bluuebunny/crossref_metadata_2025_split huggingface dataset:
Butyrobetaine availability in liver is a regulatory factor for carnitine biosynthesis in rat
Authors: SANDOR Attila , HOPPEL Charles L. 
Published: March 2005

Urinary excretion of total carnitine in 48‐h fasted rats dropped to 0.30 ± 0.01 μmol/day from 2.23 ± 0.4 μmol/day found in fed, control animals (mean ± SEM). Despite this marked retention, the total carnitine content of the whole body remained constant, about 83 μmol, predicting a slow‐down in biosynthesis.The conversion of butyrobetaine into carnitine takes place only in the liver in rats. 48 h of starvation caused a decrease in the liver butyrobetaine level from 11.6 ± 1.19 nmol/g to 9.30 ± 1.19 nmol/g, which in whole livers corresponds to a decrease from 138 nmol to 61.3 nmol. The conversion rate of butyrobetaine into carnitine was studied with radiolabelled butyrobetaine. 30 min after injection of [3H]butyrobetaine the carnitine pool in the liver of fasted rats was labelled to about the same extent as that in fed rats, but from a butyrobetaine pool with higher specific radioactivity. Therefore, the conversion rate of butyrobetaine into carnitine was reduced. The newly formed carnitine found in the whole body of fasted rats was estimated to be 59% of controls. We conclude that the biosynthesis of carnitine in fasted rats slows down, for which a decreased availability of butyrobetaine in the liver is responsible.Urinary excretion of butyrobetaine in the fasted group decreased to 74.1 nmol/day from the 222‐nmol/day control value while the butyrobetaine content of whole body did not significantly decrease (2.85 μmol vs. 3.04 μmol). Urinary excretion of trimethyllysine was also depressed.
The cDNA structure and expression analysis of the genes for the cysteine proteinase inhibitor cystatin C and for β2‐microglobulin in rat brain
Authors: COLE Timothy , DICKSON Phillip W. , ESNARD Frederic , AVERILL Sharon , RISBRIDGER Gail P. , GAUTHIER Francis , SCHREIBER Gerhard 
Published: March 2005

Tissue patterns of gene expression were analyzed by measuring mRNA levels and incorporation of radioactive amino acids for cystatin C and β2‐microglobulin, the two extracellular proteins in the brain with the highest ratio of concentration in cerebrospinal fluid over that in blood plasma.The primary structure of rat cystatin C mRNA from choroid plexus was determined by nucleotide sequencing of cloned cDNA and the tissue patterns of gene expression were analysed by RNA blot analysis and in situ hybridization. Cystatin C was found to be composed of 120 amino acids and to contain a potential site for N‐linked glycosylation. The tissue with the highest cystatin C mRNA level was the choroid plexus of the brain. Cystatin C mRNA was also detected in lower levels in other areas of the brain, testis, epididymis, seminal vesicles, prostate, ovary, submandibular gland, and, in trace amounts, in liver. Choroid plexus pieces in culture secreted radioactive cystatin C when incubated with radioactive leucine.Rat β2‐microglobulin cDNA was cloned and identified by nucleotide sequencing and comparison of the obtained sequence with that of mouse and human β2‐microglobulin cDNA. Tissue levels of β2‐microglobulin mRNA in the rat were measured by hybridization to rat β2‐microglobulin cDNA. The highest levels of β2‐microglobulin mRNA were observed in liver and choroid plexus. Other parts of the brain and testis contained lower levels of β2‐microglobulin mRNA.
Regulation of cytosolic aspartate aminotransferase mRNAs in the Fao rat hepatoma cell line by dexamethasone, insulin and cyclic AMP
Authors: BAROUKI Robert , PAVÉ‐PREUX Muriel , BOUSQUET‐LEMERCIER Bernadette , POL Stanislas , BOUGUET Jacqueline , HANOUNE Jacques 
Published: March 2005

Glucocorticoid hormones increase the activity of cytosolic aspartate aminotransferase (cAspAT) in the Fao rat hepatoma cell line. Maximal increase (6–10‐fold) was observed 48 h following the addition of the glucocorticoid agonist dexamethasone at a concentration of 0.1 μM. The effect of dexamethasone was specific since it was not mimicked by sex steroids and was inhibited by the glucocorticoid antagonist RU 486. Insulin (0.1 μM) inhibited by more than 50% the induction of cAspAT by glucocorticoids. The cAMP analog, 8‐bromoadenosine 3′,5′‐monophosphate (Br8cAMP, 0.5 mM), potentiated the effect of dexamethasone (2–3‐fold) and partially relieved the inhibitory effect of insulin on the induction by dexamethasone. Both insulin and Br8‐cAMP had no significant effect on basal activity. The mitochondrial isoenzyme was insensitive to the various hormonal treatments.Northern blot analysis revealed the presence of two major (2.1‐kb and 1.8‐kb) and one minor (4‐kb) mRNA species hybridizing with a rat cAspAT probe. The regulation of these mRNAs by glucocorticoids, insulin and cAMP correlated with the variation of the cAspAT activity, suggesting that these hormones act at the pretranslational level. We compared the regulation of cAspAT mRNAs with those of tyrosine aminotransferase mRNA. Both were similarly increased by dexamethasone but the latter was also increased by cAMP even in the absence of the glucocorticoid agonist. In addition, the increase in tyrosine aminotransferase mRNA was inhibited by cycloheximide whereas the increase in cAspAT mRNAs was not. These results show that there are significant differences in the regulation of cAspAT and tyrosine aminotransferase by glucocorticoids and other hormones, although both enzymes probably contribute to the same metabolic pathway.
Secondary structure of 7SK and 7‐2 small RNAs
Authors: SUH Dick , YUAN Yan , HENNING Dale , REDDY Ram 
Published: March 2005

Pseudogenes having homology to small RNAs, like 7SL, 7SK, 6S, 4.5S, U1, U2, and U3 RNAs, are abundant and dispersed in the genomes of higher eukaryotes [reviewed in Weiner et al. (1986) Annu. Rev. Biochem. 55, 631–661]. To understand better the possible origin of these pseudogenes, we studied the abilities of cytoplasmic 7SL, 7SK, and nucleolar 7‐2 RNAs to self‐prime and result in the synthesis of cDNAs. When rat 7SK RNA was used as substrate, a 294‐nucleotide‐long cDNA was synthesized in vitro by reverse transcriptase, indicating that the 3′ end of 7SK RNA can act in a self‐priming manner to generate 7SK cDNA. When 7‐2 RNA was used as a substrate, a cDNA of approximately 235 nucleotides was observed; 7SL RNA did not act as a self‐primer.Earlier studies have shown that DNAs homologous to 7SK RNA are represented by a moderately reiterated family in the mammalian genomes and many of these sequences were found to be truncated 7SK pseudogenes [Murphy et al. (1984) J. Mol. Biol. 177, 575–590]. In this study, one 7SK clone from the rat genome was characterized by sequencing. This clone contained 243 base pairs homologous to the 5′ end of 7SK RNA, and was flanked by direct repeats. These data suggest that, as previously proposed for some U3 pseudogenes [Bernstein et al. (1983) Cell 32, 461–472], one mechanism for the generation of truncated 7SK pseudogenes may be the integration of self‐primed reverse transcripts of 7SK RNA at random genomic sites.
Resonance Raman study on the structure of the active sites of microsomal cytochrome P‐450 isozymes LM2 and LM4
Authors: HILDEBRANDT Peter , GREINERT Rüdiger , STIER Anton , TANIGUCHI Hisaaki 
Published: March 2005

The isozymes 2 and 4 of rabbit microsomal cytochrome P‐450 (LM2, LM4) have been studied by resonance Raman spectroscopy. Based on high quality spectra, a vibrational assignment of the porphyrin modes in the frequency range between 100–1700 cm−1 is presented for different ferric states of cytochrome P‐450 LM2 and LM4. The resonance Raman spectra are interpreted in terms of the spin and ligation state of the heme iron and of heme—protein interactions. While in cytochrome P‐450 LM2 the six‐coordinated low‐spin configuration is predominantly occupied, in the isozyme LM4 the five‐coordinated high‐spin form is the most stable state. The different stability of these two spin configurations in LM2 and LM4 can be attributed to the structures of the active sites. In the low‐spin form of the isozymes LM4 the protein matrix forces the heme into a more rigid conformation than in LM2. These steric constraints are removed upon dissociation of the sixth ligand leading to a more flexible structure of the active site in the high‐spin form of the isozyme LM4.The vibrational modes of the vinyl groups were found to be characteristic markers for the specific structures of the heme pockets in both isozymes. They also respond sensitively to type‐I substrate binding. While in cytochrome P‐450 LM4 the occupation of the substrate‐binding pocket induces conformational changes of the vinyl groups, as reflected by frequency shifts of the vinyl modes, in the LM2 isozyme the ground‐state conformation of these substituents remain unaffected, suggesting that the more flexible heme pocket can accomodate substrates without imposing steric constraints on the porphyrin. The resonance Raman technique makes structural changes visible which are induced by substrate binding in addition and independent of the changes associated with the shift of the spin state equilibrium: the high‐spin states in the substrate‐bound and substrate‐free enzyme are structrually different. The formation of the inactive form, P‐420, involves a severe structural rearrangement in the heme binding pocket leading to drastic changes of the vinyl group conformations. The conformational differences of the active sites in cytochromes P‐450 LM2 and LM4 observed in this work contribute to the understanding of the structural basis accounting for substrate and product specificity of cytochrome P‐450 isozymes.
Coenzyme specificity of mammalian liver d‐glycerate dehydrogenase
Authors: Van SCHAFTINGEN Emile , DRAYE Jean‐Pierre , Van HOOF François 
Published: March 2005

d‐Glycerate dehydrogenase (glyoxylate reductase) was partially purified from rat liver by anion‐ and cation‐exchange chromatography. When assayed in the direction of d‐glycerate or glycolate formation, the enzyme was inhibited by high (≥0.5 mM), unphysiological concentrations of hydroxypyruvate or glyoxylate much more potently in the presence of NADPH than in the presence of NADH. However, the dehydrogenase displayed a much greater affinity for NADPH (Km < 1 μM) than for NADH (Km= 48–153 μM). Furthermore, NADP was over 1000‐fold more potent than NAD in inhibiting the enzyme competitively with respect to NADH. NADP also inhibited the reaction competitively with respect to NADPH whereas NAD, at concentrations of up to 10 mM had no inhibitory effect. When measured by the formation of hydroxypyruvate from d‐glycerate, the enzyme also displayed a much greater affinity for NADP than for NAD. These properties indicate that liver d‐glycerate dehydrogenase functions physiologically as an NADPH‐specific reductase.In agreement with this conclusion, the addition of hydroxypyruvate or glyoxylate to suspensions of rat hepatocytes stimulated the pentose‐phosphate pathway. The coenzyme specificity of d‐glycerate dehydrogenase is discussed in relation to the biochemical findings made in d‐glyceric aciduria and in primary hyperoxaluria type II (l‐glyceric aciduria).
Arginyl‐tRNA synthetase from yeast
Authors: FREIST Wolfgang , STERNBACH Hans , CRAMER Friedrich 
Published: March 2005

For discrimination between arginine and 19 other amino acids in aminoacylation of tRNAArg‐C‐C‐A by arginyl‐tRNA synthetase from baker's yeast, discrimination factors (D) have been determined from kcat and Km values. The lowest values were found for Trp, Cys, Lys (D= 800–8500), showing that arginine is 800–8500 times more often incorporated into tRNAArg‐C‐C‐A than noncognate acids at the same amino acid concentrations. The other noncognate amino acids exhibit D values between 10000 and 60000.In aminoacylation of tRNAArg‐C‐C‐A(3′NH2) discrimination factors D1 are in the range 10–600. From these values and AMP formation stoichiometry, pretransfer proof‐reading factors II1 were determined; from D values and AMP stoichiometry in aminoacylation of tRNAArg‐C‐C‐A, posttransfer proof‐reading factors II2 could be calculated. II1 values between 2 and 120 show that pretransfer proof‐reading is the main correction step, posttransfer proof‐reading (II2∼ 1–10) plays a marginal role.Initial discrimination factors due to different Gibbs free energies of binding between arginine and the noncognate amino acids were calculated from discrimination and proof‐reading factors. According to a two‐step binding process, two factors (I1 and I2) were determined. They can be related to hydrophobic interaction forces and hydrogen bonds that are especially formed by the arginine side chain. A hypothetical ‘stopper’ model of the amino acid recognition site is discussed.
Primary structure of horse serotransferrin glycans
Authors: CODDEVILLE Bernadette , STRATIL Antonin , WIERUSZESKI Jean‐Michel , STRECKER Gérard , MONTREUIL Jean , SPIK Geneviève 
Published: March 2005

Three serotransferrin variants Tf 2a, Tf 4b and Tf 5b were isolated in an homogeneous form from a preparation of homozygous horse serotransferrin Tf 0. On the basis of the results concerning molecular mass determination and the carbohydrate analysis, it is concluded that the serotransferrin variant Tf 2a contains only one glycan while variants Tf 4b and Tf 5b contain two glycans. The structure of all of the glycans has been established by combining methylation analysis, mass spectrometry and 400‐MHz 1H‐NMR spectroscopy. From the obtained results, it appears that the two glycans of Tf 5b variant are, like in human serotransferrin, of the N‐acetyllactosaminic biantennary type, fully sialylated by two residues of N‐acetylneuraminic acid (Neu5Ac; glycan type I).In contrast, in addition to this structure, two N‐acetyllactosaminic biantennary isomeric structures named type II‐A and type II‐B sialylated by one Neu5Ac residue and one N‐acetyl‐4‐O‐acetylneuraminic acid [Neu(4,5)Ac2] residue located either at Gal6 or 6′ and one N‐acetyllactosaminic biantennary structure (named type III) sialylated by two residues of Neu(4,5)Ac2, were identified in variants Tf 2a and Tf 4b.These results demonstrate that in an homozygous preparation of horse serotransferrin Tf 0, the heterogeneity is dependent, on the one hand, on the nature of the neuraminic acid substituting a N‐acetyllactosaminic biantennary structure and, on the other hand, on the number of glycans bound to the polypeptide chain.Moreover, the differences which exist in the molecular mass of 77.5 kDa, 80 kDa and 82 kDa for serotransferrin variants Tf 2a, Tf 4b and Tf 5b, respectively, are not completely explained by the structure and the number of the glycans suggesting that the three variants should also differ in their polypeptide chain.
Purification and characterization of 55‐kDa protein with 3,5,3′‐triiodo‐l‐thyronine‐binding activity and protein disulfide‐isomerase activity from beef liver membrane
Authors: HORIUCHI Rynya , YAMAUCHI Kiyoshi , HAYASHI Hiroaki , KOYA Sakuji , TAKEUCHI Yoshio , KATO Koichi , KOBAYASHI Masiyuki , TAKIKAWA Hiroo 
Published: March 2005

Beef liver membranes were shown to have different kinds of 3,5,3′‐triiodo‐l‐thyronine binding proteins including the 55‐kDa protein which had been reported to have this activity in many cells by affinity labelling with N‐bromoacetyl‐3,5,3′‐[125I]triiodo‐l‐thyronine. In order to characterize the molecular features of these binding proteins, the 55‐kDa protein was purified from a beef liver membrane fraction abundant in the plasma membrane. The protein was solubilized with 0.5% Chaps and purified by chromatography on gel filtration, hydroxyapatite. and Mono Q anion‐exchange columns. The purity was confirmed with reversed‐phase HPLC and SDS/PAGE. Consequently, 0.4% of the total proteins in the membrane fraction was recovered as the 55‐kDa protein. One fourth of the amino acid composition of this protein was Glx (14.6%) plus Asx (11.7%) and the pI of this protein was 4.5. The purified protein has triiodothyronine‐binding activity with a Kd of 57 nM which is similar to the high‐affinity binding site of the membranes.The anti‐(55‐kDa protein) sera specifically recognized the 55‐kDa protein of beef, rat and human cells. The immunoglobulin G fraction of the anti‐(55‐kDa protein) sera inhibited triiodothyronine binding to the beef liver membrane fraction. The purified protein also showed the activity of protein disulfide‐isomerase (EC 5.3.4.1) as determined by reactivating scrambled ribonuclease. These data strongly suggested that the multi‐functional 55‐kDa protein which has triiodothyronine‐binding activity and the activity of protein disulfide‐isomerase, which is also reported to be the β subunit of prolyl‐4‐hydroxylase, glycosylation‐site‐binding protein of oligosaccharyl transferase and iodothyronine 5′‐monodeionidase, could be significant in the action of triiodothyronine towards the target cells.
Biochemical and biophysical analysis of pseudoknot‐containing RNA fragments
Authors: van BELKUM Alex , WIERSEMA Pieter Jan , JOORDENS Jos , PLEIJ Cornelis , HILBERS Cornelis W. , BOSCH Leendert 
Published: March 2005

Three overlapping RNA fragments containing the pseudoknot, as found in the tRNA‐like structure of turnip yellow mosaic virus (TYMV) RNA, have been isolated and purified. Site‐directed cleavage of TYMV RNA by RNase H, followed by ammonium sulphate precipitation and ion‐exchange HPLC, yielded a pure preparation of a 3′‐terminal, 112‐nucleotide TYMV RNA fragment. Transcription of TYMV cDNA by T ‐ RNA polymerase, resulted in the isolation of an 88‐nucleotide fragment. Finally, a 44‐nucleotide fragment containing the TYMV RNA pseudoknot and strongly resembling the aminoacyl acceptor arm of the viral RNA was also synthesised using T7 RNA polymerase. The three fragments were isolated in milligram amounts and used for biochemical structure mapping, ultraviolet melting studies and NMR spectroscopy.Chemical modification with diethyl pyrocarbonate and sodium bisulphite and enzymatic digestion with RNase T1 confirmed the presence of the pseudoknot in the 44‐nucleotide fragment. Also the analogue of the T‐stem and T‐loop of the tRNA‐like structure of TYMV RNA was found. The results of modification at various temperatures in Mg2+ ‐containing buffers were in general agreement with optical melting studies. Ultraviolet melting analysis of the longer fragments revealed their greater complexity and the results appear similar to those obtained for some tRNA species.To obtain direct biophysical evidence for base‐pairing and stacking interactions in the pseudoknot. NMR studies were initiated. The first proton‐NMR spectra ever obtained for plant viral RNA fragments are presented. NMR spectra were recorded at various buffer conditions and at various temperatures. The spectra for the 112‐nucleotide and 88‐nucleotide fragment are too complicated to be solved at present. In the case of the 44‐nucleotide fragment, however, the imino proton resonances are well separated and this system turns out to be most promising for structural studies.
Purification and characterization of trimming glucosidase I from pig liver
Authors: BAUSE Ernst , SCHWEDEN Jürgen , GROSS Achim , ORTHEN Bruno 
Published: March 2005

Trimming glucosidase I has been purified about 400‐fold from pig liver crude microsomes by fractional salt/detergent extraction, affinity chromatography and poly(ethylene glycol) precipitation. The purified enzyme has an apparent molecular mass of 85 kDa, and is an N‐glycoprotein as shown by its binding to concanavalin A—Sepharose and its susceptibility to endo‐β‐N‐acetylglucosaminidase (endo H). The native form of glucosidase I is unusually resistant to non‐specific proteolysis. The enzyme can, however, be cleaved at high, that is equimolar, concentrations of trypsin into a defined and enzymatically active mixture of protein fragments with molecular mass of 69 kDa, 45 kDa and 29 kDa, indicating that it is composed of distinct protein domains. The two larger tryptic fragments can be converted by endo H to 66 kDa and 42 kDa polypeptides, suggesting that glucosidase I contains one N‐linked high‐mannose sugar chain.Purified pig liver glucosidase I hydrolyzes specifically the terminal α1–2‐linked glucose residue from natural Glc3‐Man9‐GlcNAc2, but is inactive towards Glc2‐Man9‐GlcNAc2 or nitrophenyl‐/methyl‐umbelliferyl‐α‐glucosides. The enzyme displays a pH optimum close to 6.4. does not require metal ions for activity and is strongly inhibited by 1‐deoxynojirimycin (Ki∼ 2.1 μM), N,N‐dimethyl‐1‐deoxynojirimycin (Ki∼ 0.5 μM) and N‐(5‐carboxypentyl)‐1‐deoxynojirimycin (Ki∼ 0.45 μM), thus closely resembling calf liver and yeast glucosidase I.Polyclonal antibodies raised against denatured pig liver glucosidase I, were found to recognize specifically the 85 kDa enzyme protein in Western blots of crude pig liver microsomes. This antibody also detected proteins of similar size in crude microsomal preparations from calf and human liver, calf kidney and intestine, indicating that the enzymes from these cells have in common one or more antigenic determinants. The antibody failed to cross‐react with the enzyme from chicken liver, yeast and Volvox carteri under similar experimental conditions, pointing to a lack of sufficient similarity to convey cross‐reactivity.
Occurrence of S‐(1,2‐dicarboxyethyl)‐cysteine at position 77 in mutant human lysozyme secreted by Saccharomyces cerevisiae
Authors: KIKUCHI Masakazu , TANIYAMA Yoshio , KANAYA Shigenori , TAKAO Toshifumi , SHIMONISHI Yasutsugu 
Published: March 2005

A mutant human lysozyme P110, in which Val110 was replaced with Pro, was secreted by Saccharomyces cerevisiae; modification of the cysteine residue at position 77 was found in a purified mutant protein (P110‐B) upon primary structure analysis. A peptide fragment containing 15 amino acid residues from Thr70 to Leu84 was obtained by proteolytic digestion of the protein and subsequently isolated by reverse‐phase HPLC. This fragment was analyzed by high‐resolution fast‐atom‐bombardment (FAB) mass spectrometry, which showed that 1,2‐dicarboxyethyl group was attached to the thiol group of Cys77. This modification was confirmed by comparing it with a sample of chemically synthesized S‐(1,2‐dicarboxyethyl)‐L‐cysteine.It was found that the modification caused a disruption of the disulfide bond Cys77–Cys95 in the mutant molecule. These observations, plus structural considerations, suggest that Cys77 and Cys95 either remain uncrosslinked or the disulfide bond Cys77–Cys95, once formed, is opened during the final step in the folding of human lysozyme in vivo.
Characterisation of the Klebsiella pneumoniae nitrogen‐fixation regulatory proteins NIFA and NIFL in vitro
Authors: AUSTIN Sara , HENDERSON Neville , DIXON Ray 
Published: March 2005

Transcriptional activation by the Klebsiella pneumoniae nitrogen‐fixation‐specific positive control protein, NIFA, (nifA gene product) has been demonstrated in vitro in S30 extracts from cells which overproduce this protein. The activity of NIFA was dramatically reduced in vitro in the presence of the negative regulatory protein NIFL (nifL gene product) but was not inhibited by the presence of a mutant NIFL protein, NIFL2404. Transcriptional activation from the nifH promoter by NIFA was dependent on the alternative σ factor, σ54, and also required the presence of an upstream activator sequence. NIFA activity was temperature‐sensitive in vitro (as it is in vivo) which is due, at least in part, to the intrinsic lability of the protein itself.The majority of overproduced NIFA and NIFL was insoluble after low‐speed centrifugation and was inactive in vitro. A low level of less aggregated NIFA protein present in cell extracts was responsible for in vitro activity and this fraction was partially purified.