bluuebunny/crossref_metadata_2025_split huggingface dataset:
Modified branched‐chain amino acid pathways give rise to acyl acids of sucrose esters exuded from tobacco leaf trichomes
Authors: KANDRA Lili , SEVERSON Ray , WAGNER George Joseph 
Published: March 2005

A major diversion of carbon from branched‐chain amino acid biosynthesis/catabolism to form acyl moieties of sucrose esters (6‐O‐acetyl‐2,3,4‐tri‐O‐acyl‐α‐D‐glucopyranosyl‐β‐D‐fructofuranosides) was observed to be associated with specialized trichome head cells which secrete large amounts of sucrose esters. Surface chemistry and acetyl and acyl substituent groups of tobacco (T.I. 1068) sucrose esters were identified and quantified by gas chromatography/mass spectrometry. Sucrose esters were prominent surface constituents and 3‐methylvaleric acid, 2‐ and 3‐methylbutyric acid, and methylpropionic acid accounted for 60%, 25% and 9%, respectively, of total C3–C7 acyl substituents. Radiolabeled Thr, Ile, Val, Leu, pyruvate and Asp, metabolites of branched‐chain amino acid pathways, were compared with radioactively labeled acetate and sucrose as donors of carbon to sucrose, acetyl and acyl components of sucrose esters using epidermal peels with undisturbed trichomes. Preparations of biosynthetically competent trichome heads (site of sucrose ester formation) were also examined. Results indicate that 3‐methylvaleryl and 2‐methylbutyryl groups are derived from the Thr pathway of branched‐chain amino acid metabolism, 3‐methylbutyryl and methylpropionyl groups are formed via the pyruvate pathway, and that acetyl groups are principally formed directly via acetyl‐CoA. Arguments are presented which rule out participation of fatty acid synthase in the formation of prominent acyl acids.Results suggest that the shunting of carbon away from the biosynthesis of Val, Leu and Ile may be due to a low level of amino acid utilization in protein synthesis in specialized glandular head cells of trichomes. This would result in the availability of corresponding oxo acids for CoA activation and esterification to form sucrose esters. Preliminary evidence was found for the involvement of cycling reactions in oxo‐acid‐chain lengthening and for utilization of pyruvate‐derived 2‐oxobutyrate to form straight‐chain acyl substituents.
The role of carbohydrate in recombinant human erythropoietin
Authors: TSUDA Eisuke , KAWANISHI Gosei , UEDA Masatsugu , MASUDA Seiji , SASAKI Ryuzo 
Published: March 2005

Recombinant human erythropoietin has N‐linked sugar [Tsuda et al., (1988) Biochemistry 27, 5646–5654]. Here we have demonstrated the presence of O‐linked sugar (0.85 mol/mol erythropoietin) composed of sialic acid and Galβ(1–3)GalNAc.
To investigate the role of these sugars, erythropoietins deglycosylated to different extents were prepared using specific glycosidases. Sugars are not essential for in vitro biological activity of erythropoietin, because the fully deglycosylated erythropoietin had the full activity when assayed with in vitro bioassay methods. Asialylation yielded erythropoietin with higher affinity to the receptor than the undigested hormone and therefore an increased in vitro activity. Although erythropoiein from which N‐linked or total sugars were removed also had higher affinity for the receptor, their in vitro activity remained unchanged compared with that of the undigested erythropoietin for unknown reasons. On the other hand, removal of sialic acids or N‐linked sugar abolished the in vivo biological activity completely, indicating that the presence of N‐linked sugar with terminal sialic acids is required for the hormone to reach target sites; full deglycosylation resulted in total loss of the in vivo biological activity of erythropoietin.
Incubation of asialo‐erythropoietin and fully deglycosylated recombinant human erythropoietin at 70 °C for 15 min decreased the biological activity to 35% and 11% of the initial activity, respectively, while the undigested erythropoietin lost no activity. Thus resistance of erythropoietin to thermal inactivation is largely due to the presence of sugars, and terminal sialic acids greatly contribute to the stability.
Cloning and sequencing of mammalian glutathione reductase cDNA
Authors: TUTIC Mevlida , LU Xiang , SCHIRMER R. Heiner , WERNER Dieter 
Published: March 2005

The molecular cloning of a partial cDNA to mouse glutathione reductase mRNA and of a full‐length cDNA to the mRNA of the human enzyme is described. An initial cDNA clone designated λGRM‐B11 was isolated by plaque‐screening of an induced mouse cDNA expression library in the λgt11 vector with a rabbit antibody probe to human glutathione reductase. 125Iodine‐labelled whole anti‐rabbit immunoglobulin was used as second antibody. EcoRI digestion of the λGRM‐B11 clone released a 720‐bp fragment which was identified as a partial mouse glutathione reductase cDNA by the following techniques. (a) Escherichia coli Y1089 lysogenized with λGRM‐B11 could be induced to synthesize a recombinant polypeptide whose antigenicity to anti‐(glutathione reductase) serum was established by SDS/polyacrylamide gel electrophoresis and subsequent immunoblotting. (b) The GRM‐B11 sequence, recloned in the Bluescript vector to give the plasmid pGRM‐B11, was found to code for a polypeptide consisting of 242 amino acid residues exhibiting 82% identities with the known amino acid sequence of the human glutathione reductase from position 77 to 318.The insert of the pGRM‐B11 plasmid was used as a bona fide nucleic acid probe to screen mouse and human cDNA libraries prepared in the λgt11 or in the λgt10 vector. The first full‐length cDNA clone (λGRH‐Mev10) was identified in a human cDNA library based on RNA of human placental cells. Its insert was composed of three EcoRI fragments of 720, 613 and 336 bp. The three fragments were recloned in the Bluescript vector and sequenced. The largest fragment (pGRH‐B) is colinear with the mouse sequence cloned in the pGRM‐B11 plasmid. The fragment of intermediate size (pGRH‐CT) comprises the 3′ end of the mRNA and the poly(A) tail while the short fragment (pGRH‐NT) corresponds to the 5′ region of the mRNA. The amino acid sequence deduced from the nucleotide sequences of the three subclones is identical with the known sequence of the mature glutathione reductase from human erythrocytes in all 478 positions.
Further studies on the role of glycogenin in glycogen biosynthesis
Authors: SMYTHE Carl , WATT Peter , COHEN Philip 
Published: March 2005

About 90% of the glycogenin in skeleta muscle extracts prepared from fed, 24‐h starved or alloxan‐diabetic rabbits sedimented at 140 000xg with the glycogen/sarcovesicular fraction, from which it was released by glycogenolysis, but not by 1% SDS. Glycogenin in the glycogen/sarcovesicular fraction is therefore bound covalently to glycogen, and not associated (covalently or non‐covalently) with the sarcoplasmic reticulum. The same proportion of glycogen synthase was also recovered in the glycogen/sarcovesicle fraction, was solubilised by glycogenolysis, and copurified with glycogenin to yield a heterodimer composed of a 1:1 complex between these proteins. Glycogen synthase and glycogenin are therefore present in equimolar amounts in skeletal muscle and there is an average of one glycogen synthase catalytic subunit associated with each molecule of glycogen in vivo.Glycogenin and glycogen synthase released into the muscle cytosol by degradation of glycogen did not form a complex initially, and only 50% reassociation took place after storage for several hours or overnight dialysis. This suggests that the muscle cytosol may contain a factor(s) which regulates glycogen biogenesis by modulating the association of glycogenin and glycogen synthase. Only glycogen synthase that was complexed to glycogenin was capable of elongating the primer formed by incubation of glycogenin with Mn2+and micromolar concentration of UDP‐glucose, demonstrating the critical importance of this complex for glycogen biogenesis.
In vitro hydrolysis of oligomannosyl oligosaccharides by the lysosomal α‐d‐mannosidases
Authors: MICHALSKI Jean‐Claude , HAEUW Jean‐François , WIERUSZESKI Jean‐Michel , MONTREUIL Jean , STRECKER Gérard 
Published: March 2005

In vitro incubation of the oligomannosyl oligosaccharides Man9GlcNAc and Man5GlcNAc with isolated disrupted lysosomes yields different oligosaccharide isomers resulting from mannosidase hydrolysis. These isomers were isolated by HPLC and characterized by 1H‐NMR spectroscopy. The first steps of the degradation involve an (α1‐2)mannosidase activity and lead to the formation of one Man8GlcNAc, one Man7GlcNAc, two Man6GlcNAc and two Man5GlcNAc isomers. These reactions do not require Zn2+ as activator.On the other hand, the following steps, which lead to the formation of Man3GlcNAc and Man2GlcNAc, are Zn2+‐dependent. This process is characterized by the preferential action of an (α1‐3)mannosidase activity, and the formation of Man(α1‐6)Man(α1‐6)Man(β1‐4)GlcNAc and Man(α1‐6)Man(β1‐4)GlcNAc. Therefore, the digestion of Man9GlcNAc inside the lysosome appears to follow a very specific pathway, since only nine intermediate compounds can be identified instead of the 38 possible isomers.Our results are consistent both with the existence of several specific enzymes for α1‐2, α1‐3 and α1‐6 linkages, and with the presence of a unique enzyme whose specificity would be dependent either on Zn2+ or on the spatial conformation of the glycan. Nevertheless, previous work on the structural analysis of oligosaccharides excreted in the urine of patients suffering from mannosidosis, demonstrates the absence of the core α1‐6‐linked mannosyl residue in the major storage product derived from oligomannosyl oligosaccharides. This observation indicates the presence of a specific (α1‐6)mannosidase form, unaffected in mannosidosis.
Regulation of mannose 6‐phosphate/insulin‐like growth factor II receptor distribution by activators and inhibitors of protein kinase C
Authors: BRAULKE Thomas , TIPPMER Stefanie , CHAO Huey‐Jiun , von FIGURA Kurt 
Published: March 2005

The tumor‐promotor phorbol dibutyrate (PDBt) increases the binding of a neoglycoprotein containing mannose 6‐phosphate (Man6P) and of insulin‐like growth factor II (IGF‐II) to the Man6P/IGF‐II receptor at the cell surface. This effect is dependent on time and concentration and is also seen with synthetic 1‐oleoyl‐2‐acetyl‐sn‐glycerol, but not with 4α‐phorbol, an inactive tumor‐promoter. The increase is due to a 3–4 fold increase in the number of cell‐surface, receptors, accompanied by a 1.6‐fold increase in ligand‐binding affinity. The internalization rate of the Man6P/IGF‐II receptor is not affected by PDBt, suggesting that the redistribution of these receptors to the cell surface is due to an accelerated externalization rate. The redistribution of Man6P/IGF‐II receptors did not impair the sorting of newly synthesized Man6P‐containing ligands while uptake of these ligands is 2–4‐fold increased. Inactivation or down regulation of protein kinase C decreased the binding of the Man6P‐containing neoglycoprotein to 65% of controls. Incubation of cells with Man6P, IGF‐I, IGF‐II or epidermal growth factor induces a rapid redistribution of Man6P/IGF‐II receptors to the plasma membrane [Braulke, T., Tippmer, S., Neher, E. & von Figura, K. (1989) EMBO J. 8, 681–686]. Incubation with PDBt prevented the effect of growth factors but not that of Man6P on receptor redistribution. Inactivation of protein kinase C did not affect the Man6P/IGF‐II receptor redistribution induced by Man6P and growth factors.These data suggest that Man6P, growth factors and activation of protein kinase C by phorbol esters and diacylglycerols modulate Man6P/IGF‐II receptor cell‐surface binding by at least two independent mechanisms, receptor redistribution as well as an increase of binding affinity, which might be involved in regulation of endocytosis of ligands.
Guanine nucleotides and Ca2+‐dependent lysosomal secretion in electropermeabilised human platelets
Authors: ATHAYDE C. M. , SCRUTTON M. C. 
Published: March 2005

Metabolically stable analogues of GTP, e.g. guanosine 5′‐[γ‐thio]trio]triphosphate (GTP[S]) and guanosine 5′‐[β‐γ‐imido]triphosphate (pp[NH]pG), enhance the extent of Ca2+‐dependent secretion of β‐N‐acetylgluco‐saminidase and β‐galactosidase from electropermeabilised human platelets in the presence of less than 5 μM Ca2+.A similar effect is observed on addition either of 1,2‐dioctanoin or of GTP in the presence or absence of thrombin.
In the presence of higher Ca2+ concentrations the extent of enhancement of lysosomal secretion declines and little, or no, enhancement is observed at a [Ca2+] of 30–40 μM. Addition of leupeptin or antipain prevents this decrease in lysosomal secretion and enhances the extent of Ca2+‐dependent lysosomal secretion obtained in the presence or absence of guanine nucleotides, thrombin or 1,2‐dioctanoin.
The concentration of GTP[S] or pp[NH]pG required to obtain half‐maximal enhancement of lysosomal secretion is dependent on [Ca2+] for secretion of 5‐hydroxytryptamine, β‐N‐acetylglucosaminidase and β‐galactosidase. At two fixed [Ca2+] the median effective concentration (EC50) values for GTP[S] and pp[NH]pG which characterise enhancement of 5‐hydroxytryptamine secretion are significantly different from those characterising enhancement of the secretion of β‐N‐acetylglucosaminidase and β‐galactosidase.
In the presence of a saturating concentration of GTP[S] marked 5‐hydroxytryptamine and β‐N‐acetylglucosaminidase secretion is observed at nanomolar [Ca2+] and these responses show little dependence on [Ca2+] over the attainable range. Secretion of β‐N‐acetylglucosaminidase is also induced at nanomolar Ca2+ concentrations by addition of activators of protein kinase C.
Guanosine 5′‐[β‐thio]diphosphate inhibits enhancement of β‐N‐acetylglucosaminidase secretion induced by GTP[S] but has no effect on secretion of this enzyme induced by Ca2+ when added alone.
Our data provide some support for a model in which addition of metabolically stable guanine nucleotides enhances Ca2+‐dependent platelet lysosomal secretion by activating a guanine‐nucleotide‐binding protein (GE) located close to the exocytotic site. However, not all the data are consistent with this postulate.
Complete amino acid sequence of tenebrosin‐C, a cardiac stimulatory and haemolytic protein from the sea anemone Actinia tenebrosa
Authors: SIMPSON Richard J. , REID Gavin E. , MORITZ Robert L. , MORTON Craig , NORTON Raymond S. 
Published: March 2005

The complete amino acid sequence of the cardiac stimulatory and haemolytic protein tenebrosin‐C, from the Australian sea anemone Actinia tenebrosa, has been determined by Edman degradation of the intact molecule and fragments produced by treatment of the polypeptide chain with cyanogen bromide and enzymatic cleavage with endoproteinase Asp‐N, thermolysin and trypsin.The molecule is a single‐chain polypeptide consisting of 179 amino acid residues with a calculated molecular mass of 19 797 Da. Tenebrosin‐C shows a high degree of amino acid sequence similarity (63%) with Stoichactis helianthus cytolysin III [Blumenthal, K. M. and Kem, W. R. (1983) J. Biol. Chem. 258, 5574–5581] and is identical to a partial sequence (90 residues) reported for equinatoxin, a cardiostimulatory and haemolytic protein isolated from the European sea anemone Actinia equina [Ferlan, I. and Jackson, K. (1983) Toxicon Suppl. 3, 141–144]. No amino acid sequence similarity was detected between tenebrosin‐C and other protein sequences stored in available databases. The predicted secondary structure of tenebrosin‐C suggests that it is a compact, highly structured molecule.
The control of lipase‐catalysed transesterification and esterification reaction rates
Authors: GOLDBERG Michel , THOMAS Daniel , LEGOY Marie‐Dominique 
Published: March 2005

The reaction rate of two lipase‐catalysed reactions, esterification and transesterification, were studied in a liquid/solid two‐phase system in order to investigate the effect of water partition between the enzyme preparation and the liquid phase composed of only the reactants, i.e. without the conventional solvents.Lipase from Candida cylindracea was used for these studies. The enzyme was inactive in dehydrated systems. In the case of monoester synthesis, the reaction rate increased with increasing water activity. The reaction rates of the non‐specific C. cylindracea lipase‐catalysed reactions were very sensitive to the nature of the substrates in this unusual system. For instance, the transesterification reaction rate of ethyl propionate was 48 times higher with nonanol than heptanol in the case of dehydrated substrates, but only 2.2 times higher in the case of watersaturated substrates. The results presented here demonstrate the absolute necessity to consider the polarity of every substrate, because of its ability to modify the water partition between the solid phase (enzyme preparation) and the liquid phase (substrate and product), which results in drastic changes in enzyme activity.Contrary to esterification, which is known to be activated by the water produced, the rate of transesterification remained constant at the beginning of the reaction. However, when transesterification and esterification were carried out in the same liquid phase, the transesterification reaction rate was controlled by the water produced by the concomitant esterification. Activation effects of the water molecules produced during the enzymatic reaction were of exactly the same order of magnitude for both reactions.
Osmotic stress and abscisic acid induce expression of the wheat Em genes
Authors: MORRIS Peter C. , KUMAR Amar , BOWLES Dianna J. , CUMING Andrew C. 
Published: March 2005

The early‐methionine‐labelled (Em) polypeptide is the single most abundant cytosolic protein of dry wheat embryos. It is encoded by messenger RNA which accumulates during the later (maturation) stages of embryogenesis. The accumulation of Em mRNA can be induced in isolated developing embryos, in culture, by the application of the plant growth regulator, abscisic acid, which prevents precocious germination.Precocious germination is also inhibited by the culture of embryos under conditions of osmotic stress when accumulation of Em mRNA is induced. This induction occurs in the absence of any significant increase in the endogenous levels of embryonic abscisic acid although there is a requirement for the continued presence of the growth regulator. Additionally, expression of Em genes can be repeated during early germination, if imbibing embryos are subjected to osmotic stress. Induction of Em‐gene expression by osmotic stress is consistent with the proposed role of the Em polypeptide in mediating the remarkable tolerance of cereal embryos to the programmed desiccation undergone during their maturation.
Cloning and DNA sequencing of the fbc operon encoding the cytochrome bc1 complex from Rhodobacter sphaeroides
Authors: YUN Chang‐Hyon , BECI Rose , CROFTS Antony R. , KAPLAN Samuel , GENNIS Robert B. 
Published: March 2005

The ubiquinol: cytochrome‐c oxidoreductase (cytochrome bc1 complex) is a central component of the mitochondrial respiratory chain as well as the respiratory and/or photosynthetic systems of numerous prokaryotic organisms. In Rhodobacter sphaeroides, the bc1 complex has a dual function. When the cells are grown photosynthetically, the bc1 complex is present in the intracytoplasmic membrane and is a critical component of the cyclic electron transport system. When the cells are grown in the dark in the presence of oxygen, the same bc1 complex is a necessary component of the cytochrome‐c2‐dependent respiratory chain. The fact that the bc1 complex from R. sphaeroides has been extensively studied, plus the ability to manipulate this organism genetically, makes this an ideal system for using site‐directed mutagenesis to address questions relating to the structure and function of the bc1 complex.In the current work, the cloning and complete sequence of the fbc operon from R. sphaeroides is reported. As in other bacteria, this operon contains three genes, encoding the Rieske 2Fe–2S subunit, the cytochrome b subunit, and the cytochrome c1 subunit. Recombination techniques were used to delete the entire fbc operon from the chromosome. The resulting strain cannot grow photosynthetically, but can grow aerobically utilizing a quinol oxidase. Photosynthetic growth is restored by providing fbc operon on a plasmid, and the reappearance of the protein subunits and the spectroscopic features due to the bc1 complex are also demonstrated. Finally, a mutation is introduced within the gene encoding the cytochrome b subunit which is predicted to confer resistance to the inhibitor myxothiazol. It is shown that the resulting strain contains a functional bc1 complex which, as expected, is resistant to the inhibitor.Hence, this system is suitable for the detailed characterization of the bc1 complex, combining site‐directed mutagenesis with the biochemical and biophysical techniques which have been previously developed for the study of photosynthetic bacteria.
Thermodynamics of information transfer between subunits in oligomeric enzymes and kinetic cooperativity
Authors: RICARD Jacques , GIUDICI‐ORTICONI Marie‐Thérèse , BUC Jean 
Published: March 2005

The strength of quatenary constraints between two subunits of a polymeric enzyme depends upon the number of neighboring subunits and upon whether these subunits are liganded or not. These quaternary constraints between two subunits of a complex polymeric enzyme may be expressed, however, in terms of quaternary constraints that exist within ideal dimers. The influence of quaternary constraints on the reaction rate of a complex polymeric enzyme may thus be expressed in terms of the intersubunit strain that exists within dimers. This conclusion, that was far from evident, appears to be the consequence of the postulates of structural kinetics, and derive as well from usual thermodynamic principles.The structural steady‐state equations may be expressed in terms of partition and sub‐partition functions. As applied to structural kinetic models, a partition function expresses how, during the steady state, the energy of a population of enzyme molecules is distributed over n states. Similarly a sub‐partition function describes how, during the steady state, the energy of these enzyme molecules is partitioned among only n–k of these states. Although the concept of partition function was initially formulated for equilibrium processes, it may be extended without any loss of generality to non‐equilibrium processes. Moreover it is reminiscent of the concept of binding polynomial presented some years ago by Wyman for the equilibrium binding of a ligand to a protein. With this formalism derived from statistical mechanics, a structural rate equation may be derived from the ratio of a subpartition function of degree n–1 and of a partition function of degree n. Again these properties are the consequence of the postulates of structural kinetics associated with simple ideas derived from statistical thermodynamics.