bluuebunny/crossref_metadata_2025_split huggingface dataset:
Studies on the active site of rat glutathione S‐transferase isoenzyme 4–4
Authors: van OMMEN Ben , PLOEMEN Jan H. T. M. , RUVEN Hendrik J. , VOS Ria M. E. , BOGAARDS Jan J. P. , van BERKEL Willem J. H. , van BLADEREN Peter J. 
Published: March 2005

The active site of glutathione S‐transferase isoenzyme 4–4, purified from rat liver, was studied by chemical modification. Tetrachloro‐1,4‐benzoquinone, a compound previously shown to inactivate glutathione S‐transferases very efficiently by covalent binding in or close to the active site, completely prevented the alkylation of the enzyme by iodoacetamide, indicating that the reaction had taken place with cysteine residues. Both from radioactive labeling and spectral quantification experiments, evidence was obtained for the covalent binding of three benzoquinone molecules per subunit, i.e. equivalent to the number of cysteine residues present. This threefold binding was achieved with a fourfold molar excess of the benzoquinone, illustrating the high reactivity of this compound. Comparison of the number of amino acid residues modified by tetrachloro‐1,4‐benzoquinone with the decrease of catalytic activity revealed an almost complete inhibition after modification of one cysteine residue.Chemical modification studies with diethylpyrocarbonate indicated that all four histidine residues of the subunit are ethoxyformylated in an at least partially sequential manner. Modification of the second histidine residue resulted in complete loss of catalytic activity. Preincubation of the transferase with the glutathione conjugate of tetrachloro‐1,4‐benzoquinone resulted in 78% protection against this modification. However, glutathione itself hardly protected against the reaction with diethylpyrocarbonate.The intrinsic fluorescence properties of the enzyme were affected by covalent binding of tetrachloro‐1,4‐benzoquinone. The concentration dependency of the fluorescence quenching is strongly correlated with the inactivation of the enzyme, indicating that covalent binding of the benzoquinone occurs in the vicinity of at least one tryptophan residue.Finally, the binding of bilirubin, as measured by means of circular dichroism, was inhibited by preincubation of the enzyme with tetrachloro‐1,4‐benzoquinone in a manner which strongly correlated with the loss of enzymatic activity, the protection against inactivation by diethylpyrocarbonate, and the fluorescence quenching. All processes showed a 70–80% decrease after incubation of the enzyme with an equimolar amount of the benzoquinone. Thus, evidence is presented for the presence of a cysteine, a histidine and a tryptophan residue in, or in the vicinity of, the active site of the glutathione S‐transferase 4 subunit.
Mycolic acid metabolic filiation and location in Mycobacterium aurum and Mycobacterium phlei
Authors: LACAVE Charlotte , LANEELLE Marie‐Antoinette , DAFFE Mamadou , MONTROZIER Henri , LANEELLE Gilbert 
Published: March 2005

Synchronous cultures of Mycobacterium aurum were used to prove a close relationship between cellular division and active synthesis of mycolic acids (characteristic long‐chain 3‐hydroxyacids, branched at position 2), confirming previous proposals.Mycolic acid biosynthesis was studied in two species (Mycobacterium phlei and M. aurum) each producing three types of mycolic acids: di‐unsatured mycolates, oxomycolates and wax‐ester mycolates (ester of dicarboxymycolic acid and 2‐icosanol or 2‐octadecanol). It was shown that unsaturated mycolates and oxomycolic acids were not directly related, whereas a metabolic filiation was confirmed between oxomycolate and wax ester mycolate: the latter derived, whereas a metabolic filiation was confirmed between oxomycolate and wax ester mycolate: the latter derived from the former by a Baeyer‐Villiger oxidation step, as has been proposed on the basis of structural considerations.By observing the labelling of the different mycolate pools in the cell, i.e. the organic‐solvent‐extractable fraction (essentially containing esters of trehalose and of glycerol) and the cell residue (assumed to be the cell‐wall polymers), it was clear that oxomycolates and unsaturated mycolates appeared first in the extractable lipids, then in the wall‐linked mycolates while wax‐ester mycolates appeared first as wall‐linked derivatives. Thus, it is proposed that mycolates could follow separate routes involving differently located enzymes to reach their complex forms either in extractable lipids or in the wall‐linked arabino‐galactan.
Assembly kinetics and identification of precursor proteins of complex I from Neurospora crassa
Authors: VIDEIRA Arnaldo , WERNER Sigurd 
Published: March 2005

Complex I from Neurospora crassa was fractionated using chaotropic agents and various chromatographic techniques. Several subunits were isolated. Polyclonal antibodies directed against the holocomplex or individual subunits were raised in rabbits, and employed to analyse the composition and assembly of this respiratory chain enzyme in vivo. N. crassa cells were pulse‐labelled with radioactive amino acids. The time course of incorporation of radioactivity into complex‐I polypeptides was studied by immunoprecipitation. The labelling kinetics of whole complex I was found to be similar to that of cytochrome oxidase, displaying a half‐maximal labelling time of 10 min. Newly synthesized individual polypeptide subunits (about 23 species) assembled into the holoenzyme at markedly different rates. Two mitochondrially synthesized proteins, a 29‐kDa polypeptide (the ND‐1 gene product) and a 12‐kDa polypeptide were the fastest components to appear in the enzyme. We estimate that the precursor pool sizes of all components range between 1–25% of the amounts present in the final complex.Precursors of polypeptides of complex I were synthesized in an heterologous cell‐free system and immunoprecipitated with subunit specific antibodies. Six isolated precursors were compared with the corresponding mature proteins. It appears that four subunits (apparent molecular masses of 22, 25, 31 and 33 kDa) are initially synthesized as larger‐molecular‐mass precursors. Two subunits (apparent molecular masses of 12.5 and 14 kDa) are made with the same size as their mature forms.
A novel role for aminoacyl‐tRNA synthetases in the regulation of polypeptide chain initiation
Authors: POLLARD Jeffrey W. , GALPINE Angela R. , CLEMENS Michael J. 
Published: March 2005

Exposure of the temperature‐sensitive leucyl‐tRNA synthetase mutant of Chinese hamster ovary cells, tsH1, to the non‐permissive temperature of 39.5°C results in a rapid inhibition of polypeptide chain initiation. This inhibition is caused by a reduced ability of the eukaryotic initiation factor eIF‐2 to participate in the formation of eIF‐2 · GTP · Met‐tRNAf ternary complexes and thus in the formation of 43S ribosomal pre‐initiation complexes. Associated with this decreased eIF‐2 activity is an increased phosphorylation of the eIF‐2α subunit. It has previously been shown in other systems that phosphorylation of eIF‐2α slows the rate of recycling of eIF‐2 · GDP to eIF‐2 · GTP catalysed by the guanine nucleotide exchange factor eIF‐2B. We show here that phosphorylation of eIF‐2α by the reticulocyte haem‐controlled repressor also inhibits eIF‐2B activity in cell‐free extracts derived from tsH1 cells. Thus the observed increased phosphorylation of eIF‐2α at the non‐permissive temperature in this system is consistent with impaired recycling of eIF‐2 in vivo.Using a single‐step temperature revertant of tsH1 cells, TR‐3 (which has normal leucyl‐tRNA synthetase activity at 39.5°C), we demonstrate here that all inhibition of eIF‐2 function reverts together with the synthetase mutation. This establishes the close link between synthetase function and eIF‐2 activity. In contrast, recharging tRNALeuin vivo in tsH1 cells at 39.5°C by treatment with a low concentration of cycloheximide failed to reverse the inhibition of eIF‐2 function. This indicates that tRNA charging per se is not involved in the regulatory mechanism.Our data indicate a novel role for aminoacyl‐tRNA synthetases in the regulation of eIF‐2 function mediated through phosphorylation of the α subunit of this factor. However, in spite of the fact that cell‐free extracts from Chinese hamster ovary cells contain protein kinase and phosphatase activities active against either exogenous or endogenous eIF‐2α, we have been unable to show any activation of kinase or inactivation of phosphatase following incubation of the cells at 39.5°C.
The rotational diffusion of chloroplast phosphate translocator and of lipid molecules in bilayer membranes
Authors: WAGNER Richard , APLEY Enno C. , GROSS Armin , FLÜGGE U. Ingo 
Published: March 2005

The rotational mobility of the phosphate translocator from the chloroplast envelope and of lipid molecules in the membrane of unilamellar azolectin liposomes has been investigated. The rotational dynamics of the liposome membrane were investigated by measuring the rotational diffusion of eosin‐5‐isothiocyanate(EITC)‐labeled l‐α‐dipalmitoylglycerophosphoethanolamine (Pam2GroPEtn) in the lipid phase of the vesicles, either in the presence or absence of the reconstituted phosphate translocator.The temperature dependence of the anisotropy decay showed that above 25°C the main contribution to the anisotropy decay was caused by uniaxial anisotropic rotation of the labelled lipid molecules around the axis normal to the membrane plane. The rate of rotation of the labelled lipid molecules was strongly dependent on the viscosity of the medium (η1). Extrapolation to η1= 0 Pa · s yielded a correlation time of φ= 20 ± 5 ns, t= 30°C, for lipid rotation with respect to the membrane normal. The rotational diffusion coefficient of the lipid molecules was calculated to be Dr= 2.0 × 109 rad2· s−1 and the apparent microviscosity in the vesicle membrane, as derived from the rotational correlation time, was η2∼ 12 mPa · s.The rotational correlation time of the phosphate translocator in the membrane was only slightly dependent on the viscosity of the medium. The temperature dependence of the protein rotation also indicated that the rotation of the protein in the membrane was largely restricted and occurred mainly about the axis normal to the membrane plane. Measurements at a medium viscosity of η1= 1 mPa · s yielded a value of φr∼ 450 ns corresponding to Dr= 8.8 × 107 rad2· s−1 for protein rotation with respect to the membrane normal. From this value and the data of the lipid rotation, the cross‐sectional area of the protein part embedded in the membrane was calculated to be ∼ 9 nm2. This cross‐sectional area is large enough to include at most 14 membrane‐spanning helices.Our result also indicated that at lipid/protein molar ratios > 1.5 × 104:1 aggregation occurred in the model membranes below 30°C. However, above 30°C and at a high dilution of the protein in the membrane it appeared that the membrane viscosity monitored by lipid and protein rotational diffusion were identical.
Functional analysis of the signal‐sequence processing site of yeast acid phosphatase
Authors: MONOD Michel , HAGUENAUER‐TSAPIS Rosine , RAUSEO‐KOENIG Isabelle , HINNEN Albert 
Published: March 2005

A systematic study of the signal peptidase cleavage site of the main cell‐wall‐repressible Saccharomyces cerevisiae acid phosphatase encoded by the PHO5 gene is presented. The last amino acid of the signal sequence, the chromosomally encoded alanine of the wild‐type gene, was changed by any of 19 other amino acids in the chromosomal DNA by using in vitro mutagenesis in Escherichia coli and the technique of gene replacement. Processing and secretion are normal when the amino acid at this position is a small neutral amino acid, i.e. alanine, glycine, cysteine, serine or threonine. Processing glycosylation, and secretion of regulated acid phosphatase are distinctly affected with other amino acid substitutions and core‐glycosylated protein accumulates in the cell.Surprisingly, PHO5 protein is still secreted to the cell wall and into the growth medium but at a lower rate and without cleavage of the signal sequence. The same features are exhibited by a mutated acid phosphatase with a deletion of four amino acids at the end of the signal peptide (−7 to −4 relative to the processing site) thus preserving the important −3 to −1 region.
Mathematical modelling of metabolic pathways affected by an enzyme deficiency
Authors: SCHUSTER Ronny , JACOBASCH Gisela , HOLZHÜTTER Hermann‐Georg 
Published: March 2005

The effects of various forms of glucose‐6‐phosphate dehydrogenase deficiency on erythrocyte metabolism have been studied on the basis of a complex mathematical model which comprises the main pathways of this cell: glycolysis, pentose pathway, reactions of the glutathione and adenine nucleotide metabolism.The calculated flux rates through the oxidative pentose pathway with and without methylene blue are in good accord with experimental results. The degree of deficiency as predicted by the model on the basis of calculated upper oxidative load boundaries, as well as of maximal methylene blue stimulation, correlates with the individual clinical manifestation of the metabolic disease. Therefore, the model allows one to judge the degree of metabolic disorder in the presence of glucose‐6‐phosphate dehydrogenase enzymopathies if the kinetic properties of the defect enzyme are known. Experimentally accessible parameters for an assessment of the oxidative load capacity of cells in vivo are proposed.It is pointed out that the threshold of tolerance as to energetic load is drastically reduced in the case of severe glucose‐6‐phosphate dehydrogenase deficiency.
Conformational analysis of brominated pA2′‐5′A2′‐5′A analogs
Authors: van den HOOGEN Yvonne Th. , HILGERSOM Coen M. A. , BROZDA Danuta , LESIAK Krystyna , TORRENCE Paul F. , ALTONA Cornelis 
Published: March 2005

NMR and model‐building studies were carried out on PA2′‐5′A2′‐5′A and analogs in which one or more of the A residues were replaced by 8‐bromoadenosine. Chemical shifts, coupling constants and NOE data were used to obtain structural information. The N/S equilibrium constant of the ribose rings as well as the phase angles and puckering amplitudes were determined from the experimental coupling constants with the aid of an improved version of the PSEUROT program. Chemical shifts in combination with NOE data were used to monitor basebase interactions and the orientation of the bases (syn or anti).The combined data suggest that different types of stacking interactions are present in the various compounds. Bromination of the first or second residue in the trimers results in a preference for N‐type sugar and syn orientation of the base in these residues. When A(3) is brominated, an S‐type sugar conformation together with a syn orientation of the base is favoured at the 2′ terminus. Energy‐minimized models of the different stacking interactions are presented which fit the present collection of data. The possible correlation between biological activity of these compounds and their conformation is briefly discussed.
Crystallin gene expression during rat lens development
Authors: AARTS Henk J. M. , LUBSEN Nicolette H. , SCHOENMAKERS John G. G. 
Published: March 2005

The analysis of the developmental pattern of the αA‐, αB‐, βB1‐, βB2‐, βB3‐, βA3/A1‐, and βs‐crystallin genes during fetal and postnatal development of the rat shows that the differential regulation of crystallin synthesis relies on differential gene shutdown rather than differential gene activation; that is, all crystallin gene are active during early development but turn off at different stages. The only two exceptions to this rule are the αB‐ and βs‐crystallin genes. The αB‐crystallin gene transcript becomes first detectable at 18 days of fetal development, while the βs‐crystallin gene appears to be active only in the postnatal period.We also determined the absolute numbers of the αA‐, αB‐, βB1‐, βB2‐, βB3‐, βA3/A1‐, βs‐, and γ‐crystallin gene transcripts present in the lens at various times after birth. Comparison of these RNA data with the published protein data shows that the αB‐ and βB2‐crystallin RNAs are relatively overrepresented, suggesting the possibility that these two RNA species are not used as efficiently as other crystallin mRNAs. Examination of the known (hamster) αB‐crystallin sequence and elucidation of the (rat) βB2‐crystallin sequence yielded no evidence for aberrant codon usage. These two RNAs have one sequence motif in common: they are the only crystallin mRNAs in which the translation initiation codon is preceded by CCACC.
NMR analysis of site‐specific mutants of yeast phosphoglycerate kinase
Authors: FAIRBROTHER Wayne J. , WALKER Philip A. , MINARD Philippe , LITTLECHILD Jennifer A. , WATSON Herman C. , WILLIAMS Robert J. P. 
Published: March 2005

Site‐specific mutants of yeast phosphoglycerate kinase have been produced in order to investigate the roles of the ‘basic‐patch’ residues, arginine 168 and histidine 170. The fully‐conserved residue, arginine 168, has been replaced with a lysine (R168K) and a methionine (R168M) residue, while the non‐conserved histidine 170 has been replaced with an aspartate (H170D). Comparison of the 500‐MHz 1H‐NMR spectra of the mutant proteins with that of wild‐type phosphoglycerate kinase shows that the overall fold of the mutants remains essentially unaltered from that of the native emzyme. Results of NOE experiments indicate that there are only very minor changes in structure in the vicinity of the mutations. These mutations have also led to firm sequence‐specific resonance assignments to histidines 62, 167 and 170.NMR studies of 3‐phosphoglycerate binding show that decreasing the positive charge in the sequence 168–170 reduces the binding of this substrate (by about 15‐fold and 4‐fold for mutants R168M and H170D respectively). Mutant R168K binds 3‐phosphoglycerate with an affinity about twofold less than that of the native enzyme. Significantly, the activity of mutant H170D, measured at saturating substrate concentrations, is unchanged from that of the wild‐type emzyme. This indicates that this residue is not of major importance in the binding or reaction of 3‐phosphoglycerate. The observation is in agreement with results obtained for the wild‐type enzyme, which indicate that 3‐phosphoglycerate interacts most strongly with histidine 62 and least strongly with histidine 170, as would be predicted from the X‐ray crystal structure. Substitution of positively charged arginine 168 with neutral methionine (or positively charged lysine) does not cause a detectable change in the pKa values of the neighbouring histidine groups, in as much as they remain below 3. The results reported here indicate that the observed reduction in catalytic efficiency relates less to direct electrostatic effects than to the mutants' inability to undergo 3‐phosphoglycerate‐induced conformational changes.
Dehydrogenases of the pentose phosphate pathway in rat liver peroxisomes
Authors: ANTONENKOV Vasily D. 
Published: March 2005

Subcellular distribution of pentose‐phosphate cycle enzymes in rat liver was investigated, using differential and isopycnic centrifugation. The activities of the NADP+‐dependent dehydrogenases of the pentose‐phosphate pathway (glucose‐6‐phosphate dehydrogenase and phosphogluconate dehydrogenase) were detected in the purified peroxisomal fraction as well as in the cytosol. Both dehydrogenases were localized in the peroxisomal matrix. Chronic administration of the hypolipidemic drug clofibrate (ethyl‐α‐p‐chlorophenoxysobutyrate) caused a 1.5–2.5‐fold increase in the amount of glucose‐6‐phosphate and phosphogluconate dehydrogenases in the purified peroxisomes. Clofibrate decreased of phosphogluconate dehydrogenase, but did not alter glucose‐6‐phosphate dehydrogenase activity in the cytosolic fraction.The results obtained indicate that the enzymes of the non‐oxidative segment of the pentose cycle (transketolase, transaldolase, triosephosphate isomerase and glucose‐phosphate isomerase) are present only in a soluble form in the cytosol, but not in the peroxisomes or other particles, and that ionogenic interaction of the enzymes with the mitochondrial and other membranes takes place during homogenization of the tissue in 0.25 M sucrose.Similar to catalase, glucose‐6‐phosphate dehydrogenase and phosphogluconate dehydrogenase are present in the intact peroxisomes in a latent form. The enzymes have Km values for their substrates in the millimolar range (0.2 mM for glucose‐6‐phosphate and 0.10–0.12 mM for 6‐phophogluconate). NADP+, but not NAD+, serves as a coenzyme for both enzymes. Glucose‐6‐phosphate dehydrogenase was inhibited by palmitoyl‐CoA, and to a lesser extent by NADPH. Peroxisomal glucose‐6‐phosphate and phosphogluconate dehydrogenases have molecular mass of 280 kDa and 96 kDa, respectively. The putative functional role of pentose‐phosphate cycle dehydrogenases in rat liver peroxisomes is discussed.
Cloning of cDNA encoding the membrane‐bound form of bovine β1,4‐galactosyltransferase
Authors: D'AGOSTARO Giacomo , BENDIAK Brad , TROPAK Michael 
Published: March 2005

UDPgalactose: N‐acetyl‐d‐glucosamine 4‐β‐d‐galactosyltransferase (EC 2.4.1.38) (GalT) is a Golgi‐membranebound enzyme that participates in the biosynthesis of the oligosaccharide structures of glyscoproteins and glycolipids.Synthetic DNA oligomers representing segments of the published partial cDNA sequence for bovine GalT were used as molecular probes to isolate from bovine‐liver cDNA libraries overlapping cDNA clones that span 1728 nucleotides and potentially code for the entire polypeptide chain of bovine galactosyltransferase. The cDNA sequence for bovine GalT reveals a 1206‐base‐pair open reading frame that codes for 402 amino acids, including a presumptive N‐terminal membrane anchoring domain of 20 hydrophobic amino acids. The colinearity between the cDNA sequence and 29 non‐overlapping amino acid residues which were positively identified by N‐terminal sequencing of two polypeptides isolated form the soluble form of the enzyme was consistent with the translation frame and confirmed the authenticity of the cDNA clones.The finding of an N‐terminal hydrophobic segment which serves as the membrane anchor and signal sequence suggests that the C‐terminal region of the GalT polypeptide is oriented within the lumen of the Golgi membranes. This conclusion is in agreement with previous biochemical studies which indicated that the 51‐kDa and 42‐kDa soluble forms of the enzyme which encompass the C‐terminal 324 and 297 amino acid residues of the entire GalT polypeptide, respectively, include the catalytic site.
Analysis of the inactivation of liver alcohol dehydrogenase during storage in Aerosol‐OT/isooctane microemulsions
Authors: LARSSON Karin M. , OLDFIELD Christopher , FREEDMAN Robert B. 
Published: March 2005

Changes in the enzymatic properties of horse liver alcohol dehydrogenase (HLADH; EC 1.1.1.1) were studied as a function of incubation time in Aerosol‐OT/isooctane microemulsions. The enzyme was characterized by fluorimetric binding studies of the inhibitor isobutyramide to the binary complex, HLADH‐NADH and by determination of Km,app and Vmax,app values for cyclohexanone.The Km,app values for cyclohexanone and the Kd,app for isobutyramide stay constant throughout a 48‐h incubation, whereas the Vmax,app and the total number of inhibitor binding sites decrease. Thus the inactivation process previously described corresponds to progressive loss of functional sites, while the properties of the remaining functional sites are unchanged.If no co‐enzyme is added to the system, the enzyme loses catalytic activity within less than an hour, but if co‐enzyme is added, a fraction of the HLADH enzyme population retains enzyme activity over a long period of time. Hence the presence of bound co‐enzyme significantly inhibits the process(es) leading to inactivation of the enzyme in the microemulsions.