bluuebunny/crossref_metadata_2025_split huggingface dataset:
Intracellular retention and degradation of human mutant variant of a α1‐antitrypsin in stably transfected Chinese hamster ovary cell lines
Authors: CICCARELLI Ernesto , ALONSO Maria Angeles , CRESTEIL Danielle , BOLLEN Alex , JACOBS Paul , ALVAREZ Fernando 
Published: March 2005

Normal (PiM) and mutant (PiZ) variants of human α1‐antitrypsin (α1‐AT) cDNA, cloned into the pTnd eucaryotic expression vector, were used to derive recombinant Chinese hamster ovary cell lines permanently expressing the corresponding proteins.Secretion, accumulation and glycosylation of PiM and PiZ α1‐AT proteins were studied in the presence of various transport‐impairing drugs. Pulse‐chase, followed by immunoprecipitation as well as immunofluorescence experiments showed that the PiZ α1‐AT undergoes continuous degradation that was prevented by Brefeldin A but not by incubation of cells at 16 °C. Moreover, monensin partially impaired the glycosylation of both PiM and PiZ α1‐AT but not their secretion nor the degradation of PiZ α1‐AT. Those results suggest that PiZ α1‐AT degradation occurs in the cis‐Golgi network, a compartment located between the endoplasmic reticulum and the Golgi stack. The process did not apparently involve lysosomes since it was insensitive to chloroquine. In addition, inhibition of PiM and PiZ α1‐AT glycosylation and secretion by tunicamycin did not result in the accumulation of the protein, but instead in its rapid lag‐free degradation. Treatment of cells with the A23187 ionophore, for a short (60 min) but not a long (24 h) period, improved the secretion of PiZ α1‐AT in a similar way as it affects retention of naturally endoplasmic‐reticulum‐resident proteins, suggesting that the small proportion of PiZ α1‐AT which is not degraded or secreted, but accumulates in the endoplasmic reticulum, is back transported as a partially glycosylated species from the post endoplasmic reticulum compartment in which degradation takes place.
Exchange reactions catalyzed by group‐transferring enzymes oppose the quantitation and the unravelling of the identity of the pentose pathway
Authors: FLANIGAN Ian , COLLINS J. Grant , ARORA Krishan K. , MacLEOD John K. , WILLIAMS John F. 
Published: March 2005

The distributions and rates of transfer of carbon isotopes from a selection of specifically labelled ketosugar‐phosphate substrates by exchange reactions catalyzed by the pentose and photosynthetic carbon‐reduction‐pathway group‐transferring enzymes transketolase, transaldolase and aldolase have been measured using 13C‐NMR spectroscopy.
The rates of these exchange reactions were 5, 4 and 1.5 μmol min−1 mg−1 for transketolase exchange, transaldolase exchange and aldolase exchange, respectively.
A comparison of the exchange capacities contributed by the activities of these enzymes in three in vitro liver preparations with the maximum non‐oxidative pentose pathway flux rates of the preparations shows that transketolase and aldolase exchanges exceeded flux by 9–19 times in liver cytosol and acetone powder enzyme preparations and by 5 times in hepatocytes. Transaldolase was less effective in the comparison of exchange versus flux rates: transaldolase exchange exceeded flux by 1.6 and 5 in catalysis by liver cytosol and acetone powder preparations, respectively, but was only 0.6 times the flux in hepatocytes.
Values of group enzyme exchange and pathway flux rates in the above three preparations are important because of the feature role of liver and of these particular preparations in the establishment, elucidation and measurement of a proposed reaction scheme for the fat‐cell‐type pentose pathway in biochemistry.
It is the claim of this paper that the excess of exchange rate activity (particularly transketolase exchange) over pathway flux will overturn attempts to unravel, using isotopically labelled sugar substrates, the identity, reaction sequence and quantitative contribution of the pentose pathway to glucose metabolism.
The transketolase exchange reactions relative to the pentose pathway flux rates in normal, regenerating and foetal liver, Morris hepatomas, mammary carcinoma, melanoma, colonic epithelium, spinach chloroplasts and epididymal fat tissue show that transketolase exchange may exceed flux in these tissues by factors ranging over 5–600 times.
The confusion of pentose pathway theory by the effects of transketolase exchange action is illustrated by the 13C‐NMR spectrum of the hexose 6‐phosphate products of ribose 5‐phosphate dissimilation, formed after 30 min of liver enzyme action, and shows 13C‐labelling in carbons 1 and 3 of glucose 6‐phosphate with ratios which range over 2.1–6.4 rather than the mandatory value of 2 which is imposed by the theoretical mechanism of the pathway.
Transcriptional control of AAC3 gene encoding mitochondrial ADP/ATP translocator in Saccharomyces cerevisiae by oxygen, heme and ROX1 factor
Authors: ŠABOVÁ Ludmila , ZEMAN Igor , SUPEK Frantisek , KOLAROV Jordan 
Published: March 2005

The AAC3 gene of Saccharomyces cerevisiae encodes a mitochondrial ADP/ATP translocator which is subject to oxygen repression. Evidence is presented here, that the repression of AAC3 expression is dependent upon heme and the ROX1 factor. The promoter region of the AAC3 gene was isolated, sequenced, and deletion analysis was performed using lacZ as a reporter gene to determine the cis‐acting regions responsible for the regulation of AAC3 expression. The results of the deletion analysis show that the negative control of the AAC3 gene by oxygen and ROX1 factor is mediated by an upstream repression sequence consisting of a T‐rich segment adjacent to the consensus elements that are present in the 5′ flanking regions of several other yeast genes. An additional upstream repressor site was located within the AAC3 promoter which, however, is not related either to oxygen or to ROX1 factor.The data presented here delineate the main cellular factors and DNA sequences involved in the regulatory mechanism by which an essential function for anaerobic cells growth, ADP/ATP translocation is ensured. In addition, they show that the AAC3 gene belongs to the family of yeast genes including TIF51B, COX5b, HEM13 and CYC7 that are negatively regulated by oxygen and heme.
Responses of metabolic systems to large changes in enzyme activities and effectors
Authors: SMALL J. Rankin , KACSER Henrik 
Published: March 2005

We extend the analysis of unbranched chains (preceding paper) to large parameter changes in branched systems using linear kinetic assumptions. More complex relationships between flux control coefficients and deviation indices are established. In particular, the deviation index in such systems depends on more than one control coefficient as well as on the magnitude of the enzyme change. Non‐additivity of the indices is the general rule. Combined changes of groups of enzymes, whether co‐ordinate or not, have also been formulated. Control coefficients can be estimated from a small number of independent large‐change experiments. Alternatively, the amplification factors can be calculated given the knowledge of the control coefficients. A ‘case study’ using published data is presented.The movement of intermediate metabolites as a consequence of large parameter changes can be dealt with in a similar manner.Experimental methods for showing the admissibility of assuming the simplifying assumptions used are summarised. Some simulation studies show possible limits of the application of the approach and some aspects of the general, non‐linear, case are discussed. It is concluded that, although metabolic systems are in principle non‐linear, many behave, in practice, as quasi‐linear systems. The relationships established between deviation indices and control coefficients therefore provide a practical way of predicting the effects of large‐scale changes in parameters for many metabolic systems.
Modification of the E1ATP binding site of Na+/K+‐ATPase by the chromium complex of adenosine 5′‐[β,γ‐methylene]triphosphate blocks the overall reaction but not the partial activities of the E2 conformation
Authors: HAMER Evelyn , SCHONER Wilhelm 
Published: March 2005

The chromium complex of adenosine 5′‐[β,γ‐methylene]triphosphate, Cr(H2O)4AdoPP[CH2]P, inactivates Na+/K+‐ATPase from pig kidney at 37°C with an inactivation velocity constant of 7.1X10‐3min‐1 by binding to the high‐affinity ATP site (E1ATP site). The dissociation constant (Kd) of the analogue at this site is 26 μM, and of ATP 0.8 μM. Inactivation of the overall reaction of Na+/K+‐ATPase by Cr(H2O)4AdoPP[CH2]P did not alter the activities of the E2 conformational state such as K+‐activated p‐nitrophenylphosphatase, 86Rb+ occlusion and [3H]ouabain binding by the ‘backdoor’ phosphorylation. However, [3H]ouabain binding via the forwards reaction from E1ATP in the presence of Na++ Mg2+ is inhibited.K+‐activated p‐nitrophenylphosphatase activity of the Cr(H2O)4AdoPP[CH2]P‐inactivated enzyme decreases when an MgATP analogue, the tetraammine cobalt complex of ATP, Co(NH3)4ATP, is used additionally to inactivate the E2ATP site. The enzyme activity of K+‐activated phosphatase is also lost if the β,γ‐bidentate chromium(III) complex of ATP, Cr(H2O)4ATP, which may form a stable E1‐chromo‐phosphointermediate, is used for the inactivation of Na+/K+‐ATPase.We conclude that the phenomenon of a blockade of the overall reaction of Na+/K+‐ATPase by the formation of a stable E1· CrAdoPP[CH2]P complex, leading thereby to a loss of the partial activities of the E1 conformation, but not of the E2 conformation, is consistent with the postulate of an (αβ)2 diprotomeric nature of the sodium pump. The observation, moreover, that treatment of the sodium pump with Cr(H2O)4ATP but not with Cr(H2O)4AdoPP[CH2]P leads to an inactivation of K+‐activated phosphatase seems to indicate that the formation of a E1‐phosphointermediate affects the E2ATP site.
Chemical structures of the core regions of Campylobacter jejuni serotypes O:1, O:4, O:23, and O:36 lipopolysaccharides
Authors: ASPINALL Gerald O. , McDONALD Armando G. , RAJU T. Shantha , PANG Henrianna , MORAN Anthony P. , PENNER John L. 
Published: March 2005

Complete structures, including the location of N‐acetylneuraminic acid (Neu5Ac) residues, were assigned for the core regions of Campylobacter jejuni serotypes O:1, O:4, and O:23 and O:36 lipopolysaccharides (LPS). In continuation of earlier studies, structure determinations of liberated oligosaccharides and, where necessary, of intact LPS, were by 1H‐NMR spectroscopy, Smith degradation, chromium trioxide and enzymic degradations, in conjunction with methylation studies supported by fast‐atom‐bombardment mass spectrometry and linkage analyses by gas chromatography/mass spectrometry. It was concluded on the basis of the following structures, in which each was linked 1→5 to a terminal 3‐deoxy‐D‐manno‐octulosonic acid residue, that the core regions with qualititatively similar sugar compositions showed serotypic differences in one or more of their sequences, linkage types, and anomeric configurations:

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The outer regions of each structure carry Neu5Ac residues linked 2→3 to available β‐D‐Galp residues and show striking similarities with various glycosphingolipids of the ganglioside family. However, Neu5Ac epitopes are not apparently involved in determining serospecificity.
Factors effecting the thermostability of cysteine proteinases from Carica papaya
Authors: SUMNER Ian G. , HARRIS Gillian W. , TAYLOR Mark A. J. , PICKERSGILL Richard W. , OWEN A. Jane , GOODENOUGH Peter W. 
Published: March 2005

Thermal denaturation of four Carica papaya cysteine proteinases (papain, chymopapain, papaya proteinases 3 and 4) was studied as a function of pH using high‐sensitivity differential scanning calorimetry.The ratios of calorimetric enthalpy to Van't Hoff enthalpy suggest that, for all these proteins, denaturation occurs as a non two state process, via an intermediate structure.Differences in the thermal stabilities of the proteinases; chymopapain > papaya proteinase 3 > papain > papaya proteinase 4, were correlated to their amino acid sequence to explain the observations in terms of mobility and specific residue mutation.Three‐dimensional structures of papain and papaya proteinase 3 were similarly used to illustrate the influence of atomic mobility on stability.
Isolation and characterization of microsomal acyl‐CoA thioesterase
Authors: ALEXSON Stefan E. H. , MENTLEIN Rolf , WERNSTEDT Christer , HELLMAN Ulf 
Published: March 2005

We have isolated and characterized an acyl‐CoA thioesterase from rat liver microsomes. The enzyme consists mainly of a monomer of 59 kDa. However, the final preparation was found to contain minor amounts of a trimeric form of the protein. The enzyme was purified more than 85‐fold from isolated microsomes and used for NH2‐terminal sequence analysis and for analysis of peptides isolated after proteolytic digestion. The NH2‐terminal sequence was unique but highly conserved compared to those of other carboxylesterases. Internal sequence data, covering almost 20% of the protein, showed high similarity to the deduced amino acid sequences from a cDNA encoding a carboxylesterase synthesized in the liver and subsequently secreted to the blood [Alexson, S. E. H., Finlay, T. H., Hellman, U., Diczfalusy, U. & Eggertsen, G., unpublished results] and nonspecific rat liver microsomal carboxylesterase with isoelectric point of 6.1 [Robbi, M., Beaufay, H. & Octave, J.‐N. (1990) Biochem. J. 269, 451–458], thus confirming earlier suggestions that this enzyme is a member of the microsomal carboxylesterase multigene family. The peptide sequences contained two of the four conserved cysteic acid residues found in other carboxylesterases. Amino acid analysis indicated that the protein contains five cysteine residues in contrast to most other described carboxylesterases which contain four highly conserved cysteins.The purified protein was used for immunization and the antiserum was used to detect the protein as well as its trimeric form, which is a minor component, in isolated rat liver microsomes. The antiserum recognized proteins of similar sizes in microsomes and 100000Xg supernatant prepared from hamster brown adipose tissue, a tissue known to contain very high activity of carboxylesterase, and to recognize carboxylesterases isolated from porcine and rabbit liver.
Yeast seryl‐tRNA synthetase expressed in Escherichia coli recognizes bacterial serine‐specific tRNAs in vivo
Authors: WEYGAND‐DURAŠEVIĆ Ivana , BAN Nenad , JAHN Dieter , SÖLL Dieter 
Published: March 2005

The Saccharomyces cerevisiae serS gene which encodes seryl‐tRNA synthetase (SerRS) was expressed in Escherichia coli from the promoter and the ribosome binding sequences contained in its own 5′‐flanking region. The low level of yeast SerRS in the prokaryotic host was sufficient to permit in vivo complementation of two temperature‐sensitive E. coli serS mutants at the nonpermissive temperature. Thus, yeast SerRS can aminoacylate E. coli tRNASer species in vivo.Yeast SerRS, isolated from an overexpressing E. coli strain by a rapid two‐step purification on FPLC, aminoacylated E. coli tRNA with serine much more poorly (relative kcat/Km= 2 × 10−4) than its homologous tRNAs. DL‐Serine hydroxamate, an inhibitor of E. coli SerRS, inhibits yeast SerRS in vivo and in vitro with an inhibition constant (K1) of 2.7 mM, a value 90‐fold higher than that for E. coli SerRS.
Localization of two myosin‐subfragment‐1 binding contacts in the 96–132 region of actin subdomain‐1
Authors: LABBÉ Jean‐Pierre , BOYER Mireille , MÉJEA Catherine , ROUSTAN Claude , BENYAMIN Yves 
Published: March 2005

Many direct observations and indirect experimental approaches have pin‐pointed two segments (sequences 1–28 and 360–372) in actin subdomain‐1 which bind to myosin subfragment‐1. In a previous investigation [Labbé, J. P., Méjean, C., Benyamin, Y. & Roustan, C. (1990) Biochem. J. 271, 407–413], we have observed competition between myosin subfragment‐1 and anti‐actin anti‐bodies specific to epitopes including Thr103. A multisite interface model has also been proposed to take into account myosin‐head binding to the N‐terminal and C‐terminal regions and to more central 40–113 sequence of actin.In the present study, two limited actin segments encompassing residues 96–103 and 112–125 were identified as myosin‐head‐binding sites. Myosin subfragment‐1 competed for monomeric actin with the antibodies directed against sequences 96–105 and 114–120 and its binding to the tryptic 96–113 and synthetic 112–125 actin peptides was prevented by magnesium pyrophosphate but not by calcium pyrophosphate. In the presence of ATP‐Mg2+, myosin subfragment‐1 was dissociated by filamin from its complex with monomeric actin or with peptide 105–120. Contact points of filamin on actin were previously located in the 105–120 and 360–372 actin sequence [Méjean, C., Lebart, M. C., Boyer, M., Roustan, C. & Benyamin, Y. (1992) Eur. J. Biochem. 209, 555–562].The in vitro inhibitory effect of filamin on actin‐activated Mg2+‐ATPase would thus be explained by this competition. Furthermore, the (27‐kDa–50‐kDa–20‐kDa) trypsin‐split myosin subfragment‐1 which could no longer be activated by actin, did not bind at all to the two sites located in the 96–125 region, but it still interacted with the 360–372 segment.Our results regarding the position of the myosin head on actin monomers in rigor conditions provide evidence on the presence of two topologically independant contact points in the myosin‐head/actin interface. One group exposed residues in the 1–7, 21–29, 77–95 and 96–103 actin segment, another, on the opposite side of subdomain‐1, included residues from 112–125 and 360–372 sequences.
The existence of two mitochondrial isoforms of 2,4‐dienoyl‐CoA reductase in the rat
Authors: HAKKOLA Elina H. , HILTUNEN J. Kalervo 
Published: March 2005

Isoforms of 2,4‐dienoyl‐CoA reductase (EC 1.3.1.34), which is the key enzyme in the β‐oxidation of fatty acids with double bonds, have been studied in rat heart and liver. Electrofocusing and adsorption chromatography on hydroxyapatite were used to separate the reductase activity in tissue homogenates into two peaks, one peak in each experiment being identified as the previously purified mitochondrial reductase.The novel activity was partially purified from rat liver by means of ammonium sulphate precipitation, anion‐exchange chromatography on DEAE‐cellulose (DE‐52), hydrophobic chromatography on Phenyl‐Sepharose and dye‐ligand binding chromatography (Blue Sepharose). Taking into account the contribution of the different reductases to the total activity in rat liver, the overall purification for the novel isoform was 1900‐fold. Ultracentrifugation on a sucrose gradient gave an Mr of 50000 and size‐exclusion chromatography on Superdex 200 an Mr of 60000.The antibody against the previously characterised reductase did not cross‐react with this novel isoform, but the distribution of the activity peaks in heart and liver tissue, and an electrofocusing experiment with isoled mitochondria, both pointed to a mitochondrial origin. The novel reductase was estimated to account for 80% (50%) of the total reductase activity in rat heart (liver) homogenate measured with 2,4‐hexadienoyl‐CoA. The present results, together with those previously published, suggest that mammals have at least three reductase isoforms: two in mitochondria and a third one in peroxisomes, but the peroxisomal activity has not been characterised so far.
NMR assignments and solution conformation of the DNA · RNA hybrid duplex d(GTGAACTT) · r(AAGUUCAC)
Authors: LANE Andrew N. , EBEL Susanne , BROWN Tom 
Published: March 2005

Nearly complete assignments of the 1H‐NMR spectrum of the DNA · RNA hybrid d(GTGAACTT) · r(AAGUUCAC) have been obtained by two‐dimensional methods. Three‐bond coupling constants measured from cross‐sections of two‐dimensional NOE spectra and double quantum‐filtered correlation spectra showed that the sugars in the DNA strand are predominantly in the S domain of puckers, whereas the ribofuranoses are mainly C3′ ‐endo. Analysis of time‐dependent NOE intensities from one‐and two‐dimensional experiments showed that the glycosidic torsion angles in the DNA strand are near −120°, whereas those in the RNA strand are in the range –140° to –160°. These nucleotide conformations correspond to those typically found in B‐DNA and A‐RNA, respectively.The circular dichroism of the duplex is similar to that of A‐form RNA, consistent with a global A‐like conformation. A large number of duplex structures was generated in which the nucleotides were fixed in the experimentally determined conformations. A subset of these structures was found that satisfied the internucleotide NOE intensities, backbone constraints and had acceptable Lennard‐Jones energies. The base pairs in the duplex were found to have positive inclinations, a translation (Dx) of about 0.4 nm from the helix axis, and more than 10bp/turn on average. This implies a helical structure in the A family of conformations.
L‐Serine and L‐threonine dehydratase from Clostridium propionicum Two enzymes with different prosthetic groups
Authors: HOFMEISTER Antje E. M. , GRABOWSKI Reiner , LINDER Dietmar , BUCKLER Wolfgang 
Published: March 2005

l‐Serine dehydratase from the Gram‐positive bacterium Peptostreptococcus asaccharolyticus is novel in the group of enzymes deaminating 2‐hydroxyamino acids in that it is an iron‐sulfur protein and lacks pyridoxal phosphate [Grabowski, R. and Buckel, W. (1991) Eur. J. Biochem. 199, 89–94]. It was proposed that this type of l‐serine dehydratase is widespread among bacteria but has escaped intensive characterization due to its oxygen lability. Here, we present evidence that another Gram‐positive bacterium, Clostridium propionicum, contains both an iron‐sulfur‐dependent l‐serine dehydratase and a pyridoxal‐phosphate‐dependent l‐threonine dehydratase. These findings support the notion that two independent mechanisms exist for the deamination of 2‐hydroxyamino acids. l‐Threonine dehydratase was purified 400‐fold to apparent homogeneity and revealed as being a tetramer of identical subunits (m= 39kDa). The purified enzyme exhibited a specific activity of 5 μkat/mg protein and a Km for l‐threonine of 7.7 mM. l‐Serine (Km= 380 mM) was also deaminated, the V/Km ratio, however, being 118‐fold lower than the one for l‐threonine. l‐Threonine dehydratase was inactivated by borohydride, hydroxylamine and phenylhydrazine, all known inactivators of pyridoxal‐phosphate‐containing enzymes. Incubation with NaB3H4 specifically labelled the enzyme. Activity of the phenylhydrazine‐inactivated enzyme could be restored by pyridoxal phosphate. l‐Serine dehydratase was also purified 400‐fold, but its extreme instability did not permit purification to homogeneity. The enzyme was specific for l‐serine (Km= 5mM) and was inhibited by l‐cysteine (Ki= 0.5 mM) and d‐serine (Ki= 8 mM). Activity was insensitive towards borohydride, hydroxylamine and phenylhydrazine but was rapidly lost upon exposure to air. Fe2+ specifically reactivated the enzyme. l‐Serine dehydratase was composed of two different subunits (α, m= 30kDa; βm= 26 kDa), their apparent molecular masses being similar to the ones of the two subunits of the iron‐sulfur‐dependent enzyme from P. asaccharolyticus. Moreover, the N‐terminal sequences of the small subunits from these two organisms were found to be 47% identical. In addition, 38% identity with the N‐terminus of one of the two l‐serine dehydratase of Escherichia coli was detected.