bluuebunny/crossref_metadata_2025_split huggingface dataset:
Changes in calpain and calpastatin mRNA induced by β‐adrenergic stimulation of bovine skeletal muscle
Authors: PARR Timothy , BARDSLEY Ronald G. , GILMOUR R. Stewart , BUTTERY Peter J. 
Published: March 2005

Administration of β‐adrenergic agonists to mammals can produce skeletal muscle hypertrophy in some species and muscle types. The growth‐promoting effect appears to be due to suppression of protein breakdown rather than stimulation of synthesis, although evidence from turnover studies is equivocal. In ovine muscle, changes in the activity of the calcium dependent neutral proteinases (calpains I and II) and their specific inhibitor (calpastatin) accompany β‐agonist‐induced hypertrophy. These observations suggest that the calpain system is involved in myofibrillar protein turnover in some way. Alternatively, the relationship with hypertrophy may be indirect, since the calpains also interact with hormone and growth‐factor receptors, protein kinase C and transcription factors, in addition to a range of membrane, cytoskeletal and nuclear proteins.In the present study, attempts have been made to determine if the β‐agonist‐induced effects on the calpain system are associated with corresponding changes in specific mRNA. The activity of both calpain isoforms and calpastatin was measured in bovine longissimus dorsi samples from trials in which test animals were treated with the β agonist cimaterol. Total RNA was extracted from the muscle samples. A cDNA probe for calpastatin mRNA was generated from bovine RNA by the polymerase chain reaction. This cDNA and a human calpain‐II large‐subunit cDNA were used to detect specific mRNA by Northern‐blot analysis.β‐agonist treatment of Friesian steers caused significant longissimus dorsi hypertrophy. Increases in muscle mass (+ 37%, P < 0.005), calpain‐II specific activity (+ 27%, P < 0.05) and calpastatin‐specific activity (+ 76%, P < 0.05) were found in treated animals. Total RNA was unchanged, but there was a 96% overall increase in calpastatin mRNA and a 30% increase in calpain‐II large‐subunit mRNA in muscle from treated animals. The mRNA changes are similar in direction and degree to the activity changes. Both calpain‐II large subunit and inhibitor expression may therefore be stimulated by agonist action at the level of transcription or mRNA stabilisation. Multiple calpastatin mRNA species were detected in steers, as reported for other species. Differential changes in these messages, induced by the β agonist, suggest that expression or stability of alternative mRNA species may be a factor in calpastatin regulation.
Biosynthesis and intracellular transport of α‐2,6‐sialyltransferase in rat hepatoma cells
Authors: BOSSHART Herbert , BERGER Eric G. 
Published: March 2005

We investigated biosynthesis, intracellular transport and release of β‐galactoside α‐2,6‐sialyltransferase in a dexamethasone‐inducible rat hepatoma cell line. Confluent cells were induced by 10 μM dexamethasone for 24 h, and metabolically labelled with [35S]methionine/cysteine, followed by immunoprecipitation of sialyltransferase and electrophoretic/fluorographic analysis. The 35S‐labelled enzyme was synthesized as a 46‐kDa precursor, converted to an intermediate 47‐kDa form after 1 h, and gradually to a mature form of 48 kDa within the following 3 h. By means of either tunicamycin inhibition of N‐glycosylation or cleavage of N‐glycans from isolated sialyltransferase using N‐glycosidase F, the sizes of the precursor and the mature form were reduced to 41 kDa and 43 kDa, respectively. After a 4‐h chase, treatment with endoglycosidase H revealed two distinct molecular forms of sialyltransferase, bearing either two N‐acetyllactosamine‐type or one oligomannose‐type and one N‐acetyllactosamine‐type N‐linked sugar chain. In addition, sialyltransferase became sensitive to neuraminidase digestion after a 4‐h chase. The half‐life of intracellular [35S]sialyltransferase was estimated at 3 h. A soluble form was detectable in the supernatant, 2 h after the pulse. Only 12% of the initially labelled sialyltransferase was found in the medium after 12 h, while 73% of the enzyme was degraded intracellularly. To characterize a possible intracellular degradation site, we studied intracellular transport in the presence of either secretion‐blocking or acidotropic agents or protease inhibitors. Degradation was significantly delayed by all treatments.Our results show that sialyltransferase follows the secretory pathway as a membrane protein and is retained at a late Golgi stage. We suggest that the bulk of sialyltransferase in rat hepatoma cells is diverted to a post‐Golgi degradation pathway. This route contrasts with the post‐Golgi trafficking of β‐1,4‐galactosyltransferase in HeLa cells, which is constitutively secreted [Strous, G. J. A. M. & Berger, E. G. (1982) J. Biol. Chem. 257, 7623–7628].
Kinase inhibition by a phosphorylated peptide corresponding to the major insulin receptor autophosphorylation domain
Authors: CHAVANIEU Alain , CALAS Bernard , VAGLIO Philippe , GRIGORESCU Florin 
Published: March 2005

We studied the inhibitory effect of non‐phosphorylated and triphosphorylated synthetic peptides, corresponding to amino acids 1143–1155 of the insulin proreceptor (domain 1151) on autophosphorylation and kinase of the insulin receptor. Tyrosine‐phosphorylated peptides were synthesized using the N(9‐fluorenylmethoxycarbonyl)‐O‐dibenzylphosphono‐l‐ tyrosine.The triphosphorylated peptide (1151‐P3) and the non‐phosphorylated peptide (1151‐NP), respectively, inhibited insulin receptor autophosphorylation by 65% and 70%, in a dose‐dependent and additive manner. When the receptor was pre‐phosphorylated for 1 min with [γ‐32P]ATP, 1151‐P3 decreased autophosphorylation to 60% of maximun, whereas 1151‐NP had no further effect. In both non‐activated and preactivated receptors, 1151‐P3 inhibition of receptor autophosphorylation was prevented by adding 2 mM vanadate. Kinase activity towards exogenous substrate poly(Glu4, Tyr) was dose‐dependently inhibited by both analogues. This effect was independent of the state of receptor phosphorylation or the addition of vanadate. Since 1151‐P3 inhibited the exogenous kinase without altering receptor endogenous autophosphorylation after the addition of vanadate, we investigated 1151‐NP and 1151‐P3 competition for the phosphorylation of a resin‐immobilized 1151 peptide. While 1151‐NP (at 2 mM) was highly competitive, inhibiting phosphate incorporation by 70%, 1151‐P3 caused a four‐fold increase in the phosphorylation of 1151‐NP–resin. The receptor underwent conformational changes during autophosphorylation and an antibody directed against a peptide corresponding to amino acids 1314–1330 of the proreceptor (1322Ab) was previously shown to immunoprecipitate specifically the non‐phosphorylated receptor forms. Nevertheless, the 1322Ab immunoprecipitated a fully autophosphorylated receptor in the presence of 1151‐NP, but not of 1151‐P3, thus suggesting a conformational change induced by the non‐phosphorylated peptide.In conclusion, kinase inhibition was still observed after the addition of phosphate groups to three 1151‐peptide tyrosines, but the peptide effect on receptor autophosphorylation, phosphorylation of homologous 1151‐NP–resin and conformational changes induced in the receptor was altered dramatically. These data may provide a basis for further understanding the role of tyrosine phosphorylation in insulin receptor kinase activation or regulation.
Mechanistic studies of 3‐deoxy‐d‐manno‐2‐octulosonate‐8‐phosphate synthase from Escherichia coli
Authors: KOHEN Amnon , JAKOB Abla , BAASOV Timor 
Published: March 2005

The anomeric specificity and the steady‐state kinetic mechanism of homogeneous 3‐deoxy‐d‐manno‐2‐octulosonate‐8‐phosphate (KDO8P) synthase were investigated. The open‐chain 4‐deoxy analogue of arabinose‐5‐phosphate (Ara5P), which is structurally prohibited from undergoing ring closure, was synthesized and tested as a substrate for the synthase. It was found that the analogue functions as a substrate with a similar kcat value to that of the original substrate. The kcat/Km value for the natural substrate is seven‐times greater than that of the 4‐deoxy analogue. However, taking into account the 9.5% and approximately 1% concentrations of the aldehyde forms of the 4‐deoxy analogue and Ara5P in solution, then the ‘true’Km values must be in the range 31.5 μM and 0.26 μM, respectively, requiring about a 3 kcal/mol contribution to the binding energy by the 4‐hydroxyl group of Ara5P. The data provides evidence that the enzyme acts upon the acyclic form of the natural substrate.The steady‐state kinetic study of KDO8P synthase was analyzed via inhibition using the products KDO8P and inorganic phosphate, and d‐ribose‐5‐phosphate as a dead‐end inhibitor. First, intersecting lines in double‐reciprocal plots of initial‐velocity data at substrate concentrations in the micromolar range suggest a sequential mechanism for the enzyme‐catalyzed reaction. The inhibition by d‐ribose‐5‐phosphate is competitive for Ara5P and uncompetitive for phosphoenolpyruvate (P‐pyruvate). These inhibition patterns are consistent with the model wherein P‐pyruvate binding precedes that of Ara5P binding. Furthermore, this order of substrate binding was supported by the observations that KDO8P is a competitive inhibitor for P‐pyruvate binding, supporting the concept that KDO8P and P‐pyruvate bind to the same enzyme form, and noncompetitively with respect to Ara5P. In addition, the inhibition by inorganic phosphate is noncompetitive with respect to both P‐pyruvate and Ara5P, suggesting an apparent ordered release of products such that Pi first, followed by KDO8P. In conclusion, these data suggest a steady‐state kinetic mechanism for KDO8P synthase where P‐pyruvate binding precedes that of Ara5P, followed by the ordered release of inorganic phosphate and KDO8P.
Matrix NADH dehydrogenases of plant mitochondria and sites of quinone reduction by complex I
Authors: MENZ R. Ian , GRIFFITH Megan , DAY David A. , WISKICH Joseph T. 
Published: March 2005

In order to distinguish the pathways involved in the oxidation of matrix NADH in plant mitochondria, the oxidation of NADH and nicotinamide hypoxanthine dinucleotide (reduced form) was investigated in submitochondrial particles prepared from beetroot (Beta vulgaris L. cv. Derwent Globe) and soybeans (Glycine max L. cv. Bragg). Nicotinamide‐hypoxanthine‐dinucleotide(reduced form)‐oxidase activity was more strongly inhibited by rotenone than the NADH‐oxidase activity but both of the rotenone‐inhibited activities could be stimulated by adding ubiquinone‐1. The corresponding ubiquinone‐1‐reductase activities were inhibited by rotenone (to 69%) and further inhibited by N,N′‐dicyclohexylcarbodiimide (to 79%), whilst the K3Fe(CN)6‐reductase activities were not sensitive to either rotenone or N,N′‐dicyclohexylcarbodiimide. Immunological analysis of mitochondrial proteins using an antiserum raised against purified beetroot complex I indicated very few differences between soybean and fresh and aged beetroot mitochondria, despite their varying sensitivities to rotenone.We confirm that there are two dehydrogenases capable of oxidising internal NADH and that only one of these, namely complex I, is inhibited by rotenone. Further, we conclude that complex I has two potential sites of quinone reduction, both sensitive to N,N′‐dicyclohexycarbodiimide inhibition but only one of which is sensitive to rotenone inhibition.
Analysis of glycosaminoglycan chains from different proteoglycan populations in human embryonic skin fibroblasts
Authors: SCHMIDTCHEN Artur , FRANSSON Lars‐Åke 
Published: March 2005

1. The structure of chondroitin/dermatan and heparan‐sulphate chains from various proteoglycan populations derived from cultured human skin fibroblasts have been examined. Confluent cell cultures were biosynthetically labelled with [3H]‐glucosamine and 35SO42‐, and proteoglycans were purified according to buoyant density, size and charge density [Schmidtchen, A., Carlstedt, I., Malmström, A. & Fransson, L.‐Å. (1990) Biochem. J. 265, 289–300]. Some proteoglycan fractions were further fractionated according to hydrophobicity on octyl‐Sepharose in Triton X‐100 gradients. The glycosaminoglycan chains, intact or degraded by chemical or enzymic methods were then analysed by gel chromatography on Sepharose CL‐6B, Bio‐Gel P‐6, ion exchange HPLC and gel electrophoresis.2. Three types of dermatan‐sulphate chains were identified on the basis of disaccharide composition and chain length. They were derived from the large proteoglycan, two small proteoglycans and a cell‐associated proteoglycan with core proteins of 90 kDa and 45 kDa. Intracellular, free dermatansulphate chains were very similar to those of the small proteoglycans.3. Heparan‐sulphate chains from different proteoglycans had, in spite of small but distinct differences in size, strikingly similar compositional features. They contained similar amounts of d‐glucuronate, l‐iduronate (with or without sulphate) and N‐sulphate groups. They all displayed heparin‐lyase‐resistant domains with average molecular mass of 10–15 kDa. The heparan‐sulphate chains from proteoglycans with 250‐kDa and 350‐kDa cores were the largest (> 50 kDa), containing an average of four or five domains, in contrast to heparan‐sulphate chains from the small heparan‐sulphate proteoglycans which had average molecular mass of 45 kDa and consisted of three or four such domains. Free, cell‐associated heparan‐sulphate chains were heterogeneous in size (5–45 kDa).4. These results suggest that the core protein may have important regulatory functions with regard to dermatan‐sulphate synthesis. On the other hand, synthesis of heparan sulphate may be largely controlled by the cell that expresses a particular proteoglycan core protein.
In vivo and in vitro modulation of the mRNA‐binding activity of iron‐regulatory factor
Authors: MÜLLNER Ernst W. , ROTHENBERGER Sylvia , MÜLLER Anna Maria , KÜHN Lukas C. 
Published: March 2005

The mRNA‐binding protein, iron‐regulatory factor (IRF) has a central role in iron metabolism. It coordinately increases transferrin‐receptor mRNA stability and inhibits translation of ferritin and erythroid δ‐aminolevulinate synthase mRNA by binding to specific mRNA structures, the iron‐responsive elements (IRE): In gel‐retardation assays, IRF had a broad tissue distribution, showing activity in cytosolic extracts from 12 mouse organs tested. In all these extracts, IRF could be further activated in vitro by 2‐mercaptoethanol. In cultured mouse 3T6 fibroblasts, growth stimulation after low serum arrest increased IRF activity 10‐fold, mainly through activation of existing inactive IRF. No change was observed during progresion of 3T6 cells through the cell cycle.IRF activation by iron chelators has been postulated to result in the reduction of an intramolecular sulfhydryl group. In a search for redox conditions that regulate IRE binding of IRF, we studied several compounds in vitro or in vivo. Hemin, known to inactivate IRF in vivo, showed a similar, reversible effect in vitro, presumably by oxidizing IRF. However, this did not appear to be relevant for the mode of IRF regulation in vivo. Addition of protoporphyrin IX to intact cells induced IRF activity almost to the same extent as desferrioxamine. This effect was inhibited by iron salts, indicating that IRF is activated in vivo through depletion of a chelatable iron pool. In vitro activation by reductants other than 2‐mercaptoethanol suggested some selectivity in their access to relevant sulfhydryl groups, but did not reveal which natural redox‐sensitive compound might regulate IRF in vivo. However, in cultured cells, inactivation of free IRF by the sulfhydryl‐specific oxidizing agent diamide was much more rapidly reversed than inactivation by iron salts. This indicates the direct involvement of a cellular reductant in setting IRF activity and suggests a rate‐limiting IRF conformation that is reached only in the presence of iron, but not after diamide oxidation.
Mechanistic studies on rhodopsin kinase
Authors: BROWN Neil G. , FOWLES Charles , SHARMA Ram , AKHTAR Muhammad 
Published: March 2005

The phosphorylation of a synthetic peptide, corresponding to the C‐terminal 11 amino acids of bovine rhodopsin (VII, residues 338–348), was studied under different conditions. The peptide was only phosphorylated in the presence of photoactivated rhodopsin. Using the same protocol, 12 other peptides, mapping in the rhodopsin C‐terminal, were screened for their effectiveness as substrates for rhodopsin kinase. It was found that the peptides became poorer substrates with increasing length, and the best substrates comprised the most C‐terminal 9–12 amino acids as opposed to other parts of the C‐terminus. It was noted that the absence of the two‐terminal residues Pro347 and Ala348 impaired peptide phosphorylation.The effect of the decay of metarhodopsin II on the phosphorylation of rhodopsin and the peptides was determined, and it was found that the rhodopsin and peptide phosphorylations decayed with half times of approximately 33 min and 28 min, respectively.The sites of phosphorylation on the peptides were determined and in all cases the phosphorylation was found to be predominantly on serine residues. Only the 11‐residue peptide (VII, residues 338–348) contained significant threonine phosphorylation, which was about 25% that on serine residues. Cumulatively, the results suggest that Ser343 is the preferred site of phosphorylation in vitro.The reason for the poor substrate effectiveness of the larger peptides was examined by competitive experiments in which it was shown that a poorly phosphorylated larger peptide successfully inhibited the phosphorylation of a ‘good’ peptide substrate. The studies above support a mechanism for rhodopsin kinase that we have termed the ‘kinase‐activation hypothesis’. This requires that the kinase exists in an inactive form and is activated only after binding to photoactivated rhodopsin.
Occurrence of α1‐2‐fucosylation in membrane glycoproteins of Morris hepatoma 7777 but not in liver
Authors: NUCK Rolf , ORTHEN Bruno , REUTTER Werner 
Published: March 2005

A comparative study was undertaken to characterize the linkages of l‐fucose in N‐glycans of plasma membrane glycoproteins from Morris hepatoma 7777, host liver and kidney cortex, as well as from rat serum.After in‐vivo radiolabelling of rats with l‐[6‐3H]fucose, the asparagine‐linked carbohydrate chains were released from delipidated plasma membrane glycoproteins, as well as from serum glycoproteins, by enzymic digestion with peptide‐N4‐(N‐acetyl‐β‐glucosaminyl)asparagine amidase from Flavobacterium meningosepticum. They were then converted to their corresponding oligosaccharide alditols by reduction with sodium borohydride.Two specific α‐l‐fucosidases from almond emulsin and from Aspergillus niger, combined with affinity HPLC on immobilized Aleuria aurantia lectin were used to study the linkage of l‐fucose in the oligosaccharide chains.Fucose α1‐2 linked to galactose, was present only in the plasma membrane of hepatoma 7777 (18% of total l‐[3H]fucose in N‐glycans), but was not expressed in host liver, kidney cortex and serum. None of the investigated sources contained an appreciable amount of fucose α1‐3/4 linked to N‐acetyl‐d‐glucosamine. All the radioactively labelled oligosaccharides from host liver, kidney cortex and serum, but only 82% of these oligosaccharides from hepatoma, contained α‐fucosyl residues linked at the C6 position of the proximal N‐acetyl‐d‐glucosamine.
A high‐resolution solid‐state 13C‐NMR study on crystalline bovine heart cytochrome‐c oxidase and lysozyme
Authors: TUZI Satoru , SHINZAWA‐ITOH Kyoko , ERATA Tomoki , NAITO Akira , YOSHIKAWA Shinya , SAITÔ Hazime 
Published: March 2005

We have recorded 100.6‐MHz high‐resolution solid‐state 13C‐NMR spectra of crystalline cytochrome‐c oxidase from bovine heart muscle and hen egg‐white lysozyme, to compare conformation and dynamics of a typical membrane‐protein complex with those of lysozyme. The absence of severe interference with the solid‐state 13C‐NMR spectra, from both the line broadenings from paramagnetic centers and overlapping of intense detergent signals, provided spectral resolution of 13C‐NMR feature of cytochrome‐c oxidase crystals comparable to that of lysozyme crystal and better than that of dissolved or lyophilized samples. In fact, the observed peak intensities of the polar heads of the detergents BL8SY and Brij 35 were only about 10% and 3% of the anticipated values, respectively.The dynamic behavior of the backbone and side chains of cytochrome‐c oxidase was compared with that of lysozyme on the basis of the 13C spin‐lattice relaxation times (T1): the backbone of the cytochrome‐c oxidase turned out to be more flexible than that of lysozyme. Molecular motions of the detergent molecules attached to the proteins are found to be highly heterogeneous. Detergent molecules undergo rapid tumbling motions in the crystals in about 10 ns as detected by T1. In addition to rapid motions, slow motions were detected by 1H spin‐lattice relaxation time in the rotating frame (TH1Q) and cross‐polarization time (TCH), together with data from static spectra, indicating that the aliphatic portion of the detergent interacts more strongly with hydrophobic protein surfaces than do the polar heads.
Purification, characterization and structure of protein phosphatase 1 from the cilia of Paramecium tetraurelia
Authors: FRIDERICH Gerald , KLUMPP Susanne , RUSSELL Chris B. , HINRICHSEN Robert D. , KELLNER Roland , SCHULTZ Joachim E. 
Published: March 2005

A type 1 serine/threonine protein phosphatase (PP1) which is mostly localized in the excitable ciliary membranes from the protozoan Paramecium, was purified to homogeneity. Approximately 4 μg enzyme of 37 kDa was isolated from 100 1 axenic culture. The enzymic properties were characterized using phosphorylase a from rabbit skeletal muscle as a substrate and several known effectors of mammalian PP1. The protozoan PP1 was enzymically indistinguishable from its mammalian congener.The amino acid sequence of the Paramecium PP1 was deduced from its cDNA. The full‐length clone was obtained in several steps starting with a pair of degenerate primers made according to the two most conserved peptides of rabbit PP1 and PP2A. The gene encodes a protein of 36 392 Da. The identity of the cloned gene and the isolated ciliary PP1 was unequivocally established by microsequencing of four tryptic and cyanogen‐bromide peptides which were generated from the purified protein. Paramecium PP1 shows 75% amino‐acid‐sequence identity with rabbit PP1α. Areas of major differences are the C‐termini and N‐termini and a sequence between residues 219–242.