bluuebunny/crossref_metadata_2025_split huggingface dataset:
S‐Adenosylmethionine synthetase in the thermophilic archaebacterium Sulfolobus solfataricus.
Authors: PORCELLI Marina , CACCIAPUOTI Giovanna , CARTENÌ‐FARINA Maria , GAMBACORTA Agata 
Published: March 2005

Two isoforms of methionine adenosyltransferase (S‐adenosylmethionine synthetase), A and B, have been partially purified from Sulfolobus solfataricus, a thermophilic archaebacterium optimally growing at 87°C. The chromatographic procedure, involving hydrophobic chromatography on a phenyl‐Sepharose column as a major step. results in 330‐fold and 150‐fold purification of adenosylmethionine synthetase A and B respectively. The apparent molecular masses, estimated by gel filtration, are 180 kDa for A and 75 kDa for B.The A and B isoforms follow Michaelis‐Menten kinetics with apparent Km values of 10 μM nd 20 μM for L‐methionine and of 50 μM and 150 μM for ATP respectively. Adenosylmethionine, a product of the reaction, acts as a powerful non‐competitive inhibitor (Ki= 50 μM) of the A isoform while it inhibits only slightly the B isoform. Both isozymes exhibit tripolyphosphatase activity but only that associated with the form A is stimulated by 5 μM adenosylmethionine concentration. The two enzymes absolutely require a divalent cation for the activity, but are not affected by monovalent ions and reducing agents.The optimum temperature is 90°C and no significant loss of activity is observable after incubation of the two isoforms at 100°C in the presence of ATP. The Arrhenius plots observed for both isozymes are biphasic, indicating different activation energies below and above 75°C.The cytoplasmic levels of ATP, methionine and adenosylmethionine are evaluated.
cDNA cloning and functional expression in yeast Saccharomyces cerevisiae of β‐naphthoflavone‐induced rabbit liver P‐450 LM4 and LM6
Authors: POMPON Denis 
Published: March 2005

A cDNA library was constructed from liver mRNA of a β‐naphthoflavone‐induced rabbit. Two clones pLM4‐1 and pLM6‐1 containaing 2.2‐kbp inserts that hybridized at low stringincy with a mouse P1 P‐450 probe were selected. The clone pLM4‐1 was fully sequenced and found to contain a full‐length cDNA coding for cytochrome P‐450 LM4. Partial sequence and restriction mapping made it possible to identify pLM6‐1 as coding for the major part of cytochrome P‐450 LM6. Cloned LM4‐1 cDNA was reformed by deletion of the 5′ and 3′ non‐coding regions before insertion into yeast expression vectors PYe DP1/10. A similar operation was performed on pLM6–1 cDNA after replacement of the missing N‐terminus‐coding sequences by homologous sequences form the pLM4–1 clone resulting in a chimeric cytochrome P‐450 coding sequence.Expression of cloned rabbit cytochrome P‐450 into transformed yeast was optimized by studying the effect of the nature of the DNA sequence just preceding the initiation codon on the level of cytochrome P‐450 production. Yeast synthesized cytochromes P‐450 were characterized by immunoblotting, spectra and catalytic activity determinations. Cloned cytochrome P‐450 LM4 was found by all criteria to be identical to the authentic rabbit one. The chimeric cytochrome P‐450 that contains the 143 N‐terminal amino acids of cytochrome P‐450 LM4 and the remaining 375 amino acids of cytochrome P‐450 LM6 was found to exhibit most of the authentic cytochrome P‐450 LM6 catalytic properties. Enzymatic and evolutionary implications of these results are discussed.
Properties of a 19‐kDa Zn2+‐binding protein and sequence of the Zn2+‐binding domains
Authors: BRAND Ingeborg Anke , HEINICKEL Antje , KRATZIN Hartmut , SÖLING Hans‐Dieter 
Published: March 2005

A novel 19‐kDa protein has been described recently [Brand, I. A. and Söling, H.‐D. (1986) J. Biol. Chem. 261, 5895 – 5900] which is able to inactivate 6‐phosphofructo‐1‐kinase reversibly in a Zn2+‐dependent manner. We present now additional biochemical and physicochemical data concerning this protein. It is extremely acidic with 40% glutamic and 15% aspartic acid residues. It contains no sulfur, aromatic amino acids, histidine or isoleucine. The protein has four binding sites for Zn2+ with an apparent Kd of about 6 μM. Two of these binding sites are called unspecific as Zn2+ is displaced from these binding sites at physiological concentrations of free Mg2+ (0.75 mM) and at high salt concentrations (100 mM NaCl). Whereas Mg2+‐binding to the two other so‐called specific Zn2+‐binding sites occurs only at Mg2+ concentrations at about 5 mM. The four Zn2+‐binding sites were detected on a tryptic peptide (T8) of 43 amino acid residues, which still possessed biological activity. This peptide has been sequenced and is characterized by four clusters of acidic amino acids separated by only a few neutral amino acids. The two specific Zn2+‐binding sites could be detected in the C‐terminal portion of T8, the two unspecific Zn2+‐binding sites must therefore be located at the N‐terminal portion.The Zn2+‐binding domains of the 19‐kDa Zn2+‐binding protein described here are completely different from those of the ‘zinc finger’ discovered in several DNA‐binding proteins.
Low‐density‐lipoprotein‐receptor mRNA content of fibroblasts from normal and familial hypercholesterolaemic subjects
Authors: KNIGHT Brian L. , PATEL Dilip D. , GAVIGAN Sean J. P. , SOUTAR Anne K. 
Published: March 2005

The amount of mRNA for the low‐density‐lipoprotein (LDL) receptor in cultured human fibroblasts was estimated by hybridization of the poly(A)‐rich RNA fraction with a DNA probe, using the recovery of β‐actin mRNA to correct for losses. During incubation of the cells with lipoprotein‐deficient serum (LPDS) both the LDL‐receptor mRNA content and the rate of receptor protein synthesis increased fourfold during the first 16 h and then fell by approximately 50% during the next 24 h. The content of β‐actin mRNA fell by a similar amount, so that the ratio of receptor/β‐actin mRNAs rose and then remained constant. The fall in β‐actin mRNA content during incubation with LPDS was not prevented by the addition of cholesterol to the medium.In cells from a homozygous familial hypercholesterolaemic (FH) subject that bound 20% of the normal amount of LDL, the content of LDL‐receptor mRNA and the changes during incubation with LPDS or free sterols were similar to normal. Cells from a familial hypercholesterolaemic subject that produced no immunodetectable receptor protein produced a small amount of receptor mRNA of apparently normal size which responded in the same way as in normal cells to LPDS and free sterols.
The binding of double‐stranded RNA and adenovirus VAI RNA to the interferon‐induced protein kinase
Authors: GALABRU Julien , KATZE Michael G. , ROBERT Nadine , HOVANESSIAN Ara G. 
Published: March 2005

The protein kinase from human cells dependent on double‐stranded (ds) RNA is a 68‐kDa protein (p68 kinase), the level of which is enhanced significantly in cells treated with interferon. When activated by low concentrations of dsRNA, the p68 kinase becomes phosphorylated and thereby catalyzes the phosphorylation of the protein‐synthesis initiation factor, eIF2. Here, we have purified the p68 kinase to homogeneity using a specific monoclonal antibody to investigate its capacity to bind dsRNA, poly(I). poly(C). Our study suggest that p68 kinase has high‐ and low‐affinity binding sites: the high‐affinity binding site is responsible for the activation and the low‐affinity binding site for the inhibition of kinase activity. This is in accord with the fact that autophosphorylation of p68 kinase occurs at low concentrations of dsRNA whereas high concentrations of dsRNA inhibit its autophosphorylation.We have also investigated the binding of adenoviral VAI RNA to the purified p68 kinase and have found that the affinity of this binding is lower than that of poly(I). poly(C). We show that VAI RNA can activate or inhibit autophosphorylation of p68 kinase in a dose‐dependent manner, i.e. activation at ≤1 μg/ml or inhibition at >1 μg/ml of VAI RNA. In spite of its lower affinity of binding, VAI RNA cannot be displaced by poly(I). poly(C) or reovirus dsRNA. These data confirm our previous results to illustrate that VAI RNA can bind p68 kinase and cause its inactivation irreversably.
Inhibition of mammalian spermine synthase by N‐alkylated‐1,3‐diaminopropane derivatives in vitro and in cultured rat hepatoma cells
Authors: BAILLON Jean G. , KOLB Michael , MAMONT Pierre S. 
Published: March 2005

A number of N‐alkylated‐1,3‐diaminopropane derivatives[H2N‐(CH2)3‐NH‐(CH2)nH, where n= 1–9] have been tested as potential inhibitors of partially purified rat hepatoma (HTC) cell or pure bovine spleen spermine synthase.Among the compounds described in this paper, the most potent competitive inhibitor of spermine synthase with respect to spermidine, is N‐butyl‐1,3‐diaminopropane with Ki values of 11.9 nM and 10.4 nM for the HTC cell and bovine spleen enzymes respectively. Inhibition of spermine synthase by this alkylated amine is selective since spermidine synthase activity is not affected up to 100 μM N‐butyl‐1,3‐diaminopropane at a range of 5–200 μM putrescine.Added to the culture medium of growing HTC cells, N‐butyl‐1,3‐diaminopropane causes the expected changes in the polyamine levels with a marked decrease of spermine and an increase of spermidine. Under these conditions cell growth continues unabated.Such N‐alkylated‐1,3‐diaminopropane derivatives may have considerable potential as tools for studying the role of polyamines and in particular the functions of spermine in cell multiplication and differentiation.
Sequence analysis of the lpdV gene for lipoamide dehydrogenase of branched‐chain‐oxoacid dehydrogenase of Pseudomonas putida
Authors: BURNS Gayle , BROWN Tracy , HATTER Kenneth , SOKATCH John R. 
Published: March 2005

The production of two lipoamide dehydrogenases by Pseudomonas is so far unique. One, LPD‐val, is the specific E3 component of the branched‐chain‐oxoacid dehydrogenase and the second, LPD‐glc, is the E3 component of 2‐oxoglutarate dehydrogenase and the L‐factor of the glycine oxidation system. The objective of the present research was to determine the nucleotide sequence of the structural gene for LPD‐val in order to compare its deduced amino acid structure with that of other redox‐active disulfide flavoproteins. Northern blots using mRNA isolated from P. putida grown in media with branched‐chain amino acids identified a transcript of 6.2 kb which is long enough to encode all the structural genes for the complex. The nucleotide sequence of the structural gene for LPD‐val, lpdV, was determined and consists of 459 codons plus the stop codon. The open reading frame begins two bases after the stop codon for the E2 subunit and is composed of 66.3% G+C. Codon usage is characteristic of moderately strongly expressed genes. There is a ribosome‐binding site preceding the ATG start codon and a strong candidate for a rho‐independent terminator at the 3′ end of the reading frame. The Mr of the protein encoded is 48164 and when the Mr of FAD is added, the total Mr is 48949, which is very close to the value of 49000 obtained by SDS‐polyacrylamide gel electrophoresis.Similarity comparisons of LPD‐val with sequences of three lipoamide dehydrogenases showed that LPD‐val was somewhat more distantly related. It is probable that the lipoamide dehydrogenases and the glutathione and mercuric reductases evolved from a common ancestral flavoprotein.
Conformational forces affecting the folding pathways of dendrotoxins I and K from black mamba venom
Authors: HOLLECKER Michelle , LARCHER Dominique 
Published: March 2005

The conformations of the major intermediates trapped during the folding of dendrotoxins I and K from venom of black mamba snakes, have been investigated by circular‐dichroism spectroscopy. Local alterations to the native, folded conformations are observed in toxins I and K and in a protein of similar sequence, bovine pancreatic trypsin inhibitor. The inability of intermediates (30–51, 14–38) to complete refolding by forming directly the 5–55 disulphide bond is explained. The following observations on the role of secondary structure in the folding of the three proteins are of interest.1. It is not necessary for the three proteins to acquire elements of secondary structure at the same stage of folding in order to attain similar three‐dimensional conformations.2. The stability of the final folded state is not directly correlated to an early appearance of secondary structure.3. The degree of secondary structure already present in intermediates (30–51) seems to determine the pathway of refolding preferred by the corresponding protein.
Characterization of the S′‐subsite specificity of porcine pancreatic elastase
Authors: SCHELLENBERGER Volker , SCHELLENBERGER Ute , MITIN Yuri V. , JAKUBKE Hans‐Dieter 
Published: March 2005

A number of maleyl peptide p‐nitrobenzyl esters have been synthesized to study elastase‐catalyzed hydrolysis reactions. These new substrates were used as acyl donors to investigate the S′‐subsite specificity of porcine pancreatic elastase by partitioning of the acyl enzyme between various added nucleophiles and water. The following results were obtained.1. Porcine pancreatic elastase prefers amino acid residues with small side chains in the P′1 position.2. The nucleophile binding is improved by a positively charged P′1 side chain, whereas a negatively charged function results in a very low binding tendency.3. Elongation of the nucleophile to the P′2 position leads to higher aminolysis rates.4. S′ specificity is substantially influenced by the P1 residue of the acyl enzyme.
Proteinase K from Tritirachium album Limber
Authors: GUNKEL F. Andreas , GASSEN H. Günter 
Published: March 2005

The cDNA and the chromosomal gene encoding proteinase K from Tritirachium album Limber have been cloned in Escherichia coli and the entire nucleotide sequences of the coding region, as well as 5′‐ and 3′‐flanking regions have been determined. The deduced primary translation product consisting of 384 amino acid residues (molecular mass = 40 231 Da) contains an N‐terminal region of 105 amino acids not present in the mature protein. By analogy to the evolutionary‐related bacterial subtilisins and other serine proteinases it is inferred that the primary secreted product is a zymogen containing a 15‐amino‐acid signal sequence and a 90‐amino‐acid propeptide. The propeptide is presumably removed in the later steps of the secretion process or upon secretion into the medium.The nucleotide‐sequence analysis of the gene and its flanking regions has revealed that the proteinase‐K gene is composed of two exons and one 63‐bp‐long intron located in the proregion. Furthermore, a putative promoter sequence and a capping site have been identified, suggesting that the transcription‐start site is located 103‐bp upstream of the ATG initiation codon. To express the proproteinase‐K gene in E. coli, proproteinase‐K cDNA was cloned in a plasmid vector under control of the tac promoter. The hybrid plasmid pSPPRO, constructed for this purpose, contained the cDNA coding for proproteinase K [from Ala (‐91) to the C‐terminal Ala (279)] fused to the N‐terminal‐signal‐peptide sequence of the alkaline‐phosphatase gene preceded by the tac promoter. E. coli BMH71‐18, harbouring this plasmid, exhibited slight proteolytic activity when tested on skimmed‐milk plates, suggesting that some fusion proteins were correctly secreted into the periplasm and processed to the mature proteinase K.
The genes for the frog skin peptides GLa, xenopsin, levitide and caerulein contain a homologous export exon encoding a signal sequence and part of an amphiphilic peptide
Authors: KUCHLER Karl , KREIL Günther , SURES Irmi 
Published: March 2005

From genomic libraries of Xenopus laevis, parts of the genes coding for the precursors of the skin peptides GLa (peptide with amino‐terminal glycine and carboxy‐terminal leucinamide), xenopsin and levitide have been isolated and sequenced. The gene for prepropeptide GLa comprises four exons, separated by relatively small introns. The gene for preproxenopsin is composed of five exons, of which all but the last one have been analyzed. This is a large gene encompassing at least 25000 base pairs. In addition, two exons of the gene for preprolevitide have been isolated.A comparison of these genes reveals the presence of a homologous exon. This exon contains 161 bp, starts one base pair prior to the initiation codon and encodes a signal peptide and part of a pro region with processing sites. In addition, the two genes for preprocaerulein analyzed previously [Vlasak et al. (1987) Eur. J. Biochem. 169, 53–58] also contain a similar exon. This demonstrates the existence of a homologous export exon in genes encoding the precursors of different skin peptides.
Intracellular localization of inositol‐phospholipid‐metabolizing enzymes in rabbit fast‐twitch skeletal muscle
Authors: VARSÁNYI Magdolna , MESSER Michael , BRANDT Neil R. 
Published: March 2005

Rabbit fast‐twitch skeletal muscle microsomes have been separated by isopycnic centrifugation on a linear sucrose gradient into triads and light sarcoplasmic reticulum. In both fractions phosphatidylinositol‐kinase activity is found [Varsànyi et al. (1986) Biochem. Biophys. Res. Commun. 138, 1395]. In contrast, phosphatidylinositol‐4‐phosphate kinase is nearly exclusively associated with triads. The phosphatidylinositol‐4,5‐bisphosphate‐phosphodiesterase activity shows a biphasic distribution: approximately 50% of the activity is associated with triads and 50% appears in the overlay.Triads have been broken mechanically into transverse tubules and terminal cisternae, then separated by isopycnic sucrose‐gradient centrifugation. Both fractions exhibit phosphatidylinositol‐kinase activity; the activities of phosphatidylinositol‐4‐phosphate kinase and phosphatidylinositol‐4,5‐bisphosphate phosphodiesterase are associated mainly with the transverse tubules. Consequently, in rabbit fast‐twitch skeletal muscle all necessary enzymes for production of D‐myo‐inositol 1,4,5‐trisphosphate are associated with transverse tubules.Phosphatidylinositol‐4,5‐bisphosphate phosphodiesterase associated with triads shows a pH optimum at 6.8. The enzyme is maximally active between pCa 5 and pCa 4.Mg2+ inhibits the enzyme activity half‐maximally at about 1 mM. Guanine‐nucleotide‐binding proteins seem not to be involved in the regulation of enzyme activity; guanosine 5′‐[γ‐thio]triphosphate does not influence phosphatidylinositol‐4,5‐bisphosphate phosphodiesterase activity. It correlates well with the observation that neither α1‐adrenergic nor muscarinic receptors have been found in fast‐twitch rabbit skeletal muscle. On basis of the respective enzyme activities estimations on maximal phosphatidylinositol turnover were made and a possible involvement of this signal pathway in excitation‐contraction coupling has been discussed. Furthermore, calculations show that during a single twitch D‐myo‐inositol 1,4,5‐trisphosphate concentration does not reach more than 2 nM. However, during a 4‐s tetanus D‐myo‐inositol 1,4,5‐trisphosphate can accumulate to a level which could effect force generation [Thieleczek and Heilmeyer (1986) Biochem. Biophys. Res. Commun. 135, 662] and aldolase distribution (Thieleczek et al., unpublished results).
Expression of human lactotransferrin receptors in phytohemagglutinin‐stimulated human peripheral blood lymphocytes
Authors: MAZURIER Joël , LEGRAND Dominique , Li HU Wei , MONTREUIL Jean , SPIK Geneviève 
Published: March 2005

In the resting rate, the human peripheral blood lymphocytes did not show detectable surface and intracellular receptors for human lactotransferrin. However, both types of lactotransferrin receptors were expressed during stimulation of lymphocytes with phytohemagglutinin. The appearance of receptors was time‐dependent and the number of receptors reached a plateau after at least two days of mitogen stimulation. These results suggest that the presence of surface receptors on mitogen‐stimulated lymphocytes is not consecutive to a modification of subcellular distribution but to an induction of biosynthesis of the receptors. As measured by incorporation of [3H]thymidine into DNA, addition of human lactotransferrin in a serum‐free medium increased the proliferative activity of phytohemagglutinin‐stimulated lymphocytes. Optimal enhancement of [3H]thymidine incorporation was obtained by adding 30% iron‐saturated lactotransferrin at a concentration of 0.17 μM. Therefore, the role of lactotransferrin in the response of lymphocytes to mitogen stimulation appears to be similar to that previously described for serotransferrin.The lactotransferrin receptor was visualized using 125I‐labeled lactotransferrin on nitrocellulose paper after electroblotting of the Triton X‐100 extract of the phytohemagglutinin‐stimulated lymphocytes as two protein bands of 100 and 110 kDa molecular mass.Purification of the lactotransferrin receptor from the Triton‐X‐100‐soluble extract of stimulated lymphocytes was performed by antiligand‐affinity chromatography. The binding of lactotransferrin to the purified receptors was reversible and dependent on concentration and pH.
The influence of purification and protein heterogeneity on the crystallization of p‐hydroxybenzoate hydroxylase
Authors: van der LAAN Jan M. , SWARTE Myra B. A. , GROENDIJK Hillie , HOL Wim G. J. , DRENTH Jan 
Published: March 2005

The structure of the enzyme p‐hydroxybenzoate hydroxylase was determined to a resolution of 0.25 nm [Wierenga et al. (1979) J. Mol. Biol. 131, 53‐73] with crystals belonging to space group C2221. Subsequently it was impossible to repeat the growth of this crystal form and only poor quality tetragonal crystals could be obtained.We have thoroughly investigated this problem and found that Cibacron‐blue‐purified enzyme appears to be heterogeneous with respect to aggregation state and Cys‐116 oxidation. Most importantly, it could be firmly established that C2221 crystals can only be grown from purely dimeric p‐hydroxybenzoate hydroxylase possessing an intact SH group. Ion‐exchange chromatography on DEAE‐Sepharose can successfully remove those forms of the enzyme which impede successful crystallization. Sulfite and dithiothreitol improve crystallization by dis‐sociating the enzyme oligomers into dimers; sulfite especially gives excellent results.
Effect of the microenvironment on the kinetic properties of immobilized enzymes
Authors: BILLE Vincent , PLAINCHAMP Dominique , LAVIELLE Solange , CHASSAING Gérard , REMACLE José 
Published: March 2005

A new immobilization method was developed in order to perform a systematic study of the influence of the microenvironment on the properties of immobilized enzymes. The enzyme, alcohol dehydrogenase, was first activated, then polypeptide arms of known composition were quantitatively grafted and finally the enzyme was covalently immobilized by co‐polymerization of the activated ends of the peptide arms with acrylamide monomers. In this way, the polypeptide linker arms fully determine the properties of the microcavity of the gel in which the enzyme is immobilized by multipoint covalent linkages.The activation energy of the reaction was determined for different microenvironments, in solution as well as after immobilization. Kinetic parameters were also calculated and a new kinetic model was developed, allowing a correction for the diffusional restrictions.The results show that the diffusional restrictions on one hand, and the nature of the microenvironment on the other hand, interact in a dynamic way with the enzyme to determine its properties. Another key point to understanding the changes in the properties of the immobilized enzyme is to consider these proteins as dynamic structures, interacting physically and chemically with their microenvironment.