bluuebunny/crossref_metadata_2025_split huggingface dataset:
An arrestin homolog of blowfly photoreceptors stimulates visual‐pigment phosphorylation by activating a membrane‐associated protein kinase
Authors: BENTROP Joachim , PLANGGER Anette , PAULSEN Reinhard 
Published: March 2005

An arrestin homolog (Arr2, 49‐kDa protein) of blowfly (Calliphora erythrocephala) retinae undergoes light‐dependent reversible binding to the photoreceptor membrane. In order to characterize this arrestin homolog and to study its function in a well‐defined experimental system, we developed a purification scheme which used microvillar photoreceptor membranes as an affinity binding matrix. Additional purification steps included ammonium sulfate precipitation, gel filtration and binding to heparin‐agarose. The molecular mass of purified Arr2, as judged by SDS/PAGE, is in the range 45–49 kDa. The isoelectric point, as judged by gel isolelectric focussing, is 8.7. Arr2 is specific to the retina, where it is subject to phosphorylation at multiple sites.Binding of purified Arr2 to isolated photoreceptor membranes efficiently activates the light‐induced phosphorylation of visual pigment. Since the assay system used is deficient in rhodopsin phosphatase activity, the arrestin‐stimulated phosphate incorporation into rhodopsin results solely from the activation of a protein kinase. Phosphorylation experiments with highly purified membrane preparations indicate that rhodopsin kinase is tightly associated with the rhabdomeric membrane or the microvillar cytoskeleton. Rhodopsin kinase is released from the membrane or inactivated upon treatment with urea. It is concluded that this arrestin is a regulator protein that controls visual‐pigment phosphorylation by affecting the interaction of metarhodopsin and rhodopsin (metarhodopsin) kinase.
Secondary structure of the DNA‐binding domain of the c‐Myb oncoprotein in solution
Authors: JAMIN Nadége , GABRIELSEN Odd S. , GILLES Nicolas , LIRSAC Pierre‐Noël , TOMA Flavio 
Published: March 2005

The DNA‐binding domain of the c‐Myb oncoprotein contains two repeats, R2 and R3, both of which have been proposed to be related to the helix‐turn‐helix(HTH) motif. As a first step towards determination of the three‐dimensional structure of this domain and of the mode of interaction with the DNA, we have undertaken multidimensional heteronuclear NMR studies using uniformly 15N‐ labeled and 13C, 15N double‐labeled R2R3 and, a selectively 15N‐enriched sample on all lysine, histidine and leucine residues of R2R3. We present almost complete assignments of the backbone 1H, 15N and 13Ca atoms and determine the secondary structure of R2R3 in solution.The R3 repeat is composed of three helices (residues 62–75, 78–85 and 91–100) while for the R2 repeat only two helices are found (residues 10–23 and 28–34). The remaining C‐terminal part of the R2 repeat, predicted to be helical and part of the HTH motif, undergoes intermediate conformational exchange processes. Stabilization of this segment might occur upon binding to DNA.
Peptidylproline cis‐trans‐isomerases: immunophilins
Authors: GALAT Andrzej 
Published: March 2005

Two sequence‐unrelated families of proteins possess peptidylproline cis‐trans‐isomerase activities (PPlase). PPlases are highly sequence conserved and multifunctional proteins which are present in many types of cells with a considerably divergent phylogenetic distribution. On the cellular level, PPlases occur in every compartment, both as free species and anchored to membranes. Diverse posttranslational modifications such as glycosylation, N‐terminal modifications and phosphorylation constitute the additional functional features of PPlase. Folding, assembly and trafficking of proteins in the cellular milieu are regulated by PPlase. These enzymes accelerate the rate of in‐vitro protein folding and they have the ability to bind proteins and act as chaperones. Some PPlases are coregulatory subunits of molecular complexes including heat‐shock proteins, glucocortcoid receptors and ion channels. Secreted forms of PPlases are inflammatory and chemotactic agents for monocytes, eosinophils and basophils. The potent and clinically useful immunosuppressants CsA, FK506 or rapamycin bind with high affinities to PPlases (immunophilins). The binding criterion allows us to sort the PPlases for the following two superfamilies of proteins: the cyclophilins (CsA‐binding proteins) and the FKBP (FK506/rapamycin‐binding proteins).Although none of PPlases appeared to be essential for the viability of haploid yeast cells some of the immunophilin/immunosuppressant complexes are toxic both for yeast and mammalian cells. At least seven unlinked genes of cyclophilins and four unlinked genes of FKBP exist in human genomic DNA. Selected immunophilins regulate two different signalling pathways in lymphoid cells, namely the secretion of growth factors by stimulated T‐cells and interleukin‐2‐induced T‐cell proliferation. Moreover, selected FKBP mediate the cytotoxic effects of rapamycin in non‐lymphoid cells. Accounts of the discovery of PPlases (immunophilins) and their functions are given in this review. A larger spectrum of proteins is analysed in relation to various signal‐transduction pathways in lymphoid cells which involve immunophilins or their complexes with the immunosuppressants CsA, FK506 or rapamycin.
Gly85 to Val substitution in proα1(I) chain causes mild osteogenesis imperfecta and introduces a susceptibility to protease digestion
Authors: VALLI Maurizia , ZOLEZZI Francesca , MOTTES Monica , ANTONIAZZI Franco , STANZIAL Franco , TENNI Ruggero , PIGNATTI PierFranco , CETTA Giuseppe 
Published: March 2005

In this paper we describe a mild moderate form of osteogenesis imperfecta caused by a point mutation in COL1A1 which converted glycine 85 to valine.The valine substitution introduced into the triple‐helical domain of type‐I collagen a conformational perturbation causing susceptibility to digestive proteases. In fact, SDS/PAGE of pepsin‐treated collagen showed the presence of a faint band, migrating between α1(I) and α2(I), both in the medium and in the cell layer. On trypsin digestion the band, a shortened form of α1(I), had a melting temperature of 39.5°C. If the triple‐helical collagen was obtained after trypsin or chymotrypsin digestion of procollagen, two shortened bands were identified; the enzymes cleaved about 40% of the trimers. The mutant procollagen was normally secreted and processed in the extracellular matrix at a normal rate. When native type‐I collagen was formed after dextran‐sulfate incubation, only chains of normal length were found, suggesting that the fibroblast proteases did not recognize the alteration introduced by the mutation.The effects of glycine 85 to valine substitution are compared with those produced by a previously described arginine substitution of the same residue (Deak et al., 1991).
In vitro binding of nuclear proteins to the barley plastocyanin gene promoter region
Authors: NIELSEN Peter S. , GAUSING Kirsten 
Published: March 2005

Plastocyanin is a nuclear‐encoded chloroplast protein participating in electron transport during photosynthesis. The plastocyanin gene is expressed in photosynthetic tissue in a developmentally regulated manner and the expression is stimulated by light. A genomic clone encoding the plastocyanin precursor was isolated from a barley (Hordeum vulgare) λ library using a barley cDNA clone as a probe and the sequence of a 1.9‐kb DNA fragment containing the plastocyanin gene was determined. TATA and CCAAT boxes are located 34‐bp and 68‐bp, respectively, upstream of the transcription start site, the 5′‐untranslated leader is 78 nucleotides long, and the intronless gene has at least two different polyadenylation sites.DNA sites in the plastocyanin gene that mediate binding of barley nuclear proteins were mapped by mobility‐shift assays with fragments of the promoter/upstream region. Two of the three specific binding sites characterised in more detail were found to form complexes with the same factor in cross‐competition experiments. One of these sites, narrowed down to a 17‐bp sequence at position –512, contains the consensus binding site for Myb‐like transcription factors. The third specific binding site, located at position –622, contains the sequence CACGTG which is a high‐affinity‐binding site for transcription factors of the basic‐region leucine‐zipper family.
Cloning, site‐specific mutagenesis, expression and characterization of full‐length chloroplast NADP‐malate dehydrogenase from Pisum sativum
Authors: RENG Winfried , RIESSLAND Robin , SCHEIBE Renate , JAENICKE Rainer 
Published: March 2005

Chloroplast NADP‐dependent malate dehydrogenase is regulated by a dithiol redox reaction. The assignment of the groups involved, requires the primary structure of the enzyme to be known. Using the polymerase chain reaction and the cDNA library of Pisum sativum, the sequence of the enzyme and its targeting signal was determined. The gene was cioned in Escherichia coli JM83 and expressed in E. coli JM83 and E. coli B at high yield. The determination of the physical properties of the gene product proves the recombinant protein to be indistinguishable from the enzyme purified from the plant. This holds true, in spite of the fact that the plant enzyme lacks 11 N‐terminal residues. The lengths of the complete polypeptide chain of the recombinant enzyme and its transit peptide are 388 and 53 residues, respectively.The comparison of the sequences of the mature enzyme with those of known chloroplast NADP‐MDH shows 83–95% identity, but with mitochondrial or bacterial MDH only approximately 20%.Reduction of the (inactive) oxidized enzyme with dithiothreitol allows mimicking of the in vivo activation. The reaction follows a consecutive second‐order‐kinetics mechanism. Guanidinium chloride (GdmCl) at concentrations below 0.4 M leads to a significant activation of the oxidized form of the enzyme. At [GdmCl] = 0.4–0.46 M, both oxidized and reduced NADP‐MDH show highly cooperative changes in the hydrodynamic and spectral properties, indicating the synchronous breakdown of the quaternary, tertiary and secondary structures.Site‐directed mutations C23A and C28A do not quench the regulatory properties of the enzyme; additional substitution of alanine for Cys206 and Cys376 renders the enzyme equally active in both the reduced and the oxidized state. Therefore, one can consider these residues, either alone or in combination with Cys23 and Cys28, as responsible for enzyme activation.
Localization of a putative magnesium‐binding site within the cytoplasmic domain of the sarcoplasmic reticulum Ca2+‐ATPase
Authors: GIRARDET Jean‐Luc , BALLY Isabelle , ARLAUD Gérard , DUPONT Yves 
Published: March 2005

The amino acid sequences of several P‐type ATPases share regions of high similarity. The functions of some of these regions, although several proposals have been made, have not yet been absolutely identified. In particular, one of these domains, located within the cytoplasmic loop in the area known as the ‘hinge’ domain, exhibits the highest degree of conservation. In the sarcoplasmic reticulum Ca2+‐ATPase (SERCA‐1), this region is located at residues 700–712.Comparison of the sequence in this domain with calcium‐binding proteins reveals similarities with the center of the helix‐loop‐helix EF‐hand structure that is known to form divalent‐cation‐binding sites. A 38‐residue polypeptide, corresponding to the domain 682–719 of the Ca2+‐ATPase was synthesized and tested for its ability to bind divalent cations.Circular‐dichroism, intrinsic‐fluorescence and fluorescence‐energy‐transfer studies performed on this polypeptide in solution support the hypothesis that this domain has, in the protein, the ability to bind a divalent cation, presumably Mg2+, with an affinity of 10–15 mM. This property is observed for the isolated polypeptide in aqueous solvent and in the presence of low concentrations of the α‐helix promoter 2,2,2‐trifluoroethanol.Substitution of either one or two critical amino acids in the sequence induces a significant reduction of the binding properties.It is proposed that this sequence is involved in the co‐ordination of a Mg2+ in the nucleotide‐binding site and/or in the phosphorylation site of P‐type ATPases.
Isolation and characterization of three cDNAs coding for Rab proteins from the albumen gland of the mollusc Lymnaea stagnalis
Authors: AGTERBERG Marja , VAN DIE Irma , YANG Hui , ANDRIESSEN J. Aino , VAN TETERING Angelique , VAN DEN EIJNDEN Dirk H. , PLOEGH Hidde L. 
Published: March 2005

Three cDNA clones encoding small GTP‐binding proteins, LS‐Rab1, LS‐Rab2 and LS‐Rab18a were isolated from a cDNA library from the albumen gland of the pulmonate snail Lymnaea stagnalis. Comparison of the deduced amino acid sequences with available sequences from the EMBL/Data Bank revealed that LS‐Rab1 and LS‐Rab2 show a sequence identity of 89–90% to the mammalian Rab1 and Rab2 proteins, and can therefore be regarded as the L. stagnalis homologs. LS‐Rab18a may be considered a new member of the Rab subfamily, closely related to mouse Rab18 (74% amino acid identity). Interestingly, LS‐Rab1 and LS‐Rab2 share a very high sequence conservation with their mammalian homologs (95–97%) over the first 178–191 N‐terminal amino acids, whereas the C‐terminal part is almost completely divergent, except for their extreme ends (2–4 amino acids). The implications of these observations for the understanding of Rab‐targeting signals are discussed.The LS‐rab cDNAs were expressed in COS‐7M6 cells. The resulting 22‐kDa products were shown to bind GTP. In the albumen gland mRNA, levels of LS‐rab1 appeared to be much higher than those of LS‐rab2 and LS‐rab18a, suggesting an important role for the LS‐Rab1 protein in the albumen gland.
Amidase coupled with low‐molecular‐mass nitrile hydratase from Rhodococcus rhodochrous J1
Authors: KOBAYASHI Michihiko , KOMEDA Hidenobu , NAGASAWA Toru , NISHIYAMA Makoto , HORINOUCHI Sueharu , BEPPU Teruhiko , YAMADA Hideaki , SHIMIZU Sakayu 
Published: March 2005

The cloned 9.4‐kb insert of plasmid pNHJ20L containing low‐molecular‐mass nitrile hydratase (L‐NHase) gene from Rhodococcus rhodochrous J1 [Kobayashi, M. et al. (1991) Biochim. Biophys. Acta 1129, 23–33] was digested with various restriction enzymes, and the trimmed fragments were inserted into pUC18 or pUC19. A 1.96‐kb EcoRI–SphI region located 1.9‐kb downstream of the L‐NHase gene was found to be essential for the expression of amidase activity in Escherichia coli; the gene arrangement of the amidase and the NHase in R. rhodochrous J1 differed from those in Rhodococcus species including N‐774 and Pseudomonas chlororaphis B23. The nucleotide‐deter‐mined sequence indicated that the amidase consists of 515 amino acids (54626 Da) and the deduced amino acid sequence of the amidase had high similarity to those of amidases from Rhodococcus species including N‐774 and P. chlororaphis B23 and to indole‐3‐acetamide hydrolase from Pseudomonas savastanoi.The amidase gene modified in the nucleotide sequence upstream from its start codon expressed 8% of the total soluble protein in E. coli under the control of lac promoter. The level of amidase activity in cell‐free extracts of E. coli was 0.468 unit/mg using benzamide as a substrate. This amidase was purified to homogeneity from extracts of the E. coli transformant with 30.4% overall recovery. The molecular mass of the enzyme estimated by HPLC was about 110 kDa and the enzyme consists of two subunits identical in molecular mass (55 kDa). The enzyme acted upon aliphatic amides such as propionamide and also upon aromatic amides such as benzamide. The apparent Km values for propionamide and benzamide were 0.48 mM and 0.15 mM, respectively. This amidase was highly specific for the S‐enantiomer of 2‐phenylpropionamide, but could not recognize the configuration of 2‐chloropropionamide. It also catalyzed the transfer of an acyl group from an amide to hydroxylamine to produce the corresponding hydroxamate.
The amino acid sequence of human ribonuclease 4, a highly conserved ribonuclease that cleaves specifically on the 3′ side of uridine
Authors: ZHOU Hai‐Meng , STRYDOM Daniel J. 
Published: March 2005

A ribonuclease (RNase) that cleaves specifically on the 3′ side of uridine [Shapiro, R., Fett, J. W., Strydom, D. J. & Vallee, B. L. (1986a) Biochemistry 25, 7255–7264] was purified from human plasma and its amino acid sequence was determined. This protein is a 119‐residue single‐chain polypeptide cross‐linked by four disulfide bonds and has an amino‐terminal pyroglutaminyl residue. No post‐translational modifications were observed during extensive sequence studies on peptide fragments, except for the amino‐terminal pyroglutamic acid and a possible deamidation of Asn66. The protein is homologous to the pancreatic ribonucleases and angiogenin, but differs substantially from both of these proteins; the protein sequence has 43% identity with human pancreatic ribonuclease and 39% identity with human angiogenin, as compared to 35% identity between human angiogenin and pancreatic ribonuclease. It is referred to as RNase 4, based on the nomenclature currently used for the genes of pancreatic RNase (RNase 1) and the eosinophil‐derived RNases (RNase 2 and RNase 3).Virtually all of the RNase active‐site components, including the catalytic residues His12, His119 and Lys41, are preserved. However, some invariant residues of RNase 1 are replaced, e.g. Lys7 by arginine, Asp14 by histidine, and Pro42 by arginine. RNase 4 contains a unique two‐residue deletion at the position corresponding to amino acids 77 and 78 of pancreatic RNase, and its carboxy‐terminal sequence is truncated at position 122. The deletion in angiogenin at position 21 is also found in RNase 4.RNase 4 is very similar to two RNases isolated from bovine and porcine liver, and together they form a new family in the RNase superfamily. The degree of inter‐species similarity (90%) is much greater than within the pancreatic RNase and angiogenin families, which suggests that this ribonuclease could possess a physiologically important function other than general RNA catabolism.
Cloning, expression and mutational analysis of the urocanase gene (hutU) fromPseudomonas putida
Authors: LENZ Martin , RÉTEY János 
Published: March 2005

The histidine‐utilizinghutUgene was isolated from a λ‐EMBL3 phage of a genomic library fromPseudomonas putidanicII and subcloned into the expression vector pT7‐7.Escherichia coliBL21 cells were transformed with the recombinant plasmid and produced a catalytically active protein, amounting to approximately 30% of the total protein in the crude cell‐free extract. The addition of NAD+to the growth medium ensured the full occupation of active sites by the cofactor. This requires a mechanism for the transport of NAD+intoE. colicells.Using the overproducing mutant a new, fast and efficient isolation procedure is described which yields electrophoretically homogeneous urocanase within two days. The yield of pure enzyme, based on the culture volume, has been improved 50–80‐fold compared with the traditional method.To investigate the possible role of cysteine residues in the catalysis or in the tight binding of the cofactor NAD+, six different mutants were prepared. In each mutant protein, one conserved cysteine was exchanged for alanine. The resulting clones were tested for the expression of urocanase with catalytic activity; theKmandVmaxvalues were determined. Only Cys410 was essential for catalysis. There was no detectable reconstitution or increase of activity after the addition of NAD+, either in the essential Cys/Ala mutant or the other mutant proteins. Electrospray‐mass spectroscopy of the wild‐type enzyme revealed that the coenzyme is not covalently bound to the protein and computational analysis showed no typical sequence for a mononucleotide‐binding domain like the Rossman fold.To obtain urocanase apoenzyme,P. putidanicII was transformed with pGP1‐2 and pTET7‐U and grown in nicotinate‐depleted medium. Like the mutant proteins, no activation of the apoform occurred after the addition of NAD+. These observations led us to postulate a new model for the non‐covalent but tight binding of NAD+to the enzyme by ‘trapping’ the cofactor while folding the nascent protein.
Higher plant mitochondria encode an homologue of the nuclear‐encoded 30‐kDa subunit of bovine mitochondrial complex I
Authors: LAMATTINA Lorenzo , GONZALEZ Daniel , GUALBERTO José , GRIENENBERGER Jean‐Michel 
Published: March 2005

We describe the structure and expression of a wheat mitochondrial gene, which codes for a subunit of mitochondrial NADH dehydrogenase. The deduced protein sequence has 70% similarity to the 30‐kDa subunit of bovine mitochondrial complex I and 65% similarity to the 31‐kDa subunit of Neurospora crassa complex I, components of the iron‐sulfur‐protein fraction, both nuclear‐encoded proteins. We named this wheat mitochondrial gene as nad9. The wheat nad9 gene is transcribed in a single mRNA of 0.9 kb that is edited (C‐to‐U conversions) in 14 positions. Transcript mapping revealed that the first ATG codon is just 20 nucleotides downstream of the mRNA 5′ end and that the 3′ end is just 23 nucleotides downstream of the nad9 stop codonThe expression of the nad9 gene in plant mitochondria was studied. Polyclonal antibodies prepared against a wheat NAD9 fusion protein specifically recognise the 30‐kDa subunit of bovine mitochondrial complex I and a 27.5‐kDa protein in the membrane fractions of wheat, maize and common bean mitochondria, whereas the same serum recognizes a 30‐kDa protein in the mitochondria of pea, chickpea and lentil.
Structural and functional properties of a multi‐enzyme complex from spinach chloroplasts
Authors: RAULT Magali , GIUDICI‐ORTICONI Marie‐Thérèse , GONTERO Brigitte , RICARD Jacques 
Published: March 2005

Antibodies have been raised specifically against chloroplast phosphoribulokinase, glyceraldehyde‐3‐phosphate dehydrogenase and ribulose 1,5‐bisphosphate carboxylase‐oxygenase. Each of these antibodies recognizes the same macromolecular entity isolated and purified from chloroplasts. This entity is a multi‐enzyme complex, previously isolated and made up of ribose‐phosphate isomerase, phosphoribulokinase, ribulose 1,5‐bisphosphate carboxylase‐oxygenase, phosphoglycerate kinase and glyceraldehyde‐3‐phosphate dehydrogenase.Under denaturing conditions the multi‐enzyme complex contains two polypeptides of 54 kDa and 15 kDa corresponding to the large and the small subunits of ribulose 1,5‐bisphosphate carboxylase‐oxygenase, the two polypeptides of the glyceraldehyde‐3‐phosphate dehydrogenase of 39 kDa and 37 kDa, one polypeptide of 40 kDa pertaining to phosphoribulokinase and one polypeptide of 30 kDa very likely pertaining to ribose‐phosphate isomerase.The combined use of immunochemical and densitometric techniques allows one to determine the number and the stoichiometry of the various types of polypeptide chains and to compare them with the quaternary structure of the corresponding isolated enzymes. Ribulose 1,5‐bisphosphate carboxylase‐oxygenase of higher plants consists of eight large and eight small subunits. Glyceraldehyde‐3‐phosphate dehydrogenase is made up of two types of polypeptide chains called A and B and its simplest quaternary structure is A2B2. Finally, phosphoribulokinase is a dimer made up of two identical subunits. Therefore, for the three isolated enzymes, the stoichiometry of the polypeptide chains is always 1:1. Within this multi‐enzyme complex, there are two subunits of phosphoribulokinase, two A and B subunits of glyceraldehyde‐3‐phosphate dehydrogenase and two large and four small subunits of ribulose 1,5‐bisphosphate carboxylase‐oxygenase. Therefore the number and the stoichiometry of the polypeptide chains of phosphoribulokinase and glyceraldehyde‐3‐phosphate dehydrogenase are the same in the multi‐enzyme complex and in the free enzymes, but those of ribulose 1,5‐bisphosphate carboxylase‐oxygenase are completely different.This conclusion that the multi‐enzyme complex contains two active sites for ribulose 1,5‐bisphosphate may be confirmed independently by kinetic inhibition studies using 6‐phosphogluconate.