bluuebunny/crossref_metadata_2025 huggingface dataset:
A 60‐kDa protein from rabbit reticulocytes specifically recognizes the capped 5′ end of β‐globin mRNA
Authors: SCHWEMMLE Martin , SCHICKINGER Jürgen , BADER Michael , SARRE Thomas F. , HILSE Kurt
Published: March 2005

The binding of proteins from rabbit reticulocyte lysate to in‐vitro‐generated β‐globin mRNA and its defined segments was investigated using ultraviolet‐cross‐linking experiments as well as gel‐retardation assays. Under stringent conditions, only three proteins (72, 60 and 50 kDa) were found associated with full‐length β‐globin mRNA at different positions. The 72‐kDa protein is most likely the poly(A)‐binding protein and binds, as expected, to the poly(A) tail, whereas the 50‐kDa protein exhibits affinity for the trailer region of β‐globin mRNA. The binding region of the 60‐kDa protein is located at the 5′ end of β‐globin mRNA. The interaction of this protein is dependent on the presence of the 5′ cap structure, as indicated by competition experiments using an uncapped β‐globin‐mRNA leader segment. Further competition experiments with β‐globin mRNA, deleted in part in the leader region, suggest that, besides the cap structure, certain sequence elements are necessary for the interaction of the 60‐kDa protein and the β‐globin mRNA leader.
An amplified insect dihydrofolate reductase gene contains a single intron
Authors: SHOTKOSKI Frank A. , FALLON Ann Marie
Published: March 2005

We have used methotrexate‐resistant mosquito (Aedes albopictus) cells as the source of DNA for cloning an 8.5‐kb EcoRI fragment containing an amplified dihydrofolate reductase (DHFR) gene. An estimated 1200 copies of the DHFR gene were represented in nuclear DNA from Mtx‐5011‐256 cells, which were 3000‐fold more resistant to methotrexate than wild‐type cells. Southern blot analysis indicated that all of the amplified DHFR genes were contained within a 1.8‐kb AccI fragment represented in the cloned DNA. In contrast to mammalian DHFR genes which span approximately 30 kb, the complete amino acid coding sequence of the mosquito DHFR gene spanned 614 nucleotides, including a single 56‐nucleotide intron that interrupted a conserved Arg codon at amino acid position 27. Additional introns characteristic of mammalian DHFR genes were absent; conservation of the first intron in the mosquito DHFR gene supports a regulatory role for this intron. The mosquito DHFR gene coded for a 186‐amino‐acid protein with 43–48% similarity to vertebrate DHFR.
Possible involvement of Lys603 from Escherichia coli glucosamine‐6‐phosphate synthase in the binding of its substrate fructose 6‐phosphate
Authors: GOLINELLI‐PIMPANEAU Béatrice , BADET Bernard
Published: March 2005

Pyridoxal 5′‐phosphate is a competitive inhibitor of glucosamine‐6‐phosphate synthase with respect to the substrate fructose 6‐phosphate. Irreversible inactivation of pyridoxal‐5′‐phosphate‐treated enzyme with [14C]‐cyanide resulted in covalent incorporation of close to 1 mol pyridoxal 5′‐phosphate/mol enzyme subunit. The enzyme‐ pyridoxal‐5′‐phosphate complex could also be inactivated by reduction with NaBH3CN. Sequence analysis of the unique radioactively labelled tryptic peptide, resulting from inactivation with [3H]NaBH3CN, identified the C‐terminal nonapeptide encompassing the modified Lys603. The presence of fructose 6‐phosphate protected this residue from pyridoxylation. Direct evidence that a lysine residue is involved in the binding of the substrate as a Schiff base came from the isolation at 4°C of a enzyme‐fructose‐6‐phosphate complex in a 1:1 molar ratio. Treatment of the enzyme‐[14C]fructose‐6‐phosphate complex with NaBH3CN revealed one site of modification in the tryptic peptide map. In contrast, trapping the same complex with potassium cyanide resulted in the isolation of several radiolabelled peptides containing lysines which could potentially bind fructose 6‐phosphate. However, since the radioactivity was not specifically associated with the lysine residues, it is suggested that these 14C‐labelled peptides resulted from the decomposition of an unstable α,α′‐dihydroxyaminonitrile adduct rather than from a lack of specificity of fructose 6‐phosphate fixation. Lys603 is then the candidate of choice for fructose 6‐phosphate binding since it lies at or near the active site as demonstrated by the trapping experiments with pyridoxal 5′‐phosphate described above, and among the lysines which belong to the sugar‐binding domain this is the only one conserved between the three members of the purF, glutamine‐dependent, amidotransferase subfamily which include the glucosamine‐6‐phosphate synthase from Escherichia coli, Saccharomyces cerevisiae and the Rhizobium nodulation protein NodM.
The conformation of porcine‐brain natriuretic peptide by two‐dimensional NMR spectroscopy
Authors: CRAIK David , MUNRO Sharon , NIELSEN Katherine , SHEHAN Philip , TREGEAR Geoff , WADE John
Published: March 2005

Two‐dimensional (2D) 1H‐NMR spectra of porcine‐brain natriuretic peptide (pBNP) have been recorded at 300 MHz and 400 MHz. Peak assignments have been made and the combined information from chemical shifts, coupling constants, temperature coefficients and NOEs have been used to determine the conformational properties of pBNP in (C2H3)2SO. Overall the peptide appears to be flexible, with the possibility of some β‐type structure near the C terminus. Some of the assignments and deduced structural features in the current study differ from those in a recent report by Inooka et al. [Inooka, H., Kikuchi, T., Endo, S., Ishibashi, Y., Wakimasu, M. and Mizuta, E. (1990) Eur. J. Biochem. 193, 127–134] which may indicate the sensitivity of the structure of this peptide to differences in solution conditions.
Yolk protein in leech
Authors: BAERT Jean‐Luc , BRITEL Mostafa , SLOMIANNY Marie‐Christine , DELBART Christiane , FOURNET Bernard , SAUTIERE Pierre , MALECHA Jean
Published: March 2005

Theromyzon tessulatum vitellin was identified as a lipoglycoprotein of 490 kDa. The insolubility of this molecule in low‐ionic‐strength media was used to extract it from the ovaries. Antiserum prepared against vitellin was shown to react with a coelomic fluid component fluid component of 520 kDa. This vitellin precursor, or vitellogenin, was purified by gel permeation and ion‐exchange column chromatography. These two lipoglycoproteins were characterized by amino acid, carbohydrate and lipid analysis and subunit composition. In spite of differences in terms of native molecular mass, solubility and isoelectric point, the lipoglycoproteins isolated from the coelomic fluid and the ovary were similar in their subunit components (a single polypeptide of 165 kDa) and in their amino acid and carbohydrate compositions. However, vitellogenin was found to be more highly lipidated (31.8% by mass) than vitellin (24% by mass) and lipid analysis indicated a higher amount of sterols and phospholipids in vitellogenin. From these data, we conclude that vitellogenin and vitellin are probably dimers of two identical subunit polypeptides plus lipid and that, after vitellogenin is sequestered in the oocyte, part of its lipid component is stripped from the molecule to give vitellin. Furthermore, electrophoretic analysis seems to indicate that vitellogenin synthesis and secretion is initiated following the third and last blood meal of the animal but that vitellogenin significantly accumulates in the coelomic fluid before being incorporated in the oocytes suggesting a complex mode of vitellogenesis regulation.
Structural studies of yeast Iso‐1 cytochrome c mutants by resonance Raman spectroscopy
Authors: HILDEBRANDT Peter , PIELAK Gary J. , WILLIAMS Robert J. P.
Published: March 2005

The Ser82 and Phe82 variants of yeast iso‐1 cytochrome c were studied by resonance Raman spectroscopy. In both oxidation states, distinct spectral changes were observed for some of those bands in the low‐frequency region, which sensitively respond to conformational perturbations of the protein environment of the heme. These bands can be assigned to modes which include strong contributions of vibrations largely localized in the propionate‐carrying pyrrole rings A and D. This indicates structural differences in the deeper part of the heme crevice, remote from the mutation site. This conclusion is in line with previous results from X‐ray crystallography and NMR spectroscopy. No differences in the resonance‐Raman spectra were observed which can be directly correlated with conformational changes of the heme pocket in the vicinity of the mutation site. Temperature‐dependent resonance Raman experiments of the oxidized mutants revealed spectral changes which are closely related to those observed for cytochrome c upon adsorption to charged silver surfaces by surface‐enhanced resonance Raman spectroscopy. These spectral changes can be attributed to an opening of the heme crevice accompanied by a weakening of the iron‐methionine ligand bond. The temperature‐dependent conformational transition occurs at approximately 30°C for the Ser82 variant and at about 45°C for the Phe82 variant, implying that the Phe → Ser substitution significantly lowers the thermal stability of the heme pocket. The reduced forms of both mutants are stable up to 65°C.
N‐Myristoyl‐transferase activity in cancer cells
Authors: BOUTIN Jean Albert , CLARENC Jean‐Pierre , FERRY Gilles , ERNOULD Anne‐Pascale , REMOND Georges , VINCENT Michel , ATASSI Ghanem
Published: March 2005

The activity catalyzed by N‐myristoyl tranferase (NMT) is described for the first time in microsome‐rich fractions from the murine leukemia cell line L1210, rat brain and mouse liver as biological sources. The enzyme from each source can accomodate various types of proteins (protein kinase A, virus structural gag protein or pp60src) as modelized by the use of their N‐terminal derived peptides (GNAAAARR, GQTVTTPL and GSSKSKPKDP, respectively).

As for some other types of membrane‐bound enzymes, NMT activity can be enhanced by pretreatment with various types of detergents, amongst which Triton 770 and deoxycholate were the most potent. Further experiments on the L1210 microsome‐rich fractions demonstrate that these two detergents were able to solubilize the microsomal enzyme, without modifying its substrate specificy.

Finally, three compounds described in the literature to be inhibitors of NMT activity from other sources were tested for L1210 microsome‐associated activity. None of them show any significant potency in inhibiting this activity.

A new compound, myristoylphenylalanine, shows a slightly better inhibitory effect on the L1210 microsomal activity than the reference compounds with a median inhibitory concentration (IC50) of 0.2 mM.
Membrane proteins exposed on the external side of the intestinal brush‐border membrane have fusogenic properties
Authors: THURNHOFER Herbert , LIPKA Gert , HAUSER Helmut
Published: March 2005

The intestinal brush‐border membrance contains one or several membrane proteins that mediate fusion and/or aggregation of small unilamellar egg phosphatidylcholine vesicles. The fusion is accompained by a partial loss of vesicle contents. Proteolytic treatment of the brush‐border membrane with proteinase K abolishes the fusogenic property. This finding suggests that the fusogenic activity is associated with a membrane protein exposed on the external or luminal side of the brush‐border membrane. Activation of intrinsic proteinases of the brush‐border membrane liberates water‐soluble proteins (supernate proteins). These proteins behave in an analogous way to intact brush‐border membrane vesicles; they induce fusion of egg phosphatidylcholine vesicles and render the egg phosphatidylcholine bilayer permeable to ions and small molecules (Mr≤ 5000). Furthermore, supernate proteins mediate phosphatidylcholine and cholesterol exchange between two populations of small, unilamellar phospholipid vesicles. Supernate proteins are fractionated on Sephadex G‐75 SF yielding three protein peaks of apparent Mr≥ 70 000, Mr= 22 000 and Mr= 11 500. All three protein fractions show similar phosphatidylcholine‐exchange activity, but they differ in their effects on the stability of egg phosphatidylcholine vesicles. The protein fraction with an apparent Mr≥ 70 000 has the highest fusogenic activity while the protein fraction of apparent Mr= 11 500 appears to be most effective in rendering the egg phosphatidylcholine bilayer permeable.
Cloning of a cDNA for a new member of the class of fibril‐associated collagens with interrupted triple helices
Authors: GORDON Marion K. , CASTAGNOLA Patrizio , DUBLET Bernard , LINSENMAYER Thomas F. , VAN DER REST Michel , MAYNE Richard , OLSEN Bjorn R.
Published: March 2005

cDNA from embryonic chick skin has been isolated and characterized which encodes a novel member of the FACIT (fibril‐associated collagen with interrupted triple helices) group whose other known members are collagen type IX and XII. Nucleotide sequence analysis of the cDNA, combined with characterization of a pepsin‐resistant fragment of the protein from embryonic chick skin, demonstrates that the collagen chain is more closely related to the chain of type XII collagen than to those of type IX. It is most similar to a collagen, type XIV, recently identified in bovine skin. It is possible, therefore, that the cDNA codes for a chain of chicken type XIV collagen. From the additional data on molecular structure obtained by sequencing the cDNA, the FACIT family appears to consist of at least two classes of molecules: one of which contains the three chains of type IX collagen, and a second which includes the chains of collagen types XII and XIV.
Conformation of [8‐arginine]vasopressin and V1 antagonists in dimethyl sulfoxide solution derived from two‐dimensional NMR spectroscopy and molecular dynamics simulation
Authors: SCHMIDT Jürgen M. , OHLENSCHLÄGER Oliver , RÜTERJANS Heinz , GRZONKA Zbigniew , KOJRO Elzbieta , PAVO Imre , FAHRENHOLZ Falk
Published: March 2005

Structural and dynamic properties of [8‐arginine]vasopressin and a class of highly potent vasopressin V1 antagonists which contain 3‐mercepto‐3, 3‐cyclopentamethylene propionic acid (Mca) in position 1 of the vasopressin sequence have been determined. On the basis of two‐dimensional NMR experiments in dimethyl sulfoxide solution, interproton distances were derived according to which model conformations were built and refined using molecular dynamics simulations. The antagonistic property was found to be related to an inversed chirality of the disulfide bridge. In all investigated molecules, characteristic β‐turn structure elements were found for the backbone conformation of the endocyclic residues Tyr2–Asn5. For this portion of the peptide sequence, various conformational equilibria were detected which matched different time scales. For [Arg8]vasopressin, averaged NMR parameters were obtained which could be explained by rapid interconversion between different β‐turn geometries, whereas multiple slowly exchanging conformations were observed for the V1 antagonists. V1 antagonists containing sarcosine in position 7 exhibited multiple spectral patterns for the exocyclic part attributed tocis/trans isomerization. The X‐ray structure of deamino‐oxytocin [Wood, S. P., Tickle, I. J., Treharne, A. M., Pitts, J. E., Mascarenhas, Y., Li, J. Y., Husain, J., Cooper, S., Blundell, T. L., Hruby, V. J., Buku, A., Fischman, A. J. & Wyssbrod, H. R. (1986) Science 232, 633–636] was found to represent one sample of the conformational space covered by the multiple conformations found for [Mca1, Arg8]vasopressin.
Characterisation of the DNA‐polymerase‐α–primase complex from the silk glands of Bombyx mori
Authors: NIRANJANAKUMARI Somashekarappa , GOPINATHAN Karumathil P.
Published: March 2005

Silk gland cells ofBombyx mori undergo chromosomal endoduplication throughout larval development. The DNA content of both posterior and middle silk gland nuclei increased by 300000 times the haploid genomic content, amounting to 18 rounds of replication. The DNA doubling time is approximately 48 h and 24 h during the fourth and fifth instars of larval development. However, DNA content does not change during the interim moult. Concomitant with DNA content, DNA polymerase activity also increases as development progressed. Enzyme activity is predominantly due to DNA polymerase α with no detectable level of polymerase β. DNA polymerase α from silk gland extracts was purified to homogeneity (using a series of columns involving ionexchange, gel‐filtration and affintiy chromatography), resulting in a 4000‐fold increase in specific activity. The enzyme is a heterogeneous multimer of high molecular mass, and the catalytic (polymerase) activity is resident in the 180‐kDa subunit. The enzyme shows a PI of 6.2 and theKm values for the dNTP vary over 5–16 μM. The polymerase is tightly associated with primase activity and initiates primer synthesis in the presence of ribonucleoside triphosphates on a single‐stranded DNA template. The primase activity is resident in the 45‐kDa subunit. The enzyme is devoid of any detectable exonuclease activity. The abundance of DNA polymerase α in silk glands and its strong association with the nuclear matrix suggest a role in the DNA endoduplication process.
Characterization and 400‐MHz 1H‐NMR analysis of urinary fucosyl glycoasparagines in fucosidosis
Authors: MICHALSKI Jean‐Claude , WIERUSZESKI Jean‐Michel , ALONSO Catherine , CACHE Philippe , MONTREUIL Jean , STRECKER Gérard
Published: March 2005

Fucosyl glycoasparagines accumulating in the urine of a patient with fucosidosis were isolated using reversephase HPLC. Structural analysis of 25 glycoasparagines was carried out by combination of methylation and 400 MHz 1H‐NMR spectroscopy analyses. The compounds represent different steps in the incomplete catabolism of N‐glycosidically linked glycans, as the result of an α‐l‐fucosidase deficiency. All of the glycoasparagines possess a fucose residue α‐1,6‐linked to the GlcNAc 1 residue attached to asparagine. Fucose residues on the peripheral branches were linked either α‐1,3 to GlcNAc residues (X determinant) or α‐1,2 to galactose residues (H determinant). The present study allows precise assignments of the NMR parameters for most of the fucosyl linkages occurring inN‐glycosidically linked glycans of theN‐acetyllactosamine type.
Molecular cloning and characterisation of a novel putative protein‐serine kinase related to the cAMP‐dependent and protein kinase C families
Authors: COFFER Paul J. , WOODGETT James R.
Published: March 2005

Highly degenerate oligonucleotide primers designed from regions conserved between protein‐serine kinases have been used specifically to amplify human epithelial (HeLa) cDNA by the polymerase chain reaction (PCR). Of several novel cDNA fragments encodingputative kinases thus isolated, one was further characterised. Screening of human fibroblast and bovine brain cDNA libraries with the PCR fragment yielded several clones with an open reading frame of 479 amino acids containing all of the conserved sequence motifs of protein‐serine kinases. The predicted protein was most similar to the protein kinase C (PKC)/cAMP‐dependent protein kinase (PKA) families and its gene has thus been termedPkb. Expression of thePkb gene is general but highest in brain, heart and lung. Translation ofPkb RNAin vitro generated a 57‐kDa protein (PKB) recognised by antisera raised to a bacterially expressed PKB/TrpE fusion protein. Transfection of COS cells with the kinase cDNA resulted in the synthesis of a 60‐kDa protein which was partially purified purified by Mono Q anion‐exchange chromatography. Column fractions containing PKB‐immunoreactive protein exhibited elevated histone H1 kinase activity compared with similar fractions from control cells, demonstrating the enzymatic activity of this protein kinase.
Specificity determinants for tyrosine protein kinases
Authors: DONELLA‐DEANA Arianna , STONE Stuart R. , PINNA Lorenzo A.
Published: March 2005

Hirudin, the powerful anticoagulant agent of leech (Hirudo medicinalis) saliva, was readily phosphorylated by two spleen tyrosine protein kinases (TPK‐IIB and TPK‐III) at Tyr63 withKm values of 238 μM and 74 μM, respectively. The synthetic tridecapeptide DGDFEEIPEEYLQ, corresponding to the hirudin 53–65 C‐terminal fragment, was phosphorylated even more efficiently than hirudin itself. Four hirudin mutants, in which one or more of the glutamic acids at positions 57, 58, 61 and 62 have been replaced by glutamines, were poorer substrates than hirudin. The mutant in which all four glutamates were substituted, [Gln57, 58, 61, 62]hirudin, was virtually not phosphorylated by either TPK‐IIB. Substitution of Glu57 and Glu58 was less deleterious than substitution of the two glutamic acids adjacent to Tyr63: [Gln61,62]hirudin exhibited a 20‐fold lower phosphorylation efficiency with TPK‐IIB. With TPK‐III, however, theKm value of[Gln61,62]hirudin was slightly lower, while theVmax decreased sixfold. The substitution of Glu62 alone was also more detrimental with TPK‐IIB than with TPK‐III. The behaviour of a third spleen TPK, namedlyn TPK‐I and belonging to thesrc family, was markedly different in that it did not phosphorylate hirudin but exhibited significant activity towards [Gln57, 58, 61, 62]hirudin.

Taken together, these data confirm and extend with a protein substrate the results obtained with short model peptides which indicated the stringent substrate requirements of TPK‐IIB (and of TPK‐III to a lesser extent) for N‐terminal acidic residues. In contrast, such residues are deleterious withlyn TPK‐I. These observations also support the concept that tyrosine protein kinases recognize specificity determinants situated in the vicinity of the target residue rather than requiring higher‐order structural features.
Mechanism of NADPH oxidation catalyzed by horse‐radish peroxidase and 2,4‐diacetyl‐[2H]heme‐substituted horse‐radish peroxidase
Authors: DE SANDRO Virginie , DUPUY Corinne , KANIEWSKI Jacques , OHAYON Renée , DÈME Danielle , VIRION Alain , POMMIER Jacques
Published: March 2005

The mechanism of NADPH oxidation catalyzed by horse‐radish peroxidase (HRP) and 2,4‐diacetyl‐[2H]heme‐substituted horse‐radish peroxidase (DHRP) was studied. The roles of the different H2O2/peroxidase compounds were examined by spectral studies. The oxidized NADPH species were identified using the superoxide dismutase effect and by measuring the stoichiometry between NADPH oxidized and H2O2 used.

In the presence of a mediating molecule, like scopoletin, both enzymes acted via a similar mechanism, producing only NADP°, which in turn reacted with O2 producing O2. Consequently H2O2 was completely regenerated in the presence of superoxide dismutase and partially regenerated in its absence.

In the absence of a mediating molecule, the H2O2 complex of both enzymes (compound I) catalysed NADPH oxidation by single‐electron transfer, producing NADP°; compound II of these enzymes catalyzed NADPH oxidation more slowly by a direct two‐electron transfer, producing NADP+.

There were difference between HRP and DHRP. HRP compound II was produced by the oxidation of 1 mol NADPH/mole compound I, while DHRP compound II was formed by the spontaneous conversion of compound I to compound II. The NADPH oxidation catalyzed by DHRP compound I did not lead to the formation of compound II. When H2O2 was produced slowly by the glucose/ glucose‐oxidase system, compound II was never formed and a pure O2 adduct of DHRP (compound III) accumulated.
The superoxide‐generating oxidase of phagocytic cells
Authors: MOREL Françoise , DOUSSIERE Jacques , VIGNAIS Pierre V.
Published: March 2005

Professional phagocytes (neutrophils, eosinophils, monocytes and macrophages) possess an enzymatic complex, the NADPH oxidase, which is able to catalyze the one‐electron reduction of molecular oxygen to superoxide, O2−. The NADPH oxidase is dormant in non‐activated phagocytes. It is suddenly activated upon exposure of phagocytes to the appropriate stimuli and ther by contributes to the microbicidal activity of these cells. Oxidase activation in phagocytes involves the assembly, in the plasma membrane, of membrane‐bound and cytosolic components in phagocytes involves the assembly, in the plasma membrane, of membrane‐bound and cytosolic components of the oxidase complex, which were disassembled in the resting state. One of the membrane‐bound components in resting phagocytes has been identified as a low‐potential b‐type cytochrome, a heterordimer composed of two subunits of 22‐kDa. The link between NADPH and cytochrome b is probably a flavoprotein whose subcellular localizaion in resting phagocytes remains to be determined. Genetic defects in the cytochrome b subunits and in the cytosolic factors have been shown to be the molecular basis of chronic granulomatous disease, a group of inherited disorders in the host defense, characterized by severe, recurrent bacterial and fungal infections in which phagocytic cells fail generate O2− upon stimulation. The present review is focused on recent data concerning the singnaling pathway which leads to oxidase activation, including specific receptors, the producion of second messengers, the organization of the oxidase complex and the molecular defects responsible for granulomatous disease.
Purification and cDNA cloning of Xenopus laevis skin peptidylhydroxyglycine N‐C lyase, catalyzing the second reaction of C‐terminal α‐amidation
Authors: IWASAKI Yasuno , KAWAHARA Takashi , SHIMOI Hiroko , SUZUKI Kenji , GHISALBA Oreste , KANGAWA Kenji , MATSUO Hisayuki , NISHIKAWA Yoshiki
Published: March 2005

The α‐amidation of glycine‐extended peptides is a two‐step reaction catalyzed by peptidylglycine α‐hydroxylating monooxygenase (PHM) and peptidylhydroxyglycine N‐C lyase (PHL). PHL was purified to homogeneity from Xenopus laevis skin and its partial amino acid sequene (including the N‐terminal 35 residues) was determined. A cDNA, termed AE‐III, encoding X leavis PHL, was cloned and its complete nucleotide sequence was determined. It was found that the cDNA codes for a 935‐residue precursor protein (AE‐III protein), containing the PHM and PHL sequences at its N terminus and C terminus, respectively. The PHM sequence in AE‐III protein is completely identical to that deduced from the nucleotide sequence of X. laevis AE‐I cDNA, which encodes only PHM, except that the AE‐I protein has an extra 10 residues at its C terminus. It is suggested that AE‐I and AE‐III mRNA are encoded by the same gene and produced by alternative splicing.
Identification and characterization of the lantibiotic nisin Z, a natural nisin variant
Authors: MULDERS John W. M. , BOERRIGTER Ingrid J. , ROLLEMA Harry S. , SIEZEN Roland J. , de VOS Willem M.
Published: March 2005

Lactococcus lactis strain NIZO 22186 produces an extracellular, lanthinine‐containing 3.5‐kDa polypeptide with antimicrobial activity. Its retention time on reversed‐phase (RP) HPLC and its amino acid composition showed high similarities but no complete identity to nisin. The gene for this lantibiotic, designated nisZ, has been cloned and its nucleotide sequence was found to be identical to that of the precursor nisin gene apart from a single mutation resulting in the substitution His27Asn in the mature polypeptide. NMR studies of the natural nisin variant, which has been designated nisin Z, confirmed the His27Asn substitution and idicated that it has a similar structure to nisin.
Structure/activity relationship of eel calcitonin
Authors: INOUE Akira , SHIKANO Mayumi , KOMATSU Yasuhiko , OBATA Junko , OCHIAI Junko , NISHIDE Hiroko , ITO Noriko , NAGAO Hiromasa , KONDO Kiyoshi , TUNEMOTO Daiei , HEMMI Hiromichi , NUMAO Naganori
Published: March 2005

A series of analogs of eel calcitonin (eCT) was synthesized according to a newly devised scheme, ‘the insertion‐inactivation method’, to clarify the structure/activity relationship of a given peptide. This method consists of two steps: the deletion of a residue of the peptide is first chosen and then a series of analogs with the residue reinserted into serial positions is synthesized and biological activities are assessed in each step.

An analog lacking Lys18 (dK), selected as a deleted analog for the first step, showed marked loss of activities determined by inhibition of 125I‐eCT binding, growth inhibition, and cAMP production in a porcine kidney cell line LLC‐PK1. Activities of a set of 20 analogs with the reinserted lysine residue at serial positions from 12 to 32 (K12–K32) were then evaluated. The results showed the following three patterns of the expression of activities according to the position of the reinsertion: (a) analogs K12–K16 (positions 12–16) and K25 (position 25) showed lower activities than eCT in all assays; (b) K17–K24 (positions 17–24) showed slightly lower activities than eCT in the receptor binding and the growth inhibition and similar level in cAMP production; (c) K26–K32 (positions 26–32) showed considerably lower activities in the former two assays and slightly lower activity in cAMP production. Further, analogs considerably less active than eCT showed unchanged α‐helix contents and destroyed amphiphilicity by the insertion of a lysine residue, indicating that amphiphilicity is one of important factors for expressing the activity.

The results obtained here lead to a conclusion on the significance of each region of eCT molecule as follows: (a) the presence of Lys18 is necessary for the complete expression of biological activity; (b) the length of amphiphilic α‐helix to be required for the activity is at most 10 residues ranging from position 8 to position 17; (c) the receptor binding region is located within 9 residues ranging from position 24 to position 32.
Multiple mRNA species code for two non‐allelic forms of ovine αs2‐casein
Authors: BOISNARD Monique , HUE Dominique , BOUNIOL Christine , MERCIER Jean‐Claude , GAYE Pierre
Published: March 2005

The two non‐allelic forms of αs2‐casein, occurring in ovine milk, differ by an internal deletion of nine amino acid residues, at positions 34 and 42 in the mature chain. Sequencing of several αs2‐casein cDNA, isolated from the mammary cDNA library of a single lactating ewe, showed three new types which differed from that previously studied. In addition to the expected deletion of codons + 34 to + 42 affecting 30–40% of mRNA, another structural difference involving an internal stretch of 44 nucleotides in the 5′untranslated region, was found. S1‐nuclease protection assays confirmed the existence of several types of the relevant mRNA and sequencing of in‐vitro‐amplified genomic DNA demonstrated the presence of the 44‐nucleotide stretch in the αs2‐casein transcriptional unit, thus ruling out the possibility of a cloning artefact.

The different αs2‐casein mRNA, four in terms of deletion and two in terms of nucleotide substitutions for a given ewe, can be readily explained by partial exon skipping and allelic differences, respectively. This assumption is well supported by the following observations: 5′ and 3′ ends of both deleted DNA fragments are similar to those of exons; sequences neighbouring the 44‐nucleotide stretch of the genomic DNA perfectly match consensus sequences described for 3′ and 5′ ends of introns; the rather simple patterns observed on Southern blots of different enzymatic digests of genomic DNA strongly suggest the occurrence of only 1 copy αs2‐casein gene/haploid genome. During the course of evolution, the αs2‐casein‐encoding gene has undergone many mutations and some of them might have occurred in regions corresponding to consensus splicing regions of the pre‐mRNA. Thus, complete skipping of some exons might be responsible for the shorter sizes of rat and mouse αs2‐casein mRNA. If so, the overall organization of the αs2‐casein gene in the different species might be more similar than expected from structural comparisons of the cognate mRNA or caseins.
1H‐NMR conformational analysis of a high‐affinity antigenic 11‐residue peptide from the tryptophan synthase β2 subunit
Authors: DELEPIERRE Muriel , LARVOR Marie‐Pierre , BALEUX Françoise , GOLDBERG Michel E.
Published: March 2005

Two synthetic peptides from the β2 subunit of tryptophan synthase have been studied by 1H‐NMR spectroscopy at 300 MHz. One peptide, His‐Gly‐Arg‐Val‐Gly‐Ile‐Tyr‐Phe‐Gly‐Met‐Lys (peptide 11; Ile, isoleucine) is antigenic and binds with a high affinity to a monoclonal antibody that recognizes the native β2 subunit. The second peptide, His‐Gly‐Arg‐Val‐Gly‐Ile‐Tyr‐Phe (peptide 8) reacts very weakly with the antibody. The 1H‐NMR spectra of the two peptides have been assigned from two‐dimensional techniques in H2O, 2H2O and (2H6) dimethyl sulfoxide [(2H6)Me2SO]. The structure has been evaluated through analysis of nuclear Overhauser effects, coupling constants, amide‐proton exchange rates and their temperature coefficients, and chemical shifts.

In aqueous solvent, the C‐terminal part of peptide 11 presents some structure centered around residues Phe‐Gly‐Met. The relationship between the structure found in peptide 11 and its antigenic nature is discussed.
Spectroscopic characterization of true enzyme‐substrate intermediates of aspartate aminotransferase trapped at subzero temperatures
Authors: STERK Michael , GEHRING Heinz
Published: March 2005

Absorption and circular dichroism spectra of stable enzyme‐substrate intermediates of aspartate aminotransferase were recorded at subzero temperatures (down to ‐65°C) in the cryosolvent water/methanol. The intermediates were formed either between the pyridoxal from of the enzyme and its amino acid substrates, or between the pyridoxamine form and its oxo acid substrates. K′d values determind by spectroscopic titration were very close to the Km values reported for the different substrates. The adsorption complex of the pyridoxal form was probably obtained on addition of cysteine sulfinate. This complex is characterized by an increased absorption at 430nm together with a positive Cotton effect, as also observed in the case of the complex with the competitive inhibitor maleate indicating protonation of the internal aldimine. Addition of the substrates aspartate or glutamate to the pyridoxal form seemed to result in the direct accumulation of the external aldimine which showed a slight decrease in both the absorbance and the Cotton effect at 360nm. Additionally, a bathochromic shift of 5nm was observed in the case of glutamate. At 430nm, only a minor increase in absorbance, but not in circular dichroism, was observed with aspartate, and no changes were found with glutamate and the substrate analog 2‐methylaspartate, indicating a deprotonated external aldimine. Presumably, the ketimine intermediate was obtained on addition of the oxo acids 2‐oxoglutarate or oxalacetate to the pyridoxamine from. The intemediate showed a slight bathochromic shift (2nm) of the absorption band and decreased circular dichroism. On formation of the ketimine, a tyrosine residue, probably active‐site Tyr225, becomes partly ionized.

The finding that the external aldimine can probably be accumulated in the conversion of the pyridoxal to the pyridoxamine from with the natural substrates would confirm the proton abstraction at Cα to be the rate‐limiting step in the tautomerization, although with cysteine sulfinate, the formation of the external aldimine might contribute to the rate limitation. Accumulation of the ketimine in the reverse direction would indicate that the proton abstraction at C4′ is rate‐limiting in this half‐reaction. The results demonstrate the feasibility of further structural investigations of true enzyme‐substrate intermediates.
Characterization, stability and refolding of recombinant hirudin
Authors: OTTO Angelika , SECKLER Robert
Published: March 2005

A recombinant variant of hirudin, the blood‐clotting inhibitor of the leech Hirudo medicinalis, has been characterized employing spectroscopic and hydrodynamic techniques. Conditions have been defined for efficient reconstitution of the native, disulfide‐bonded inhibitor from completely unfolded, reduced polypeptide chains.

The spectral properties of the native inhibitor are consistent with previous results on the solution structure of hirudin. Extremely low circular dichroism in the far ultraviolet () indicates a very low content of regular secondary structure. Although both tyrosine residues of the recombinant inhibitor titrate around pH 10.6, typical for solvent‐exposed tyrosines, fluorescence emission and near‐ultraviolet circular dichroism suggest that at least one of the tyrosines is partially shielded from solvent quench, and immobilized in an asymmetric environment. Reversible thermal unfolding of hirudin around 65° C is indicated by the disappearance of its dichroic absorption in the near ultraviolet and by a fourfold increase in ellipticity at 225 nm. The transition can be approximated by a two‐state model with a transition enthalpy of Hvan't Hoff= 159 kJ/mol and a transition entropy of 464 J · mol−1· K−1. Reduced hirudin at room temperature is largely unfolded and inactive as an inhibitor of thrombin assayed with a low‐molecular‐mass substrate. Refolding and reoxidation are observed at alkaline pH in the presence of a mixture of glutathione and glutathione disulfide. Spectroscopy, thrombin inhibition, and reversed‐phase HPLC indicate reconstitution yields close to 100% and that the reconstituted inhibitor is identical to the native starting material.
Expression of rat heme oxygenase in Escherichia coli as a catalytically active, full‐length form that binds to bacterial membranes
Authors: ISHIKAWA Kazunobu , SATO Michihiko , YOSHIDA Tadashi
Published: March 2005

A plasmid, pKK‐RHO, was constructed by incorporating the coding sequence of a cDNA for rat heme oxygenase into the expression vector pKK233‐2. Escherichia coli strain XL1‐blue transformed with pKK‐RHO produced a catalytically active, full‐length heme oxygenase. The 32‐kDa native enzyme expressed, was localized in the bacterial membranes, possibly due to the spontaneous membrane‐binding properties of a hydrophobic segment in its C‐terminal region. During cultivation, a few degraded forms of heme oxygenase that had lost their membrane‐associative properties appeared. Probably, some bacterial proteases cut the native heme oxygenase at sites near its C‐terminus and so release hydrophilic peptides of heme oxygenase from the membranes. A 30‐kDa polypeptide, one of the degraded forms of heme oxygenase, retained ability to accept electrons from NADPH‐cytochrome P450 reductase and also activity for catalyzing breakdown of heme to biliverdin.

The cultured cells were pale green. From them we extracted green pigment(s), of which the absorption spectrum closely resembled that of biliverdin, suggesting that a large amount of the endogenous heme of E. coli was actually degraded to biliverdin by the expressed heme oxygenase.
Specificity of glucose transport in Trypanosoma brucei
Authors: SEYFANG Andreas , DUSZENKO Michael
Published: March 2005

Glucose transport in the bloodstream form of the protozoan parasite Trypanosoma brucei was characterized by enzymatically measuring the d‐glucose uptake. Uptake kinetics showed a concentration‐dependent saturable process, typical for a carrier‐mediated transport system, with an apparent Km= 0.49 ± 0.14 mM and Vmax= 252 ± 43 nmol · min−1· mg cell protein−1 (equal to 2.25 × 108 trypanosomes). The specificity of glucose transport was investigated by inhibitor studies. Glucose uptake was shown to be sodium independent; neither the Na+/K+‐ATPase inhibitor ouabain (1 mM) nor the ionophor monensin (1 μM) inhibited uptake. Transport was also unaffected by the H+‐ATPase inhibitor N,N′‐dicyclohexylcarbodiimide (DCCD; 20 μM) and the uncoupler carbonylcyanide‐4‐(trifluoromethoxy)phenylhydrazone (FCCP; 1 μM). However, highly significant inhibition was obtained with both phloretin (82% at 0.13 mM; Ki= 64 μM) and cytochalasin B (77% at 0.3 mM; Ki= 0.44 mM), and partial inhibition with phlorizin (14% at 0.5 mM; Ki= 3.0 mM). In each case, inhibition was noncompetitive, partially reversible (45%) for phloretin and completely reversible for cytochalasin B and phlorizin. Measurement of the temperature‐dependent glucose uptake between 25°C and 37°C resulted in a temperature quotient of Q10= 1.97 ± 0.02 and an activation energy of Ea= 52.12 ± 1.00 kJ/mol for glucose uptake. We conclude that glucose uptake in T. brucei bloodstream forms occurs via a facilitated diffusion system, clearly distinguished from the human erythrocyte‐type glucose transporter with about a 10‐fold higher affinity for glucose and about a 1000‐fold decreased sensitivity to the inhibitor cytochalasin B.
Role of aspartic acid 38 in the cofactor specificity of Drosophila alcohol dehydrogenase
Authors: CHEN Zhuo , LEE William R. , CHANG Simon H.
Published: March 2005

Drosophila alcohol dehydrogenase (ADH), an NAD+‐dependent dehydrogenase, shares little sequence similarity with horse liver ADH. However, these two enzymes do have substantial similarity in their secondary structure at the NAD+‐binding domain [Benyajati, C., Place, A. P., Powers, D. A. & Sofer, W. (1981) Proc. Natl Acad. Sci. USA 78, 2717–2721]. Asp38, a conserved residue between Drosophila and horse liver ADH, appears to interact with the hydroxyl groups of the ribose moiety in the AMP portion of NAD+. A secondary‐structure enzymes also suggests that Asp38 could play an important role in cofactor specificity. Mutating Asp38 of Drosophila ADH into Asn38 decreases Km(app)NADP 62‐fold and increases kcat/Km(app)NADP 590‐fold at pH 9.8, when compared with wild‐type ADH. These results suggest that Asp38 is in the NAD+‐binding domain and its substituent, Asn38, allows Drosophila ADH to use both NAD+ and NADP+ as its cofactor. The observations from the experiments of thermal denaturation and kinetic measurement with pH also confirm that the repulsion between the negative charges of Asp38 and 2′‐phosphate of NADP+ is the major energy barrier for NADP+ to serve as a cofactor for Drosophila ADH.
Atrial natriuretic peptide (ANP) is a high‐affinity substrate for rat insulin‐degrading enzyme
Authors: MÜLLER Dieter , BAUMEISTER Hans , BUCK Friedrich , RICHTER Dietmar
Published: March 2005

A cytosolic protein specifically binding to and degrading atrial natriuretic peptide (ANP) was purified from rat brain homogenate. Based on partial amino acid sequences and enzymatic properties, this protein with an apparent molecular mass of 112 kDa has been identified as the rat insulin‐degrading enzyme (IDE). In addition to the known substrates, insulin and transforming‐growth‐factor α IDE binds also with high affinity (apparent Kd 60 nM) to ANP. Competition studies with structural variants of ANP demonstrate that both the C terminus and the disulfide loop of the molecule are essential for high‐affinity binding. The data suggest that IDE might be involved in the cellular processing and/or metabolic clearance of ANP.
The effect of water content and nature of organic solvent on enzyme activity in low‐water media
Authors: van ERP Saskia H. M. , KAMENSKAYA Elvira O. , KHMELNITSKY Yuri L.
Published: March 2005

A simple theoretical model was suggested to describe quantitatively the effect of water content and nature of organic solvents on catalytic behavior of enzymes suspended in low‐water media. The model was based on a generally accepted notion that the destruction of the protein hydration shell is one of the main reasons for protein denaturation by organic solvents. The validity of the model was confirmed by the example of catalytic behavior of immobilized laccase suspended in water/organic mixtures of different compositions. In addition, the results were used to demonstrate that the effect of organic solvents and/or water content on catalytic behavior of enzymes in low‐water media can be adequately assessed only in terms of the full kinetic description based on properly determined Vm and Km values.
Biochemical and biophysical investigations of the ferrocene‐iron‐loaded rat
Authors: WARD Roberta J. , FLORENCE Anne L. , BALDWIN Dianne , ABIAKA Clifford , ROLAND Francine , RAMSEY Michael H. , DICKSON Dominic P. E. , PETERS Timothy J. , CRICHTON Robert R.
Published: March 2005

Male Wistar rats fed with ferrocene had high hepatic iron loading (7.24 ± 1.97 mg Fe/g tissue) after 6 weeks, principally located in lysosomes, which was comparable to the levels and distribution determined in human haemochromatosis.

The two iron‐storage proteins, ferritin and haemosiderin were isolated from the livers of the ferrocene‐loaded rats and their iron cores were investigated by Mössbauer spectroscopy and inductively coupled plasma‐emission spectrometry. Ferrihydrite was the predominant form of iron present in both ferritin and haemosiderin, while haemosiderin contained higher amounts of phosphorus, magnesium, calcium and barium, then either normal or ferrocene‐loaded ferritin.

Free‐radical‐mediated damage in the iron‐loaded livers was inferred by the significant depletion of α‐tocopherol in both the livers and subcellular hepatic lysosomal fraction, which inversely correlated with the increasing iron content (r=–0.61; P < 0.05) and was associated with increased fragility of the lysosomal membranes.
Efficient expression of bovine α‐lactalbumin in Saccharomyces cerevisiae
Authors: VIAENE Ann , VOLCKAERT Guido , JONIAU Marcel , De BAETSELIER Annie , Van CAUWELAERT Frans
Published: March 2005

A synthetic gene encoding the mature bovine α‐lactalbumin fused to the preproregion of the yeast α‐mating factor has been expressed and secreted at high level in Saccharomyces cerevisiae under the control of the α‐mating promotor. Growth conditions were found to be critical for the expression: recombinant α‐lactalbumin could only be detected in the medium provided the culture was grown at neutral pH. The secreted bovine α‐lactalbumin is enzymatically active and identical to the whey protein, as confirmed by SDS/PAGE, IEF, ultraviolet and CD spectral analysis, and amino‐terminal sequence determination.
Synthesis of dinucleoside polyphosphates catalyzed by firefly luciferase
Authors: Günther SILLERO Maria A. , GURANOWSKI Andrzej , SILLERO Antonio
Published: March 2005

In the presence of ATP, luciferin (LH2), Mg2+ and pyrophosphatase, the firefly (Photinus pyralis) luciferase synthesizes diadenosine 5′,5″′‐P1, P4‐tetraphosphate (Ap4A) through formation of the E‐LH2‐AMP complex and transfer of AMP to ATP. The maximum rate of the synthesis is observed at pH 5.7. The Km values for luciferin and ATP are 2–3 μM and 4 mM, respectively. The synthesis is strictly dependent upon luciferin and a divalent metal cation. Mg2+ can be substituted with Zn2+, Co2+ or Mn2+, which are about half as active as Mg2+, as well as with Ni2+, Cd2+ or Ca2+, which, at 5 mM concentration, are 12–20‐fold less effective than Mg2+. ATP is the best substrate of the above reaction, but it can be substituted with adenosine 5′‐tetraphosphate (p4A), dATP, and GTP, and thus the luciferase synthesizes the corresponding homo‐dinucleoside polyphosphates: diadenosine 5′,5″′‐P1, P5‐pentaphosphate (Ap5A), dideoxyadenosine 5′,5″′‐P1, P4‐tetraphosphate (dAp4dA) and diguanosine 5′,5″′‐P1, P4‐tetraphosphate (Gp4G). In standard reaction mixtures containing ATP and a different nucleotide {p4A, dATP, adenosine 5′‐[α,β‐methylene]‐triphosphate, (Ap[CH2]pp), (S)‐adenosine‐5′‐[α‐thio]triphosphate ((Sp)ATP[αS]) and GTP}, luciferase synthesizes, in addition to Ap4A, the corresponding hetero‐dinucleoside polyphosphates, Ap5A, adenosine 5′,5″′‐P1, P4‐tetraphosphodeoxyadenosine (Ap4dA), diadenosine 5′,5″′‐P1, P4‐[α,β‐methylene] tetraphosphate (Ap[CH2]ppA), (Sp‐diadenosine 5′,5″′‐P1, P4‐[α‐thio]tetraphosphate ((Sp)Ap4A[αS]) and adenosine‐5′,5″′‐P1, P4‐tetraphosphoguanosine (Ap4G), respectively. Adenine nucleotides, with at least a 3‐phosphate chain and with an intact α‐phosphate, are the preferred substrates for the formation of the enzyme‐nucleotidyl complex. Nucleotides best accepting AMP from the E‐LH2‐AMP complex are those which contain at least a 3‐phosphate chain and an intact terminal pyrophosphate moiety. ADP or other NDP are poor adenylate acceptors as very little diadenosine 5′,5″′‐P1, P3‐triphosphate (Ap3A) or adenosine‐5′,5″′‐P1, P3‐triphosphonucleosides (Ap3N) are formed. In the presence of NTP (excepting ATP), luciferase is able to split Ap4A, transferring the resulting adenylate to NTP, to form hetero‐dinucleoside polyphosphates. In the presence of PPi, luciferase is also able to split Ap4A, yielding ATP. The cleavage of Ap4A in the presence of Pi or ADP takes place at a very low rate. The synthesis of dinucleoside polyphosphates, catalyzed by firefly luciferase, is compared with that catalyzed by aminoacyl‐tRNA synthetases and Ap4A phosphorylase.