferritin by its firm association with lipid and by the absence of heat-stability at 75 degrees C. The present study demonstrates that cytosolic ferritin and lipid-associated ferritin in guinea-pig livers have distinctly different rates of turnover. Cytosolic ferritin has a rate of turnover approx. 3.5 times as high as lipid-associated ferritin. The apparent metabolic heterogeneity suggests that the two forms of ferritin may have different functional roles.
Kallikrein-gene expression in the rat gastrointestinal tract
Authors: Fuller P J , Verity K , Matheson B A , Clements J A
Published: August 2015

The serine proteinase glandular kallikrein has been demonstrated in the gastrointestinal tract, although there is some doubt as to whether it is synthesized there or derives from exocrine-gland secretions. Using a rat pancreatic kallikrein cRNA probe we have demonstrated kallikrein-like gene expression in the corpus, duodenum, jejunum, ileum, caecum and colon, and compared the pattern of expression with that of the gastrointestinal peptides somatostatin, gastrin and glucagon. In addition, using a panel of oligonucleotide probes specific for various members of the rat kallikrein-gene family, we have shown that the kallikrein-like gene expressed appears to be expressed as true kallikrein.
High-yield isolation of functionally competent endosomes from mouse lymphocytes
Authors: Beaumelle B D , Hopkins C R
Published: August 2015

A discontinuous-sucrose-gradient procedure for isolating endosomes from mouse lymphoma cells has been developed. After centrifugation, most organelles (especially mitochondria and lysosomes) are recovered in the denser fractions of the gradient, whereas a mixture of plasma membrane and endosomes is present at lighter densities. The endosome recovery in this fraction can be increased (by 100%) by (a) a mild trypsin treatment of the postnuclear supernatant and (b) loading the cell endosomes with a saturating concentration of low-density lipoproteins. Removal of the plasma-membrane contamination was achieved by preincubating the cells with a gold-ricin complex at 4 degrees C. On centrifugation, the gold-loaded membranes sediment to the bottom of the gradient. The endosome preparation isolated by these procedures is less than 6% contaminated by other organelles and contains 42% of internalized 125I-transferrin. We show that these isolated endosomes are functional, as displayed by their ability to fuse and to acidify in a cell-free system. Endosome fusion was studied by a new assay based on the use of fluorescence resonance energy transfer. This fusion is dependent on ATP and on a cytosolic, thermoresistant but trypsin- and N-ethylmaleimide-sensitive, protein factor. Early endosomes fuse more actively among themselves than with late-endocytic vesicles, and they fuse only slowly with plasma-membrane vesicles.
The non-fluorescence of 4-fluorotryptophan
Authors: Hott J L , Borkman R F
Published: August 2015

The derivative 4-fluorotryptophan was confirmed to have negligible fluorescence at 25 degrees C and 285 nm (tryptophan/4-fluorotryptophan quantum-yield ratio greater than 100:1). However, photolysis experiments on tryptophan and 4-fluorotryptophan, in which loss of starting material was measured by reverse-phase h.p.l.c., demonstrated that 4-fluorotryptophan was significantly more photochemically active than the parent tryptophan, with the 4-fluorotryptophan photolysis quantum yield being 7 times larger than that of tryptophan at 25 degrees C and 285 nm. In addition, at 77 K and 275 nm 4-fluorotryptophan displayed strong fluorescence and phosphorescence, with emission quantum yields comparable with those of tryptophan at 77 K and 275 nm.
The quantitative spectrum of inositol phosphate metabolites in avian erythrocytes, analysed by proton n.m.r. and h.p.l.c. with direct isomer detection
Authors: Radenberg T , Scholz P , Bergmann G , Mayr G W
Published: August 2015

The spectrum of inositol phosphate isomers present in avian erythrocytes was investigated in qualitative and quantitative terms. Inositol phosphates were isolated in micromolar quantities from turkey blood by anion-exchange chromatography on Q-Sepharose and subjected to proton n.m.r. and h.p.l.c. analysis. We employed a h.p.l.c. technique with a novel, recently described complexometric post-column detection system, called ‘metal-dye detection’ [Mayr (1988) Biochem. J. 254, 585-591], which enabled us to identify non-radioactively labelled inositol phosphate isomers and to determine their masses. The results indicate that avian erythrocytes contain the same inositol phosphate isomers as mammalian cells. Denoted by the ‘lowest-locant rule’ [NC-IUB Recommendations (1988) Biochem. J. 258, 1-2] irrespective of true enantiomerism, these are Ins(1,4)P2, Ins(1,6)P2, Ins(1,3,4)P3, Ins(1,4,5)P3, Ins(1,3,4,5)P4, Ins(1,3,4,6)P4, Ins(1,4,5,6)P4, Ins(1,3,4,5,6)P5, and InsP6. Furthermore, we identified two inositol trisphosphate isomers hitherto not described for mammalian cells, namely Ins(1,5,6)P3 and Ins(2,4,5)P3. The possible position of these two isomers in inositol phosphate metabolism and implications resulting from absolute abundances of inositol phosphates are discussed.
Decreased activity and impaired hormonal control of protein phosphatases in rat livers with a deficiency of phosphorylase kinase
Authors: Toth B , Bollen M , Stalmans W
Published: August 2015

1. Livers from gsd/gsd rats, which do not express phosphorylase kinase activity, also contain much less particulate type-1 protein phosphatases. In comparison with normal Wistar rats, the glycogen/microsomal fraction contained 75% less glycogen-synthase phosphatase and 60% less phosphorylase phosphatase activity. This was largely due to a lower amount of the type-1 catalytic subunit in the particulate fraction. In the cytosol, the synthase phosphatase activity was also 50% lower, but the phosphorylase phosphatase activity was equal. 2. Both Wistar rats and gsd/gsd rats responded to an intravenous injection of insulin plus glucose with an acute increase (by 30-40%) in the phosphorylase phosphatase activity in the liver cytosol. In contrast, administration of glucagon or vasopressin provoked a rapid fall (by about 25%) in the cytosolic phosphorylase phosphatase activity in Wistar rats, but no change occurred in gsd/gsd rats. 3. Phosphorylase kinase was partially purified from liver and subsequently activated. Addition of a physiological amount of the activated enzyme to a liver cytosol from Wistar rats decreased the V of the phosphorylase phosphatase reaction by half, whereas the non-activated kinase had no effect. The kinase preparations did not change the activity of glycogen-synthase phosphatase, which does not respond to glucagon or vasopressin. Furthermore, the phosphorylase phosphatase activity was not affected by addition of physiological concentrations of homogeneous phosphorylase kinase from skeletal muscle (activated or non-activated). 4. It appears therefore that phosphorylase kinase plays an essential role in the transduction of the effect of glucagon and vasopressin to phosphorylase phosphatase. However, this inhibitory effect either is specific for the hepatic phosphorylase kinase, or is mediated by an unidentified protein that is a specific substrate of phosphorylase kinase.
The expression of canine cardiac phospholamban in heterologous systems
Authors: Cook E A , Huggins J P , Sathe G , England P J , Piggott J R
Published: August 2015

A synthetic phospholamban gene has been cloned and expressed in Escherichia coli, producing both native phospholamban and a fusion protein with 81 amino acids of the influenza virus NS1 protein. Both the native phospholamban and fusion proteins produced extensive cell lysis upon induction of gene expression, but only the native protein underwent spontaneous pentamer formation in E. coli. Translation in vitro of mRNA produced by transcription in vitro of phospholamban cDNA was used to demonstrate the spontaneous aggregation of phospholamban to form pentamers in this system also, both in the presence and absence of exogenous microsomes from canine pancreas or heart. Phospholamban produced by translation in vitro was apparently susceptible to proteolysis by enzymes present in the particulate fractions in these experiments.
Functional properties of membrane cofactor protein of complement
Authors: Seya T , Atkinson J P
Published: August 2015

Membrane cofactor protein (MCP or gp45-70) of the complement system is a cofactor for factor I-mediated cleavage of fluid-phase C3b and C3b-like C3, which opens the thioester bond. In the present study the activity of MCP was further characterized. Unexpectedly, in the absence of factor I, MCP stabilized the alternative- and, to a lesser extent, the classical-pathway cell-bound C3 convertases and thereby enhanced C3b deposition. Soluble MCP, if added exogenously, hardly functioned as cofactor for the cleavage of erythrocyte-bound C3b to iC3b; i.e. its activity, compared with the cofactor activity of factor H, was inefficient, since less than 10% of the bound C3b was MCP-sensitive. Further, exogenously added soluble MCP was also a weak cofactor for the cleavage of C3b bound to zymosan. Likewise, factor I, in the presence of cells bearing MCP, cleaved fluid-phase C3b inefficiently. These results imply that MCP has very little extrinsic cofactor activity for factor I. In contrast, exogenously added MCP and factor I mediated efficient cleavage of erythrocyte-bound C3b if the concentration of Nonidet P40 was sufficient to solubilize the cells. Interestingly, soluble MCP and factor I degraded C3b attached to certain solubilized acceptor membrane molecules more readily than others. The cleavage reaction of fluid-phase and cell-bound C3b by soluble MCP and factor I produced iC3b, but no C3c and C3dg. These and prior data indicate that soluble MCP has potent cofactor activity for fluid-phase C3b or C3b bound to solubilized molecules, but acts inefficiently towards C3b on other cells. This functional profile is unique for a C3b/C4b binding protein and, taken together with its wide tissue distribution, suggests an important role for MCP in the regulation of the complement system.
Mechanisms of luteinizing-hormone exocytosis in Staphylococcus aureus-α-toxin-permeabilized sheep gonadotropes
Authors: van der Merwe P A , Millar R P , Wakefield I K , Davidson J S
Published: August 2015

We have used primary gonadotropes permeabilized with the pore-forming protein Staphylococcus aureus alpha-toxin to investigate luteinizing hormone (lutropin, LH) exocytosis. The diameter of the alpha-toxin pores (2-3 nm) allows the exchange of small molecules, whereas larger cytosolic proteins are retained. Because of the slow exchange of small molecules through the pores, we have developed a protocol which combines prolonged pre-equilibration of the permeabilized cells at 0 degrees C before stimulation with strong Ca2+ buffering. Under these conditions, increasing the free Ca2+ concentration from 0.1 microM to 10 microM [EC50 (concentration effecting half-maximal response) 2-3 microM] resulted in a 15-20-fold increase in LH exocytosis. LH exocytosis was maximal in the first 3 min and completed by 12 min. When permeabilized cells were equilibrated for prolonged periods in the absence of MgATP, Ca2(+)-stimulated LH secretion gradually declined (greater than 90% decrease by 60 min). Addition of MgATP (5 mM) rapidly restored full Ca2(+)-stimulated LH secretion. MgATP supported Ca2(+)-stimulated LH secretion at a half-maximal concentration of 1.5 mM. UTP and adenosine 5′-[gamma-thio]triphosphate were 40 and 31% as effective as MgATP, whereas other nucleotide triphosphates were ineffective. The protein kinase C (PKC) activator phorbol 12-myristate 13-acetate (PMA; 50 nM) stimulated LH exocytosis at free Ca2+ concentrations as low as 1 nM and was additive with Ca2+ at higher free Ca2+ concentrations. PMA-stimulated exocytosis required MgATP at concentrations similar to those required for Ca2(+)-stimulated LH exocytosis. These results demonstrate that LH exocytosis can be triggered both by micromolar Ca2+ concentrations or, in the virtual absence of Ca2+, by PKC activation. Both mechanisms of stimulated exocytosis have an absolute requirement for millimolar ATP. Because they retain cytosolic proteins, alpha-toxin-permeabilized cells may have advantages over alternative permeabilization methods provided that conditions are used that compensate for slow diffusion through alpha-toxin pores.
Identification of the haem-binding subunit of cytochrome b−245
Authors: Nugent J H A , Gratzer W , Segal A W
Published: August 2015

Cytochrome b-245 from neutrophil plasma membranes contains two types of subunit with apparent molecular masses from gel electrophoresis in the presence of SDS of 23 kDa and 76-92 kDa. Radiation-inactivation analysis revealed a single-exponential decay process for the visible absorption of the haem chromophore in the membrane, corresponding to a molecular mass of 21 +/- 5 kDa for the haem-containing polypeptide chain. Sedimentation equilibrium of the cytochrome solubilized by the detergent Triton N101 showed that the protein was polydisperse, with a molecular mass of approx. 350 kDa for the smallest detectable species. In another detergent, n-octyl beta-O-glucopyranoside (octyl glucoside), the molecular mass of the haem-containing particle was found to be 20-30 kDa. Thus the quaternary structure of the protein breaks down in this detergent. The haem group is inferred to be attached to the smaller subunit.
Participation of two structurally related enzymes in rat hepatic microsomal androstenedione 7α-hydroxylation
Authors: Waxman D J , Lapenson D P , Nagata K , Conlon H D
Published: August 2015

Rat hepatic cytochrome P-450 form 3 (testosterone 7 alpha-hydroxylase; P-450 gene IIA1) and P-450 form RLM2 (testosterone 15 alpha-hydroxylase; P-450 gene IIA2) are 88% identical in primary structure, yet they hydroxylate testosterone with distinct and apparently unrelated regioselectivities. In this study, androstenedione and progesterone were used to assess the regioselectivity and stereospecificity of these two P-450 enzymes towards other steroid substrates. Although P-450 RLM2 exhibited low 7 alpha-hydroxylase activity with testosterone or progesterone as substrate (turnover number less than or equal to 1-2 nmol of metabolite/min per nmol of P-450), it did catalyse androstenedione 7 alpha-hydroxylation at a high rate (21 min-1) which exceeded that of P-450 3 (7 min-1). However, whereas P-450 3 exhibited a high specificity for hydroxylation of these steroids at the 7 alpha position (95-97% of total activity), P-450 RLM2 actively metabolized these compounds at four or more major sites including the nearby C-15 position, which dominated in the case of testosterone and progesterone. The observation that androstenedione is actively 7 alpha-hydroxylated by purified P-450 RLM2 suggested that this P-450 enzyme might make significant contributions to microsomal androstenedione 7 alpha-hydroxylation, an activity that was previously reported to be associated with immunoreactive P-450 3. Antibody inhibition experiments were therefore carried out in liver microsomes using polyclonal anti-(P-450 3) antibodies which cross-react with P-450 RLM2, and using a monoclonal antibody that is reactive with and inhibitory towards P-450 3 but not P-450 RLM2. P-450 3 was thus shown to catalyse only around 35% of the total androstenedione 7 alpha-hydroxylase activity in uninduced adult male rat liver microsomes, with the balance attributed to P-450 RLM2. The P-450-3-dependent 7 alpha-hydroxylase activity was increased to approximately 65% of the total in phenobarbital-induced adult male microsomes, and to greater than 90% of the total in untreated adult female rat liver microsomes. These observations are consistent with the inducibility of P-450 3 by phenobarbital and with the absence of P-450 RLM2 from adult female rat liver respectively. These findings establish that P-450 RLM2 and P-450 3 can both contribute significantly to microsomal androstenedione 7 alpha-hydroxylation, thus demonstrating that the 7 alpha-hydroxylation of this androgen does not serve as a specific catalytic monitor for microsomal P-450 3.
Synergistic activation of 2-deoxy-d-glucose uptake in rat and murine peritoneal macrophages by human macrophage colony-stimulating factor-stimulated coupling between transport and hexokinase activity and phorbol-dependent stimulation of pentose phosphate-shunt activity
Authors: Rist R J , Jones G E , Naftalin R J
Published: August 2015

1. Transport and accumulation of 2-deoxy-D-glucose (2dGlc) in rat and murine peritoneal macrophages were investigated by using C-1-3H-labelled and C-2,6-3H-labelled 2dGlc. 2. There was active accumulation of both C-1- and C-2,6-labelled 2dGlc by quiescent rat and murine macrophages via a phloretin-inhibitable transport system. 3. The rate of uptake and accumulation of 2dGlc (C-1 label) was increased by exposure to human macrophage colony-stimulating factor (mCSF-1) (1000 units/ml) in both murine and rat macrophages. This indicates that mCSF-1 enhances coupling between hexokinase activity and glucose transport at the endofacial surface of the transporter. 4. Phorbol 12-myristate 13-acetate (‘phorbol’) at 40 nM stimulated 2dGlc in rat macrophages entirely by increasing the C-2,6 label uptake. This indicates that phorbol stimulates 2dGlc uptake mainly by increasing the activity of the pentose phosphate pathway. 5. Simultaneous exposure to phorbol and mCSF-1 stimulates 2dGlc uptake to a greater extent than found with either phorbol or mCSF-1 alone. This result is explained by a simultaneous enhancement of pentose phosphate-pathway activity and of hexokinase activity acting at the endofacial surface of the cell membrane. The dual activation of these serial processes coupled to the loss of the reaction products of the pentose phosphate-shunt pathway from the cells in the form of reactive oxygen intermediates, protons and CO2 could explain the synergistic action of phorbol and mCSF-1 in activation of sugar transport in macrophages.
Calcium-binding affinity and calcium-enhanced activity of Clostridium thermocellum endoglucanase D
Authors: Chauvaux S , Beguin P , Aubert J P , Bhat K M , Gow L A , Wood T M , Bairoch A
Published: August 2015

Clostridium thermocellum endoglucanase D (EC 3.2.1.4: EGD), which is encoded by the celD gene, was found to bind Ca2+ with an association constant of 2.03 x 10(6) M-1. Ca2+ stimulated the activity of EGD towards swollen Avicel by 2-fold. In the presence of Ca2+, the Kd of the enzyme towards p-nitrophenyl-beta-D-cellobioside and carboxymethylcellulose was decreased by 4-fold. Furthermore, Ca2+ increased the half-life of the enzyme at 75 degrees C from 13 to 47 min. Since the 3′ sequence of celD encodes a duplicated region sharing similarities with the Ca2+-binding site of several Ca2+-binding proteins, a deleted clone was constructed and used to purify a truncated form of the enzyme which no longer contained the duplicated region. The truncated enzyme was very similar to EGD expressed from the intact gene with respect to activity, Ca2(+)-binding kinetics and Ca2+ effects on substrate binding and thermostability. Thus the latter parameters do not appear to be mediated through the duplicated conserved region.
The contributions of Ca2+, phospholipids and tissue-factor apoprotein to the activation of human blood-coagulation factor X by activated factor VII
Authors: Bom V J J , Bertina R M
Published: August 2015

In the extrinsic pathway of blood coagulation, Factor X is activated by a complex of tissue factor, factor VII(a) and Ca2+ ions. Using purified human coagulation factors and a sensitive spectrophotometric assay for Factor Xa, we could demonstrate activation of Factor X by Factor VIIa in the absence of tissue-factor apoprotein, phospholipids and Ca2+. This finding allowed a kinetic analysis of the contribution of each of the cofactors. Ca2+ stimulated the reaction rate 10-fold at an optimum of 6 mM (Vmax. of 1.1 x 10(-3) min-1) mainly by decreasing the Km of Factor X (to 11.4 microM). In the presence of Ca2+, 25 microM-phospholipid caused a 150-fold decrease of the apparent Km and a 2-fold increase of the apparent Vmax. of the reaction; however, both kinetic parameters increased with increasing phospholipid concentration. Tissue-factor apoprotein contributed to the reaction rate mainly by an increase of the Vmax., in both the presence (40,500-fold) and absence (4900-fold) of phospholipid. The formation of a ternary complex of Factor VIIa with tissue-factor apoprotein and phospholipid was responsible for a 15 million-fold increase in the catalytic efficiency of Factor X activation. The presence of Ca2+ was absolutely required for the stimulatory effects of phospholipid and apoprotein. The data fit a general model in which the Ca2(+)-dependent conformation allows Factor VIIa to bind tissue-factor apoprotein and/or a negatively charged phospholipid surface resulting into a decreased intrinsic Km and an increased Vmax. for the activation of fluid-phase Factor X.