bluuebunny/crossref_metadata_2025 huggingface dataset:
Altered stoichiometry of the human leucocyte Na+/H+ antiport with decreasing pH
Authors: Ng L L , Bomford J
Published: August 2015

The Na+/H+ antiport is an important regulator of cellular volume, pH and Na+ concentration in mammalian cells. The stoichiometry of this antiporter has previously been shown to be a 1:1 exchange of internal H+ for external Na+. We have investigated this stoichiometry in human leucocytes by using a novel intracellular pH-clamping technique and measuring 22Na+ influx and H+ efflux in the same cells. As internal pH was lowered, the stoichiometry of H+/Na+ exchange rose to a mean +/- S.D. of 2.23 +/- 0.69. This mechanism allows a higher H+ efflux in the face of intracellular acid stress without causing excessive intracellular Na+ overload.
The cilia of Paramecium tetraurelia contain both Ca2+-dependent and Ca2+-inhibitable calmodulin-binding proteins
Authors: Evans T C , Nelson D L
Published: August 2015

To identify protein targets for calmodulin (CaM) in the cilia of Paramecium tetraurelia, we employed a 125I-CaM blot assay after resolution of ciliary proteins on SDS/polyacrylamide gels. Two distinct types of CaM-binding proteins were detected. One group bound 125I-CaM at free Ca2+ concentrations above 0.5-1 microM and included a major binding activity of 63 kDa (C63) and activities of 126 kDa (C126), 96 kDa (C96), and 36 kDa (C36). CaM bound these proteins with high (nanomolar) affinity and specificity relative to related Ca2+ receptors. The second type of protein bound 125I-CaM only when the free Ca2+ concentration was below 1-2 microM and included polypeptides of 95 kDa (E95) and 105 kDa (E105). E105 may also contain Ca2+-dependent binding sites for CaM. Both E95 and E105 exhibited strong specificity for Paramecium CaM over bovine CaM. Ciliary subfractionation experiments suggested that C63, C126, C96, E95, and E105 are bound to the axoneme, whereas C36 is a soluble and/or membrane-associated protein. Additional Ca2+-dependent CaM-binding proteins of 63, 70, and 120 kDa were found associated with ciliary membrane vesicles. In support of these results, filtration binding assays also indicated high-affinity binding sites for CaM on isolated intact axonemes and suggested the presence of both Ca2+-dependent and Ca2+-inhibitable targets. Like E95 and E105, the Ca2+-inhibitable CaM-binding sites showed strong preference for Paramecium CaM over vertebrate CaM and troponin C. Together, these results suggest that CaM has multiple targets in the cilium and hence may regulate ciliary motility in a complex and pleiotropic fashion.
Polyphosphoinositide breakdown and subsequent exocytosis in the Ca2+/ionophore-induced acrosome reaction of mammalian spermatozoa
Authors: Roldan E R S , Harrison R A P
Published: August 2015

An investigation was made of the modifications in phospholipids that occur during the exocytotic event known as the ‘sperm acrosome reaction’. Phospholipids were prelabelled with 32P, and exocytosis was induced with Ca2+ and the ionophore A23187. When incubated with [32P]Pi in various media suitable for supporting sperm survival or fertilization in vitro, spermatozoa from all five species examined (ram, boar, guinea pig, mouse and human) incorporated 32P rapidly into the components of the phosphoinositide cycle. There were differences both between species and between media with respect to the actual rate of incorporation of label, and also between species with respect to other phospholipids labelled. Treatment of spermatozoa with Ca2+ and A23187 to induce the acrosome reaction resulted in a rapid breakdown of phosphatidylinositol 4, 5-bisphosphate and phosphatidylinositol 4-phosphate, which was complete within 3 min; there was also a great increase in labelling of phosphatidate. Occurrence of acrosome reactions in the sperm population was only observed after 5-10 min and reached a maximum response of greater than 90% after more than 30 min. The phosphoinositide breakdown was related to subsequent exocytosis: after EGTA/ionophore treatment, neither inositide breakdown nor exocytosis took place; however, later addition of Ca2+ resulted in immediate inositide breakdown, and exocytosis followed, with a delay relative to Ca2+ addition exactly similar to that following standard Ca2+/ionophore treatment. Neomycin inhibited both inositide breakdown and subsequent exocytosis provided it was added together with Ca2+ and ionophore; however, if the drug was added 3 min after Ca2+ and ionophore (by which time inositide breakdown was already complete), exocytosis was not inhibited. Ca2+ seemed to have several consecutive roles in the acrosome reaction. Low (micromolar) levels of free Ca2+ were needed both for phosphoinositide breakdown and for an event downstream of this breakdown; no other bivalent cation could substitute for Ca2+ in either event, and inositide breakdown was actually inhibited by Mg2+. In addition, millimolar levels of Ca2+ were needed for later stages of exocytosis, although this requirement could be satisfied by Sr2+. We conclude that breakdown of polyphosphoinositides is an essential early process after Ca2+ entry in the chain of events that lead to exocytosis in the mammalian sperm acrosome reaction.
Primary sequence and domain structure of chicken vinculin
Authors: Price G J , Jones P , Davison M D , Patel B , Bendori R , Geiger B , Critchley D R
Published: August 2015

We have determined the complete sequence of chick vinculin from two overlapping cDNA clones. The vinculin mRNA consists of 262 bp of 5' untranslated sequence, an open reading frame of 3195 bp (excluding the initiation codon) and a long 3' untranslated sequence (greater than 2 kb). Chick vinculin contains 1066 amino acid residues, and has a deduced molecular mass of 116,933 Da. Analysis of the domain structure of vinculin shows that the molecule can be cleaved by V8 proteinase into a 90 kDa globular head and a 32 kDa tail region, the latter of which could further be cleaved into a 27 kDa polypeptide. The 90 kDa globular head contains the N-terminus of vinculin, three 112-residue repeats (residues 259-589), and extends to approximately residue 850. Gel overlay experiments show that it also contains a binding site for the cytoskeletal protein talin. The talin-binding domain was further localized to the N-terminal 398 amino acid residues of the protein by expression in vitro of this region from a vinculin cDNA cloned into the Bluescript SK+ vector. The head and tail domains are apparently separated by a proline-rich region that contains V8-proteinase-cleavage sites and a candidate tyrosine (822)-phosphorylation site. Secondary-structure prediction suggests that the head and tail domains contain alpha-helical regions separated by short stretches of turn/coil. Comparison of the chick with a partial human sequence reveals that vinculin is a highly conserved protein. In chickens Southern-blot analysis is consistent with a single vinculin gene, and it is therefore likely that vinculin, and its higher-molecular-mass isoform termed metavinculin, arise through alternative splicing.
Effects of angiotensin II and of phorbol ester on protein kinase C activity and on prostacyclin production in cultured rat aortic smooth-muscle cells
Authors: Lang U , Vallotton M B
Published: August 2015

The role of the Ca2+-sensitive phospholipid-dependent protein kinase C (PKC) was studied in cultured rat aortic smooth-muscle cells, known to respond to angiotensin II (Ang II) by producing prostacyclin, determined by the release of 6-oxo-prostaglandin F1 alpha. PKC activity was measured in the cytosol and the solubilized membrane fraction after DEAE-cellulose chromatography using a linear NaCl gradient. Ang II stimulated the activity of PKC in the cytosolic and in the membrane fractions of aortic smooth-muscle cells. These increases in PKC activity were concentration-dependent and occurred rapidly, reaching a plateau within 10 min. In contrast, phorbol 12-myristate 13-acetate (PMA) rapidly decreased cytosolic PKC activity and at the same time increased membrane PKC activity to reach a plateau after 20 min. Cytosolic PKC activity from control and Ang II-stimulated cells was found to be less dependent on [Ca2+] than was the highly [Ca2+]-dependent membrane PKC activity from the same cells. In contrast, membrane PKC activity from PMA-treated cells was largely [Ca2+]-independent. In the presence of 10 nM-PMA, the sensitivity of cultured smooth-muscle cells towards Ang II was increased, and maximal values of Ang II-induced prostacyclin production were enhanced by about 60%. In cells incubated with both Ang II and PMA, an additive effect on membrane PKC activity was observed, whereas cytosolic PKC activity was suppressed as in cells treated with PMA alone. These results suggest that an increase of the membrane, but not the cytosolic, PKC activity represents a positive signal in the prostacyclin production induced by Ang II stimulation of aortic smooth-muscle cells. PMA seems to induce a state of activation of membrane PKC which does not need increased intracellular [Ca2+] to be fully expressed, whereas Ang II-stimulated membrane PKC activity requires higher Ca2+ concentrations. The possibility exists that the addition of both signals leads to the augmentation of Ang II-stimulated prostacyclin production.
Epidermal-growth-factor-stimulated phosphorylation of calpactin II in membrane vesicles shed from cultured A-431 cells
Authors: Blay J , Valentine-Braun K A , Northup J K , Hollenberg M D
Published: August 2015

Membrane vesicles shed from intact A-431 epidermoid carcinoma cells and harvested in the presence of Ca2+ contained epidermal-growth-factor (EGF) receptor/kinase substrates of apparent molecular masses 185, 85, 70, 55, 38 and 27 kDa. The 38 kDa substrate (p38) was recognized by an antibody that had been raised against the human placental EGF receptor/kinase substrate calpactin II (lipocortin I). The A-431 and placental substrates, isolated by immunoprecipitation after phosphorylation in situ, yielded identical phosphopeptide maps upon limited proteolytic digestion with each of five different enzymes. The A-431-cell vesicular p38 is therefore calpactin II. EGF treatment of the intact A-431 cells before inducing vesiculation was not necessary for the substrate to be present within the vesicles. Our data thus indicate that receptor internalization is not a prerequisite for receptor-mediated phosphorylation of calpactin II. The ability of the protein to function as a substrate for the receptor/kinase depended upon the continued presence of Ca2+ during the vesicle-isolation procedure. EGF-stimulated phosphorylation of calpactin II was much less pronounced in vesicles prepared from A-431 cells in the absence of Ca2+, although comparable amounts of the protein were detectable by immunoblotting. Calpactin II therefore appears to be sequestered in a Ca2+-modulated manner within shed vesicles, along with at least four other major targets for the EGF receptor/kinase. The vesicle preparation may be a useful model system in which to study the phosphorylation and function of potentially important membrane-associated substrates for the receptor.
X-ray-absorption-spectroscopic evidence for a novel iron cluster in hydrogenase II from Clostridium pasteurianum
Authors: George G N , Prince R C , Stokley K E , Adams M W , Stockley K E
Published: August 2015

Hydrogenase II from Clostridium pasteurianum contains three different iron-sulphur clusters. Two are [4Fe-4S](2+.1+) clusters, whereas the other, which is thought to be the site of interaction with H2 and is known as the ‘H cluster’, is of unknown structure and possesses unusual spectroscopic properties. Analysis of the iron e.x.a.f.s. spectra shows that the H cluster contains iron co-ordinated mostly to sulphur and possesses 2.8 A (1 A = 0.1 nm) Fe--Fe separations when oxidized and 3.3 A Fe--Fe separations when reduced with H2. The data suggest that the reduced H cluster represents a new structural type of iron-sulphur cluster.
Effect of sulphate on glutamate synthesis by intact spinach (Spinacia oleracea) chloroplasts
Authors: Dumas R , Joyard J , Douce R
Published: August 2015

During the course of NH4+ (or NO2-)-plus-alpha-oxoglutarate-dependent O2 evolution in spinach (Spinacia oleracea) chloroplasts, glutamate was continuously excreted out of the chloroplasts. Under these conditions, for each molecule of NO2- or NH4+ which disappeared, one molecule of glutamate accumulated in the medium and the concentration of glutamate in the stroma space was maintained constant. SO4(2-) (or SO3(2-) behave as inhibitors of NH4+ incorporation into glutamate by intact chloroplasts. This considerable inhibition of glutamate synthesis by SO4(2-) was correlated with a rapid decline in the stromal Pi concentration. The reloading of stromal Pi with either external Pi or PPi4- relieved SO4(2-)-induced inhibition of glutamate synthesis by intact chloroplasts. It was concluded that SO4(2-) induced a rapid efflux of stromal Pi out of the chloroplast, leading to a limitation of ATP synthesis and therefore to an arrest of ATP-dependent glutamine synthetase functioning.
Characterization of the bis(5′-nucleosidyl) tetraphosphate pyrophosphohydrolase from encysted embryos of the brine shrimp Artemia
Authors: Prescott M , Milne A D , McLennan A G
Published: August 2015

The P1P4-bis(5′-nucleosidyl) tetraphosphate asymmetrical-pyrophosphohydrolase from encysted embryos of the brine shrimp Artemia has been purified over 11,000-fold to homogeneity. Anion-exchange chromatography resolves two major species with very similar properties. The enzyme is a single polypeptide of Mr 17,600 and is maximally active at pH 8.4 and 2 mM-Mg2+. It is inhibited by Ca2+ (IC50 = 0.9 mM with 2 mM-Mg2+) but not by Zn2+ ions. It preferentially hydrolyses P1P4-bis(5′-nucleosidyl) tetraphosphates, e.g. P1P4-bis(5′-adenosyl) tetraphosphate (Ap4A) (kcat. = 12.7 s-1; Km = 33 microM) and P1P4-bis(5′-guanosyl) tetraphosphate (Gp4G) (kcat. = 6.2 s-1; Km = 5 microM). With adenosine 5′-P1-tetraphospho-P4-5‴-guanosine (Ap4G) as substrate, there is a 4.5-fold preference for AMP and GTP as products and biphasic reaction kinetics are observed giving Km values of 4.7 microM and 34 microM, and corresponding rate constants of 6.5 s-1 and 11.9 s-1. The net rate constant for Ap4G hydrolysis is 7.6 s-1. The enzyme will also hydrolyse nucleotides with more than four phosphate groups, e.g. Ap5G, Ap6A and Gp5G are hydrolysed at 25%, 18% and 10% of the rate of Ap4A respectively. An NTP is always one of the products. Ap2A and Gp2G are not hydrolysed, while Ap3A and Gp3G are very poor substrates. When the enzyme is partially purified from embryos and larvae at different stages of development by sedimentation through a sucrose density gradient, its activity increases 3-fold during the first 12 h of pre-emergence development. This is followed by a slow decline during subsequent larval development. The similarity of this enzyme to other asymmetrical-pyrophosphohydrolases suggests that it did not evolve specifically to degrade the large yolk platelet store of Gp4G which is found in Artemia embryos, but that it probably serves the same general function in bis(5′-nucleosidyl) oligophosphate metabolism as in other cells.
Alkylation, reduction, solubilization and enrichment of binding activity do not impair the ability of insulin receptors to convert from a rapid- into a slow-dissociating state
Authors: Lipson K E , Kolhatkar A A , Donner D B
Published: August 2015

Hormone binding promotes conversion of rat hepatic insulin receptors from a rapid-dissociating into a slow-dissociating affinity state. Solubilization into detergent does not impair the ability of receptors to convert into a slow-dissociating state, and this property is retained as receptor-binding activity is enriched 11,000-13,000-fold during purification. Hormone binding also induces two conformational changes (alterations of tryptic lability) in the insulin receptor. The first change is rapid and exposes parts of the receptor to tryptic degradation. The second, slower, change occurs with the same time course, and probably mediates the conversion into the slower-dissociating binding state. Reduction of disulphide bonds with dithiothreitol does not prevent conversion of binding sites into a slower-dissociating state, and reduced receptors retain the ability to undergo conformational changes in response to hormone binding. Alkylation with N-ethylmaleimide also does not affect the insulin-induced conversion into a slow-dissociating state. These observations suggest that the conversion into a slow-dissociating state is an intrinsic property of the insulin receptor. Free thiol groups in the insulin receptor and disulphide bonds between the alpha-subunits are not essential to this process.
Contraction-mediated inactivation of glycogen synthase is accompanied by inactivation of glycogen synthase phosphatase in human skeletal muscle
Authors: Kida Y , Katz A , Lee A D , Mott D M
Published: August 2015

Activities of glycogen synthase (GS) and GS phosphatase were determined on human muscle biopsies before and after isometric contraction at 2/3 maximal voluntary force. Total GS activity did not change during contraction (4.92 +/- 0.70 at rest versus 5.00 +/- 0.42 mmol/min per kg dry wt.; mean +/- S.E.M.), whereas both the active form of GS and the ratio of active form to total GS decreased by approximately 35% (P less than 0.01). GS phosphatase was inactivated in all subjects by an average of 39%, from 5.95 +/- 1.30 to 3.63 +/- 0.97 mmol/min per kg dry wt. (P less than 0.01). It is suggested that at least part of the contraction-induced inactivation of GS is due to an inactivation of GS phosphatase.
The use of permeabilized cells to study the ion requirements of receptor–ligand dissociation in endosomes
Authors: Diaz R , Wileman T E , Anderson S J , Stahl P
Published: August 2015

The mannose receptor mediates the transport of high-mannose glycoproteins from the cell surface to lysosomes in macrophages. The binding of ligand to the receptor is dependent on both pH and Ca2+. Upon internalization, ligands enter an acidic pre-lysosomal compartment where receptor-ligand dissociation takes place. Acidification is driven by an endosomal proton pump and anion transport is coupled to this acidification step. A permeabilized-cell assay has been designed to characterize the ionic requirements for receptor-ligand dissociation in endosomes. The plasma membrane of macrophages has been permeabilized selectively with digitonin without affecting endosomal membranes. Receptor-ligand dissociation in permeabilized cells required ATP and was blocked by proton ionophores. Di-isothiocyanostilbene-disulphonic acid and N-ethylmaleimide also blocked dissociation, but mitochondrial ATPase inhibitors and vanadate were ineffective. To explore the nature of the anion requirement for acidification, the ability of different anions to compensate for Cl- was tested. For the halide series, Br- was as equally effective as Cl- in supporting receptor-ligand dissociation, but I- was inhibitory. Citrate and gluconate were only partially effective, while SO4(2-), NO3- and PO4(2-) blocked dissociation. Addition of Ca2+ to permeabilized-cell preparations impaired ATP-dependent dissociation without affecting endosome acidification. These results suggest that the endosomal membrane has a Ca2+ conductance that would permit the rapid efflux of Ca2+ from endosomes during acidification, and this would appear to be a necessary step for efficient sorting of Ca2+-dependent receptors from their ligands.
Effects of sepiapterin and 6-acetyldihydrohomopterin on the guanosine triphosphate cyclohydrolase I of mouse, rat and the fruit-fly Drosophila
Authors: Jacobson K B , Manos R E
Published: August 2015

The regulation of GTP cyclohydrolase I would lead to the regulation of tetrahydrobiopterin, an important cofactor for synthesis of neurotransmitters. In an attempt to extend a previous finding [Bellahsene, Dhondt, & Farriaux (1984) Biochem. J. 217, 59-65] that GTP cyclohydrolase I of rat liver is inhibited by subnanomolar concentrations of reduced biopterin and sepiapterin, we found that this could not be verified with the enzyme from mouse liver, fruit-fly (Drosophila) heads or, indeed, from rat liver. It was shown, however, that 12 microM-sepiapterin inhibited mouse liver GTP cyclohydrolase I. Another compound, namely 6-acetyldihydrohomopterin, was also employed in the present study to explore its effect on enzymes that lead to its synthesis in Drosophila and for effects on mammalian systems; at 2-5 microM this compound was shown to stimulate one form of mouse liver GTP cyclohydrolase I and then to inhibit at higher concentrations (40 microM). Neither sepiapterin nor 6-acetyldihydrohomopterin caused any effect on the Drosophila head enzyme. On the other hand, the sigmoid GTP concentration curve for the Drosophila enzyme may indicate a regulatory characteristic of this enzyme. Another report, on the lower level of GTP cyclohydrolase I in mutant mouse liver [McDonald, Cotton, Jennings, Ledley, Woo & Bode (1988) J. Neurochem. 50, 655-657], was confirmed and extended. Instead of having 10% activity, we find that the hph-1 mouse mutant has less than 2% activity in the liver. These studies demonstrate that micromolar levels of reduced pterins may have regulatory effects on GTP cyclohydrolase I and that a mouse mutant is available that has low enough activity to be considered as a model for human atypical phenylketonuria.
Glucocorticoids modulate mRNA levels for G-protein β-subunits
Authors: Ros M , Watkins D C , Rapiejko P J , Malbon C C
Published: August 2015

Adrenalectomy decreases, whereas glucocorticoid treatment increases, the steady-state levels of G-protein beta-subunits (G beta) in rat fat-cells. A DNA-excess solution-hybridization assay was established to define the steady-state mRNA levels for G beta [5.8 +/- 0.4 amol/micrograms of RNA (n = 5) in control fat-cells]. G beta mRNA levels decrease by 20% after adrenalectomy; dexamethasone treatment reverses the decline. Dexamethasone treatment itself increases G beta mRNA levels by 50%.
Prostaglandin E2 inhibits phosphoinositide metabolism in isolated pancreatic islets
Authors: Laychock S G
Published: August 2015

Isolated islets of the rat labelled with myo-[3H]inositol showed decreased accumulation of total inositol phosphates (InsPs) and [3H]polyphosphoinositide hydrolysis in response to glucose after preincubation with prostaglandin E2 (PGE2). The response was concentration-dependent and specific for PGE2. PGE2 did not affect basal [3H]phosphoinositide hydrolysis or InsPs accumulation. Pertussis-toxin pretreatment antagonized the response to PGE2, whereas 8-bromo cyclic AMP was without effect. The PGE2-induced decrease in InsPs may contribute to the suppression of insulin secretion.
Novel monoclonal antibodies against human uterine progesterone receptor. Mapping of receptor immunogenic domains
Authors: Vu Hai M T , Jolivet A , Ravet V , Lorenzo F , Perrot-Applanat M , Citerne M , Milgrom E
Published: August 2015

Progesterone receptor was purified in a single step from human uteri using immunoaffinity chromatography with monoclonal antibodies raised against the rabbit receptor. A total of 39 monoclonal antibodies were prepared against the human receptor and characterized. Immunoblot experiments using crude uterine cytosol or purified receptor showed that the antibodies belonged to three groups: they recognized either a single receptor species (apparent molecular mass 110 kDa), two species (110 and 79 kDa) or three species (110, 79 and 65 kDa). The species specificity of the antibodies was very variable; some recognized only the human receptor, others interacted with several mammalian receptors (rabbit, guinea pig and rat), while a single one also cross-reacted with the chick receptor. The epitopes recognized by 15 of the antibodies showing the strongest affinity for the human receptor were mapped using a method recently described [Lorenzo, Jolivet & Milgrom (1988) Eur. J. Biochem. 176, 53-60] which involves immunoprecipitation of C-terminally truncated proteins obtained by transcription and translation of cloned cDNA in vitro. The antibodies recognized five different regions of the receptor, all localized on the N-terminal half of the protein. None of the antibodies interacted with an epitope present in the DNA-binding or steroid-binding regions of the receptor. Comparison of the pattern of receptor species recognized by the antibodies and the localization of their epitopes showed that the 79 and 65 kDa receptor species were derived from the 110 kDa form by deletion of its N-terminal part. The N-terminus of the 79 kDa species was found to lie between amino acids 121 and 208, and that of the 65 kDa species between amino acids 208 and 296.
Metabolism of platelet-activating factor (PAF; 1-O-alkyl-2-acetyl-sn-glycero-3-phosphocholine) and lyso-PAF (1-O-alkyl-2-lyso-sn-glycero-3-phosphocholine) by cultured rat Kupffer cells
Authors: Chao W , Siafaka-Kapadai A , Hanahan D J , Olson M S
Published: August 2015

The metabolism of platelet-activating factor (PAF; identified as AGEPC: 1-O-alkyl-2-acetyl-sn-glycero-3-phosphocholine) and lyso-PAF (lyso-GEPC: 1-O-alkyl-2-lyso-sn-glycero-3-phosphocholine) was investigated in cultured rat Kupffer cells. The rat Kupffer cells accumulated [3H]AGEPC and deacetylated this compound to the corresponding [3H]lyso-GEPC, which was the major metabolic product of [3H]AGEPC. [3H]Lyso-GEPC was distributed primarily in the supernatant fraction of incubated cells throughout the experimental interval. Only a very small portion of the [3H]lyso-GEPC was further converted to 1-O-alkyl-2-acyl-sn-glycero-3-phosphocholine (alkylacyl-GPC), indicating that this acylation process was not particularly active in these cells. When [3H]lyso-GEPC was incubated with Kupffer cells, the conversion of lyso-GEPC to AGEPC via the acetyltransferase reaction increased up to 30 min and declined thereafter. Bovine serum albumin (BSA) had a substantial influence on both the cellular uptake and the metabolism of [3H]AGEPC. An increase in the BSA concentration in the incubation media reduced the cellular uptake of [3H]AGEPC and the subsequent formation of lyso-GEPC. The results of this study suggest that the hepatic Kupffer cells play an important role in the metabolism of PAF. Moreover, these results infer that the regulation of the PAF level in certain hepatic pathophysiological situations may be a consequence of the production and subsequent metabolism of this potent lipid autacoid in the Kupffer cells of the liver.
Metabolism of inositol phosphates in α1-adrenoceptor-stimulated and homogenized cardiac myocytes of adult rats
Authors: Guse A H , Berg I , Gercken G
Published: August 2015

Previous studies demonstrated the accumulation of inositol mono- and poly-phosphates in carbamoylcholine-stimulated cultured cardiac ventricular myocytes of adult rats [Berg, Guse & Gercken (1989) Biochim. Biophys. Acta 1010, 100-107]. Stimulation with noradrenaline (50 microM) in the presence of propranolol (10 microM) led to a time-dependent pattern of inositol mono- and poly-phosphates in cultured cardiac-ventricular myocytes. Ins(1,4,5)P3 and Ins(1,3,4,5)P4 increased in a rapid initial phase. The degradation products of Ins(1,4,5)P3, namely Ins(1,4)P2 and Ins(4)P, accumulated between 1 and 15 min. Ins(1,3,4,5)P4 was rapidly dephosphorylated to Ins(1,3,4)P3, which was then metabolized to Ins(1,3)P2 and Ins(3,4)P2. The last two InsP2 isomers and their degradation products, Ins(1)P and Ins(3)P (determined as an enantiomeric mixture), increased at extended stimulation periods. To confirm the pathway of Ins(1,3,4,5)P4 degradation, homogenates of isolated ventricular myocytes were incubated with [3H]INs(1,3,4,5)P4. The subsequent products were Ins(1,3,4)P3, Ins(3,4)P2, Ins(1,3)P2 and InsP. The effect of noradrenaline was antagonized by prazosin (0.1 microM), but not by yohimbine (0.1 microM), indicating phosphoinositidase activation via the alpha 1-adrenoceptor.
Comparative effects of tumour necrosis factor-α (cachectin), interleukin-1-β and tumour growth on amino acid metabolism in the rat in vivo. Absorption and tissue uptake of α-amino[1-14C]isobutyrate
Authors: Argilés J M , López-Soriano F J , Wiggins D , Williamson D H
Published: August 2015

The effects of acute administration of either tumour necrosis factor-alpha (cachectin) (TNF) or interleukin-1-beta (IL-1), or of tumour growth (Walker-256 carcinosarcoma), on blood amino acid concentrations and tissue alpha-amino[1-14C]isobutyrate (AIB) uptake in virgin and lactating rats were compared. Both monokines decreased the blood concentrations of those amino acids (serine, glycine, alanine and proline) transported via the A system. Tumour growth decreased the blood concentrations of serine, proline and histidine, whereas the concentrations of glutamine and leucine were increased. IL-1 decreased the intestinal absorption of AIB in all groups studied; TNF or tumour growth had no effect. Tissue AIB uptake was increased (1.5-2.5-fold) in liver, whereas it was decreased in heart and skeletal muscle of the three treatment groups (except skeletal muscle of the IL-1-treated rats). Lactating rats had lower hepatic uptake of AIB compared with livers of virgin rats. IL-1 increased the hepatic uptake of AIB in lactating rats, but not to the values seen in virgin rats treated with IL-1; there was no effect of the cytokine on muscle or mammary-gland uptake. In adrenalectomized rats, the stimulatory effect of IL-1 on hepatic AIB uptake was diminished, whereas that of TNF still persisted. IL-1 caused a marked decrease of AIB uptake in muscle and heart of adrenalectomized rats, which was accompanied by an increase in the blood concentrations of branched-chain amino acids. These effects did not occur with TNF. It is concluded that the effects of the cytokines on tissue amino acid metabolism may depend on a differential endocrine response involving glucagon and/or glucocorticoids.
Identification of a cysteine residue at the active site of Escherichia coli isocitrate lyase
Authors: Nimmo H G , Douglas F , Kleanthous C , Campbell D G , MacKintosh C
Published: August 2015

Escherichia coli isocitrate lyase was inactivated by iodacetate in a pseudo-first-order process. Complete inactivation was associated with the incorporation of only one carboxymethyl group per enzyme subunit. The substrate and products of the enzyme protected against inactivation, suggesting that the reactive group may be located at the active site. Isolation and sequencing of a carboxymethylated peptide showed that the modified residue was a cysteine, in the sequence Cys-Gly-His-Met-Gly-Gly-Lys. The reactivity of isocitrate lyase to iodoacetate declined with pH, following a titration curve for a group of pKa 7.1. The Km of the enzyme for isocritrate declined over the same pH range.
Passive diffusion of non-electrolytes across the lysosome membrane
Authors: Iveson G P , Bird S J , Lloyd J B
Published: August 2015

An osmotic-protection method has been used to study the permeability of rat liver lysosomes to 43 organic non-electrolytes of formula weights ranging from 62 to 1000. A lysosome-rich centrifugal fraction of rat liver homogenate was resuspended in an unbuffered 0.25 M solution of test solute, pH 7.0, and incubated at 25 degrees C for 60 min. The free and total activities of 4-methylumbelliferyl N-acetyl-beta-D-glucosaminidase were measured after incubation for 0, 30 and 60 min. Three patterns of results were seen. In pattern A the percentage free activity remained low throughout the 60 min incubation, indicating little or no solute entry into the lysosomes. In pattern B, the percentage free activity was initially low, but rose substantially during the incubation, indicating solute entry. In pattern C there was not even initial osmotic protection, indicating very rapid solute entry. The rapidity of solute entry into the lysosomes showed no correlation with the formula weight, but a perfect inverse correlation with the hydrogen-bonding capacity of the solutes. The results, which can be used to predict the ability of further compounds to cross the lysosome membrane by unassisted diffusion, are discussed in the context of metabolite and drug release from lysosomes in vivo.
Lysophospholipase A2 activity in guinea-pig heart microsomal fractions displaying high activities with 2-acylglycerophosphocholines with linoleic and arachidonic acids
Authors: Arthur G
Published: August 2015

Lysophospholipases A1 which catalyse the hydrolysis of acyl groups from 1-acylglycerophosphocholine (GPC) have been characterized in a number of mammalian tissues and do not exhibit any acyl specificity. In the present study lysophospholipase activity in guinea-pig heart microsomes (microsomal fractions) that hydrolyses 2-acyl-GPC was detected and characterized. The enzyme showed a high degree of acyl specificity. The relative rates of hydrolysis of individual 2-acyl-GPCs with different fatty acids was as follows: C18:2/C20:1/C18:1/C16:0, 14:6:1:1. When substrates were presented in pairs, the hydrolysis of each substrate by the enzyme was inhibited, but to very different extents. Of each pair of lysolipids examined (2-arachidonoyl- and 2-palmitoyl-GPC; 2-arachidonoyl- and 2-linoleoyl-GPC), the one with the expected higher rate of hydrolysis was more severely inhibited and the degree of inhibition was dependent on the concentration of the other lysolipid. The characteristics of the lysophospholipase A2 suggest the enzyme could work in concert with phospholipase A1 to release arachidonic and linoeic acids for further metabolism. The properties of lysophospholipase A2 and A1 suggest that they are different enzymes.
The low C5 convertase activity of the C4A6 allotype of human complement component C4
Authors: Kinoshita T , Dodds A W , Law S K A , Inoue K
Published: August 2015

We have compared the C5-convertase-forming ability of different C4 allotypes, including the C4A6 allotype, which has low haemolytic activity and which has previously been shown to be defective in C5-convertase formation. Recent studies suggest that C4 plays two roles in the formation of the C5 convertase from the C3 convertase. Firstly, C4b acts as the binding site for C3 which, upon cleavage by C2, forms a covalent linkage with the C4b. Secondly, C4b with covalently attached C3b serves to form a high-affinity binding site for C5. Purified allotypes C4A3, C4B1 and C4A6 were used to compare these two activities of C4. Covalently linked C4b-C3b complexes were formed on sheep erythrocytes with similar efficiency by using C4A3 and C4B1, indicating that the two isotypes behave similarly as acceptors for covalent attachment of C3b. C4A6 showed normal efficiency in this function. However, cells bearing C4b-C3b complexes made from C4A6 contained only a small number of high-affinity binding sites for C5. Therefore a lack of binding of C5 to the C4b C3b complexes is the reason for the inefficient formation of C5 convertase by C4A6. The small number of high-affinity binding sites created, when C4A6 was used, were tested for inhibition by anti-C3 and anti-C4. Anti-C4 did not inhibit C5 binding, whereas anti-C3 did. This suggests that the sites created when C4A6 is used to make C3 convertase may be C3b-C3b dimers, and hence the low haemolytic activity of C4A6 results from the creation of low numbers of alternative-pathway C5-convertase sites.
Purification and characterization of glucose dehydrogenase from the thermoacidophilic archaebacterium Thermoplasma acidophilum
Authors: Smith L D , Budgen N , Bungard S J , Danson M J , Hough D W
Published: August 2015

Glucose dehydrogenase was purified to homogeneity from the thermoacidophilic archaebacterium Thermoplasma acidophilum. The enzyme is a tetramer of polypeptide chain Mr 38,000 +/- 3000, it is catalytically active with both NAD+ and NADP+ cofactors, and it is thermostable and remarkably resistant to a variety of organic solvents. The amino acid composition was determined and compared with those of the glucose dehydrogenases from the archaebacterium Sulfolobus solfataricus and the eubacteria Bacillus subtilis and Bacillus megaterium. The N-terminal amino acid sequence of the Thermoplasma acidophilum enzyme was determined to be: (S/T)-E-Q-K-A-I-V-T-D-A-P-K-G-G-V-K-Y-T-T-I-D-M-P-E.
X-ray-scattering of turkey skeletal-muscle troponin C in solution at low pH
Authors: Wachtel E J , Sverbilova T , McCubbin W D , Kay C M
Published: August 2015

The solution structure of troponin C from turkey skeletal muscle was studied at low pH by small-angle X-ray-scattering. We find that troponin C at pH 5.3 in the presence of Mg2+ has a triaxial radius of gyration and maximum dimension comparable with those of the crystallized protein. However, the relative disposition of domains is more similar to that found for the highly homologous rabbit protein in solution at pH 7.4.
Studies on some specific Ap4A-degrading enzymes with the use of various methylene analogues of P1P4-bis-(5′,5‴-adenosyl) tetraphosphate
Authors: Guranowski A , Starzyńska E , Taylor G E , Blackburn G M
Published: August 2015

Six new methylenephosphonate analogues of P1P4-bis-(5′,5‴-adenosyl) tetraphosphate, Ap4A, having P2-P3 carbon bridges CF2, CCl2 and CH2CH2 or P1-P2 and P3-P4 carbon bridges CF2, CCl2 and CH2CH2 in the tetraphosphate chain, were examined as substrates or inhibitors for two specific Ap4A-degrading enzymes: (asymmetrical) Ap4A hydrolase (EC 3.6.1.17) from yellow-lupin seeds and (symmetrical) Ap4A hydrolase (EC 3.6.1.41) from Escherichia coli. All analogues in which the central oxygen atom was replaced by a stable carbon bridge were hydrolysed by the asymmetrical hydrolase (CF2 greater than CCl2 greater than O greater than CHBr greater than CH2 greater than CH2CH2). As expected, these analogues were not hydrolysed by the symmetrical hydrolase, which was also unable to act on analogues having P1-P2 and P3-P4 carbon bridges.
Measurement of the acyl-CoA intermediates of β-oxidation by h.p.l.c. with on-line radiochemical and photodiode-array detection. Application to the study of [U-14C]hexadecanoate oxidation by intact rat liver mitochondria
Authors: Watmough N J , Turnbull D M , Sherratt H S A , Bartlett K
Published: August 2015

The quantitative isolation of acyl-CoA esters of chain length C2-C17 from mitochondrial incubations and their analysis by reverse-phase radio-h.p.l.c. is described. Photodiode-array detection was used to characterize 2-enoyl-CoA esters. The chromatographic behaviour of all 27 intermediates of the beta-oxidation of hexadecanoyl-CoA is documented. Only C16, C14 and C12 intermediates were detected in uncoupled mitochondria oxidizing [U-14C]hexadecanoyl-CoA in the presence of fluorocitrate and carnitine, providing evidence for some organization of the enzymes of beta-oxidation [Garland, Shepherd & Yates (1965) Biochem. J. 97, 587-594; Sumegi & Srere (1984) J. Biol. Chem. 259, 8748-8752]. Rotenone increased concentrations of 3-hydroxyacyl-CoA and 2-enoyl-CoA esters and inhibited flux. These experiments provide the first direct unambiguous measurements of acyl-CoA esters in intact respiring rat liver mitochondrial fractions.
Down-regulation of protein kinase C potentiates angiotensin II-stimulated polyphosphoinositide hydrolysis in vascular smooth-muscle cells
Authors: Pfeilschifter J , Ochsner M , Whitebread S , De Gasparo M
Published: August 2015

In smooth-muscle cells (SMC) isolated from rat aorta, angiotensin II stimulates a phospholipase C with subsequent formation of inositol trisphosphate (InsP3). Short-term (10 min) pretreatment of SMC with 12-O-tetradecanoylphorbol 13-acetate (TPA; 100 nM) decreases the angiotensin II-induced InsP3 formation. However, this inhibition is not observed after incubating the cells for 2 h with TPA. Longer-term pretreatments even lead to an enhanced generation of InsP3. This increased response to angiotensin II occurs without a significant change in the receptor number or Kd value of angiotensin II binding to the cells. The biologically inactive phorbol ester 4 alpha-phorbol 12,13-didecanoate was without effect on angiotensin II-stimulated InsP3 generation, irrespective of the time of preincubation. In parallel with this potentiation of angiotensin II-induced generation of InsP3 by TPA, a down-regulation of protein kinase C activity is observed. A 24 h pretreatment of SMC with TPA decreases protein kinase C activity to less than 10% of that of control cells. Longer-term pretreatment also increases the angiotensin II-induced release of Ca2+ and delays the decay of the transient Ca2+ increase. All these data suggest that protein kinase C exerts a negative feedback control on angiotensin II-stimulated polyphosphoinositide turnover, and that protein kinase C is an important factor in limiting the production of InsP3 in stimulated cells.
Regulation of very-low-density-lipoprotein lipid secretion in hepatocyte cultures derived from diabetic animals
Authors: Duerden J M , Bartlett S M , Gibbons G F
Published: August 2015

Hepatocytes were derived from 2-3-day streptozotocin-diabetic rats and maintained in culture for up to 3 days. Compared with similar cultures from normal animals, these hepatocytes secreted less very-low-density-lipoprotein (VLDL) triacylglycerol, but the decrease in the secretion of VLDL non-esterified and esterified cholesterol was not so pronounced. This resulted in the secretion of relatively cholesterol-rich VLDL particles by the diabetic hepatocytes. Addition of insulin for a relatively short period (24 h) further decreased the low rates of VLDL triacylglycerol secretion from the diabetic hepatocytes. The secretion of VLDL esterified and non-esterified cholesterol also declined. These changes occurred irrespective of whether or not exogenous fatty acids were present in the culture medium. Little or no inhibitory effect of insulin was observed after longer-term (24-48 h) exposure to the hormone. Both dexamethasone and a mixture of lipogenic precursors (lactate plus pyruvate) stimulated VLDL triacylglycerol and cholesterol secretion, but not to the levels observed in hepatocytes from normal animals. The low rate of hepatic VLDL secretion in diabetes contrasts with the increase in whole-body VLDL production rate. This suggests that the intestine is a major source of plasma VLDL in insulin-deficient diabetes.
Cholera toxin treatment produces down-regulation of the α-subunit of the stimulatory guanine-nucleotide-binding protein (Gs)
Authors: Milligan G , Unson C G , Wakelam M J
Published: August 2015

The effect of activation of the alpha-subunit(s) of the stimulatory guanine-nucleotide-binding protein, Gs, on levels of this polypeptide(s) associated with the plasma membrane of L6 skeletal myoblasts was ascertained. Incubation of these cells with cholera toxin led to a time- and concentration-dependent ‘down-regulation’ of both 44 and 42 kDa forms of Gs alpha as assessed by immunoblotting with an anti-peptide antiserum (CS1) able to identify the extreme C-terminus of Gs. The effect of cholera toxin was specific for Gs; levels of Gi alpha in membranes of cholera toxin-treated cells were not different from untreated cells. Down-regulation of Gs was absolutely dependent upon prior ADP-ribosylation, and hence activation of Gs and was not mimicked by other agents which elevate intracellular levels of cyclic AMP. Pretreatment with pertussis toxin, which catalyses ADP-ribosylation of Gi but not of Gs, did not down-regulate either Gi or Gs, demonstrating that covalent modification by ADP-ribosylation is alone not a signal for removal of G-proteins from the plasma membrane.
Participation of peroxisomes in lipid biosynthesis in the harderian gland of guinea pig
Authors: Horie S , Suga T
Published: August 2015

Peroxisomal enzyme activities in the guinea-pig harderian gland, which has a unique lipid composition, were studied. Activities of catalase, acyl-CoA oxidase and the cyanide-insensitive acyl-CoA beta-oxidation system in this tissue were comparable with those in rat liver. The activities of dihydroxyacetone phosphate acyltransferase (DHAPAT, EC 2.3.1.42) and alkyl-DHAP synthase (EC 2.5.1.26) were appreciable, and the distributions of both activities were consistent with that of sedimentable catalase activity. Glycerol-3-phosphate acyltransferase (GPAT, EC 2.3.1.15), which is localized in both microsomes (microsomal fractions) and mitochondria in the rat liver, was a peroxisomal enzyme in the harderian gland, though the activity was only about one-tenth of the DHAPAT activity. These enzymes had different pH profiles and substrate specificity. The existence of high activities of enzymes of the acyl-DHAP pathway in peroxisomes suggests the physiological significance of peroxisomes in the biosynthesis of glycerol ether phospholipid and 1-alkyl-2,3-diacylglycerol in the guinea-pig harderian gland.
Developmentally regulated expression of a 78 kDa erythroblast membrane glycoprotein immunologically related to the platelet thrombospondin receptor
Authors: Kieffer N , Bettaieb A , Legrand C , Coulombel L , Vainchenker W , Edelman L , Breton-Gorius J
Published: August 2015

We have previously described a monoclonal antibody (FA6-152), obtained by immunizing mice with fetal human erythrocytes [Edelman, Vinci, Villeval, Vainchenker, Henri, Miglierina, Rouger, Reviron, Breton-Gorius, Sureau & Edelman (1986) Blood 67, 56-63]. The antibody labelled fetal, but not adult, erythrocytes and bound to both fetal and adult platelets and monocytes. In the present study we have characterized the antigen recognized by FA6-152 on human platelets and on cells of the erythroid lineage at different stages of maturation. FA6-152 precipitated a chymotrypsin-resistant 88 kDa sialoglycoprotein from both iodinated and periodate/NaB3H4-surface-labelled platelets which corresponds to glycoprotein IV, the platelet thrombospondin (TSP) receptor. After neuraminidase treatment, a shift of the apparent molecular mass from 88 kDa to 85 kDa was observed. Scatchard analysis revealed that 125I-FA6-152 bound saturably with high affinity to a single class of platelet binding sites (Kd 6.4 +/- 0.6 nM). The number of FA6-152 IgG molecules bound per platelet was 25,400 +/- 8,800 (n = 4) and did not change upon thrombin activation of platelets. At low doses of alpha-thrombin (0.025 unit), FA6-152 inhibited platelet aggregation as well as endogenous TSP binding to the platelet surface. Immunofluorescence labelling of bone-marrow cells and of cultures in vitro of burst-forming units-erythroid (BFU-E) and colony-forming units-erythroid (CFU-E) revealed that that FA6-152 antigen is a very early marker of erythroid differentiation and that its expression declines during maturation. Immunochemical identification of the FA6-152 antigen on fetal erythroblasts and fetal mature erythrocytes revealed a 78 kDa glycoprotein migrating just in front of the glycophorin A dimer. The antigen, which was absent from adult mature erythrocytes, was also detected in human erythroleukaemic (HEL) cells where FA6-152 precipitated two bands of molecular mass 85 and 88 kDa. Our data establish the existence of a previously unidentified 78 kDa erythroblast cell-surface glycoprotein whose expression is developmentally regulated during erythroid differentiation and which is immunologically related to the 88 kDa platelet TSP receptor.