bluuebunny/crossref_metadata_2025 huggingface dataset:
Analysis and physiological implications of renal 2-oxoglutaramate metabolism
Authors: Nissim I , Wehrli S , States B , Nissim I , Yudkoff M
Published: August 2015

The relative significance of the flux through the glutamine aminotransferase (glutaminase II) pathway to renal ammoniagenesis is poorly understood. A basic and unresolved question is whether 2-oxoglutaramate (2-OGM), a product of the glutaminase II reaction, is deamidated to yield 2-oxoglutarate and NH3, or whether 2-OGM accumulates as an unreactive lactam, depending on the environmental pH. In the current studies we utilized 13C n.m.r. as well as 15N n.m.r. as well as 15N n.m.r. to demonstrate that 2-OGM occurs as a lactam, i.e. 5-hydroxypyroglutamate, regardless of the environmental pH. Our additional aims were to determine whether human kidney cells (HK cells) in culture can produce 2-OGM and to ascertain a pH-dependent relationship between NH3 and 2-OGM production from glutamine. We therefore developed an isotope dilution assay for 2-OGM utilizing 5-hydroxy[4-13C,1-15N]pyroglutamate as the labelled species. Incubations of HK cells in minimal essential medium supplemented with 1 mM-[2-15N]glutamine demonstrated significantly higher production of 2-OGM at pH 6.8 and lower production at pH 7.6 compared with pH 7.4. Similarly both 15NH3 and [15N]alanine formation were significantly higher in acute acidosis (pH 6.8) and lower in acute alkalosis (pH 7.6) compared with that at physiological pH. Addition of 1 mM-amino-oxyacetate to the incubation medium at pH 7.4 significantly diminished [15N]alanine and 2-OGM production, but the production of 15NH3 via the glutamate dehydrogenase pathway was significantly stimulated. The current observations indicate that the glutaminase II pathway plays a minor role and that flux through glutamate dehydrogenase is the predominant site for regulation of ammoniagenesis in human kidney.
Rat and human neutrophil N-formyl-peptide chemotactic receptors. Species difference in the glycosylation of similar 35-38 kDa polypeptide cores
Authors: Remes J J , Petäjä-Repo U E , Rajaniemi H J
Published: August 2015

Rat and human neutrophil N-formyl-peptide chemotactic receptors were subjected to glycosidase and proteinase treatments to determine the extent and species differences of glycosylation and the carbohydrate requirement in the high-affinity ligand binding. N-Formyl-Nle-Leu-Phe-Nle-125I-Tyr-Lys was attached to rat and human neutrophils either before or after glycosidase and proteinase treatments, and the labelled receptors were solubilized after glutaraldehyde cross-linking and analysed by SDS/PAGE and autoradiography. Both the rat and human N-formyl-peptide chemotactic receptors contain only N-linked oligosaccharides, as demonstrated by their sensitivity to peptide N-glycosidase F (PNGase F) and resistance to O-glycanase treatment. The N-linked oligosaccharides seem to be of the complex type rather than the high-mannose or hybrid type and lack terminal sialic acid, as demonstrated by their resistance to endoglycosidases D and H and neuraminidase treatments. This sensitivity pattern was similar in both species, and the shift in the molecular size of the receptors to 35-38 kDa after PNGase F treatment occurred through one intermediate product, suggesting that both receptors contain a similar 35-38 kDa polypeptide core with two N-linked complex-type oligosaccharides, the heterogeneity of which is responsible for the species difference in receptor size. Papain treatment alone or followed by PNGase F produced in both species a 33-36 kDa membrane-bound fragment that was still able to bind the ligand, suggesting that the oligosaccharides are located on the approx. 2 kDa papain-cleavable polypeptide fragment of the receptors. The cleavage sites for both papain and PNGase F were hidden in occupied receptors, suggesting a conformational or topographical change in these upon ligand binding. Scatchard analyses and cross-linking experiments demonstrated that carbohydrates are not required for high-affinity ligand binding and that the 33-36 kDa membrane-bound papain fragment of both receptors contains the ligand-binding site.
Structural modifications of human β2 microglobulin treated with oxygen-derived radicals
Authors: Capeillere-Blandin C , Delaveau T , Descamps-Latscha B
Published: August 2015

Treatment of human beta 2 microglobulin (beta 2m) with defined oxygen-derived species generated by treatment with gamma-radiation was studied. As assessed by SDS/PAGE, the hydroxyl radicals (.OH) caused the disappearance of the protein band at 12 kDa that represents beta 2m, and cross-linked the protein into protein bands stable to both SDS and reducing conditions. However, when .OH was generated under oxygen in equimolar combination with the superoxide anion radical (O2.-), the high-molecular-mass protein products were less represented, and fragmented derivatives were not obviously detectable. Exposure to .OH alone, or to .OH + O2.- in the presence of O2, induced the formation of beta 2m protein derivatives with a more acidic net electrical charge than the parent molecule. In contrast, O2.- alone had virtually no effect on molecular mass or pI. Changes in u.v. fluorescence during .OH attack indicated changes in conformation, as confirmed by c.d. spectrometry. A high concentration of radicals caused the disappearance of the beta-pleated sheet structure and the formation of a random coil structure. Loss of tryptophan and significant production of dityrosine (2,2′-biphenol type) were noted, exhibiting a clear dose-dependence with .OH alone or with .OH + O2.-. The combination of .OH + O2.- induced a pattern of changes similar to that with .OH alone, but more extensive for c.d. and tryptophan oxidation (2 Trp/beta 2m molecule), and more limited for dityrosine formation. Lower levels of these oxidative agents caused the reproducible formation of species at 18 and 25 kDa which were recognized by antibodies against native beta 2m. These findings provide a model for the protein pattern observed in beta 2m amyloidosis described in the literature.
Potentiation of hyperthermia-induced haemolysis of human erythrocytes by photodynamic treatment. Evidence for the involvement of the anion transporter in this synergistic interaction
Authors: Prinsze C , Tijssen K , Dubbelman T M A R , Van Steveninck J
Published: August 2015

Heat treatment of human erythrocytes led to increased passive cation permeability, followed by haemolysis. K+ leakage was linear up to a loss of about 80% in the temperature range 46-54 degrees C. Kinetic analysis of the results revealed an activation energy of 246 kJ/mol, implicating a transition in the membrane as critical step. Pretreatment of erythrocytes with 4,4′-di-isothiocyano-2,2′-stilbenedisulphonate, chymotrypsin or chlorpromazine caused a potentiation of subsequent heat-induced K+ leakage. Photodynamic treatment of erythrocytes with Photofrin II, eosin isothiocyanate or a porphyrin-Cu2+ complex as sensitizer also induced an increase in passive cation permeability, ultimately resulting in colloid osmotic haemolysis. The combination of photodynamic treatment immediately followed by hyperthermia had a synergistic effect on K+ leakage. Analysis of the results by the Arrhenius equation revealed that both the activation energy and the frequency factor of heat-induced K+ leakage were decreased significantly by preceding photodynamic treatment, suggesting that hyperthermia and photodynamic treatment have a common target for the induction of K+ leakage. Several lines of reasoning indicate that this common target is band 3. A model is thus proposed for the observed potentiation of hyperthermically induced K+ leakage by photodynamic treatment, in which photo-oxidation of band 3 results in increased sensitivity to subsequent thermal denaturation. These phenomena may be of more general significance, as photodynamic treatment and hyperthermia interacted synergistically with respect to K+ leakage with L929 fibroblasts also.
Sequence analysis of heparan sulphate indicates defined location of N-sulphated glucosamine and iduronate 2-sulphate residues proximal to the protein-linkage region
Authors: Turnbull J E , Gallagher J T
Published: August 2015

A strategy that we originally used to identify an N-acetylated domain adjacent to the protein-linkage sequence of heparan sulphate proteoglycan (HSPG) [Lyon, Steward, Hampson & Gallagher (1987) Biochem. J. 242, 493-498] has been adapted for analysis of the location of GlcNSO3-HexA and GlcNSO3(+/- 6S)-IdoA(2S) units most proximal to the core protein. [3H]Glucosamine-labelled HSPG from human skin fibroblasts was depolymerized by using HNO2 or heparinase under conditions that allowed cleavage of all susceptible linkages. The degraded PG was coupled to Sepharose beads through the protein component, enabling specific recovery of protein-linked resistant oligosaccharides. These were released by treatment with alkaline borohydride and analysed by gel filtration and gradient PAGE. This strategy allowed investigation of the sequence of sugar residues along the chain relative to a common reference point (i.e. the reducing end of the chain). HNO2 scission confirmed the presence of a well-defined N-acetylated sequence predominantly 9-12 disaccharide units in length proximal to the core protein. Heparinase scission produced two classes of oligosaccharides (Mr approx. 7000 and 15,000) with the general formula: IdoA(2S)-GlcNSO3-[HexA-GlcNR]n-HexA-GlcNSO3-[Hex A-GlcNAc]9 12-GlcA-Gal-Gal-Xyl in which the average value for n is 1-2 for the 7000-Mr species and approx. 22 for the 15,000-Mr species. The latter oligosaccharides extend to about one-third of the total length of the HS chains (Mr approx. 45,000). HNO2 scission of these oligosaccharides enabled hypothetical models for their sequence to be proposed. The general arrangement of N-sulphated and N-acetylated disaccharides between the proximal GlcNSO3 and terminal IdoA(2S) residues of the 15,000-Mr fragment was similar to that in the original polysaccharide, suggesting the possibility of a tandemly repeating pattern in the sequence of HS.
Studies of inositol analogues as inhibitors of the phosphoinositide pathway, and incorporation of 2-deoxy-2-fluoro-myo-inositol to give analogues of phosphatidylinositol intermediates
Authors: McPhee F , Downes C P , Lowe G
Published: August 2015

The incorporation of [3H]Ins into PtdIns by exchange of free and lipid-bound inositol moieties occurs via the action of at least two types of Mg2+/Mn(2+)-dependent enzymes in turkey erythrocytes. One is a nucleotide-independent PtdIns/Ins exchange enzyme and its function is, as yet, unknown, whereas the other is CMP-dependent and appears to be an exchange reaction catalysed by PtdIns synthase. The effects of analogues with modifications of the substituent at the 1-, 2-, 3-, 4- and 5-positions on the incorporation of [3H]Ins into PtdIns under both synthase and exchange reaction conditions were investigated in turkey erythrocytes. Analogues causing substantial inhibition of [3H]Ins incorporation were then used in kinetic experiments to determine the type of inhibition involved. The analogues 1-deoxy-1-fluoro-scyllo-inositol and 5-O-methyl-myo-inositol exhibited the greatest effects on the incorporation of [3H]Ins via both the synthase and exchange reactions, and the kinetic analysis indicated that they were competitive inhibitors of Ins. Ki values of 0.37 mM and 2.87 mM were observed for 1-deoxy-1-fluoro-scyllo-inositol under exchange and synthase reaction conditions respectively; similar Ki values of 0.26 mM and 2.80 mM were observed for 5-O-methyl-myo-inositol in the exchange and synthase reactions respectively. The ability of 1-deoxy-1-fluoro-scyllo-inositol and its diastereoisomer, 2-deoxy-2-fluoro-myo-inositol, to act as substrates for the synthase and exchange reactions in turkey erythrocytes was investigated. The radiolabelled derivative of the former analogue was not incorporated into phospholipids, whereas the radiolabelled derivative of the latter analogue was a poor substrate for the synthase and exchange enzymes. In the presence of ATP, the labelled analogue of PtdIns, derived from 2-deoxy-2-fluoro-myo-[2-3H]inositol, appeared to be converted into phosphorylated PtdIns analogues, presumably by the enzymes of the phosphoinositide pathway.
Properties and structural requirements for substrate specificity of cytochrome P-450-dependent obtusifoliol 14 α-demethylase from maize (Zea mays) seedlings
Authors: Taton M , Rahier A
Published: August 2015

The biochemical properties of cytochrome P-450-dependent obtusifoliol 14 alpha-demthylase (P-450OBT.14DM) from maize (Zea mays) seedlings were defined. In particular, the enzyme was shown by differential centrifugation to be localized in the endoplasmic reticulum. P-450OBT.14DM had an apparent Km of 160 +/- 5 microM and an apparent Vmax of 65 +/- 5 pmol/min per mg of protein for its best substrate, obtusifoliol. The substrate specificity of P-450OBT.14DM was thoroughly investigated by comparing the demethylation of obtusifoliol with that of a series of 15 natural or novel synthetic analogues of obtusifoliol. The results obtained clearly indicate that three distinct domains of the sterol substrate are governing obtusifoliol demethylation by P-450OBT.14DM. They revealed that (i) P-450OBT.14DM has probably a specific apolar binding site for the side chain, (ii) the delta 8-double bond is an absolute requirement for substrate demethylation and (iii) the 3-hydroxy group plays a critical role in the enzyme-substrate interaction. Interestingly the binding site, beyond the C-3 position, contains a cleft which cannot accommodate a 4 beta-methyl substituent present in lanosterol or eburicol, the precursors of 14-desmethylsterols respectively in mammals and yeast. This result indicates that P-450OBT.14DM is a novel constitutive cytochrome P-450 with a high degree of substrate and product specificity.
Tartrate-resistant acid phosphatase from human osteoclastomas is translated as a single polypeptide
Authors: Hayman A R , Dryden A J , Chambers T J , Warburton M J
Published: August 2015

Tartrate-resistant acid phosphatases have been isolated from a number of sources. These enzymes consist of one subunit (Mr 30,000-40,000) or two dissimilar subunits (Mr 15,000-20,000). Previously we isolated the enzyme from human osteoclastomas, as a two-subunit protein. By Northern blotting and hybridization with radiolabelled oligonucleotides corresponding to the N-terminal sequences of the two subunits, we demonstrate here that the enzyme is transcribed as one mRNA which is translated in vitro to produce a single polypeptide of approx. Mr 33,000. Transcription as a single mRNA species is also the case in other tissues. These results suggest that the osteoclastoma enzyme undergoes post-translational modification in the form of cleavage of a single peptide bond to give a disulphide-bonded two-subunit protein.
Lipid peroxidation in electroporated hepatocytes occurs much more readily than does hydroxyl-radical formation
Authors: Hallinan T , Gor J , Rice-Evans C A , Stanley R , O'Reilly R , Brown D
Published: August 2015

1. Rat hepatocytes suspended in 0.25 M-sucrose were electropermeabilized. This completely disrupted their plasma-membrane permeability barrier. 2. The endoplasmic reticulum in electroporated hepatocytes appeared morphologically preserved and maintained its permeability barrier as evidenced by electron-microscopic examination and latency measurements on luminal reticular enzymes. 3. Upon aerobic incubation with an NADPH-generating system and iron/ADP, porated hepatocytes peroxidized their membrane lipids at rates similar to those of matched microsomal preparations. 4. When hepatocytes were incubated with iron/EDTA and azide, radical formation detectable with dimethyl sulphoxide (DMSO) was only 10-20% that shown by microsomes. Omitting azide abolished hepatocyte reactivity with DMSO completely. Effects of hydroxyl-radical (.OH) scavengers and of added catalase suggest that the radical detected by DMSO is .OH. 5. Cytosolic inhibitor(s) from hepatocytes seemed to be a major factor limiting .OH formation. These were macromolecular, but showed a degree of heat-stability. Dialysis largely abolished inhibition, but this could be restored again by adding GSH. 6. Since .OH formation in hepatocytes seems to be much more stringently prevented than lipid peroxidation, free-radical damage originating from intracellular redox systems seems more likely to take the form of lipid peroxidation.
Identification of inducible damage-recognition proteins that are overexpressed in HeLa cells resistant to cis-diamminedichloroplatinum (II)
Authors: Chao C C K , Huang S L , Lee L Y , Lin-Chao S
Published: August 2015

Two cis-diamminedichloroplatinum (II) (cisplatin)-inducible proteins [b 130 (approximately 130 kDa) and b95 (approximately 95 kDa]) in HeLa cells that recognize both the cisplatin-modified and u.v.-modified DNA were identified in this study. These damage-recognition proteins were overexpressed in cisplatin-resistant HeLa cells. The results suggest that the damage-recognition proteins are regulated in the cells in response to DNA damage, and they may be important for DNA repair and probably the emergence of cisplatin resistance.
Redistribution of 23 kDa tubulovesicle-associated GTP-binding proteins during parietal cell stimulation
Authors: Basson M D , Goldenring J R , Tang L H , Lewis J J , Padfield P , Jamieson J D , Modlin I M
Published: August 2015

Small GTP-binding proteins are important regulators of intracellular traffic. The presence of several small GTP-binding proteins was documented in subfractions of rabbit parietal cells. Upon maximal stimulation of the cells with a combination of histamine and forskolin, one 23 kDa GTP-binding band was observed to decrease in a 50,000 g membrane fraction while increasing in 4000 g membranes. The 23 kDa band resolved into one major and two minor species on two-dimensional gels. GTP-binding species of 23 kDa, 24 kDa and 25 kDa were present in purified preparations of tubulovesicles. The three isoelectric species of the 23 kDa proteins observed in parietal cell 50,000 g microsomes were enriched in tubulovesicle preparations. None of the tubulovesicle-associated GTP-binding proteins were substrates for ADP-ribosylation by a preparation of botulinum D toxin. These results indicate that tubulovesicles contain discrete small GTP-binding proteins which redistribute during parietal cell stimulation.
Purification to homogeneity of Charonia lampas α-fucosidase by using sequential ligand-affinity chromatography
Authors: Butters T D , Scudder P , Rotsaert J , Petursson S , Fleet G W J , Willenbrock F W , Jacob G S
Published: August 2015

An alpha-fucosidase from the liver of the marine gastropod Charonia lampas was purified to homogeneity using a procedure that included cation-exchange and gel-filtration chromatography, chromatofocusing and a final series of affinity-chromatography steps which involved the following gel-immobilized ligands: N-(5-carboxy-1-pentyl)-1,5-dideoxy-1,5-imino-L-fucitol, N-(5-carboxy-1-pentyl)-2-acetamido-1,5-imino-1,2,5-trideoxy-D-glucitol and thio-beta-D-galactoside. The enzyme was found to be a tetrameric glycoprotein with a native Mr of 208,000, and to exist in a number of isoforms displaying pI values in the range 6.0-6.4. Substrate-specificity studies using a number of fucosylated oligosaccharides of the lacto-N and lacto-N-neo series and a synthetic disaccharide confirmed that the enzyme catalyses the hydrolysis of a broad range of fucosidic linkages, and established the following hierarchy of susceptibility: Fuc alpha 2Gal beta 4Glc much much greater than Fuc alpha 6GlcNAc greater than Fuc alpha 2Gal beta 4GlcNAc greater than Gal beta 3(Fuc alpha 4)GlcNAC much much greater than Gal beta 4(Fuc alpha 3)GlcNAc. Similar relative rates of hydrolysis were also demonstrated using biantennary oligosaccharide alditols as substrates which contained fucose linked either alpha 3 or alpha 6 to the N-acetylglucosaminitol residue of the chitobiosyl core.
Analysis of leukotriene B4 metabolism in human promyelocytic HL-60 cells
Authors: Kasimir S , Schönfeld W , Hilger R A , König W
Published: August 2015

We previously reported that human alveolar macrophages rapidly metabolize the chemotactic active lipid mediator leukotriene B4 (LTB4) into the dihydro-LTB4 by reduction of one of the conjugated double bonds. We herein report that human HL-60 cells (a myeloid precursor which can be differentiated into granulocyte- as well as monocyte-like cells by dimethyl sulphoxide or phorbol myristate acetate) express a highly active LTB4 reductase in the undifferentiated state. Differentiation by dimethyl sulphoxide (1.3%) along the granulocyte lineage, as confirmed by light microscopy, conversion of NitroBlue Tetrazolium into formazan, failed to induce a substantial capacity for omega-oxidation of LTB4; this reaction is exclusively found in mature granulocytes. Studies with the cell homogenate of undifferentiated HL-60 cells indicated that the activity of the enzyme depends on the presence of NADPH, Ca2+ and Mg2+, with a pH optimum of 7.5 at 37 degrees C. The enzyme was not released into the supernatant after stimulation of HL-60 cells with phorbol myristate acetate (100 ng) or Ca2+ ionophore (7.5 microM). Subcellular fractionation revealed evidence that the LTB4 reductase is located within the membrane fraction. Purification of the enzyme by gel filtration and gel electrophoresis suggests an apparent molecular mass of 40 kDa.
Increased gene expression of lipogenic enzymes and glucose transporter in white adipose tissue of suckling and weaned obese Zucker rats
Authors: Pénicaud L , Ferré P , Assimacopoulos-Jeannet F , Perdereau D , Leturque A , Jeanrenaud B , Picon L , Girard J
Published: August 2015

Previous experiments have shown that insulin-induced glucose utilization is increased in white adipose tissue of young obese Zucker rats. We have investigated the possible role of over-expression of the muscle/fat glucose transporter (Glut 4) and key lipogenic enzymes in this increased insulin-responsiveness. The amount or activity and the mRNA concentrations of Glut 4, fatty acid synthase (FAS) and acetyl-CoA carboxylase (ACC) were measured before and after weaning in white adipose tissue of obese and lean Zucker rats. Comparison of the levels of Glut 4 and lipogenic-enzyme expression in 15-day-old suckling and 30-day-old weaned rats on a high-carbohydrate diet shows a marked increase in the latter group. The increase was, in lean and obese rats respectively, 6- and 7-fold for the amount of Glut 4 and 2- and 3-fold for its mRNA concentrations, 40- and 100-fold for the activity of lipogenic enzymes (FAS and ACC) and 30- and 10-fold for their mRNA concentrations. Furthermore, all these parameters, except the amount of Glut 4, were 2-5-fold higher in obese rats, both before and after weaning. Changes at weaning were largely blunted when rats were weaned on to a high-fat diet, although the differences between lean and obese rats persisted, and even became significant for the amount of Glut 4. Whatever the experimental conditions, plasma insulin levels were significantly higher in obese than in lean rats. These results indicate the existence of an enhanced expression of Glut 4, FAS and ACC in white adipose tissue of young obese fa/fa rats which could be related to the increased plasma insulin levels.
Myosin heavy-chain mRNA is present in both myofibrillar and subsarcolemmal regions of muscle fibres
Authors: Hesketh J , Campbell G , Loveridge N
Published: August 2015

Hybridization in situ with riboprobes to the myosin heavy-chain slow isoform showed that, in the rat soleus muscle, the myosin heavy-chain mRNA was distributed throughout the myofibres. There was greater density of autoradiographic grains in the subsarcolemmal regions of the fibres, but there was also a considerable number of grains in the core myofibrillar region of the fibres. Microdensitometry showed that the grain density in the myofibrillar region was approximately half that in the subsarcolemmal rim; this would correspond to some 70% of the mRNA being present in the myofibrillar region. The results are consistent with the hypothesis that myosin is synthesized on polyribosomes present in the intermyofibrillar cytoplasm.
Evidence for coupling of resynthesis to hydrolysis in the phosphoinositide cycle
Authors: Monaco M E , Adelson J R
Published: August 2015

Previous data suggest that agonist-induced hydrolysis of phosphatidylinositol bisphosphate is accompanied by resynthesis through phosphatidylinositol such that these metabolic events function in a cyclic manner. However, it is not known whether resynthesis depends on the presence of agonist or is a direct result of agonist-induced breakdown. In the present study we demonstrate that: (1) increasing the intracellular free inositol concentration will not stimulate phosphatidylinositol synthesis, as measured by assessing the amount of [32P]Pi incorporation; (2) regeneration of free inositol is required for resynthesis; however, addition of exogenous inositol can sustain resynthesis under conditions which inhibit the regeneration of endogenous inositol; (3) resynthesis can take place in the absence of agonist provided that cells have been previously incubated under conditions which prevent resynthesis; and (4) the presence of agonist does not increase the rate of resynthesis. Thus the resynthetic phase of the phosphoinositide cycle is a compensatory event triggered either by the decrease in the level of phosphatidylinositol or by an increase in precursor substrates. The agonist itself appears to have no direct effect on the resynthesis process.
Pectin methylesterase, metal ions and plant cell-wall extension. The role of metal ions in plant cell-wall extension
Authors: Moustacas A M , Nari J , Borel M , Noat G , Ricard J
Published: August 2015

The study of pectin methylesterase and wall-loosening enzyme activities in situ, as well as the estimation of the electrostatic potential of the cell wall, suggest a coherent picture of the role played by metal ions and pH in cell-wall extension. Cell-wall growth brings about a decrease of local proton concentration because the electrostatic potential difference (delta psi) of the wall decreases. This in turn activates pectin methylesterase, which restores the initial delta psi value. This process is amplified by the attraction of metal ions in the polyanionic cell-wall matrix. The amplification process is basically due to the release of enzyme molecules that were initially bound to ‘blocks’ of carboxy groups. This increase of metal-ion concentration also results in the activation of wall-loosening enzymes. Moreover, the apparent ‘inhibition’ of pectin methylesterase by high salt concentrations may be considered as a device which prevents the electrostatic potential from becoming too high.
Kinetic and regulatory properties of cytosolic pyruvate kinase from germinating castor oil seeds
Authors: Podestá F E , Plaxton W C
Published: August 2015

The kinetic and regulatory properties of cytosolic pyruvate kinase (PKc) isolated from endosperm of germinating castor oil seeds (Ricinus communis L.) have been studied. Optimal efficiency in substrate utilization (in terms of Vmax/Km for phosphoenolpyruvate or ADP) occurred between pH 6.7 and 7.4. Enzyme activity was absolutely dependent on the presence of a bivalent and a univalent metal cation, with Mg2+ and K+ fulfilling this requirement. Mg2+ binding showed positive and negative co-operativity at pH 6.5 (h = 1.6) and pH 7.2 (h = 0.69) respectively. Hyperbolic saturation kinetics were observed with phosphoenolpyruvate (PEP) and K+, whereas ADP acted as a mixed-type inhibitor over 1 mM. Glycerol (10%, v/v) increased the S0.5(ADP) 2.3-fold and altered the pattern of nucleotide binding from hyperbolic (h = 1.0) to sigmoidal (h = 1.79) without modifying PEP saturation kinetics. No activators were identified. ATP, AMP, isocitrate, 2-oxoglutarate, malate, 2-phosphoglycerate, 2,3-bisphosphoglycerate, 3-phosphoglycerate, glycerol 3-phosphate and phosphoglycolate were the most effective inhibitors. These metabolites yielded additive inhibition when tested in pairs. ATP and 3-phosphoglycerate were mixed-type inhibitors with respect to PEP, whereas competitive inhibition was observed for other inhibitors. Inhibition by malate, 2-oxoglutarate, phosphorylated triose sugars or phosphoglycolate was far more pronounced at pH 7.2 than at pH 6.5. Although 32P-labelling studies revealed that extensive phosphorylation in vivo of soluble endosperm proteins occurred between days 3 and 5 of seed germination, no alteration in the 32P-labelling pattern of 5-day-germinated endosperm was observed after 30 min of anaerobiosis. Moreover, no evidence was obtained that PKc was a phosphoprotein in aerobic or anoxic endosperms. It is proposed that endosperm PKc activity of germinating castor seeds is enhanced after anaerobiosis through concerted decreases in ATP levels, cytosolic pH and concentrations of several key inhibitors.
Endotransglycosylation of xyloglucans in plant cell suspension cultures
Authors: Smith R C , Fry S C
Published: August 2015

1. A xyloglucan-derived nonasaccharide ([3H]XG9; Glc4,Xyl3,Gal,Fuc) was neither taken up by cultured plant cells nor appreciably hydrolysed by them, but a proportion of it became incorporated into extracellular polymers in all cultures tested (Spinacia, Daucus, Rosa, Acer, Capsicum, Zea and Festuca). 2. In Spinacia these polymers were soluble in 20% (w/v) trichloroacetic acid, had apparent Mr 20,000-30,000, were able to bind reversibly to cellulose powder and were susceptible to hydrolysis by endo-beta-(1→4)-D-glucanase, indicating that they were xyloglucans. 3. The linkage formed between [3H]XG9 and the xyloglucan was alkali-stable and glucanase-labile, indicating that the reaction responsible for the incorporation was a transglycosylation. 4. The reducing terminus of the XG9 moiety remained reducing (convertible into [3H]glucitol by NaBH4) after incorporation into the polymer, showing that the XG9 was the glycosyl acceptor and the polysaccharide the donor. 5. The results provide the first evidence that polymeric xyloglucans are subject in vivo to cleavage followed by transfer of the cut end on the other xyloglucan-related molecules. 6. Similar endotransglycosylation reactions could occur within the primary cell wall, between pairs of high-Mr structural xyloglucan molecules. Such a reaction would provide a mechanism for reversible wall loosening and may thus be relevant to our understanding of plant cell growth.
Structural characterization of a novel glycosyl-phosphatidylinositol from the protozoan Tetrahymena mimbres
Authors: Weinhart U , Thomas J R , Pak Y , Thompson G A , Ferguson M A J
Published: August 2015

A glycolipid metabolically labelled with [14C]GlcN was isolated from the free-living protozoan Tetrahymena mimbres. The glycolipid was sensitive to a bacterial phosphatidylinositol-specific phospholipase C, and the headgroup was shown to contain a phosphorylated Man alpha 1-2Man alpha 1-4Man alpha 1-4GlcN glycan. The Tetrahymena glycolipid is structurally unique among the glycosylphosphatidylinositols that have so far been characterized, including those from several protozoan parasites of humans.
Enzyme catalysis as a chain reaction
Authors: Szedlacsek S E , Duggleby R G , Vlad M O
Published: August 2015

A new type of enzyme kinetic mechanism is suggested by which catalysis may be viewed as a chain reaction. A simple type of one-substrate/one-product reaction mechanism has been analysed from this point of view, and the kinetics, in both the transient and the steady-state phases, has been reconsidered. This analysis, as well as literature data and theoretical considerations, shows that the proposed model is a generalization of the classical ones. As a consequence of the suggested mechanism, the expressions, and in some cases even the significance of classical constants (Km and Vmax.), are altered. Moreover, this mechanism suggests that, between two successive enzyme-binding steps, more than one catalytic act could be accomplished. The reaction catalysed by alcohol dehydrogenase was analysed, and it was shown that this chain-reaction mechanism has a real contribution to the catalytic process, which could become exclusive under particular conditions. Similarly, the mechanism of glycogen phosphorylase is considered, and two partly modified versions of the classical mechanism are proposed. They account for both the existing experimental facts and suggest the possibility of chain-reaction pathways for any polymerase.
Analysis of mutations in the putative nuclear localization sequence of interleukin-1β
Authors: Grenfell S , Smithers N , Witham S , Shaw A , Graber P , Solari R
Published: August 2015

Previous studies have shown that, after receptor-mediated endocytosis, interleukin-1 alpha (IL1 alpha) and interleukin-1 beta (IL1 beta) are translocated to the nucleus, where they appear to accumulate. It has been suggested that nuclear translocation may be involved in the biological responsiveness of target cells to IL1 stimulation. The human IL1 beta molecule contains a seven-amino-acid sequence (-Pro208-Lys-Lys-Lys-Met-Glu-Lys-) that shows some sequence identity with the nuclear localization sequence of the simian-virus-40 large T-antigen. The effects of point mutations within this putative nuclear localization sequence on IL1 beta binding, receptor-mediated endocytosis and biological activity have been characterized. Mutants M49 (Lys210→Ala), M50 (Lys211→Ala) and M51 (Pro208→Ala) all retained the ability to bind to the IL1 receptor, albeit with lower affinity than the wild-type molecules. However, mutants M49, M50 and M51 showed greater biological potency than wild-type IL1 alpha or IL1 beta, as measured by the induction of IL2 secretion. However, receptor-mediated endocytosis and nuclear accumulation of M50 were comparable with those in the wild-type. These observations suggest that the putative nuclear localization sequence may play an important role in the generation of biological responses to IL1 stimulation, even though it may not influence internalization of the ligand.
Polyamine catabolism in rodent and human cells in culture
Authors: Carper S W , Tome M E , Fuller D J M , Chen J R , Harari P M , Gerner E W
Published: August 2015

N1-Acetylspermidine (N1AcSpd) accumulates in late exponential phase, or after certain stresses such as heat shock, in both human tumour (A549) and rodent (HTC, CHO) cells, grown in medium containing an inhibitor of the FAD-dependent polyamine oxidase (PAO). Inhibition of PAO has little effect on cell growth or on the cellular content of the major polyamines, putrescine, spermidine or spermine, found in proliferating cells in culture, but decreases cellular putrescine content in heat shocked cells. Putrescine and spermidine are generated when N1AcSpd or N1-acetylspermine (N1AcSpm) respectively is added to either human or rodent cells depleted of the former amines by alpha-difluoromethylornithine. N1AcSpm is formed in polyamine-depleted human A549 cells when N1AcSpd is added to cultures treated with the PAO inhibitor. This reaction does not occur in either rodent line, suggesting that N1AcSpd can be converted directly into N1AcSpm in human, but not rodent, cells under specific conditions. The data presented demonstrate that a variety of human and rodent cells express PAO activity and catabolize polyamines by a mechanism which includes PAO. PAO activity is of little consequence to proliferating A549, HTC or CHO cells in culture, but does produce new putrescine in both late-exponential-phase and heat-shocked cells. These findings suggest that polyamine catabolism is part of a general response of both rodent and human cells to a variety of environmental and physiological stresses.
Selective uptake of cholesteryl esters from apolipoprotein-E-free high-density lipoproteins by rat parenchymal cells in vivo is efficiently coupled to bile acid synthesis
Authors: Pieters M N , Schouten D , Bakkeren H F , Esbach B , Brouwer A , Knook D L , van Berkel T J C
Published: August 2015

[3H]Cholesteryl ester-labelled human high-density lipoprotein (HDL) was injected into rats and its decay, intrahepatic cellular distribution and the kinetics of biliary secretion were determined. At 10 min after injection the hepatic uptake of cholesteryl esters from HDL was 3-fold higher as compared with the apolipoprotein. Selective uptake was exerted only by parenchymal cells (5.6-fold more cholesteryl esters than apolipoprotein) and not by liver endothelial or Kupffer cells. The kinetics of biliary secretion of processed cholesteryl esters initially associated with HDL or low-density lipoprotein (LDL) were compared in unrestrained rats, equipped with permanent catheters in bile duct, duodenum and heart. At 72 h after injection of [3H]cholesteryl oleate-labelled HDL, 51.0 +/- 2.5% of the injected dose was recovered as bile acids, which is about twice as high as the secretion of biliary radioactivity after injection of [3H]cholesteryl oleate-labelled LDL. Oestradiol treatment stimulated only liver uptake of LDL cholesteryl esters, and resulted in a 2-fold higher liver uptake than with HDL. However, the rate of radioactive bile acid formation from [3H]cholesteryl oleate-labelled HDL was still more rapid than for LDL. It is concluded that the selective uptake pathway for cholesteryl esters from HDL in parenchymal cells is more efficiently coupled to the formation of bile acids than is the cholesteryl ester uptake from LDL. This efficient coupling may facilitate the role of HDL in reverse cholesterol transport.
Amino acid uptake by liver of genetically obese Zucker rats
Authors: Ruiz B , Felipe A , Casado J , Pastor-Anglada M
Published: August 2015

Alanine and glutamine uptake by the liver of 50-52-day-old genetically obese Zucker rats and their lean littermates has been studied. The net uptake in vivo of L-alanine is 2-fold higher in the obese animals. No significant change in L-glutamine net balance was found. We also studied the Na(+)-dependent uptake of L-alanine and L-glutamine into plasma-membrane vesicles isolated from either obese- or lean-rat livers. Vmax. values of both L-alanine and L-glutamine transport were 2-fold higher in those preparations from obese rats. No change in Km was observed. As suggested by inhibition studies, this seemed to be mediated by an enhancement of the activities of systems A, ASC and N. We conclude that the liver of the obese Zucker rat is extremely efficient in taking up neutral amino acids from the afferent blood, which results in an enhanced net uptake of L-alanine in vivo. The changes in transport activities at the plasma-membrane level might contribute to increase amino acid disposal by liver, probably for lipogenic purposes, as recently reported by Terrettaz & Jeanrenaud [Biochem. J. (1990) 270, 803-807].
Interaction of the small proteoglycan decorin with fibronectin. Involvement of the sequence NKISK of the core protein
Authors: Schmidt G , Hausser H , Kresse H
Published: August 2015

Decorin, an interstitial small proteoglycan, was shown to interact with fibronectin via its core protein. In a solid-phase assay, both high-affinity (KD values between 10 and 20 nM) and low-affinity (KD values between 110 and 130 nM) binding sites were found. The central position of decorin core protein is made up of several repeats containing NKISK in positions 85-89 and similar sequences in other repeats. The pentapeptide inhibited, albeit not completely, the high-affinity interaction between decorin and fibronectin in a specific charge-independent manner. Half-maximal inhibition occurred at a peptide concentration of 10 microM. Core-protein-derived peptides that had been produced by endoproteinase Lys-C digestion were not inhibitory, but endoproteinase Arg-C-generated peptides served as inhibitors of binding. These results suggest that NKISK as a component of repetitive sequences of decorin is involved in the interaction between the proteoglycan and fibronectin.
Fluorimetric assay of the neurotensin-degrading metalloendopeptidase, endopeptidase 24.16
Authors: Dauch P , Barelli H , Vincent J P , Checler F
Published: August 2015

Mcc-Pro-Leu-Gly-Pro-D-Lys-Dnp (Mcc = 3-carboxy-7-methoxycoumarin; Dnp = dinitrophenyl), a quenched fluorimetric substrate originally designed as a probe to measure Pz-peptidase (also called endopeptidase 24.15), was examined as a putative substrate of the neurotensin-degrading neutral metalloendopeptidase, endopeptidase 24.16. During the purification of endopeptidase 24.16 the neurotensin(1-10) and neurotensin(11-13) formation due to this enzyme was coeluted with Mcc-Pro-Leu-Gly-Pro-D-Lys-Dnp-hydrolysing activity. By both fluorimetric and h.p.l.c. analyses, we observed that the latter activity was dose-dependently and completely abolished by neurotensin with an IC50 value (2.6 microM) that closely corresponds to the affinity of purified endopeptidase 24.16 for neurotensin (Km = 2.5 microM). Furthermore, Mcc-Pro-Leu-Gly-Pro-D-Lys-Dnp hydrolysis was inhibited by a series of dipeptides with a rank of order of potencies that parallels that observed in competition experiments of tritiated neurotensin hydrolysis by brain and intestinal endopeptidase 24.16. Altogether, these data clearly demonstrate that, in addition to Pz-peptidase, Mcc-Pro-Leu-Gly-Pro-D-Lys-Dnp also behaves as a substrate of endopeptidase 24.16, with a Km of about 26 microM. In addition, we show that, even in crude membrane preparations, Mcc-Pro-Leu-Gly-Pro-D-Lys-Dnp behaves as a useful tool to monitor and accurately quantify endopeptidase 24.16.
Accumulation of acyl-enzyme in dd-peptidase-catalysed reactions with analogues of peptide substrates
Authors: Jamin M , Adam M , Damblon C , Christiaens L , Frère J M
Published: August 2015

Thioester substrates can be used to study the hydrolysis and transfer reactions catalysed by beta-lactamases and DD-peptidases. With the latter enzymes, accumulation of the acyl-enzyme can be detected directly. The efficiency of various amines as acceptor substrates was in excellent agreement with previous results obtained with peptide substrates of the DD-peptidases. The results indicated the presence of a specific binding site for the acceptor substrates. Although most of the results agreed well with a simple partition model, more elaborate hypotheses will be needed to account for all the data presented.
Primer terminus recognition and highly processive replication by Epstein-Barr virus DNA polymerase
Authors: Tsurumi T
Published: August 2015

The Epstein-Barr virus (EBV) DNA polymerase is essential for viral DNA replication in the lytic phase of the EBV life cycle. It efficiently extends RNA primers on the template DNA, suggesting the possible involvement of the EBV DNA polymerase in synthesizing Okazaki fragments from RNA primers on the lagging strand template. Competition experiments revealed that the EBV DNA polymerase had significantly higher affinity for primer termini hybridized to the template DNA than for the single-stranded DNA template or the single-stranded primer itself. ATP was not required either for primer terminus recognition or for sustainment of polymerization. The stimulation of the enzyme by (NH4)2SO4 was dependent on the template/primers utilized. These observations suggest that the primary and secondary structure of the template/primers are important factors for primer terminus recognition by the EBV DNA polymerase. The enzyme elongated synthetic RNA primer annealed to circular single-stranded M13 DNA coated with Escherichia coli single-stranded DNA-binding protein without dissociation. The processivity of the EBV DNA polymerase was strikingly high (greater than 7200 nucleotides) and the rate of polymerization was 12 nucleotides/s per polymerase molecule. The high processing capacity is a desirable feature in the synthesis of multiple copies of the EBV genome in rolling-circle DNA replication.
Interaction of recombinant human cystatin C with the cysteine proteinases papain and actinidin
Authors: Lindahl P , Abrahamson M , Björk I
Published: August 2015

The interaction between recombinant human cystatin C and the cysteine proteinases papain and actinidin was studied by spectroscopic, kinetic and equilibrium methods. The absorption, near-u.v.c.d. and fluorescence-emission difference spectra for the cystatin C-proteinase interactions were all found to be similar to the corresponding spectra for chicken cystatin. The kinetics of binding of cystatin C to the two enzymes were best described by a simple reversible one-step bimolecular mechanism, like the kinetics of the reaction of chicken cystatin with several cysteine proteinases. Moreover, the second-order association rate constants at 25 degrees C, pH 7.4 and I0.15, of 1.1 x 10(7) and 2.4 x 10(6) M-1.s-1 for the reactions of cystatin C with papain and actinidin respectively, were similar to the corresponding rate constants for the chicken inhibitor and close to the value expected for a diffusion-controlled rate. The dissociation equilibrium constants, approx. 11 fM and approx. 19 nM for the binding of cystatin C to papain and actinidin respectively, were also comparable with the dissociation constants for chicken cystatin. The affinity between cystatin C and several inactivated papains or actinidins decreased with increasing size of the inactivating group in a manner similar to that in earlier studies with the chicken inhibitor. Together, these results strongly indicate that the mechanisms of the reactions of cystatin C and chicken cystatin with cysteine proteinases are identical or highly similar, but differ from that of reactions between serine-proteinase inhibitors and their target enzymes. The model for the proteinase-inhibitor interaction, based on the X-ray structure of chicken cystatin, therefore should be largely applicable also to human cystatin C.
Mitochondrial metabolism in different thyroid states
Authors: Soboll S , Horst C , Hummerich H , Schumacher J P , Seitz H J
Published: August 2015

The protonmotive force, as well as the mitochondrial and cytosolic concentrations of malate, 2-oxoglutarate, glutamate and aspartate, were determined in livers from hypo-, eu- and hyper-thyroid rats, by density-gradient centrifugation of freeze-clamped livers in non-aqueous solvents [Soboll, Akerboom, Schwenke, Haase & Sies (1980) Biochem. J. 192, 951-954]. The mitochondrial/cytosolic pH difference and the membrane potential were significantly enhanced in hyperthyroid livers compared with the hypothyroid state, resulting in an increased protonmotive force in the presence of thyroid hormones [Soboll & Sies (1989) Methods Enzymol. 174, 118-130]. The mitochondrial concentrations of 2-oxoglutarate, glutamate and aspartate were significantly higher in the euthyroid than in the hypothyroid state, but only slightly higher in the hyperthyroid state. Mitochondrial malate, on the other hand, increased significantly from the hypothyroid to the hyperthyroid state. The mitochondrial/cytosolic concentration gradients were significantly increased in the presence of thyroid hormones only for malate. The changes in steady-state metabolite concentrations reflect a higher substrate supply and a stimulation of mitochondrial metabolism. However, a clear relationship between the increased protonmotive force, as the driving force for mitochondrial metabolite transport, and the subcellular metabolite concentrations is not observable in different thyroid states.