bluuebunny/crossref_metadata_2025 huggingface dataset:
Molecular cloning of cDNA for human complement component C1s
Authors: MACKINNON Charlotte M. , CARTER Philip E. , SMYTH S. Jane , DUNBAR Bryan , FOTHERGILL John E.
Published: March 2005

The complete amino acid sequence (673 residues plus 15 residues of leader sequence) of human complement component C1s has been determined by nucleotide sequencing of cDNA clones from a human liver library probed with synthetic oligonucleotides. Much of the sequence is supported by independent amino acid sequence information. The cDNA sequence contains an anomalous “intron‐like” sequence, including a stop codon, that can be discounted because of the amino acid sequence evidence. The N‐terminal chain (422 residues) of C1s, like that of C1r with which it is broadly homologous, contains five domains: domains I and III are homologous to one another and to similar regions in C1r, domain II is homologous to the epidermal growth factor sequence found in C1r and several other proteins, and domains IV and V are homologous to one another and to the 60‐residue repeating sequence found in C1r, C2, factor B, C4‐binding protein and some apparently unrelated proteins. The sequence of the C‐terminal chain (251 residues) agrees with that already established to be the “serine protease” domain of C1s.
Physico‐chemical studies of micelle formation on sepia cartilage collagen solutions in acetate buffer and its interaction with ionic and nonionic micelles
Authors: MANDAL Asit B. , RAMESH D. Vijaya , DHAR Susil C.
Published: March 2005

Sepia cartilage collagen (pepsin‐extracted) in acetate buffer (pH = 2.98) forms micelles at a particular concentration below which they do not normally form. The critical micelle concentration (cmc) of the collagen was determined in buffer as well as in SDS, cetyltrimethylammonium bromide (CTAB) and Tween‐80 micellar environments at different temperatures. Mutual interaction of collagen micelles with the ionic and nonionic micelles through the formation of the mixed micelle concept has also been found. The cmc of collagen decreased in the presence of SDS and Tween‐80 micelles whereas it increased in the presence of CTAB micelles. This clearly suggests that the micelle formation of collagen is facilitated by the presence of SDS and Tween‐80 and hindered by CTAB micelles. The various thermodynamic parameters were estimated from viscosity measurements and the transfer of collagen into the micelles of various surfactants and the reverse phenomenon was analyzed. This analysis has also been modelled conceptually as a different phase and the results have supported the above phenomenon. Our thermodynamic results are also able to predict the exact denaturation temperature as well as the structural order of water in the collagen in various environments. The hydrated volumes, Vh, of collagen in the above environments and intrinsic viscosity were also calculated. The low intrinsic viscosity, [η], of collagen in an SDS environment compared to buffer and other surfactant environments suggested more workable systems in cosmetic and dermatological skin care preparations. The one and two‐hydrogen‐bonded models of this collagen in various environments have been analyzed. The calculated thermodynamic parameters varied with the concentration of collagen. The change of thermodynamic parameters from coil‐coil to random‐coil conformation upon denaturation of collagen were calculated from the amount of proline and hydroxyproline residues and compared with viscometric results. Thermodynamic results suggest that the stability of the collagen in the additive environments is in the following order: SDS > Tween‐80 > buffer > CTAB.
A new type of prosome‐like particle, composed of small cytoplasmic RNA and multimers of a 21‐kDa protein, inhibits protein synthesisin vitro
Authors: AKHAYAT Omar , INFANTE Anthony A. , INFANTE Dzintra , MARTINS de SA Cezar , GROSSI de SA M.‐Fatima , SCHERRER Klaus
Published: March 2005

A large fraction of the translationally repressed non‐globin messenger RNA in duck erythroblasts is present in non‐polyribosomal free mRNP structures which sediment in the 30–40‐S range (‘35 S’). In 0.5 M KCl, they form core complexes which show a pronounced peak at about 32 S containing mRNA and a discrete spherical RNP particle with a diameter of about 12 nm and the typical morphology of a prosome [H.‐P. Schmid et al. (1984)EMBO J. 3, 29–34]. Buoyant density measurements and chromatography on oligo(dT)‐cellulose indicate that this particle is bound to mRNA; it can be released from the mRNA by treatment of the free mRNP fraction with SDS.

This prosome‐like particle inhibits the translation of mRNAin vitro.It is composed primarily of multimers of a single 21‐kDa protein and at least one species of RNA of about 80–100 nucleotides. It is resistant to dissociation by 2 M Cs2SO4and 1% SDS; the 21‐kDa protein is not attacked by proteinase K unless the particle is extracted with phenol prior to treatment with the protease. The small RNA moiety of the particle hybridizes to the poly(A)‐rich mRNA derived from the free mRNPs, as well as to polyribosomal mRNA.

These data indicate that prosomes may serve to regulate mRNA translation; they show furthermore that prosome‐like particles (about 600 kDa mass) may be built of up to 25 molecules of a single specific protein, rather than of the entire set of about 20 prosomal proteins previously indentified.
Isolation and characterization of three different carbohydrate chains from melanoma tissue plasminogen activator
Authors: POHL Gunnar , KENNE Lennart , NILSSON Bo , EINARSSON Monica
Published: March 2005

Electrophoretic analysis of endoglycosidase‐treated tissue plasminogen activator obtained from human melanoma cells showed that the heterogeneity observed for the protein in these preparations is caused by an N‐glycosidically linked N‐acetyllactosamine type of carbohydrate chain which is present in about 50% of the molecules. An oligomannose type and an N‐acetyllactosamine type of glycan is present in all molecules.

Three glycopeptides were isolated and characterized by 1H‐NMR, sugar determination, methylation analysis and amino acid determination. The exact attachment site for each of the three glycans could be deduced from the amino acid compositions of the glycopeptides. Asn‐117 carries the oligomannose type of glycan, the structure of which was completely determined. Asn‐184 is the site where the presence or absence of a biantennary N‐acetyllactosamine type of glycan causes the size heterogeneity. The third N‐glycosylation site, Asn‐448, was found to carry a triantennary or tetraantennary N‐acetyllactosamine type of carbohydrate chain.
ω‐Oxidation of cysteine‐containing leukotrienes by rat‐liver microsomes
Authors: ÖRNING Lars
Published: March 2005

Leukotriene E4 was metabolized to two polar products by rat liver microsomes. These products were characterized by physico‐chemical and chemical techniques. The chemical structures, (5S, 6R)‐5,20‐dihydroxy‐6S‐cysteinyl‐7,9‐trans‐11,14‐cis‐icosatetraenoic acid (ω‐hydroxy‐leukotriene E4) and (5S, 6R)‐5‐hydroxy‐6S‐cysteinyl‐7,9‐trans‐11,14‐cis‐icosatetraen‐1,20‐dioic acid (ω‐carboxy‐leukotriene E4) suggested that leukotriene E4 was transformed by an ω‐hydroxylase and ω‐hydroxyleukotriene E dehydrogenase in sequence. N‐Acetyl‐leukotriene E4 was also transformed by these enzymes, but at a rate six times lower than leukotriene E4. The products formed from N‐acetylleukotriene E4 were characterized as being N‐acetyl‐ω‐hydroxy‐leukotriene E4 and N‐acetyl‐ω‐carboxy‐leukotriene E4. Other substrates were 11‐trans‐leukotriene E4 and N‐acetyl‐11‐trans‐leukotriene E4. In contrast, leukotrienes C4 and D4 were not converted into ω‐oxidized metabolites. The leukotriene E ω‐hydroxylase reaction required NADPH and molecular oxygen as cofactors, and was most rapidly catalyzed by liver microsomes. Liver cytosol, fortified with NAD+, converted ω‐hydroxyleukotriene E4 and N‐acetyl‐ω‐hydroxy‐leukotriene E4 into ω‐carboxy metabolites. Microsomes contained at least 18 times less ω‐hydroxy‐leukotriene E dehydrogenase activity than did cytosol. Liver microsomes supplemented with acetyl‐coenzyme A converted ω‐hydroxy and ω‐carboxy‐leukotriene E4 into the corresponding N‐acetyl derivatives. The novel enzyme, leukotriene E ω‐hydroxylase, which is described here is distinct from a previously described leukotriene B ω‐hydroxylase based on substrate competition and kinetic data.
Increased efficiency of translation of ornithine decarboxylase mRNA in mitogen‐activated lymphocytes
Authors: WHITE Michael W. , KAMEJI Takaaki , PEGG Anthony E. , MORRIS David R.
Published: March 2005

Ornithine decarboxylase (ODC) mRNA was elevated ninefold by 6 h following concanavalin A (ConA) stimulation of bovine lymphocytes. Comparison of the increases in ODC mRNA and ODC activity revealed a fivefold discrepancy, which is consistent with a change in efficiency of translation of ODC mRNA. In resting cells, 45% of the total ODC mRNA was associated with particles sedimenting at about 40 S, and therefore was not translated. The untranslated ODC mRNA in resting cells could be completely shifted into polysomes by a 15‐min treatment of the cells with appropriate concentrations of cycloheximide. In activated cells, the proportion of ODC mRNA in untranslated material was reduced to 18%. This shift in distribution of ODC mRNA occurred between 6 h and 12 h following mitogen stimulation with no increase in the cellular level of this message. The rate of synthesis of ODC protein was found in increase twofold between 6 h and 12 h, paralleling the increase in the amount of ODC mRNA associated with polysomes. Thus, in this time frame, a decrease in the amount of untranslated ODC mRNA with a corresponding increase in the amount associated with polysomes leads to an increase in the biosynthesis of ODC with no change in the cellular level of the message. These changes in translational efficiency were not observed with actin mRNA.
Catalase gene of the yeast Candida tropicalis
Authors: OKADA Hirofumi , UEDA Mitsuyoshi , SUGAYA Takeshi , ATOMI Haruyuki , MOZAFFAR Sabiha , HISHIDA Tadashi , TERANISHI Yutaka , OKAZAKI Koei , TAKECHI Takanori , KAMIRYO Tatsuyuki , TANAKA Atsuo
Published: March 2005

A clone harbouring the genomic DNA sequence for the peroxisomal catalase of an n‐alkane‐utilizable yeast, Candida tropicalis, has been isolated by the hybrid‐selection method and confirmed with a probe of catalase partial cDNA. Nucleotide sequence analysis of the cloned DNA disclosed that the gene fragment coding for catalase had a length of 1455 base pairs (corresponding to 485 amino acids; m= 54937 Da), and that the size of this enzyme was the smallest among all catalases reported hitherto. No intervening sequence was found in this coding region and some portions coincided with the amino acid sequences obtained from the analysis of the purified catalase. The comparison with three peroxisomal catalases from rat liver, bovine liver and human kidney, and one cytosolic catalase from Saccharomyces cerevisiae has revealed that catalase from C. tropicalis was more homologous to the peroxisomal enzymes than to the cytosolic one. C. tropicalis used the codons of the high‐expression type. Amino acid residues were all conserved at the active and heme‐binding sites. In the N and C‐terminal regions there was no characteristic signal sequence or consensus sequence. However, a noticeable region, which can be discriminated between peroxisomal and cytosolic catalases, was proposed.
Structural relationship between α1‐microglobulin from man, guinea‐pig, rat and rabbit
Authors: ÅKERSTRÖM Bo , LÖGDBERG Lennart , BABIKER‐MOHAMED Hassabo , LOHMANDER Stefan , RASK Lars
Published: March 2005

Rabbit α1‐microglobulin was purified from the urine of sodium‐chromate‐treated animals by the use of gel chromatography on Sephadex G‐100, affinity chromatography on concanavalin‐A – Sepharose and ion‐exchange chromatography on DEAE‐Sephadex. Rabbit α1‐microglobulin had a molecular mass of 25.6 kDa on SDS/polyacrylamide gel electrophoresis. α1‐microglobulin has previously been purified from the urine of humans, guinea‐pigs and rats by similar methods, and the molecular masses of the four homologues were compared by SDS/polyacrylamide gel electrophoresis and gel chromatography in a denaturing medium. By these two methods the human homologue was 6 kDa and 3 kDa larger, respectively, than the other three proteins. Endoglycosidase F digestion of α1‐microglobulin, followed by SDS/polyacrylamide gel electrophoresis, revealed three protein bands in the human α1‐microglobulin sample, and only two bands in guinea‐pig, rat and rabbit α1‐microglobulin, with a gap between each band of 2.6–2.9 kDa. The amino‐terminal amino acid sequences of the four homologues were determined and between 72% and 81% homology was seen. The five amino‐terminal amino acids present in the other species were missing in guinea‐pig α1‐microglobulin. Our results indicate that human α1‐microglobulin is substituted with two N‐linked oligosaccharides, while only one is attached to each of the other α1‐microglobulins, and that the extra glycosylamine‐linked oligosaccharide in the human protein is attached to asparagine in position 17. Finally it is shown that all four homologues inhibit antigen stimulation of human lymphocytes, a finding which is consistent with our previous suggestion that the N‐linked oligosaccharides carry the immunosuppressive activity of α1‐microglobulin.
Human 27‐kDa calbindin complementary DNA sequence
Authors: PARMENTIER Marc , LAWSON David Erik Millar , VASSART Gilbert
Published: March 2005

Human 27‐kDa calbindin cDNA clones were selected by antibody screening from λgt11 brain libraries. The sequence revealed an open reading frame coding for a protein of 261 amino acids, containing four active calcium‐binding domains, and two modified domains that had presumably lost their calcium‐binding capability. Comparison with chick and bovine calbindins showed that the protein was highly conserved in evolution (evolutionary rate: 0.3 × 10−9 amino acid−1 year−1) and that active and inactive domains were equally conserved. From the data we postulate that calbindin has an important physiological function involving protein–protein interactions. Comparison of calcium‐binding domains from various proteins suggested that all members of the troponin C superfamily derive from a common two‐domained ancestor, but that duplications leading to calbindin and to the four‐domained calcium‐binding proteins took place independently on different branches of the evolutionary tree. Preliminary data showed that another calcium‐binding protein, homologous to calbindin, is present in the brain and encoded by a different gene.
An NMR study of polymorphous behaviour of the mismatched DNA octamer d(m5C‐G‐m5C‐G‐A‐G‐m5C‐G) in solution
Authors: ORBONS Leonard P. M. , van der MAREL Gijs A. , van BOOM Jacques H. , ALTONA Cornelis
Published: March 2005

By means of one‐ and two‐dimensional NMR spectroscopy the solution structures of the partly self‐complementary octamer d(m5C‐G‐m5C‐G‐A‐G‐m5C‐G) were investigated. It is shown that this DNA fragment, under conditions of high DNA concentration (8 mM DNA) and/or high ionic strength prefers to adopt a duplex structure. At low DNA concentration (0.4 mM DNA), the duplex exists in a 1:1 slow equilibrium with a monomeric hairpin form. Addition of salt destabilizes the hairpin structure in favour of the dimer. At high temperatures the hairpin form, as well as the dimer structure, exist in a fast equilibrium with the random‐coil form. For the hairpin/random‐coil equilibrium a Tm of 329 K and a ΔH° of ‐121 kJ · mol−1 were deduced. These thermodynamic parameters are independent of the DNA concentration, as is expected for a monomeric structure. For the dimer to coil transition a Tm of 359 K(1 M DNA) and a ΔH° of ‐285 kJ · mol duplex−1 were derived. The thermodynamic data of the hairpin‐coil transition mutually agree with those recently reported for the hairpin to random coil equilibrium of the DNA octamer d(m5C‐G‐m5C‐G‐T‐G‐m5C‐G) [Orbons, L. P. M., van der Marel, G. A., van Boom, J. H. & Altona, C. (1987) J. Biomol. Struct. Dyns. 4, 939–963].

It is demonstrated that the dimer structure exhibits B‐DNA characteristics, as is witnessed by the NOESY experiments and the analysis of the proton‐proton coupling data. It is shown that the base‐pair formation of the G × A mismatches is anti‐anti. A comparison of 1H and 31P chemical‐shift data of the title compound with those of a well‐characterized B‐DNA structure reveals large differences in the dm5C(3)‐dG(4)‐dA(5) part of the mismatched dimer structure. These differences apparently indicate some major local structural changes due to the incorporation of the G × A mismatches.

Under the most extreme conditions used (i.e. up to 3 M NaCl or 75% CH3OH in the presence of 10 mM MgCl2) no Z‐DNA structure was observed.

It is shown that the structural features of the hairpin form of the title compound mimic those of the hairpin structure of d(m5C‐G‐m5C‐G‐T‐G‐m5C‐G). An energy‐minimized model of the hairpin form is given.
The nucleotide sequence of the nifM gene of Klebsiella pneumoniae and identification of a new nif gene: nifZ
Authors: PAUL Wyatt , MERRICK Mike
Published: March 2005

A 1.4‐kb PstI–HpaI DNA fragment carrying the Klebsiella pneumoniae nifM gene has been sequenced; nifM has been shown to encode a 30.6‐kDa polypeptide. Two other open‐reading frames were identified upstream of nifM. The one immediately upstream of nifM encodes a 16.6‐kDa polypeptide which has been identified by in vitro transcription/translation in an Escherichia coli 30000 × g supernatant system; we propose to designate this gene nifZ. The sequence of the second open reading frame is incomplete but it does not correspond to nifV, the gene previously thought to be immediately upstream of nifM, and may therefore identify another new nif gene. Both nifM and nifZ have functional nif promoters with the characteristic ‐24, ‐12 consensus sequence, we find no evidence for a nifM upstream activator sequence. The role of nifZ in nitrogenase biosynthesis is unknown but its identification calls into question previous assertions that only nifM and nifH are required for the synthesis of nitrogenase Fe protein.
Complexes of DNA hairpins and a single‐stranded oligonucleotide detected by affinity chromatography and mung bean nuclease cleavage
Authors: BAUMANN Ulrich , LEHMANN Urs , SCHWELLNUS Konrad , van BOOM Jaques H. , KUHN Hans
Published: March 2005

The emergence of a simple translation device consisting of an assembler strand (primordial mRNA) and RNA hairpins (primordial tRNA) is presumed to be an important step leading to the origin of life. The assumption of a non‐enzymatic interaction of primordial tRNA and mRNA is experimentally approached. DNA hairpins containing five or more adenosine residues in the loop are able to bind to complementary oligonucleotides covalently bound to cellulose. The exact number of base pairs formed between the hairpins and the assembler strand is determined by two methods applied to DNA hairpin/assembler complexes. The melting temperature of a complex is measured and the cleavage pattern by nuclease from mung bean is determined. The loop of the smallest hairpin able to bind consists of five adenosine residues and only three base pairs are formed. This supports the idea of a primordial recognition similar to the contemporary codon‐anticodon interaction.
Phenol hydroxylase from yeast
Authors: SEJLITZ Torsten , NEUJAHR Halina Y.
Published: March 2005

The binding of phenol to phenol hydroxylase was studied by equilibrium dialysis, spectrophotometric titration and by steady‐state kinetics. A binding model with two identical, negatively cooperative, effector/substrate‐binding sites per enzyme dimer is proposed. The spectral perturbation caused by phenol and the kinetics of the overall reaction were analysed with relation to the enzyme‐phenol complexes of the binding model. The main part of the spectral perturbation as well as of the increase in NADPH oxidation rate was achieved by one molecule of phenol bound per enzyme dimer. The properties of different enzyme‐phenol complexes, in terms of spectral changes, hydroxylase activity, oxidase activity and substrate inhibition are discussed. A new purification procedure is described.
Characterization and partial purification of a novel mannosylphosphodolichol phosphodiesterase from chicken liver microsomes
Authors: TOMITA Yoshio , MOTOKAWA Yutaro
Published: March 2005

Mannosylphosphodolichol phosphodiesterase, which catalyzes the release of mannose from mannosylphosphodolichol, was solubilized from chicken liver microsomes by treatment with the non‐ionic detergent, Emulgen 909. The enzyme was partially purified using ammonium sulfate precipitation, DEAE‐cellulose chromatography, and gel filtration on Sepharose 6B. The enzyme showed absolute requirement for sulfhydryl reducing agents. The enzyme activity was stimulated by the addition of CaCl2 and Emulgen 909 and exhibited a pH optimum around 5.3. The Km value for mannosylphosphodolichol was found to be 0.43 μM. The activity was competitively inhibited by dolichyl phosphate and dolichol and the Ki value for dolichyl phosphate was estimated to be 12.5 μM. The purified preparation had no activity toward N‐acetylglucosaminyldiphosphodolichol, glucosylphosphodolichol, mannose 1‐phosphate, or artificial substrates for mannosidases, glucosidases, acid phosphatase, and acid phosphodiesterase.

A heat‐stable factor which stabilizes the mannosylphosphodolichol phosphodiesterase was separated from the enzyme by DEAE‐cellulose chromatography. It was precipitated by trichloroacetic acid and not extracted into lipid solvents. The separation resulted in the complete loss of the enzyme activity and the restoration of the activity was not observed when the factor was added back to the enzyme solution.
Binding of alkaline‐earth metal cations and some anions to hosphatidylcholine liposomes
Authors: TATULIAN Suren A.
Published: March 2005

The dependence of electrophoretic mobility of multilamellar liposomes composed of egg phosphatidylcholine (PtdCho), dimyristoyl‐glycerophosphocholine (Myr2Gro‐P‐Cho) and dipalmitoyl‐glycerophosphocholine (Pam2‐Gro‐P‐Cho) on the concentration of several cations and anions has been measured. Values of surface densities of binding sites and intrinsic binding constants of ions to liposome membranes were determined by processing the results in the framework of Gouy‐Stern theory. Sharp reductions in the positive surface potential of Myr2Gro‐P‐Cho and Pam2Gro‐P‐Cho liposomes have been detected at the thermotropic transition of the lipids from the gel to liquid‐crystalline phase. Similar alterations of liposome surface potential were revealed at the temperature of pretransition, as well as at about 50°C, in the case of Pam2Gro‐P‐Cho. A model is suggested for ion binding to PtdCho membranes, according to which the ion‐binding sites are considered as point defects (vacancies) in the structure of lipid head‐groups arranged over a trigonal lattice.
Effect of phospholipid, detergent and protein‐protein interaction on stability and phosphoenzyme isomerization of soluble sarcoplasmic reticulum Ca‐ATPase
Authors: VILSEN Bente , ANDERSEN Jens P.
Published: March 2005

The purpose of the present study was to elucidate the separate roles of lipid, detergent and protein‐protein interaction for stability and catalytic properties of sarcoplasmic reticulum Ca‐ATPase solubilized in the non‐ionic detergent octa(ethylene glycol) monododecyl ether (C12E8). The use of large‐zone high‐performance liquid chromatography permitted us to define the self‐association state of Ca‐ATPase peptide at various detergent, phospholipid and protein concentrations, and also during enzymatic turnover with ATP. Conditions were established for monomerization of Ca‐ATPase in the presence of a high concentration of phospholipid relative to detergent. The lipid‐saturated monomeric preparation was relatively resistant to inactivation in the absence of Ca2+, whereas delipidated enzyme in monomeric or in oligomeric form was prone to inactivation.

Kinetics of phosphoenzyme turnover were examined in the presence and absence of Mg2+. Dephosphorylation rates were sensitive to Mg2+, irrespective of whether the peptide was present in soluble monomeric form or was membrane‐bound. C12E8‐solubilized monomer without added phospholipid was, however, characterized by a fast initial phase of dephosphorylation in the absence of Mg2+. This was not observed with monomer saturated with phospholipid or with monomer solubilized in myristoylglycerophosphocholine or deoxycholate. The mechanism underlying this difference was shown to be a C12E8‐induced acceleration of conversion of ADP‐sensitive phosphoenzyme (E1P) to ADP‐insensitive phosphoenzyme (E2P).

The phosphoenzyme isomerization rate was also found to be enhanced by low‐affinity binding of ATP, This was demonstrated both in membrane‐bound and in soluble monomeric Ca‐ATPase.

Our results indicate that a single peptide chain constitutes the target for modulation of phosphoenzyme turnover by Mg2+ and ATP, and that detergent effects, distinct from those arising from disruption of protein‐protein contacts, are the major determinants of kinetic differences between C12E8‐solubilized and membrane‐bound enzyme preparations.
Fructose metabolism via the pentose cycle in tumoral islet cells
Authors: SENER Abdullah , MALAISSE‐LAGAE Francine , MALAISSE Willy J.
Published: March 2005

In tumoral islet cells (RINm5F line) the phosphorylation of d‐fructose is catalyzed by hexokinase rather than fructokinase. Fructose 6‐phosphate appears to be preferentially channelled into the pentose cycle, as suggested by a ratio of d‐[1‐14C]fructose/d‐[U‐14C]fructose oxidation close to 2.7, the failure to generate 14C‐labelled lactate from d‐[1‐14C]fructose and a poor metabolic response to menadione. When the islet cells are exposed to both d‐fructose and d‐glucose, however, the metabolism of the former hexose is dramatically modified, fructose 6‐phosphate being now formed at a lower rate and preferentially channelled into the glycolytic pathway. These findings illustrate the existence of regulatory steps in fructose catabolism located distally to its site of phosphorylation.
Extracellular origin of the lipid lysosomal storage in cultured fibroblasts from Wolman's disease
Authors: SALVAYRE Robert , NEGRE Anne , MARET Arlette , RADOM Jean , DOUSTE‐BLAZY Louis
Published: March 2005

The experiments reported here allowed us to compare the metabolism of neutral lipids from extracellular origin (lipoproteins) and endogenous origin (triacylglycerol biosynthesis induced by feeding cells with high levels of free fatty acid) in normal and acid‐lipase‐deficient fibroblasts (Wolman's disease). When the cells were grown in hyperlipemic‐rich medium, a major neutral lipid storage appeared in normal as well as in acid‐lipase‐deficient cells; this storage disappeared rapidly in normal cells during the ‘chase’, whereas in Wolman cells, the storage of cholesteryl esters and triacylglycerols remained unchanged, or only decreased very slowly. When the cells were fed with high levels of radiolabelled oleic acid, a major accumulation of radiolabelled triacylglycerols was observed. These cytoplasmic triacylglycerols were similarly degraded in normal and Wolman fibroblasts during the ‘chase’ period. From these results it was concluded that the neutral lipids stored in lysosomes of Wolman fibroblasts are only of extracellular origin (lipoproteins), whereas triacylglycerols biosynthesized by the cells do not participate in this accumulation. Therefore, both cellular compartments involved in triacylglycerol metabolism (lysosomes containing exogenous lipids and cytoplasmic granules of endogenously biosynthesized triacylglycerols) are strictly independent.
Effects of insulin and norepinephrine on glucose transport and metabolism in rat brown adipocytes
Authors: EBNER Sylvie , BURNOL Anne‐Françoise , FERRE Pascal , de SAINTAURIN Marie‐Alice , GIRARD Jean
Published: March 2005

Glucose is an important fuel for rat brown adipose tissue in vivo and its utilization is highly sensitive to insulin. In this study, the different glucose metabolic pathways and their regulation by insulin and norepinephrine were examined in isolated rat brown adipocytes, using [6‐14C]glucose as a tracer. Glucose utilization was stimulated for insulin concentrations in the range of 40–1000 μU/ml. Furthermore, the addition of adenosine deaminase (200 mU/ml) or adenosine (10 μM) did not alter insulin sensitivity of glucose metabolism. The major effect of insulin (1 mU/ml) was a respective 7‐fold and 5‐fold stimulation of lipogenesis and lactate synthesis, whereas glucose oxidation remained very low. The 5‐fold stimulation of total glucose metabolism by 1 mU/ml of insulin was accompanied by an 8‐fold increase in glucose transport. In the presence of norepinephrine (8 μM), total glucose metabolism was increased 2‐fold. This was linked to a 7‐fold increase of glucose oxidation, whereas lipogenesis was greatly inhibited (by 72%). In addition, norepinephrine alone did not modify glucose transport. The addition of insulin to adipocytes incubated with norepinephrine, induced a potentiation of glucose oxidation, while lipogenesis remained very low. In conclusion, in the presence of insulin and norepinephrine glucose is a oxidative substrate for brown adipose tissue. However the quantitative importance of glucose as oxidative fuel remains to be determined.
Human factor VIII from heparinized plasma
Authors: GANZ Peter R. , TACKABERRY Eilleen S. , PALMER Douglas S. , ROCK Gail
Published: March 2005

Human factor VIII was purified from heparinized blood by cryoprecipitation, poly(ethyleneglycol) precipitation, Affi‐Gel blue, aminohexyl, polyelectrolyte E5 and immunoaffinity chromatography. A purification of 280000‐fold over plasma with a specific activity over 5300 units/mg was achieved. Analyses of factor VIII using HPLC indicated a molecular mass of 280–340 kDa. Variation in the native mass may reflect heterogeneity of the protein due to associated lipid since structural analysis confirmed that factor VIII contained variable amounts of free fatty acids and diglycerides and triglycerides, but no phospholipids. Additional characterization by denaturing polyacrylamide gel electrophoresis under reducing conditions, followed by silver staining, showed a major single‐chain polypeptide of factor VIII with a mass of approximately 260 kDa. To determine whether proteolyzed forms of factor VIII were present during fractionation, we analysed earlier steps in purification. This revealed additional species of factor VIII eluting faster than the single‐chain form during chromatography on polyelectrolyte E5. Gel electrophoresis showed that these species of factor VIII consisted of multiple polypeptide chains, and partial peptide mapping using Staphylococcus aureus V8 protease indicated that they were structurally related. Monoclonal and hemophilic antibodies were used in immunoadsorption experiments to demonstrate that the purified factor VIII was composed predominantly of the 260‐kDa factor VIII chain.
Two‐dimensional 1H‐NMR studies of the solution structure of plasminogen kringle 4
Authors: MABBUTT Bridget C. , WILLIAMS Robert J. P.
Published: March 2005

Native kringle 4 from human plasminogen has been studied by two‐dimensional 1H‐NMR methods in order to obtain new structural information about the kringle fold. Two‐dimensional scalar correlated spectroscopy (COSY), two‐dimensional dipolar correlated spectroscopy (NOESY) and two‐dimensional relayed coherance transfer spectroscopy (RCT) experiments were recorded, allowing most resonances arising from the aromatic and methyl‐containing residues to be assigned in the spectrum. From an analysis of NOE data, a small segment of double‐stranded β‐sheet has been identified near residues Phe63 and Thr64. Further analysis of the NOESY spectrum has allowed detailed study of the conformation of sidechains located in regions near Leu45 and Val69. A model has been constructed of the polypeptide segment comprising residues 40–49 which accounts for the observed NOE interactions.
The primary structure of the basic isoform of Acanthamoeba profilin
Authors: AMPE Christophe , SATO Masahiko , POLLARD Thomas D. , VANDEKERCKHOVE Joël
Published: March 2005

Acanthamoeba profilin‐II [Kaiser, D. A., Sato, M., Ebert, R. F. and Pollard, T. D. (1986) J. Cell. Biol. 102, 221–226] was digested with trypsin or cleaved by 2‐(2‐nitrophenylsulphenyl)‐3‐methyl‐3‐bromoindolenine. The tryptic peptides were purified by reversed‐phase‐high‐performance liquid chromatography and completely sequenced using automated gas‐phase sequence analysis. The complete profilin‐II sequence was deduced by ordering the tryptic peptides using the sequence information of the tryptophan‐cleavage products. Acanthamoeha profilin‐II was found to be homologous to the previously determined profilin‐I sequence [Ampe, C., Vandekerckhove, J., Brenner, L., Tobacman, L. and Korn, E. D. (1985) J. Biol. Chem. 260, 834–8401. Like profilin‐I, profilin‐II consists of 125 amino acids, has a blocked NH2 terminus and a trimethyllysine residue at position 103. Profilin‐II differs in at least 21 positions from one of the profilin‐I isoforms. The amino acid exchanges are mainly concentrated in the middle part of the sequence. Profilin‐II contains two more basic residues than profilin‐I, which explains its higher isoelectric point.
Ribonucleotide reductase of Brevibacterium ammoniagenes is a manganese enzyme
Authors: WILLING Andreas , FOLLMANN Hartmut , AULING Georg
Published: March 2005

Ribonucleotide reduction and not DNA replication is the site for the specific manganese requirement of DNA synthesis and cell growth in the coryneform bacterium Brevibacterium ammoniagenes. To characterize the metal effect we have isolated and purified ribonucleoside‐diphosphate reductase from overproducing bacteria that were first deprived of and then reactivated by manganese ions. Purification on columns of Sephacryl S400, DEAE‐cellulose and hydroxyapatite provided an apparently homogeneous enzyme consisting of two protein subunits. These were characterized by affinity chromatography on 2′,5′‐ADP‐Sepharose as nucleotide‐binding protein B1 (Mr= 80000) and catalytic protein B2 (M r= 100000, composed of two Mr= 50000 polypeptides), which were both necessary for activity.

In vitro the purified enzyme does not require added metal ions except for an unspecific, twofold activity increase observed in the presence of Mg2+ and other divalent cations. Enzyme activity is inhibited by hydroxyurea (I50= 2.5 mM). The electronic spectrum with maxima around 455 nm and 485 nm closely resembles that of manganese(III)‐containing pseudocatalase and of oxo‐bridged binuclear Mn(III) model complexes. Denaturation of the enzyme in trichloroacetic acid liberated an equimolar amount of Mn(II) which was detected by EPR spectroscopy. It was not possible to remove and reintroduce metal ions without loss of enzyme activity.

Manganese‐deficient cell cultures were also grown in the presence of 54MnCl2. Ribonucleotide reductase activity and radioactivity cochromatographed in several systems. Non‐denaturing polyacrylamide gel electro‐phoresis showed that protein subunit B2 was specifically 54Mn‐labeled.

All these properties suggest that the ribonucleotide reductase of B. ammoniagenes is a manganese‐containing analog of the non‐heme‐iron‐containing reductases of Escherichia coli and eukaryotes.
Influence of the protonation degree on the self‐association properties of adenosine 5′‐triphosphate (ATP)
Authors: TRIBOLET Roger , SIGEL Helmut
Published: March 2005

The concentration dependence of the chemical shifts for the protons H‐2, H‐8 and H‐1′ of ATP has been measured in D2O at 27°C under several degrees of protonation in the pD range from 1.5 to 8.4. The results at pD > 4.5 are consistent with the isodesmic model of indefinite noncooperative stacking, while those at pD < 4.5 indicate a preference for the formation of dimeric stacks. The stacking tendency follows the series, ATP4− (K= 1.3 M−1) < D(ATP)3− (2.1 M−1) < 1:1 ratio of D(ATP)3−/D2(ATP)2− (6.0 M−1) ≪ D2(ATP)2−(∼ 200M−1) ≫ D3(ATP)− (K≃ 17 M−1) (for reasons of comparison all constants are expressed in the isodesmic model). These results are compared with previous data for adenosine [Ado (K= 15 M−1) > 1:1 ratio of Ado/D(Ado)+ (6.0 M−1) > D(Ado)+ (0.9 M−1)] and AMP [AMP2− (K = 2.1 M−1) < D(AMP)− (3.4 M−1) < 1:1 ratio of D(AMP)−/D2(AMP)± (5.6 M−1) > D2(AMP)± (∼ 2M−1) > D3(AMP)+ (K ≤ 1 M−1)] to facilitate the interpretation of the results for the ATP systems. Stack formation of H2(ATP)2− is clearly favored by additional ionic interactions; this is confirmed by measuring via potentiometric pH titrations the acidity constants of H2(ATP)2− in solutions containing different concentrations of ATP. It is suggested that in the [H2(ATP)]4−2 dimer intermolecular ion pairs (and hydrogen bonds) are formed between the H + (N‐1) site of one H2(ATP)2− and the γ‐P(OH)(O)−2 group of the other; in this way (a) the stack is further stabilized, and (b) the positive charges at the adenine residues are compensated (otherwise repulsion would occur as is evident from the adenosine systems). A detailed structure for the [H2(ATP)]4−2‐dimer is proposed and some implications of the described stacking properties of ATP for biological systems are indicated.
Properties and crystallization of a genetically engineered, water‐soluble derivative of penicillin‐binding protein 5 of Escherichia coli K12
Authors: FERREIRA Luís C. S. , SCHWARZ Uli , KECK Wolfgang , CHARLIER Paulette , DIDEBERG Otto , GHUYSEN Jean‐Marie
Published: March 2005

Derivatives of the Escherichia coli penicillin‐binding protein 5 (PBPS) with truncated carboxyl terminals were obtained by altering the carboxyl‐coding end of the dacA gene. After cloning the modified dacA gene into a runaway‐replication‐control plasmid, one clone that overproduced and excreted the desired protein into the periplasm was used as a source for the isolation of a water‐soluble PBP5 (i.e. PBP5S). In PBP5S the carboxyl‐terminal 21‐amino‐acid region of the wild‐type protein was replaced by a short 9‐amino‐acid segment. Milligram amounts of PBP5S were purified by penicillin affinity chromatography in the absence of detergents or of chaotropic agents. PBP5S was stable and possessed dd‐carboxypeptidase activity without added Triton X‐100. Upon reaction with [14C]benzylpenicillin it was converted into a rather short‐lived acyl‐enzyme complex, as observed with PBPS. Both PBP5 and PBP5S were crystallized. In contrast to PBP5, PBP5S yielded enzymatically active, well‐formed prismatic crystals suitable for X‐ray analysis.
Transcription factors mediate rRNA synthesis during myogenesis
Authors: ZAHRADKA Peter , SELLS Bruce H.
Published: March 2005

This study was designed to identify factors which control rRNA transcription during terminal differentiation of rat L6 myoblasts. A cell‐free system using 100000 ×g supernatants (S100 extracts) from rat L6 myoblasts and a genomic rRNA clone was established which accurately transcribes the rat rRNA gene. The myoblast S100 extract produced a high level of transcription with accurate initiation at the rRNA promoter. Myotube S100 extracts, on the other hand, displayed a reduced capacity to transcribe the rRNA gene. Thus constituents of the cell extract, rather than rearrangements of the DNA sequences, are implicated in regulating rRNA gene expression. The RNA polymerase content of the S100 extracts was analyzed using α‐amanitin to determine the activity of each polymerase species and found to be comparable in both myoblast and myotube S100 extracts. This observation suggests that factors other than RNA polymerase I are involved in regulating rRNA transcription. Two of the factors associated with accurate rRNA gene expression in vitro, namely transcription factors B and D, occurred at comparable levels in both myoblast and myotube extracts suggesting that other components of the transcription complex are responsible for limiting rRNA transcription in myotubes. The cell‐free system should facilitate identification and characterization of these factors which regulate expression of the rRNA gene.
Site of action of a Vero toxin (VT2) from Escherichia coli O157:H7 and of Shiga toxin on eukaryotic ribosomes
Authors: ENDO Yaeta , TSURUGI Kunio , YUTSUDO Takashi , TAKEDA Yoshifumi , OGASAWARA Toshihiro , IGARASHI Kazuei
Published: March 2005

The site of action of a Vero toxin (VT2 or Shiga‐like toxin II) from enterohemorrhagic Escherichia coli and Shiga toxin from Shigella dysenteriae 1 on eukaryotic ribosomes was studied. Treatment of eukaryotic ribosomes with either toxin caused the release of a fragment of 400 nucleotides from 28S ribosomal RNA when the isolated ribosomal RNA was treated with aniline. Release of this fragment with aniline treatment was accompanied by inhibition of protein synthesis and of elongation‐factor‐1‐dependent aminoacyl‐tRNA binding to ribosomes. Analysis of the nucleotide sequence of the 3′‐terminal fragment of 553 nucleotides of 28S rRNA of rat liver 60s ribosomal subunits suggested that an adenine base at position 4324 (A‐4324) was absent in toxin‐treated 28S rRNA. Further analysis by thin‐layer chromatography demonstrated quantitative release of adenine from rat liver ribosomes on treatment with the toxins. These results indicate that both VT2 and Shiga toxin inactivate 60S ribosomal subunits by cleaving the N‐glycosidic bond at A‐4324 in 28S ribosomal RNA.
Elongation factor 1βγ from Artemia
Authors: JANSSEN George M. C. , MÖLLER Wim
Published: March 2005

The guanine nucleotide exchange factor, elongation factor 1βγ (EF‐1βγ) has been purified from Artemia cysts using an improved method. The protein consists of two distinct polypeptides with relative molecular masses of 26000 (EF‐1β) and 46000 (EF‐1γ). A nucleoside diphosphate phosphotransferase activity often found in EF‐1βγ preparations has been completely separated from the actual guanine nucleotide exchange stimulatory activity of EF‐1βγ, thus indicating that nucleotide diphosphate phosphotransferase is not an intrinsic property of EF‐1β. Both EF‐1βγ and EF‐1β have been shown to stimulate the following three reactions to a comparable degree: (a) exchange of GDP bound to EF‐1α with exogenous GDP; (b) EF‐1α‐dependent binding of Phe‐tRNA to ribosomes; (c) poly(U)‐dependent poly(phenylalanine) synthesis. However, a significantly higher nucleotide exchange rate was observed in the presence of EF‐1βγ compared to EF‐1β alone.

Concerning elongation factor 1γ (EF‐1γ) the following observations were made. In contrast to EF‐1β, pure EF‐1γ is rather insoluble in aqueous buffers, but the tendency to precipitate can be partially suppressed by the addition of detergents. In particular, EF‐1γ partitions solely into the detergent phase of Triton X‐114 solutions. EF‐1γ is also more susceptible to spontaneous, specific fragmentation. It is remarkably that about 5% of the cellular pool of EF‐1βγ was found to be present in membrane fractions, under conditions where no EF‐1α was detectable in these fractions. Furthermore it was noted that EF‐1βγ copurified strongly with tubulin on DEAE‐cellulose. Moreover, it was observed that from a mixture of EF‐1βγ and tubulin, EF‐1γ coprecipitates with tubulin using a non‐denaturating immunoprecipitation technique. These findings suggest that EF‐1γ has a hydrophobic domain and interacts with membrane and cytoskeleton structures in the cell.
Expression in Escherichia coli and characterization of human growth‐hormone‐releasing factor
Authors: VILLA Stefano , DE FAZIO Gabriele , DONINI Silvia , TARCHI Giulio , CANOSI Umberto
Published: March 2005

A chemically synthesized DNA sequence, coding for the 44 amino acid residues of human growth‐hormone‐releasing factor (GRF) preceded by a tryptophan codon, was cloned in frame with Escherichia coli trpE gene within a pBR322‐derived plasmid. GRF was expressed in E. coli as a fused polypeptide chain (TrpE‐GRF) and then the GRF amino acid sequence was released from the fused protein by specific chemical cleavage at the tryptophan residue using o‐iodosobenzoic acid. The thioether group of the methionine residue of GRF was converted in the sulfonium salt derivative, in order to prevent irreversible oxidation of methionine to the sulfone derivative by the o‐iodosobenzoic acid reagent.

GRF was purified by HPLC and characterized in terms of amino acid composition after acid hydrolysis, protein sequencing and gel electrophoretic behaviour. These data clearly established that the biosynthetic GRF was identical to the natural one, except for the lack of amidation at the carboxyl‐terminal amino acid. Far‐ultraviolet circular dichroism measurements established that both biosynthetic and natural GRF are devoid of secondary structure in aqueous solution at neutral pH, whereas both peptide samples achieve a high percentage of helical structure in the presence of trifluoroethanol.
ADP‐ribosylation of skeletal muscle and non‐muscle actin by Clostridium perfringens iota toxin
Authors: SCHERING Beate , BÄRMANN Michael , CHHATWAL Gursharan S. , GEIPEL Udo , AKTORIES Klaus
Published: March 2005

The enzymatically active component ia of Clostridium perfringens iota toxin ADP‐ribosylated actin in human platelet cytosol and purified platelet β/γ‐actin, in a similar way to that been reported for component I of botulinum C2 toxin. ADP‐ribosylation of cytosolic and purified actin by either toxin was inhibited by 0.1 mM phalloidin indicating that monomeric G‐actin but not polymerized F‐actin was the toxin substrate. Perfringens iota toxin and botulinum C2 toxin were not additive in ADP‐ribosylation of platelet actin. Treatment of intact chicken embryo cells with botulinum C2 toxin decreased subsequent ADP‐ribosylation of actin in cell lysates by perfringens iota or botulinum C2 toxin. In contrast to botulinum C2 toxin, perfringens iota toxin ADP‐ribosylated skeletal muscle α‐actin with a potency and efficiency similar to non‐muscle actin. ADP‐ribosylation of purified skeletal muscle and non‐muscle actin by perfringens iota toxin led to a dose‐dependent impairment of the ability of actin to polymerize.