bluuebunny/crossref_metadata_2025 huggingface dataset:
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.
On the non‐liner Eadie plots of the tRNA kinetics and non‐liner Dixon plots of the PPi inhibition kinetics of the aminoacyl‐tRNA synthetases
Authors: AIRAS R. Kalervo
Published: March 2005

A model of the aminoacyl‐tRNA synthetase reaction was analyzed by deriving a rate equation, and by calculating the aminoacylation rates at various values of the rate and equilibrium constants. The model specially contained the possibilities that (1) the activation of the amino acid occurs either with bound or non‐bound tRNA, and that (2) the transfer of the aminoacyl moiety from the aminoacyl adenylate to tRNA occurs either with bound or non‐bound PPi. The analysis showed that the Eadie plots (tRNA as the variable substrate) are straight lines only if the rates of the activation reactions with bound and non‐bound tRNA are equal. Otherwise the Eadie plots can be either curved upwards or downwards. The Dixon plots of the PPi inhibition are straight lines only if PPi must be dissociated from the enzyme before the tranfer reaction. The conditions under which the Kiapp values are much lower than the dissociation constants for PPi are met if the transfer reaction is relatively slow and the reverse reaction of the activation (pyrophosphorolysis) is fast, and if the tRNA concentration is low.
Ionic‐strength modulation of electrically induced permeabilization and associated fusion of mammalian cells
Authors: ROLS Marie‐Pierre , TEISSIE Justin
Published: March 2005

Application of a high electric field to cells in culture has been shown to make them both permeable and fusogenic. The molecular events involved in the phenomenon are still poorly understood. In this study we investigated the effects of the ionic strength of the pulsing buffer on the electropermeabilization and electrofusion of Chinese hamster ovary cells. Increasing the ionic strength of the pulsing medium results in an increase in sieving of transient permeant structures, but decreases the fusion index. Treatment of cells with trypsin or pronase before application of the pulses abolishes the ionic modulation of both electropermeabilization and electrofusion. A similar rate of expansion of permeabilization is obtained whatever the ionic content of the pulsing buffer, and cells fuse even at high inoic strength. This observation lends support to our hypothesis that membrane proteins play a role in electrofusion.
Photomorphogenesis in Physarum polycephalum
Authors: POETSCH Bettina , SCHRECKENBACH Thomas , WERENSKIOLD Anne K.
Published: March 2005

Photo‐induced fruiting‐body formation of the slime mold Physarum polycephalum can be divided into two stages. The first stage (I) starts with the beginning of illumination (O h) and ends with the formation of nodules (12 h). The second stage (II) is characterized by culmination, sporangiophore formation and melanization of sporangiophore heads (13–17 h). We investigated the expression of actin, α‐ and β‐tubulin during this differentiation process using Northern blot analysis and run‐off transcription in isolated nuclei. Whereas actin mRNA is irreversibly lost during stage I, we observed two peaks in mRNA concentration for α‐tubulin and one peak for that of β‐tubulin during stage I and a coordinated α/β‐tubulin mRNA induction during stage II. Stage II induction appears to be related to a presporangial mitosis. Transcriptional activity of the three genes studied shows two maxima, namely one in the middle of stage I and the other at the end of stage II. Our data suggest that the expression of the three cytoskeletal proteins investigated follows a distinct temporal pattern comprising changes in both mRNA synthesis and decay. We also propose a novel function of tubulin in Physarum which is not related to mitosis.
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.
Implication of the α1β1 interface in the hemoglobin affinity changes
Authors: ANTRI Said EL , ZENTZ Christian , ALPERT Bernard
Published: March 2005

The α1β1 interface of normal and mutated San Diego hemoglobins in their fully liganded form was investigated, through the SH vibrational absorption of β‐112 cysteine, byFourier‐transform infrared spectroscopy. The center frequency of this thiol group was significantly shifted in San Diego hemoglobin compared with normal human hemoglobin. Different dimer organization between the two proteins was also revealed by circular dichroism of the heme. These findings agree well with assessment that the α1β1 interface, far from being inert, is involved in the affinity changes of the hemoglobin molecule.
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.
Preparation of immobilized monomeric actin and its use in the isolation of protease‐free and ribonuclease‐free pancreatic deoxyribonuclease I
Authors: NEFSKY Bradley , BRETSCHER Anthony
Published: March 2005

A procedure is described for the immobilization of monomeric actin so that about 30% of the immobilized protein is competent to bind the monomeric‐actin‐binding proteins bovine pancreatic deoxyribonuclease I and chicken villin. The intact tertiary structure of the immobilized actin is required to bind these proteins. Using this resin, a method has been developed for the affinity purification of pancreatic deoxyribonuclease I on a reusable actin column. It involves the binding of deoxyribonuclease I to immobilized actin, extensive washing of the column, followed by elution of the bound deoxyribonuclease I with 10 M formamide. After removal of the formamide, the deoxyribonuclease I has a higher specific activity than the starting material and contained no detectable protease or ribonuclease contamination. This preparation should find considerable application in molecular genetic studies where the enzyme is needed free of these particular contaminants. The affinity column should also be useful for the isolation of other, physiologically relevant, monomeric‐actin‐binding proteins.
Alanine dehydrogenase from Streptomyces fradiae
Authors: VANČURA Aleš , VANČUROVÁ Ivana , VOLC Jindřich , JONES Shanagh K. T. , FLIEGER Miroslav , BASAŘOVÁ Gabriela , BĚHAL Vladislav
Published: March 2005

Alanine dehydrogenase was purified to homogeneity from a cell‐free extract of Streptomyces fradiae, which produces tylosin. The enzyme was purified 1180‐fold to give a 21% yield, using a combination of hydrophobic chromatography and ion‐exchange fast protein liquid chromatography. The relative molecular mass of the native enzyme was determined to be 210000 or 205000 by equilibrium ultracentrifugation or gel filtration, respectively. The enzyme is composed of four subunits, each of Mr 51000. Using analytical isoelectric focusing the isoelectric point of alanine dehydrogenase was found to be 6.1. The Km were 10.0 mM for L‐alanine and 0.18 mM for NAD+. Km values for reductive amination were 0.23 mM for pyruvate, 11.6 mM for NH+4 and 0.05 mM for NADH. Oxidative deamination of L‐alanine proceeds through a sequential‐ordered binary‐ternary mechanism in which NAD+ binds first to the enzyme, followed by alanine, and products are released in the order ammonia, pyruvate and NADH.
The predicted secondary structures of the nucleotide‐binding sites of six cation‐transporting ATPases lead to a probable tertiary fold
Authors: TAYLOR William R. , GREEN N. Michael
Published: March 2005

Six cation‐dependent transporting ATPases have homologous sequences in the region assigned by chemical labelling to nucleotide binding. Comparison of the most highly conserved segments with other nucleotide‐binding domains showed that the sequences were consistent with a mononucleotide‐binding fold and enabled a number of likely folding topologies to be limited to two or three alternatives. One of these possible folds was topologically equivalent to adenylate kinase; this was taken as a model in which the significance of conserved amino acids was investigated. In this model conserved amino acids were grouped around a postulated ATP‐binding cleft, satisfactorily accounting for their degree of conservation.
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.
Synthesis and characterization of 1‐substituted 5‐alkylphenazine derivatives carrying functional groups
Authors: YOMO Tetsuya , SAWAI Haruyo , URABE Itaru , YAMADA Yasuhiro , OKADA Hirosuke
Published: March 2005

The following 1‐substituted derivatives of 5‐methylphenazine and 5‐ethylphenazine were synthesized: 1‐(3‐carboxypropyloxy)‐5‐methylphenazine (1B), 1‐(3‐carboxypropyloxy)‐5‐ethylphenazine (2B), 1‐(3‐ethoxycarbo‐nylpropyloxy)‐5‐ethylphenazine (2C) and 1‐[N‐(2‐aminoethyl)carbamoylpropyloxy]‐5‐ethylphenazine (2D); their spectra, stability and reactivity as electron mediators were investigated, together with those of 5‐methylphenazine (1A) and 5‐ethylphenazine (2A). The 1‐substituted derivatives are all insensitive to light and the derivatives of 5‐ethylphenazine are more stable than those of 5‐methylphenazine under neutral and alkaline conditions; 2B is the most stable of all the derivatives. The spectral properties of the decomposed compounds showed that photodecomposition of 1A and 2A is associated with hydroxylation at position 1, alkali decomposition of 1A and 1B with elimination of the 5‐methyl group and alkali decomposition of 2A, 2B, and 2D with a ring‐opening reaction. The second‐order rate constant k1 for the reaction of the phenazine derivatives with NADH was measured under steady‐state conditions. The k1 values vary depending on the substituents at positions 1 and 5: the values for 1A, 1B, 2A, 2B, 2C and 2D are 1.83 mM−1 s−1, 3.33 mM−1 s−1, 0.75 mM−1 s−1, 1.42 mM−1 s−1, 1.68 mM−1 s−1 and 2.03 mM−1 s−1, respectively. The rate constants, k2 and k3, for the reactions of the reduced form of 2B with oxygen and with 3‐(4′,5′‐dimethylthiazole‐2‐yl)‐2,5‐diphenyltetrazolium ion, respectively, were k2= 1.21 mM−1 s−1 and k3= 91 mM−1 s−1. These phenazine derivatives have potential applications in the biochemical field.
Characterization of MSM1, the structural gene for yeast mitochondrial methionyl‐tRNA synthetase
Authors: TZAGOLOFF Alexander , VAMBUTAS Andrea , AKAI Anna
Published: March 2005

Respiratory‐deficient mutants of Saccharomyces cerevisiae assigned to pet complementation group G72 are impaired in mitochondrial protein synthesis. The loss of this activity has been correlated with the inability of the mutants to acylate the two methionyl‐tRNAs of yeast mitochondria. A nuclear gene (MSM1) capable of complementing the respiratory deficiency has been cloned by transformation of the G72 mutant C122/U3 with a yeast genomic library. In situ disruption of the MSM1 gene in a wild‐type haploid strain of yeast induces a respiratory‐deficient phenotype but does not affect the ability of the mutant to grow on fermentable substrates indicating that the product of MSM1 were functions only in mitochondrial protein synthesis. Mitochondrial extracts prepared from the mutant with the disrupted copy of MSM1 were found to be defective in acylation of the two mitochondrial methionyl‐tRNAs thereby confirming the identity of MSM1 as the structural gene for the mitochondrial methionyl‐tRNA synthetase. The sequence of the protein encoded by MSM1 is similar to the Escherichia coli and yeast cytoplasmic methionyl‐tRNA synthetases. Based on the primary‐sequence similarities of the three proteins, the mitochondrial enzyme appears to be more related to the bacterial than to the yeast cytoplasmic methionyl‐tRNA synthetase.
Refolding human serum albumin at relatively high protein concentration
Authors: BURTON Steven J. , QUIRK Alan V. , WOOD P. Carolyn
Published: March 2005

The conditions for refolding reduced and denatured human serum albumin (HSA) were investigated with a view to maximising the yield of native monomeric albumin. Refolding by dialysis was found to be preferable to dilution as a means of chaotrope (urea) and reductant (2‐mercaptoethanol) removal. Dialysis of denatured HSA solutions containing 4–8 urea and 14 mM 2‐mercaptoethanol at pH 10.0 was found to be optimal for HSA refolding. The yield of monomeric HSA was maximal (94%) for dialysis in the presence of EDTA (1 mM) and sodium palmitate (20 μM). Using this protocol it was possible to refold HSA at concentrations in excess of 5 mg · m1−1 whilst maintaining a high recovery of native monomer. These results represent a considerable improvement on established methods of HSA refolding.
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.
Structural organization of the human heme oxygenase gene and the function of its promoter
Authors: SHIBAHARA Shigeki , SATO Michihiko , MULLER Rita M. , YOSHIDA Tadashi
Published: March 2005

Human heme oxygenase is induced by its substrate heme, but not induced by heat shock [Yoshida et al. (1988) Eur. J. Biochem. 171, 457 – 461]. In order to study the molecular mechanisms of heme‐mediated induction of human heme oxygenase, we have isolated and characterized the genomic clones for heme oxygenase. The human heme oxygenase gene (HO gene) is about 14 kb long and organized into five exons. The transcription initiation site was identified by S1 nuclease mapping and primer‐extension analyses. Using HeLa whole cell extracts, we confirmed that the transcription of the cloned HO gene is initiated accurately at the assigned initiation site. In its 5′‐flanking region, a potential heat‐shock element (HSE) is present 367 bp upstream from the initiation site, although, in contrast to rat heme oxygenase, human enzyme is not induced by heat shock. We therefore analyzed the effects of heat shock on the transient expression of chimeric fusion genes harboring the promoter of the human HO gene ligated to the Escherichia coli gene gpt in mouse amelanotic melanoma cells. The 5′‐flanking region of the human HO gene bearing a potential HSE failed to confer the heat‐inducibility of gpt RNA production, suggesting that the HSE of the human HO gene is not functional.
The lipopolysaccharide‐induced stimulation of peritoneal macrophages involves at least two signal pathways
Authors: BENNINGHOFF Bernd , DRÖGE Wulf , LEHMANN Volker
Published: March 2005

Bacterial lipopolysaccharides (LPS) stimulate the ability of macrophages to convert arginine into ornithine. This effect is inhibited by the cycloxygenase inhibitor indomethacin and reconstituted by application of exogenous prostaglandin E2 (PGE2) or cholera toxin, i.e. two substances which are known to raise the intracellular concentration of cyclic AMP. Moreover, the LPS‐mediated effect is augmented in a synergistic manner by PGE2, cholera toxin and the dibutyryl‐derivative of cyclic AMP. Lipid A precursor IA, which lacks lauric, myristic and palmitic acids, and lipid A precursor IB, which is devoid of lauric and myristic acids of the complete lipid A structure, are capable of augmenting PGE2 synthesis but do not stimulate the conversion of arginine into ornithine. Taken together, our experiments suggest that the LPS‐induced production of PGE2 and the PGE2‐induced increase of the intracellular cAMP concentrations are essential elements of an autoregulatory loop that controls the LPS‐mediated stimulation of the ornithine production by macrophages. The stimulation of the autoregulatory loop is necessary but not sufficient for this effect, indicating that an additional signal is required which is provided by the complete lipid A structure but not by the incomplete lipid A structures IA and IB. This additional signal can be provided by‐a lipid A structure containing the 3‐acyloxyacyl residues with lauric and myristic acids.
Purification of nitrate reductase from Nicotiana plumbaginifolia by affinity chromatography using 5′AMP‐Sepharose and monoclonal antibodies
Authors: MOUREAUX Thérèse , LEYDECKER Marie‐Thérèse , MEYER Christian
Published: March 2005

Nitrate reductase was purified from leaves of Nicotiana plumbaginifolia using either 5′AMP‐Sepharose chromatography or two steps of immunoaffinity chromatography involving monoclonal antibodies directed against nitrate reductase from maize and against ribulose‐1,5‐bisphosphate carboxylase from N. plumbaginifolia. Nitrate reductase obtained by the first method was purified 1000‐fold to a specific activity of 9 units/mg protein. The second method produced an homogenous enzyme, purified 21 000‐fold to a specific activity of 80 units/mg protein. SDS/PAGE of nitrate reductase always resulted in two bands of 107 and 99.5 kDa. The 107‐kDa band was the nitrate reductase subunit of N. plumbaginifolia; the smaller one of 99.5 kDa is thought, as commonly reported, to result from proteolysis of the larger protein. The molecular mass of 107 kDa is close to the values calculated from the coding sequences of the two nitrate reductase genes recently cloned from tobacco (Nicotianatabacum cv Xanthi).
A high‐resolution 1H‐NMR study of human transforming growth factor α
Authors: TAPPIN Michael J. , COOKE Robert M. , FITTON John E. , CAMPBELL Iain D.
Published: March 2005

The 500‐MHz and 600‐MHz 1H‐NMR spectra of recombinant human transforming growth factor α have been recorded at pH values of 3.8, 6.5 and 9.4. Analysis of various two‐dimensional spectra has enabled sequence‐specific assignments to be made and the secondary structure to be identified. Information on the tertiary fold has also been obtained from observed nuclear Overhauser effects and titration of histidine residues. The overall fold of the protein is very similar to that of epidermal growth factor, as might be expected from the sequence similarity. However, the structure of transforming growth factor α at pH 3.8 is found to show interesting differences from those at the two higher pHs and from that of epidermal growth factor.
The carboxyltransferase activity of the sodium‐ion‐translocating methylmalonyl‐CoA decarboxylase of Veillonella alcalescens
Authors: HOFFMANN Artur , HILPERT Wilhelm , DIMROTH Peter
Published: March 2005

Methylmalonyl‐CoA decarboxylase of Veillonella alcalescens catalyzed the isotopic exchange between methylmalonyl‐CoA and [1‐14C]propionyl‐CoA or between malonyl‐CoA and [1‐14C]acetyl‐CoA. The exchange was independent of sodium ions and was abolished by avidin. The enzyme also catalyzed the carboxyl transfer reaction from methylmalonyl‐CoA to acetyl‐CoA yielding propionyl‐CoA and malonyl‐CoA, and vice versa. The β subunit was dissociated from methylmalonyl‐CoA decarboxylase by prolonged washing of the enzyme while bound via its biotin prosthetic group to monomeric avidin‐Sepharose. The β‐chain‐depleted enzyme was inactive as a methylmalonyl‐CoA decarboxylase but retained carboxyltransferase activity. The β subunits were specifically protected by Na+ ions from tryptic hydrolysis. Based on these and other observations the following functions may be assigned to the different polypeptide chains of methylmalonyl‐CoA decarboxylase: carboxyltransferase (α). carboxybiotin‐carrier‐protein decarboxylase (β), biotin carrier protein (γ). The function of the δ chain is unknown.
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.
The uncoupling protein dimer can form a disulfide cross‐link between the mobile C‐terminal SH groups
Authors: KLINGENBERG Martin , APPEL Maria
Published: March 2005

Isolated uncoupling protein (UCP) can be cross‐linked, by various disulfide‐forming reagents, to dimers. The best cross‐linking is achieved with Cu2+‐phenanthroline oxidation. Because cross‐linking is independent of UCP concentration and prevented by SDS addition, a disulfide bridge must be formed between the two subunits of the native dimer. Cross‐linking is prevented by SH reagent and reversed by SH‐reducing reagents. In mitochondria, cross‐linking of UCP with disulfide‐forming agents is even more efficient than in isolated state. It proves that UCP is a dimer in mitochondria, before isolation. Disulfide‐bridge formation does not inhibit GTP‐binding to UCP. Cross‐linked UCP re‐incorporated in proteoliposomes either before or after cross‐linking fully retains the H+‐transport function.

Rapid cross‐linking by membrane impermeant reagents indicates a surface localization of the C‐terminus in soluble UCP and projection to the outer surface in mitochondria. Intermolecular disulfide‐bridge formation in a dimer requires juxtaposition of identical cysteines at the twofold symmetry axis. A rigid juxtaposition of cysteines is unlikely, unless intended for a native disulfide bridge. The absence of such a bridge in UCP suggests that juxtaposition of cysteines is generated by high mobility. In order to localize the cysteine involved, cross‐linked UCP was cleaved by BrCN. The CB‐7 C‐terminal peptide, which contains cysteines at positions 287 and 304, disappears. Limited trypsinolytic cleavage, previously shown to occur at Lys‐292, removed cross‐linking in UCP both in the solubilized and mitochondrially bound state. The cleaved C‐terminal peptide of 11 residues contains only cystein‐304 which, thus, should be the only one (out of 7 cysteines in UCP) involved in the S—S bridge formation. Obviously, the C‐terminal location of the cysteine, because of its high mobility, permits juxtapositioning for cross‐linking. This agrees with predictions from hydrophobicity analysis that the last 14 residues in UCP protrude from the membrane.
Comparison of the amino acid sequence of the lytic enzyme from broad‐host‐range bacteriophage PRD1 with sequences of other cell‐wall‐peptidoglycan lytic enzymes
Authors: PAKULA Tiina M. , SAVILAHTI Harri , BAMFORD Dennis H.
Published: March 2005

The gene for the lytic enzyme of the lipid‐containing, broad‐host‐range bacteriophage PRD1 codes for a protein of 149 amino acids (17271 Da). The sequence of the protein is unique when compared to other lytic enzymes sequenced. However, three regions of weak similarity with other phage lytic enzymes were observed. The C‐terminal region shared seven amino acids in common with phage P22 lysozyme at a site which is conserved in phage‐type lysozymes.
Interaction of β‐very‐low‐density lipoproteins with rat liver cells
Authors: HARKES Leen , van DUIJNE Astrid , van BERKEL Theo J. C.
Published: March 2005

Cholesteryl‐ester‐rich very‐low‐density lipoproteins (β‐VLDL) are considered to be atherogenic because in vitro they can provoke cholesterol accumulation in macrophages. The greatest population of macrophages resides inside the liver and in the present study the rat β‐VLDL uptake by the various rat liver cell types is determined in vivo and compared to the uptake of rat VLDL. β‐VLDL isolated from cholesterol‐fed rats was iodinated and injected into the rat. After 10 min of circulation, 45% of the injected β‐VLDL was found in the liver. A low‐temperature cell‐isolation procedure shows that rat liver parenchymal cells form the major site for β‐VLDL uptake (96%) and, consequently, rat liver macrophages (nonparenchymal liver cells) do not perform a quantitatively significant role in the uptake of these lipoproteins. In vitro competition studies indicate that apolipoprotein (apo) E is the site recognised by liver parechymal cells and even a 600‐fold excess of apo‐E‐free human LDL was an ineffective competitor. Furthermore it can be demonstrated that induction of apo‐B,E receptors on liver parenchymal cells by estrogen treatment does not result in a significant increased uptake of β‐VLDL. These data show that recognition of β‐VLDL is presumably exerted by the remnant receptor. Intracellular processing of both the apolipoproteins and phospholipids of β‐VLDL was followed by subcellular distribution studies. It appears that, within 45 min, 75% of the apolipoproteins are degraded and subsequently released from the liver. In contrast the phospholipids remain associated with the liver for a prolonged time and a specific transfer to the mitochondrial fraction is found. It can be concluded that liver parenchymal cells form in vivo the major site for β‐VLDL uptake and it appears that recognition of β‐VLDL is coupled to internalization and processing of both the apolipoproteins and phospholipids by a route which involves the lysosomes.
Identification of proteins secreted from axons of embryonic dorsal‐root‐ganglia neurons
Authors: STOECKLI Esther T. , LEMKIN Peter F. , KUHN Thomas B. , RUEGG Markus A. , HELLER Martin , SONDEREGGER Peter
Published: March 2005

Secretion of proteins from the growth cone has been implicated in axon growth and synapse formation and might be involved in the transmission of a variety of axon‐derived regulatory signals during neurogenesis. In order to identify axonally secreted proteins, dorsal‐root‐ganglia neurons from chicken embryos were cultured in a compartmentalized cell culture system that allows separate access to neuronal cell somas and axons. The proteins synthesized by the neurons were metabolically labeled by addition of [35S]methionine to the compartment containing the cell somas; the proteins released from the axons were harvested from the culture medium of the axonal compartment. Two‐dimensional gel electrophoresis revealed two axonally secreted proteins with apparent molecular mass of 132–140 kDa and 54–60 kDa; they were termed axonin‐1 and axonin‐2, respectively. Both axonins were found to be secreted from a variety of neuronal cell cultures, but not from any of the nonneuronal cultures investigated, and hence might be neuron‐specific. Virtual absence of these proteins from the axonal protein pattern suggests constitutive secretion. The information acquired on coordinated and spot morphology of these proteins in two‐dimensional gel electrophoresis provides a useful assay for their purification.
Human insulin‐like growth‐factor‐binding protein
Authors: LUTHMAN Holger , SÖDERLING‐BARROS Jane , PERSSON Bengt , ENGBERG Catherine , STERN Ingrid , LAKE Mats , FRANZÉN Sven‐Åke , ISRAELSSON Marianne , RÅDÉN Benny , LINDGREN Björn , HJELMQVIST Lars , ENERBÄCK Sven , CARLSSON Peter , BJURSELL Gunnar , POVOA Guilherme , HALL Kerstin , JÖRNVALL Hans
Published: March 2005

Two different insulin‐like growth‐factor (IGF)‐binding proteins have been found in human blood, one of high molecular mass and dependent on growth hormone for synthesis, the other of low molecular mass and independent of growth hormone. The small IGF‐binding protein is abundant in human amniotic fluid. Its amino acid sequence has now been determined by direct analysis of the protein and its proteolytic fragments. Also, by immunoscreening a partial cDNA clone was isolated from a human hepatoma cell line. The mature protein consists of 234 amino acids and is coded for by an mRNA of approximately 1700 nucleotides in length. The primary structure of the protein reveals 18 Cys residues in N‐terminal and C‐terminal clusters and an Arg‐Gly‐Asp peptide sequence, common to extracellular proteins binding to receptors of the integrin family. A protein‐sequence polymorphism was detected at position Ile/Met‐228, indicating possible allelic variation. The 3′‐untranslated mRNA sequence has a high A + T content and shows five copies of an ATTTA sequence which has been shown to be involved in the regulation of the stability of certain mRNAs coding for growth‐regulating proteins.
Secretion‐stimulating and secretion‐inhibiting hormones stimulate high‐affinity pertussis‐toxin‐sensitive GTPases in membranes of a pituitary cell line
Authors: OFFERMANNS Stefan , SCHULTZ Günter , ROSENTHAL Walter
Published: March 2005

Different peptide hormones influence hormone secretion in pituitary cells by diverse second messenger systems. Recent data indicate that luteinizing‐hormone‐releasing hormone (LHRH) stimulates and somatostatin inhibits voltage‐dependent Ca2+ channels of GH3 cells via pertussis‐toxin‐sensitive mechanisms [Rosenthal et al. (1988) EMBO J. 7, 1627–1633]. In other pituitary cell lines, somatostatin has been shown to cause a pertussis‐toxin‐sensitive decrease in adenylate cyclase activity, and LHRH and thyrotropin‐releasing hormone (TRH) stimulate phosphoinositol lipid hydrolysis in a pertussis‐toxin‐independent manner. Whether stimulation of Ca2+ influx by TRH is affected by pertussis toxin is not known. In order to ellucidate which of the hormone receptors interact with pertussis‐toxin‐sensitive and ‐insensitive G‐proteins, we measured the effects of LHRH, somatostatin and TRH on high‐affinity GTPases in membranes of GH3 cells. In control membranes, both LHRH and TRH stimulated the high‐affinity GTPases by 20% somatostain by 25%. Maximal hormone effects were observed at a concentration of about 1 μM. Pretreatment of cells with pertussis toxin abolished pertussis‐toxin‐catalyzed [32P]ADP‐ribosylation of 39–40‐kDa proteins in subsequently prepared membranes and reduced basal GTPase activity. The toxin also reduced by more than half the increases in GTPase activity induced by LHRH and TRH; stimulation of GTPase by somatostatin was completely suppressed. Stimulation of adenylate cyclase by vasoactive intestinal peptide (VIP) was not impaired by pretreatment of cells with pertusis toxin. Somatostatin but not LHRH and TRH decreased forskolin‐stimulated adenylate cyclase activity. The results suggest that the activated receptors for LHRH and TRH act via pertussis‐toxin‐sensitive and ‐insensitive G‐proteins, whereas effects of somatostatin are exclusively mediated by pertussis toxin‐sensitive G‐proteins.
Inhibition and active‐site modelling of prolidase
Authors: KING Glenn F. , CROSSLEY Maxwell J. , KUCHEL Philip W.
Published: March 2005

Consideration of the active‐site model of prolidase led us to examine azetidine, pyrrolidine and piperidine substrate analogs as potential in vivo inhibitors of the enzyme. One of these, N‐benzyloxycarbonyl‐L‐proline, was shown to be a potent competitive inhibitor of porcine kidney prolidase (Ki= 90μM); its rapid protein‐mediated permeation of human and sheep erythrocytes suggests that it may be effective in vivo. The higher homolog, N‐benzyloxycarbonyl‐L‐pipecolic acid, was also a potent inhibitor of the enzyme while the antihypertensive drugs, captoprilR and enalaprilatR, were shown to have mild and no inhibitor effects, respectively

Analysis of inhibitor action and consideration of X‐ray crystallographic data of relevant Mn2+ complexes allowed the active‐site model of prolidase to be further refined; a new model is presented in which the substrate acts as a bidentate ligand towards the active‐site manganous ion. Various aspects of the new model help to explain why Mn2+ has been ‘chosen’ by the enzyme in preference to other biologically avialable metal ions.
Biochemical characterization of φX174‐protein‐E‐mediated lysis of Escherichia coli
Authors: WITTE Angela , LUBITZ Werner
Published: March 2005

Energetic and permeability properties of Escherichia coli cells were determined prior to and during lysis caused by expression of the cloned gene E of bacteriophage φX174. Before onset of cell lysis the transmembrane gradients for K+, Na+ or Mg2+/ions, the level of ATP and the membrane potential, were unaffected. All these parameters changed simultaneously at the time of lysis onset, as monitored by measurements of culture turbidity as well as by determining the various specifications over a period of 1 min. During cell lysis chromosomal DNA was fragmented whereas plasmid DNA was liberated in its intact supercoiled form. Cytoplasmic constituents were released almost entirely, as indicated by the activity of β‐galactosidase in the supernatant fraction of protein‐Elysed cells.

Periplasmic enzymes were only found in limited amounts in the cell supernatant and most remained associated with the cell ghosts. Such ghosts exhibited no gross cell damage or morphological alterations when compared with intact E. coli by light microscopy. All parameters investigated indicated that protein‐E‐mediated lysis of E. coli is caused by the formation of a transmembrane tunnel structure through the envelope complex of the bacterium.
Comparison of computer simulations of the F‐type and L‐type non‐oxidative hexose monophosphate shunts with 31P‐NMR experimental data from human erythrocytes
Authors: MCINTYRE Lisa M. , THORBURN David R. , BUBB William A. , KUCHEL Philip W.
Published: March 2005

Mathematical modelling was used to predict the behaviour of the two most favoured schemes for the operation of the non‐oxidative hexose monophosphate shunt (HMS), the F‐type and the L‐type pathways. The models simulate the time courses of sugar‐phosphate concentrations when various substrates are metabolized via each pathway. A 31P‐NMR technique, with which to observe time courses of concentrations of sugar phosphates in a human red cell lysate, was developed. The accuracy of each hypothesised scheme was then evaluated by comparing predicted with observed data.

The results were more consistent with time courses of sugar‐phosphate levels predicted by the F‐type (classical) pathway than those predicted by the L‐type model. However, the accumulation of sedoheptulose 1,7‐bisphosphate when a haemolysate was incubated with ribose 5‐phosphated showed that the F‐type pathway is not a complete description of the system of reactions. Transaldolase was demonstrated to be essential for the normal metabolism of sugar phosphates by haemolysates. The effects of the heat‐inactivation of transldolase on the metabolism of sugar phosphates were accurately predicted by the F‐type model.

The relevance of attempting to describe the reaction of the non‐oxidative HMS as a distinct ‘pathway’ or ‘cycle’ is discussed.