bluuebunny/crossref_metadata_2025 huggingface dataset:
Identification of the sites in the eukaryotic elongation factor 1α involved in the binding of elongation factor 1β and aminoacyl‐tRNA
Authors: van DAMME Henk T. F. , AMONS Reinout , MÖLLER Wim
Published: March 2005

In this article we report the identification of the sites which are involved in the binding of the GDP‐exchange factor EF‐1β and aminoacyl tRNA to the α‐subunit of the eukaryotic elongation factor 1 (EF‐1) from Artemia. For this purpose the polypeptide chain of EF‐1α, having 461 amino acid residues, was proteolytically cleaved into large fragments by distinct proteases. Under well defined conditions, a mixture of two large fragments, free from intact EF‐1α and with molecular masses of 37 kDa and 43 kDa, was obtained. The 37‐kDa and 43‐kDa fragments comprise the residues 129–461 and 69–461, respectively. However, in aqueous solution and under non‐denaturing conditions, the mixture still contained a short amino‐terminal peptide, encompassing the residues 1–36, that remained tightly bound. The ability of the mixture of the 37 + 43‐kDa fragments, including this amino‐terminal peptide 1–36, to bind GDP or to facilitate aminoacyl tRNA binding to salt‐washed ribosomes was severely reduced, compared to intact EF‐1α. However, both of these complexes were able to bind to the GDP‐exchange‐stimulating subunit EF‐1β. A 30‐kDa fragment, comprising the residues 1–287, was generated after treatment of the protein with endoproteinase Glu‐C. This fragment contained the complete guanine nucleotide binding pocket. Although it was able to bind GDP and to transport aminoacyl tRNA to the ribosome, no affinity towards EF‐1β was observed. We propose that the guanine‐nucleotide‐exchange stimulation by EF‐1β is induced through binding of this factor to the carboxy‐terminal part of EF‐1α. As a result, a decreased susceptibility towards trypsin of the carboxy‐terminal part of EF‐1α. As a result, a decreased susceptibility towards trypsin of the guanine‐nucleotide‐binding pocket of EF‐1α, especially in the region of its presumed effector loop is induced.
Molecular characterization of a human anti‐Rh(D) antibody with a DH segment encoded by a germ‐line sequence
Authors: CHOUCHANE Lotfi , VAN SPRONSEN Anke , BREYER Joan , GUGLIELMI Paul , STROSBERG A. Donny
Published: March 2005

The λ‐light‐chain and λ‐heavy‐chain variable‐region genes of an anti‐Rh(D) (Rh, Rhesus; D, heavy‐chain diversity region) human monoclonal antibody secreted by lymphocytes transformed by the Epstein‐Barr virus have been cloned and sequenced. Sequence comparison of the anti‐Rh(D)mAb λ‐chain variable region with those of the other available human λ chains revealed that it belonged to the human VλI (Vλ, variable region of λ chain) subgroup. The greatest sequence similarity (80%) was observed with that of another anti‐Rh antibody λ‐chain directed against the Rh(c) antigen. For the VH (VH, variable region of heavy chain) sequence, the highest similarity (86%) was observed with the germline VHG3 gene which belongs to the VHI subgroup. The expressed DH sequence of the anti‐Rh(D) antibody is also of germline origin and complementarity‐determining region 3 is thus produced by VH‐DH and DH‐JH (J, joining region) joining without recombination of multiple DH gene segments.
The polygalacturonases of Aspergillus niger are encoded by a family of diverged genes
Authors: BUSSINK Hendrik J. D. , BUXTON Frank P. , FRAAYE Bartholomeus A. , GRAAFF Leo H. de , VISSER Jaap
Published: March 2005

Aspergillusniger produces several polygalacturonases that, with other enzymes, are involved in the degradation of pectin. One of the two previously characterized genes coding for the abundant polygalacturonases I and II (PGI and PGII) found in a commercial pectinase preparation was used as a probe to isolate five more genes by screening a genomic DNA library in phage λEMBL4 using conditions of moderate stringency. The products of these genes were detected in the culture medium of Aspergillus nidulans transformants on the basis of activity measurements and Western‐blot analysis using a polyclonal antibody raised against PGI. These transformants were, with one exception, constructed using phage DNA. A. nidulans transformants secreted high amounts of PGI and PGII in comparison to the previously characterized A. niger transformants and a novel polygalacturonase (PGC) was produced at high levels by A. nidulans transformed with the subcloned pgaC gene. This gene was sequenced and the protein‐coding region was found to be interrupted by three introns; the different intron/exon organization of the three sequenced A. niger polygalacturonase genes can be explained by the gain or loss of two single introns. The pgaC gene encodes a putative 383‐amino‐acid prepro‐protein that is cleaved after a pair of basic amino acids and shows approximately 60% amino acid sequence similarity to the other polygalacturonases in the mature protein. The N‐terminal amino acid sequences of the A. niger polygalacturonases display characteristic amino acid insertions or deletions that are also observed in polygalacturonases of phytopathogenic fungi. In the upstream regions of the A. niger polygalacturonase genes, a sequence of ten conserved nucleotides comprising a CCAAT sequence was found, which is likely to represent a binding site for a regulatory protein as it shows a high similarity to the yeast CYC1 upstream activation site recognized by the HAP2/3/4 activation complex.
A strong protein‐tyrosine kinase activity is associated with a baculovirus‐expressed chicken tkl gene
Authors: GÄRTNER Thomas , KÜHNEL Herbert , RAAB Gerhard , RAAB Monika , STREBHARDT Klaus , RÜBSAMEN‐WAIGMANN Helga
Published: March 2005

We have previously described a gene named tkl (tyrosine kinase related to lck). It belongs to the src family of protein‐tyrosine kinases and among these it has significant homology to the lck gene (lymphoide cell kinase). The tkl gene product may represent the avian homolog of Lck, which is believed to participate in a lymphocyte‐specific signal transduction pathway by association with a membrane receptor.

To study the biochemical properties of the protein, a nearly complete tkl gene (isolated from a cDNA library from chicken spleen cells) was expressed in a baculovirus system. Approximately 10% of the extracted protein consisted of the soluble 51‐kDa Tkl protein (p51tkl) at 40 h post‐infection. This protein was found to be phosphorylated on tyrosine and serine residues at a ratio of 5:1. As expected, glycosylation or myristoylation could not be detected. Immunocomplex kinase assays indicated strong autophosphorylation of p51tkl at tyrosine residues and phosphorylation of exogenous substrates such as D‐glyceraldehyde‐3‐phosphate dehydrogenase (GAPDH), histones H2b and H4, and casein. This protein‐tyrosine kinase activity also exhibited a marked preference for Mn2+ compared to Mg2+. The high level expression of enzymatically active Tkl should provide an excellent tool to further study the biological functions of this class of enzymes.
A study of the hinge‐bending mechanism of yeast 3‐phosphoglycerate kinase
Authors: DRYDEN David T. F. , VARLEY Paul G. , PAIN Roger H.
Published: March 2005

The hinge‐bending mechanism proposed as part of the catalytic mechanism for phosphoglycerate kinase (PGK) has been investigated using yeast PGK and the site‐directed mutant [H388Q]PGK, where His388 is replaced by Gln. The emission and quenching of fluorescence, supported by the aromatic CD band, show that the mutation in the waist region affects the tryptophan environment in the C‐terminal domain. The mutant is also less stable to guanidine denaturation and less cooperative in its unfolding.

The effect of substrates on the conformation of PGK was studied using 8‐anilino‐1‐naphtha‐lenesulphonic acid (ANS), a competitive inhibitor of ATP binding to the C‐terminal domain, and 8‐[2‐[(iodoacetyl)ethyl]amino]naphthalene (I‐AEDANS), attached to Cys197 on the N‐terminal domain. Under the influence of substrates the novel anisotropy decay curves for ANS indicate a 1–5° change in the orientation of the probe, interpreted as a small reorientation of the domains about the waist region. The experimental data are interpreted as a small swivelling of the domains about the waist region under the influence of substrate. The results with AEDANS anisotropy decay are consistent with those for ANS.

The enzyme activity of PGK shows a break in the Arrhenius plot at 20°C mirrored by a break in the temperature dependence of tryptophan ellipticity. This is interpreted as a change in protein dynamics associated with destabilisation of the waist region. This destabilisation is shown to have already taken place in the mutant enzyme and in the wild type at pH 5.6, both of which exhibit linear Arrhenius plots. NMR titration curves show that the pH effect must be due to a group other than histidine.

The results give further support to the permissive model of hinge bending previously proposed by one of the authors, in which binding of substrate destabilises the waist region. This loosens the hinge which can then swing slightly to bring the domains closer together to make favourable interactions between the domains and the substrates, with the exclusion of water.
Regulation of the final phase of mammalian melanogenesis
Authors: AROCA Pilar , SOLANO Francisco , SALINA Cristina , GARCÍA‐BORRÓN José C. , LOZANO José A.
Published: March 2005

The regulation of the final steps of the melanogenesis pathway, after L‐2‐carboxy‐2,3‐dihydroindole‐5,6‐quinone (dopachrome) formation, is studied. It is shown that both tyrosinase and dopachrome tautomerase are involved in the process. In vivo, it seems that tyrosinase is involved in the regulation of the amount of melanin formed, whereas dopachrome tautomerase is mainly involved in the size, structure and composition of melanin, by regulating to the incorporation of 5,6‐dihydroxyindole‐2‐carboxylic acid (DHICA) into the polymer. Moreover, using L‐3,4‐dihydroxyphenylalanine (dopa) and related compounds, it was shown that the presence of dopachrome tautomerase mediates an initial acceleration of melanogenesis since L‐dopachrome is rapidly transformed to DHICA, but that melanin formation is inhibited because of the stability of this carboxylated indole compared to 5,6‐dihydroxyindole (DHI), its decarboxylated counterpart obtained by spontaneous decarboxylation of L‐dopachrome. Using L‐dopa methyl ester as a precursor of melanogenesis, it is shown that this carboxylated indole does not polymerize in the absence of DHI, even in the presence of tyrosinase. However, it is incorporated into the polymer in the presence of both tyrosinase and DHI. Thus, this study suggests that DHI is essential for melanin formation, and the rate of polymerization depends on the ratio between DHICA and DHI in the medium. In the melanosome, this ratio should be regulated by the ratio between the activities of dopachrome tautomerase and tyrosinase.
Formation of homogeneous carbohydrate–lectin cross‐linked precipitates from mixtures of D‐galactose/N‐acetyl‐D‐galactosamine‐specific lectins and multiantennary galactosyl carbohydrates
Authors: BHATTACHARYYA Lokesh , BREWER Curtis Fred
Published: March 2005

Quantitative precipitation studies have shown that the Man/Glc‐specific lectin concanavalin A (ConA) forms homogeneous (homopolymeric) cross‐linked precipitates with individual asparagine‐linked oligomannose and bisected hybrid‐type glycopeptides in the presence of binary mixtures of the carbohydrates [Bhattacharyya, L., Khan, M. I. & Brewer, C. F. (1988) Biochemistry 27, 8762–8767]. The results indicate that the ConA‐glycopeptide precipitates are highly organized cross‐linked lattices that are unique for each carbohydrate. Using similar techniques, the present study shows that the Gal‐specific lectins from Erythrina indica and Ricinus communis (agglutinin I) form homogeneous cross‐linked complexes with individual carbohydrates in binary mixtures of triantennary and tetraantennary complex‐type oligosaccharides with terminal Gal residues. Conversely, binary mixtures of Gal/GalNAc‐specific lectins from E. indica, Erythrina cristagalli, Erythrina flabelliformis, R. communis, soybean (Glycine max), and Wistaria floribunda (tetramer) in the presence of a naturally occurring or synthetic branched‐chain oligosaccharide with terminal GalNAc or Gal residues provide evidence for the formation of separate cross‐linked lattices between each lectin and the carbohydrate. The present results therefore demonstrate the formation of homogeneous lectin–carbohydrate cross‐linked lattices in (a) a mixture of branched‐chain complex‐type oligosaccharides in the presence of a specific Gal/GalNAc‐binding lectin, and (b) a mixture of lectins with similar physicochemical and carbohydrate binding properties in the presence of an oligosaccharide. These findings show that lectin–carbohydrate cross‐linking interactions provide a high degree of specificity which may be relevant to their biological functions as receptors.
The laws of cell energetics
Authors: SKULACHEV Vladimir P.
Published: March 2005

Recent progress in membrane bioenergetics studies has resulted in the important discovery that Na+ can effectively substitute for H+ as the energy coupling ion. This means that living cells can possess three convertible energy currencies, i.e. ATP, protonic and sodium potentials. Analysis of interrelations of these components in various types of living cells allows bioenergetic laws of universal applicability to be inferred.
Concepts and strategies for human gene therapy
Authors: ROEMER Klaus , FRIEDMANN Theodore
Published: March 2005

Methods of modern molecular genetics have been developed that allow stable transfer and expression of foreign DNA sequences in human and other mammalian somatic cells. It is therefore no surprise that the methods have been applied in attempts to complement genetic defects and correct disease phenotypes. Two decades of research have now led to the first clinically applicable attempts to introduce genetically modified cells into human beings to cure diseases caused at least partially by genetic defects. We discuss here some of the strategies being followed for both in vitro and in vivo application of therapeutic gene transfer and summarize some of the technical and conceptual difficulties associated with somatic‐cell gene therapy.
Site‐directed mutagenesis of elongation factor Tu
Authors: ANBORGH Pieter H. , PARMEGGIANI Andrea , JONÁK Jiří
Published: March 2005

A Cys residue located in the second consensus sequence element (DCPG) of the GTP‐binding region is highly conserved in bacterial elongation factors (EF) Tu. Chemical modification of this Cys81 in EF‐Tu from Escherichia coli by N‐tosyl‐l‐phenylalanine chloromethane [Jonák, J., Petersen, T. E., Clark, B. F. C. & Rychlík, I. (1982) FEBS Lett. 150, 485–488], and of homologous Cys residues in other bacterial EF‐Tu, selectively blocks the binding of Xaa‐tRNA. We have substituted Cys81 with Gly using site‐directed mutagenesis of the EF‐Tu‐encoding tufA gene. This substitution induces a partial inhibition (20–70%) of: (a) poly(U)‐directed poly(Phe) synthesis; (b) EF‐Tu/Xaa‐tRNA interaction, determined as protection by EF‐Tu of the non‐enzymic deacylation of Xaa‐tRNA; (c) EF‐Tu‐dependent binding of Xaa‐tRNA to the mRNA/ribosome complex and (d) the intrinsic GTPase reaction, that is also less sensitive to stimulation by Xaa‐tRNA. Our results thus provide evidence that Cys81, though important, is not essential for the binding of Xaa‐tRNA to EF‐Tu. The accuracy in poly(Phe) synthesis, measured as misincorporation of Leu, was increased. Both the binding affinity of [C81G]EF‐Tu for the nucleotide and the resistance against thermal denaturation are more strongly decreased in the case of the GDP‐bound state than in the case of the GTP‐bound state, suggesting that Cys81 plays a more specific role in the former conformation. The sensitivity to N‐tosyl‐l‐phenylalanine chloromethane is decreased by 80% but not totally lost. The inhibition by N‐tosyl‐l‐phenylalanine chloromethane treatment of the function of EF‐Tu appears to be a consequence of steric hindrance and/or of an altered conformation of EF‐Tu · GTP. The lower activities of [C81G]EF‐Tu are probably due to long‐range effects, mediated by an overall destabilization of the molecule that is particularly pronounced for the GDP‐bound state.
Preferential binding of insulin‐like growth factor‐II (IGF‐II) to a putative α2β2 IGF‐II receptor type in C2 myoblasts
Authors: DOMEYNE Anne , PINSET Christian , MONTARRAS Didier , GARANDEL Véronique , ROSENFELD Ron G. , BARENTON Bruno
Published: March 2005

We have studied insulin‐like‐growth‐factor (IGF) binding in two subclones of the C2 myogenic cell line. In the permissive parental subclone, myoblasts differentiate spontaneously into myotubes in medium supplemented with fetal calf serum. Unlike permissive myoblasts, inducible myoblasts require high concentrations of insulin (1.6 μM) or lower concentrations of IGF‐I (25 nM) to differentiate, and expression of MyoD1 is not constitutive. IGF receptors were studied in microsomal membranes of proliferating and quiescent myoblasts and myotubes. IGF‐II binding was also studied in inducible myoblasts transfected with the MyoD1 cDNA (clone EP5).

Both inducible and permissive cells exhibited a single class of binding sites with similar affinity for IGF‐I (Kd 0.8–1.2 nM). Affinity cross‐linking of [125I]IGF‐I to microsomal membranes, under reducing conditions, revealed a binding moiety with an apparent molecular mass of 130 kDa in permissive cells and 140 kDa in inducible cells, which corresponded to the α subunit of the IGF‐I receptor.

In permissive quiescent myoblasts, linear Scatchard plots suggested that [125I]IGF‐II bound to a single class of binding sites (Kd 0.6 nM) compatible with binding to the IGF‐II/M6P receptor. This was confirmed by affinity cross‐linking experiments showing a labeled complex with an apparent molecular mass of 260 kDa and 220 kDa when studied under reducing and non‐reducing conditions, respectively.

In contrast, competitive inhibition of [125I]IGF‐II binding to inducible quiescent myoblasts generated curvilinear Scatchard plots which could be resolved into two single classes of binding sites. One of them corresponded to the IGF‐II/M6P receptor (Kd 0.2 nM) as evidenced by cross‐linking experiments. The second was the binding site of highest affinity (Kd 0.04 nM) which was less inhibited by IGF‐I than by IGF‐II and was not inhibited by insulin. It migrated in SDS/PAGE at a position equivalent a molecular mass of 140 kDa, under reducing conditions, and at approximately 300 kDa, under non‐reducing conditions. The labeling of this atypical binding moiety was not inhibited by anti(IGF‐II/M6P‐receptor) immunoglobulin. It was also observed in permissive and inducible myoblasts at proliferating stage. It was absent for permissive quiescent myoblasts and from permissive and inducible myotubes. Forced expression of MyoD1 in inducible cells (EP5 cells) dramatically reduced [125I]IGF‐II binding to this atypical receptor.

It emerges from these experiments that C2 cells express a putative α2β2 IGF‐II receptor structurally related to the insulin/IGF‐I receptor family. It is present in myoblasts but not in myotubes. The possible relationship between expression of this receptor and expression of MyoD1 as well as myoblast differentiation is discussed.
Temperature effects on the MgATP‐induced electron transfer between the nitrogenase proteins from Azotobacter vinelandii
Authors: MENSINK Richard E. , HAAKER Huub
Published: March 2005

The temperature dependence of the pre‐steady‐state MgATP‐dependent electron transfer from the MoFe protein to the Fe protein of the nitrogenase from Azotobacter vinelandii has been investigated between 6°C and 31°C by stopped‐flow spectrophotometry. Below 14°C, the data are consistent with a model in which interaction of MgATP with nitrogenase is fast and irreversible, and is followed by reversible electron transfer. From the extent and from the rate of the absorbance change, the rate constants for electron transfer from Fe protein to MoFe protein and of the reverse reaction were calculated. The direct rate constant increases with temperature (6–14°C) from about 1 s−1 to about 26 s−1. The rate constant for the reverse reaction was found to be approximately 4 s−1 and invariant with the reaction temperature. Analysis of the data obtained in the temperature range between 6°C and 12°C within the framework of the transition‐state theory show that electron transfer from the Fe protein to the MoFe protein occurs via a highly disordered transition state with activation parameters ΔH°†= 289 kJ · mol−1 and ΔS°†= 792 J · K−1· mol−1. The Eyring plot of the stopped‐flow data displays an inflection point around 14°C. From the stopped‐flow data obtained between 18°C and 27°C the activation parameters ΔH°† and ΔS°† for the reduction of the MoFe protein by Fe protein are calculated to be 90 kJ · mol−1 and 99 J · K−1· mol−1 respectively. A second inflection point in the Eyring plot could exist around 28°C.
Three‐dimensional structure of phenylalanyl‐transfer RNA synthetase from Thermus thermophilus HB8 at 0.6‐nm resolution
Authors: RESHETNIKOVA Ludmila , CHERNAYA Maia , ANKILOVA Valentina , LAVRIK Olga , DELARUE Marc , THIERRY Jean‐Cloud , MORAS Dino , SAFRO Mark
Published: March 2005

The three‐dimensional structure of the heterodimeric α2β2 enzyme phenylalanyl‐tRNA synthetase from Thermus thermophilus HB8 has been determined by X‐ray crystallography, using the multiple‐isomorphous‐replacement method at 0.6 nm resolution. Trigonal crystals of space group P3221 have cell dimensions a=b= 17.6 nm and c= 14.2 nm. Assuming one heterodimeric molecule/asymmetric unit, the ratio of unit cell volume/molecular mass was V= 0.00244 nm3/Da, which is in the middle of the range normally observed. However, after a rotation‐function calculation and measurement of the density of the native crystals, we postulate the existence of only the αβ dimer in the asymmetric units. This implies 73% solvent content in the unit cell. Three heavy‐atom derivatives [K2PtCl4, KAu(CN)2 and Hg(CH3COO)2] and the solvent‐flattening procedure were used for electron‐density‐map calculations. This map confirmed our hypothesis and revealed a remarkably large space filled by solvent, with αβ dimer only in the asymmetric unit. The phenylalanyl‐tRNA synthetase from T. thermophilus molecule has a ‘quasi‐linear’ subunit organization. As can be concluded at this level of resolution, there is no contact between small α subunits in the functional heterodimer.
Conversion, by limited proteolysis, of an archaebacterial citrate synthase into essentially a citryl‐CoA hydrolase
Authors: LILL Ute , LEFRANK Sigrid , HENSCHEN Agnes , EGGERER Hermann
Published: March 2005

1. Limited proteolysis of citrate synthase from Sulfolobus solfataricus by trypsin reduced the rate of the overall reaction (acetyl‐CoA + oxaloacetate + H2O → citrate + CoASH) to 4% but did not affect the hydrolysis of citryl‐CoA. Experimental results indicate that a connecting link between the enzyme's ligase and hydrolase activity becomes impaired specifically on treatment with trypsin. Other proteolytic enzymes like chymotrypsin and subtilisin inactivated catalytic functions of citrate synthase, ligase and hydrolase, equally well.

2. Tryptic hydrolysis occurs at the N‐terminal region of citrate synthase, but a study by SDS/PAGE revealed no difference in molecular mass between native and proteolytically nicked citrate synthase. The peptide removed from the enzyme by trypsin, therefore, contains less than about 15 amino acid residues.

3. The Km values of the substrates for both native and nicked enzyme were identical, as was the state of aggregation (dimeric) of the two enzyme species. These could be separated by affinity chromatography on Blue‐Sepharose and differentiated by their isolelectric points (pI= 6.68 ± 0.08 and pI= 6.37 ± 0.03 for native citrate synthase and the large tryptic peptide, respectively) as well as by the N‐terminus which is blocked in the native enzyme only.

4. Edman degradation of the large tryptic fragment yielded the N‐terminal sequence GLEDVYIKSTSLTYIDGVNGVLRY, which is 71% identical to the N‐terminal region (positions 9–32) of citrate synthase from Thermoplasma acidophilum.

5. The conversion of citrate synthase into essentially a citryl‐CoA hydrolase is considered the consequence of a conformational change thought to occur on tryptic removal of the N‐terminal small peptide.
Detection of cGMP dependent protein kinase isozymes by specific antibodies
Authors: KEILBACH Alexandra , RUTH Peter , HOFMANN Franz
Published: March 2005

Two isozymes of cGMP‐dependent protein kinase (cGMP kinase) have been identified. Polyclonal antibodies were developed which recognize both isozymes or specifically the Iα and Iβ isoform. The specificity of these antibodies was verified by using the recombinant or purified Iα and Iβ isozymes. The antibodies cross‐reacted with the purified isozymes of cGMP kinase from bovine tracheal smooth muscle. The tissue concentration of cGMP kinase was determined by ELISA. High concentrations (> 10 pmol/g wet tissue) were present in bovine lung, rumen, trachea, aorta, uterus and stomach. The tissue distribution of the isozymes Iα and Iβ was investigated by immunoblots using crude extracts of the different tissues. The Iβ‐specific antibody yielded strong signals with extracts of trachea, aorta, stomach and uterus, whereas heart, cerebellum and lung apparently contain mainly the Iα isozyme
Cytochrome P‐450 induction in human lung tumor‐derived cell lines
Authors: STANLEY Lesley A. , CARMICHAEL James , WOLF C. Roland
Published: March 2005

Cytochrome P‐450 species (P‐450) comprise a polymorphic multigene family of heme‐containing enzymes which are essential to the phase‐I metabolism of xenobiotics. Induction of P‐450 species by drugs and carcinogens has been extensively studied; endogenous regulation of P‐450 also occurs during normal development and disease. The aim of this project was to study the in‐vitro induction of P‐450 and its modulation by inflammatory mediators in the human lung tumor‐derived cell lines NCI H322 and NCI H358. The cell lines expressed detectable levels of 7‐ethoxyresorufin O‐deethylase which could be induced by benzanthracene. After benzanthracene treatment, a protein tentatively identified as isozyme CYP1A1 was detected by Western‐blot analysis and a concommitant increase in CYP1A mRNA expression was observed. Optimal induction was observed at a benzanthracene concentration of 5 μg/ml with cells grown in RPMI medium containing 10% fetal calf serum. The effects of endotoxin, dexamethasone and five recombinant DNA‐derived cytokines, interleukin‐1ß, tumor necrosis factor, and interferons α, ß and γ, on constitutive and benzanthracene‐induced ethoxyresorufin O‐deethylase activity were examined in NCI H322 cells. Of all the lymphokines studied, only interferon γ had any marked effect. Administration of this lymphokine strongly suppressed ethoxyresorufin O‐deethylase activity in both control and benzanthracene‐treated cells.
The purification of a detergent‐soluble glucose‐6‐phosphatase from rat liver
Authors: SPETH Maria , SCHULZE Hans‐Ulrich
Published: March 2005

A highly active and soluble glucose‐6‐phosphatase has been purified to near homogeneity from rat liver. Successful purification has been initiated by covalent labeling of the enzyme in native rat liver microsomes with pyridoxal 5′‐phosphate and NaBH4, followed by solubilization of the microsomes with Triton X‐100, chromatography on phenyl‐Sepharose, hydroxyapatite, DEAE‐Sephacel and a second chromatography step on hydroxyapatite. The final enzyme preparation obtained was approximately 700‐fold purified over the activity of starting microsomes. As judged by SDS/PAGE the purified glucose‐6‐phosphatase is composed of a single protein with a molecular mass of 35 kDa. The present work demonstrates that the purified glucose‐6‐phosphatase must be arranged in the native microsomal membrane so that it is accessible to pyridoxal 5′‐phosphate from the cytoplasmic side.
Alkylacyl glycerophosphoinositol in human and bovine erythrocytes
Authors: BÜTIKOFER Peter , ZOLLINGER Markus , BRODBECK Urs
Published: March 2005

Glycosyl‐inositolphospholipid (glycosyl‐PtdIns) anchors of proteins in mammalian cells which have been analyzed so far are exclusively of the alkylacyl type. However, little is known about the putative precursor of glycosyl‐PtdIns, the alkylacyl derivative of glycerophosphoinositol (GroPIns), in these cells since it is generally believed that cellular GroPIns consists of diacyl‐type molecular species only. In this report, we describe the isolation and identification of alkylacyl GroPIns molecular species in both human and bovine erythrocytes, and compare it with the molecular species compositions of the glycosyl‐PtdIns anchors of human and bovine erythrocyte acetylcholinesterase. Diradyl GroPIns was isolated from lipid extracts of ghost membranes and treated with phospholipase C. Diradylglycerols of the glycosyl‐PtdIns anchors of affinity‐purified human and bovine erythrocyte acetylcholinesterase were generated by sequential treatment with glycoprotein phospholipase D and acidic phosphatase and by PtdIns‐specific phospholipase C, respectively. Diradylglycerols were subsequently converted into benzoate derivatives and separated into diacyl, alkylacyl, and alkenylacylglycerol subclasses. The molecular species compositions were quantitated and determined by combined HPLC/mass spectrometry. We found that human and bovine erythrocyte membrane diradyl GroPIns consist of 1.5–4.8% alkylacyl GroPIns. Molecular species analysis showed a heterogeneous species composition for both human and bovine erythrocyte alkylacyl GroPIns. Their compositions are distinctly different from those of human and bovine erythrocyte acetylcholinesterase glycosyl‐PtdIns anchors. The number of alkylacyl GroPIns molecules/cell is roughly equal with the number of glycosyl‐PtdIns‐anchored proteins in human erythrocytes.
Regulation of the activity of ribulose‐1,5‐bisphosphate carboxylase/oxygenase through cooperative binding of 6‐phosphogluconate to its regulatory sites
Authors: YOKOTA Akiho , HIGASHIOKA Masaya , WADANO Akira
Published: March 2005

This study was attempted to elucidate the mechanism of the regulation of the turnover number on the catalytic sites by the regulatory sites of spinach ribulose‐1,5‐bisphosphate carboxylase/oxygenase [Rbu(1,5)P2CO]. To this end, we analyzed the effects of the binding of 6‐phosphogluconate (6‐P‐Gln) to the regulatory sites of the enzyme on the progress in the subsequent catalysis assayed with 0.5 mM ribulose 1,5‐bisphosphate [Rbu(1,5)P2]. This concentration of Rbu(1,5)P2 hardly binds to the regulatory sites but competes with 6‐P‐Gln for the catalytic sites. An equilibrium binding assay showed that Rbu(1,5)P2CO bound 8 mol 6‐P‐Gln/mol enzyme in addition to the catalytic sites. The binding to the eight regulatory sites was positively cooperative. The biphasic reaction course, inherent in the carboxylase reaction of plant Rbu(1,5)P2CO and composed of a burst for an initial few minutes and a subsequent linear phase, became faint with increasing binding of 6‐P‐Gln to the sites. The change of the reaction progress from the biphasic to linear course was ascribed to the suppression of the functioning form of the enzyme from the high‐activity to low‐activity form and to the increased turnover number on the catalytic sites though the binding of 6‐P‐Gln to the regulatory sites.
Characterization of the cytochrome P‐450IID subfamily in bovine liver
Authors: TSUNEOKA Yutaka , MATSUO Yoshinori , HIGUCHI Ryota , ICHIKAWA Yoshiyuki
Published: March 2005

To elucidate the molecular mechanisms underlying drug detoxification, the structures of the members of the microsomal cytochrome P‐450IID subfamily were analyzed by isolating, mapping and sequencing cytochrome P‐450IID (CYP2D) cDNA clones from bovine liver. The screening was performed under nonstringent conditions so that most of the P‐450IID subfamily members could be obtained. 114 of the 147 positive clones were classified into four groups on the basis of their restriction‐enzyme maps. The maps of the four groups were highly similar, however, the clones of one group contained an insertion of approximately 500 bp in the coding region. Analysis of partial nucleotide sequences of several representative clones from each group showed that the bovine P‐450IID subfamily in liver consisted of several, not many, highly similar members, differing by less than 7% in their nucleotide sequences.

The location of the insertion found in the minor group corresponded to intron 7 and the GT/AG rule was found at the exon/intron boundary, suggesting that intron 7 was retained in this group.

The complete nucleotide sequences of two clones from the major group were examined to determine the structures of the P‐450IID subfamily in bovine liver. A full‐length cDNA clone (1615 bp) and a partial cDNA clone (1538 bp) contained open reading frames encoding 500 and 487 amino acid residues, respectively. The partial clone lacked the nucleotide sequence corresponding to the first 13 N‐terminal amino acid residues. The deduced amino acid sequences of the two clones were 98% similar, and 80% and 68% similar to those from human CYP2D6 and rat CYP2D1, respectively. Comparisons of the amino acid sequences of the P‐450IID subfamily members showed the highly conserved C‐terminal region of their molecules and the high similarity between the members in one species, especially in cattle and man.
Induction of H10‐gene expression in B16 murine melanoma cells
Authors: ROUSSEAU Denis , KHOCHBIN Saadi , GORKA Claude , LAWRENCE Jean‐Jacques
Published: March 2005

Histone H10 accumulation is associated with the terminal stage of differentiation. Unlike other H1 histones, it is able to accumulate in the absence of DNA synthesis, however the transcription of its gene is cell‐cycle dependent. The regulation of H10‐gene expression has been studied during the induced differentiation of B16 cells and during reversion of the process, which may be achieved when induced cells are released into an inducing‐agent‐free medium. During the earlier period of induced differentiation, H10 mRNA showed over‐expression when the cells were still proliferating. Then the amount of H10 mRNA decreased as the cells became arrested in G0‐G1. H10 mRNA half‐life measurements and run‐on experiments demonstrated that such modulation of the amount of mRNA originated from a transcriptional control of H10‐gene expression. When induced cells reverted to a proliferative undifferentiated state, H10 mRNA decreased very rapidly, indicating that an active process was involved in this decay. This behavior differed from that observed in rat liver hepatocytes allowed to proliferate and de‐differentiate after partial hepatectomy, or in murine erythroleukemia cells when the inducing agent was removed from the culture.
Biochemical properties of the proteasome from Thermoplasma acidophilum
Authors: DAHLMANN Burkhardt , KUEHN Lothar , GRZIWA Anja , ZWICKL Peter , BAUMEISTER Wolfgang
Published: March 2005

We have purified proteasomes to apparent homogeneity from the archaebacterium Thermoplasma acidophilum. This proteinase has a molecular mass of about 650 kDa and an isoelectric point of 5.6. The proteasome hydrolyses peptide substrates containing an aromatic residue adjacent to the reporter group, as well as [14C]methylated casein optimally at pH 8.5 and 90°C. The enzyme activity is enhanced severalford by Mg2+ and Ca2+ at 25–500 mM. This increase in activity results primarily from a change in Km. The serine‐proteinase inhibitors diisopropylfluorophosphate and 3,4‐dichlorosiocoumarin irreversibly inhibit the enzyme, obviously by modification of both the α and β subunits in the proteasome. The inhibition of proteasomal activity by the peptidylchloromethanes, Cbz‐Leu‐Leu‐CH2Cl and Cbz‐Ala‐Ala‐Phe‐Ch2Cl (Cbz, benzyloxycarbonyl), is reversible and predominantly of a competitive type. The enzyme is not activated by any of the compounds that typically stimulate the activities of the eukaryotic proteasome.
Purification and characterisation of the NADH: acceptor reductase component of xylene monooxygenase encoded by the TOL plasmid pWWO of Pseudomonas putida mt‐2
Authors: SHAW Jeffrey P. , HARAYAMA Shigeaki
Published: March 2005

The xylene monooxygenase system encoded by the TOL plasmid pWWO of Pseudomonas putida catalyses the hydroxylation of a methyl side‐chain of toluene and xylenes. Genetic studies have suggested that this monooxygenase consists of two different proteins, products of the xylA and xylM genes, which function as an electron‐transfer protein and a terminal hydroxylase, respectively. In this study, the electron‐transfer component of xylene monooxygenase, the product of xylA, was purified to homogeneity. Fractions containing the xylA gene product were identified by its NADH: cytochrome c reductase activity. The molecular mass of the enzyme was determined to be 40 kDa by SDS/PAGE, and 42 kDa by gel filtration. The enzyme was found to contain 1 mol/mol of tightly but not covalently bound FAD, as well as 2 mol/mol of non‐haem iron and 2 mol/mol of acid‐labile sulfide, suggesting the presence of two redox centers, one FAD and one [2Fe‐2S] cluster/protein molecule. The oxidised form of the protein had absorbance maxima at 457 nm and 390 nm, with shoulders at 350 nm and 550 nm. These absorbance maxima disappeared upon reduction of the protein by NADH or dithionite. The NADH: acceptor reductase was capable of reducing either one‐ or two‐electron acceptors, such as horse heart cytochrome c or 2,6‐dichloroindophenol, at an optimal pH of 8.5. The reductase was found to have a Km value for NADH of 22 μM. The oxidation of NADH was determined to be stereospecific; the enzyme is pro‐R (class A enzyme). The titration of the reductase with NADH or dithionite yielded three distinct reduced forms of the enzyme: the reduction of the [2Fe‐2S] center occurred with a midpoint redox potential of −171 mV; and the reduction of FAD to FAD˙ (semiquinone form), with a calculated midpoint redox potential of −244 mV. The reduction of FAD˙ to FAD˙˙ (dihydroquinone form), the last stage of the titration, occurred with a midpoint redox potential of −297 mV. The [2Fe‐2S] center could be removed from the protein by treatment with an excess of mersalyl acid. The [2Fe‐2S]‐depleted protein was still reduced by NADH, giving rise to the formation of the anionic flavin semiquinone observed in the native enzyme, thus suggesting that the electron flow was NADH→FAD→[2Fe‐2S] in this reductase. The resulting protein could no longer reduce cytochrome c, but could reduce 2,6‐dichloroindophenol at a reduced rate.
Redox properties of the iron‐sulfur clusters in activated Fe‐hydrogenase from Desulfovibrio vulgaris (Hildenborough)
Authors: PIERIK Antonio J. , HAGEN Wilfred R. , REDEKER Jan S. , WOLBERT Ronnie B. G. , BOERSMA Marelle , VERHAGEN Marc F. J. M. , GRANDE Hans J. , VEEGER Cees , MUTSAERS Peter H. A. , SANDS Richard H. , DUNHAM W. Richard
Published: March 2005

The periplasmic Fe‐hydrogenase from Desulfovibrio vulgaris (Hildenborough) contains three iron‐sulfur prosthetic groups: two putative electron transferring [4Fe‐4S] ferredoxin‐like cubanes (two F‐clusters), and one putative Fe/S supercluster redox catalyst (one H‐cluster). Combined elemental analysis by proton‐induced X‐ray emission, inductively coupled plasma mass spectrometry, instrumental neutron activation analysis, atomic absorption spectroscopy and colorimetry establishes that elements with Z > 21 (except for 12–15 Fe) are present in 0.001–0.1 mol/mol quantities, not correlating with activity. Isoelectric focussing reveals the existence of multiple charge conformers with pI in the range 5.7–6.4. Repeated re‐chromatography results in small amounts of enzyme of very high H2‐production activity determined under standardized conditions (∼ 7000 U/mg). The enzyme exists in two different catalytic forms: as isolated the protein is ‘resting’ and O2‐insensitive; upon reduction the protein becomes active and O2‐sensitive. EPR‐monitored redox titrations have been carried out of both the resting and the activated enzyme. In the course of a reductive titration, the resting protein becomes activated and begins to produce molecular hydrogen at the expense of reduced titrant. Therefore, equilibrium potentials are undefined, and previously reported apparent Em and n values [Patil, D. S., Moura, J. J. G., He, S. H., Teixeira, M, Prickril, B. C., DerVartanian, D. V., Peck, H. D. Jr, LeGall, J. & Huynh, B.‐H. (1988) J. Biol. Chem. 263, 18732–18738] are not thermodynamic quantities. In the activated enzyme an S= 1/2 signal (g= 2.11, 2.05, 2.00; 0.4 spin/ protein molecule), attributed to the oxidized H cluster, exhibits a single reduction potential, Em, 7=–307 mV, just above the onset potential of H2 production. The midpoint potential of the two F clusters (2.0 spins/protein molecule) has been determined either by titrating active enzyme with the H2/H+ couple (E'm=–330 mV) or by dithionite‐titrating a recombinant protein that lacks the Hcluster active site (Em7.5=–340 mV). There is no significant redox interaction between the two F clusters (n ∼ 1).
Comparison of structure of quinone redox site in the mitochondrial cytochrome‐bc1 complex and photosystem II (QB site)
Authors: SAITOH Isao , MIYOSHI Hideto , SHIMIZU Ryo , IWAMURA Hajime
Published: March 2005

A series of nitrophenolic electron‐transport inhibitors (2‐substituted 4,6‐dinitrophenols) of rat liver mitochondrial cytochrome‐bc1 complex and of photosystem II (QB site) of spinach thylakoids was synthesized. The structure/inhibitory‐activity relationship was examined to elucidate differences in the three‐dimensional structure of the quinone redox site in the two systems. These inhibitors occupy the ubiquinone redox site of cytochrome‐bc1 complex competitively with natural ubiquinol, probably at a Qo reaction center. The inhibitory activity tended to increase with the length of the 2‐substituent, which may correspond to the isoprenoid side chain of ubiquinone and plastoquinone, increased in both experimental systems. However, the strict structural requirements of the 2‐substituent for binding to the ubiquinone or plastoquinone redox site were not identical. The alkyl substituents with a branching structure at the α‐position to the benzene ring were favorable for inhibition of the cytochrome‐bc1 complex, but not of photosystem II. Molecular‐orbital calculations indicated that the main chain of 2‐substituents with an α‐branching structure was almost perpendicular to the benzene‐ring plane because of steric congestion between the α‐methyl and phenolic OH groups. The main chain of 2‐substituents without an α‐branching structure was flexible. Molecular‐orbital studies indicated that ubiquinol was most stable when the portion of the isoprenoid side chain adjacent to the quinol ring was perpendicular to the quinol‐ring plane, because of steric congestion by the vicinal OH and methyl groups. The side chain of plastoquinol was flexible because of the lack of a vicinal methyl group. Thus, the difference in the inhibitory activities between the two systems seemed to reflect the difference in the configuration of the isoprenoid side chain of ubiquinone and plastoquinone. These results suggested that the quinone redox site of the cytochrome‐bc1 complex may recognize the configuration of the side chain near the quinone ring in the strict sense, whereas that of photosystem II (QB site) may recognize it in a loose sense.
Phenotypic transformation of normal rat kidney cells by transforming growth factor β is not paralleled by enhanced production of a platelet‐derived growth factor
Authors: van ZOELEN Everardus J. J. , van ROTTERDAM Walter , WARD‐VAN OOSTWAARD Theodora M. J. , FEIJEN Alie
Published: March 2005

Phenotypic transformation of normal rat kidney (NRK) cells requires the concerted action of multiple polypeptide growth factors. Serum‐deprived NRK cells cultured in the presence of epidermal growth factor (EGF) become density‐inhibited at confluence, but they can be restimulated by a number of defined polypeptide growth factors, resulting in phenotypic cellular transformation. Kinetic data show that restimulation by transforming growth factor β (TGF‐β) and retinoic acid is delayed when compared to induction by platelet‐derived growth factor (PDGF), indicating that both TGFβ and retinoic acid may exert their growth‐stimulating action by an indirect mechanism. Northern blot analysis shows that NRK cells express the genes for various polypeptide growth factors, including TGFβ1, PDGF A‐chain and basic fibroblast growth factor, but that the levels of expression are not affected by TGFβ or retinoic acid treatment. NRK cells also secrete low amounts of a PDGF‐like growth factor into their extracellular medium, but the levels of secretion are insufficient to induce mitogenic stimulation and are unaffected by agents inducing phenotypic transformation. In combination with studies on the effects of anti‐PDGF antibodies, it is concluded that phenotypic transformation of NRK cells by TGFβ and retinoic acid is not the result of enhanced production of a PDGF‐like growth factor.
Endocytosis of thyroglobulin is not mediated by mannose‐6‐phosphate receptors in thyrocytes
Authors: LEMANSKY Peter , HERZOG Volker
Published: March 2005

Thyroglobulin, the major secretory product of thyrocytes, is the macromolecular precursor of thyroid hormones. After its synthesis, thyroglobulin follows a complex secretion, storage and recapture pathway to lysosomes. Porcine thyroglobulin was shown to carry the mannose 6‐phosphate‐(Man6P)‐recognition marker on its N‐linked glycans. Since the cation‐independent Man6P receptor could also be found on the apical plasma membrane of porcine thyrocytes, we examined the significance of the Man6P signal for the transport of thyroglobulin. Here, we present data implying that Man6P receptors are not relevant for endocytosis of thyroglobulin in thyrocytes. Instead, we provide evidence for the existence of specific, low‐affinity‐binding sites for thyroglobulin on the apical plasma membrane of thyrocytes responsible for endocytosis of thyroglobulin. Binding studies with intact, polar‐organized porcine thyrocytes grown on collagen‐coated filters revealed cooperative and saturable binding of thyroglobulin to the apical‐plasma‐membrane domain at relatively high concentrations of thyroglobulin (20 μM). These observations show that low‐affinity interactions between thyroglobulin and the apical plasma membrane play a key role in endocytosis of thyroglobulin and hormone formation in the thyroid. The data in this publication have been published as an abstract [Lemansky, P. and Herzog, V. (1991) J. Cell Biol. 115, 261a].
Effects of nutrients and hormones on gene expression of ATP citrate‐lyase in rat liver
Authors: FUKUDA Hitomi , KATSURADA Akihiko , IRITANI Nobuko
Published: March 2005

Northern‐blot analyses demonstrated a strong gene expression of ATp citrate‐lyase in liver and adipose tissue of rat and a weak expression in brain, heart, small intestine and muscle. After refeeding a carbohydrate/protein diet to fasted rats, the transcriptional rate had already increased within 2 h, the mRNA concentration reached a maximal level of approximately 30‐fold increased in 16 h, and the enzyme induction increased sixfold in 48 h. By feeding only carbohydrate without protein, the transcriptional rate was increased threefold, and the mRNA concentration and enzyme induction comparably, to the levels in the carbohydrate/protein diet. It appears that protein feeding is not necessary to induce ATP citrate‐lyase. In diabetic rats fed on a glucose diet, the transcriptional rate, mRNA concentration and enzyme level were very low in comparison with the normal. By fructose feeding, however, the transcriptional rate was more greatly increased and the mRNA concentration increased comparably to the levels reached by insulin treatment, while the enzyme induction was not so increased. Thus, it is suggested that insulin is important in regulated translation in addition to transcription. However, triiodothyronine treatment did not have much effect on the gene expression. As a result of the present experiment, it is noted that ATP citrate‐lyase‐gene expression was greatly dependent on carbohydrate.