bluuebunny/crossref_metadata_2025 huggingface dataset:
Cloning and structural analysis of α‐latroinsectotoxin cDNA
Authors: KIYATKIN Nikita , DULUBOVA Irina , GRISHIN Eugene
Published: March 2005

α‐Latroinsectotoxin (α‐LIT), purified from venom glands of the black widow spider Latrodectus mactans tredecimguttatus, is a presynaptic neurotoxin selective only for insects. A cDNA encoding the putative α‐LIT precursor was isolated from a spider venom gland cDNA library. The cDNA contains a 4236‐base‐pair open reading frame corresponding to a 157826‐Da protein composed of 1411 amino acids. The mature α‐LIT, with molecular mass approximately 130 kDa, is probably derived from double processing in the N‐terminal and C‐terminal regions of the primary translation product. The structure region, extending over residues 464–1176, is composed almost entirely of ankyrin‐like repeats which represent a motif also found in the α‐latrotoxin (α‐LTX), which has selective action on vertebrates. Total alignment of the α‐LIT and α‐LTX amino acid sequences reveals an overall similarity of 34.1%. Strong sequence divergence is observed in analogous cysteine‐rich regions situated within the ankyrin‐repeat domains of both α‐LIT and α‐LTX.
Genetic screening in Saccharomyces cerevisiae for large numbers of mitochondrial point mutations which affect structure and function of catalytic subunits of cytochrome‐c oxidase
Authors: MEUNIER Brigitte , LEMARRE Philippe , COLSON Anne‐Marie
Published: March 2005

A new search for mitochondrial respiratory deficient mutants (Mit−) has been undertaken in order to accumulate a large number of point mutations in the coding portions of cytochrome‐c‐oxidase catalytic subunits and cytochrome b. Therefore, a mitochondrial DNA which retains the exons and lacks all the introns of the cytochrome oxidase subunit I and of the cytochrome‐b split genes has been introduced into a strain carrying a nuclear recessive mutation affecting the adenine‐nucleotide translocator, the op1 mutation, which is known to prevent the accumulation of large deletion petite mutants and this was used as the parental strain. After a moderate MnCl2 mutagenesis in order to limit multiple mutations, 105 Mit− mutants were isolated from 15000 mutagenised clones in Saccharomyces cerevisiae. Mutations were mapped to the three catalytic subunits encoding genes (COX1, COX2 and COX3) of the cytochrome‐c oxidase (70 mutations) and to the cytochrome‐b gene (15 mutations). More than 50% of the mutants tested still exhibited mitochondrial translation products (subunits I, II and III), suggesting that they carry a missense mutation, rather than a nonsense mutation which would normally have led to a truncated protein. Mutations in the COX1 gene were allocated to four different subregions corresponding to exons 4 and 8 or to groups of exons, exons 1, 2, 3 or exons 5, 6, 7. Seven missense monosubstitution mutations and two frameshift mutations were also identified. The amino acid changes of the missense mutations were located in the vicinity of the CuB‐heme a3 binuclear centre ligands.
Rapid screening of cytochromes of respiratory mutants of Saccharomyces cerevisiae
Authors: BROWN Simon , COLSON Anne‐Marie , MEUNIER Brigitte , RICH Peter R.
Published: March 2005

A technique has been developed for the direct analysis by visible spectrophotometry of yeast spots growing on agar plates. This allows rapid semi‐quantitative estimations of cytochromes c, b and oxidase and permits the identification of strains with impaired respiratory electron flow. Results of screening of 105 mutants are presented. There appears to be a correlation between the exonic location of the mutation in COX1 of oxidase and the level of optically detectable enzyme. Mutations in cytochrome b of the bc1 complex also affect the level of expression of cytochrome oxidase and can cause either an increased or decreased level of expression of oxidase relative to the wild‐type strain.

Twelve strains selected by the rapid level‐1 screening were grown as lawns on sections of an agar plate and resuspended for a second level of screening. Quantitative estimates have been made of the concentrations of cytochromes, the turnover number of cytochrome oxidase and the kinetics of recombination of carbon monoxide with oxidase after flash photolysis. This confirmed the validity of the rapid screening procedure, and we have identified several strains which contain high levels of a mutant form of cytochrome oxidase with properties worthy of further investigation.
Chymotryptic cleavage of lipoprotein lipase
Authors: LOOKENE Aivar , BENGTSSON‐OLIVECRONA Gunilla
Published: March 2005

Treatment of bovine lipoprotein lipase (LPL) with chymotrypsin results in cleavage between residues Phe390‐Ser391 and between Trp392‐Ser393, indicating that this region is exposed in the native conformation of LPL. Two main fragments are generated, one large including the aminoterminus (chymotrypsin‐truncated LPL = c‐LPL) and one small, carboxy‐terminal fragment. The small fragment is not stable, but is further degraded by the protease. Isolated c‐LPL has full catalytic activity against tributyryl glycerol (tributyrin) and p‐nitrophenyl butyrate, while the activity against emulsions of long‐chain triacylglycerols and against liposomes is reduced and the activity against milk fat globules and chylomicrons is lost. Several properties of c‐LPL were investigated. It was found that c‐LPL interacts with apolipoprotein CII (apo CII) as efficiently as intact LPL. The truncated enzyme bound to liposomes and to emulsions of long‐chain triacylglycerols as well as the intact enzyme did. In contrast, c‐LPL did not bind to milk fat globules or to chylomicrons. The activity of c‐LPL was more sensitive to inhibition by other lipid‐binding proteins, e.g. apolipoprotein CIII (apo CIII), than was the intact enzyme. The affinity for heparin was as high with c‐LPL as with intact LPL. Like intact LPL, c‐LPL is dimeric in its active form, as evidenced by sucrose density gradient centrifugation.

It is concluded that the reduced catalytic and lipid‐binding properties of c‐LPL compared with intact LPL are related to the properties of the substrate interface. It is speculated that the carboxyterminal part of LPL contains a secondary lipid‐binding site, which is important for activity against chylomicrons and related substrates.
Reconstitution studies of lipid effects on insulin‐receptor kinase activation
Authors: LERAY Valérie , HUBERT Pierre , BURGUN Claude , STAEDEL Cathy , CRÉMEL Gérard
Published: March 2005

Insulin receptors extracted from human placenta were reconstituted by dialysis into well‐characterized lipid vesicles. For all types of lipids studied, vesicles were shown to be unilamellar, about 120 nm in diameter. The incorporation of lectin‐purified insulin receptors was assessed by cosedimentation of 125I‐insulin binding and [32P]phospholipids in a sucrose gradient. The insulin‐binding activity was not modified by the composition of the lipid vesicles. However, tyrosine kinase activation appeared to be more sensitive to its lipid environment. Mixtures of phosphatidylcholine/phophatidylserine or phospholipids/phosphatidylserine, in ratios of 1–4, increased the insulin‐induced tyrosine kinase activation in a dose‐dependent manner. In contrast, experiments performed in the presence of phosphatidylinositol showed a decrease in the enzyme stimulation. These results indicate an opposing involvement of these two anionic phospholipids in the kinase activation. Inclusion of cholesterol (10–30%) into phosphatidylcholine vesicles reduced kinase activation, which was drastically inhibited by 30% cholesterol. The effect of a total extract of brain gangliosides was biphasic, stimulatory at low concentration (5–10%), but with a reverse effect at higher concentrations. These results stress the importance of the lipid environment for insulin‐receptor signaling, particularly for the insulin‐induced activation of its β‐subunit kinase.
Purification and characterization of an extremely thermostable β‐glucosidase from the hyperthermophilic archaeon Pyrococcus furiosus
Authors: KENGEN Servé W. M. , LUESINK Evert J. , STAMS Alfons J. M. , ZEHNDER Alexander J. B.
Published: March 2005

Cell‐free extracts of cellobiose‐grown cells of the hypermophile Pyrococcus furiosus contain very high activities (19.8 U/mg) of a β‐glucosidase. The cytoplasmic enzyme was purified 22‐fold to apparent homogeneity, indicating that the enzyme comprises nearly 5% of the total cell protein. The native β‐glucosidase has a molecular mass of 230 ± kDa, composed of 58 ± 2‐kDa subunits. The enzyme has a pI of 4.40. Thiol groups are not essential for activity, nor is the enzyme dependent on divalent cations or a high ionic strength. The enzyme shows optimum activity at pH 5.0 and 102–105°C. From Lineweaver‐Burk plots, Vmax values of 470 U/mg and 700 U/mg were found for cellobiose (Km= 20 mM) and p‐nitrophenyl‐β‐d‐glucopyranoside (Km= 0.15 mM), respectively. The purified enzyme also exhibits high βgactosidase activity and β‐xylosidase activity, but shows no activity towards α‐linked disaccharides or β‐linked polymers, like cellulose. The purified β‐glucosidase shows a remarkable thermostability with a half life of 85 h at 100°C and 13 h at 110°C.
Overproduction, purification and characterization of the bacterioferritin of Escherichia coli and a C‐terminally extended variant
Authors: ANDREWS Simon C. , SMITH John M. A. , HAWKINS Chris , WILLIAMS John M. , HARRISON Pauline M. , GUEST John R.
Published: March 2005

The bacterioferritin (BFR) of Escherichia coli is an iron‐sequestering haemoprotein composed of 24 identical polypeptide chains forming an approximately spherical protein shell with a central iron‐storage cavity. BFR and BFR‐λ, a variant with a 14‐residue C‐terminal extension, have been amplified (120‐fold and 50‐fold, respectively), purified by a new procedure and characterized. The overproduced BFR exhibited properties similar to those of natural BFR, but the iron content (25–75 non‐haem Fe atoms/molecule) was 13–39‐fold lower. Two major assembly states of BFR were detected, a 24‐subunit protein (tetracosamer) and a novel haem‐containing subunit dimer. BFR‐λ subunits assembled into tetracosamers having the same external‐surface properties as BFR, presumably because their C‐terminal extensions project into and occupy about 60% of the central cavity. As a result, BFR‐λ failed to take up iron under conditions that allowed incorporation into BFR in vitro. The haem content of BFR‐λ (1–2 haems/tetracosamer) was lower than that of BFR (3.5–10.5 haems/tetracosamer) and this, together with a difference in the visible spectra of the two haemoproteins, suggested that the C‐terminal extensions in BFR‐λ perturb the haem‐binding pockets. A subunit dimer form of BFR‐λ was not detected. A combination of Mössbauer spectroscopy and electron diffraction showed that the BFR loaded with iron in vitro has a ferrihydrite‐like iron core, whereas the in‐vivo loaded protein has an amorphous core.
Structural requirements of phosphatidylinositol‐specific phospholipase C δ1 for enzyme activity
Authors: ELLIS Moira V. , CARNE Alexander , KATAN Matilda
Published: March 2005

Phosphatidylinositol‐specific phospholipase C δ1 isozyme of phosphoinositol‐specific phospholipase C has been used for studies of structural requirements for the catalytic function. The enzyme was expressed in a bacterial system and purified to homogeneity. Using a combination of deletion mutant analysis and limited proteolysis, it was found that the large proportion of the molecule participated in formation of a catalytic domain (residues 139–756); it included regions of high and low conservation with other phospholipase‐C molecules. These studies also showed that the residues spanning regions of conservation, designated as X and Y, were exposed and highly susceptible to proteolysis by trypsin. Two of the fragments resulting from the cleavage (30 kDa and 40 kDa) interacted and, under non‐denaturing conditions, formed a protein of 70 kDa.
Generation of phosphatidic acid during calcium‐loading of human erythrocytes
Authors: BÜTIKOFER Peter , YEE Maggie C. , SCHOTT Mary Ann , LUBIN Bertram H. , KUYPERS Frans A.
Published: March 2005

We have studied the mechanism by which calcium‐loading of human erythrocytes stimulates phospholipid turnover and generates diacylglycerol and phosphatidic acid. Using quantitative measurement of individual phospholipid classes, we have demonstrated that the amount of phosphatidic acid generated during calcium‐loading of intact red cells exceeds the amount of diacylglycerol formed by phospholipase‐C‐mediated hydrolysis of the polyphosphoinositol lipids and that addition of the diacylglycerol kinase inhibitor, R59022, only partly inhibited this increase. Thus, in contrast to current explanations, the phosphatidic acid generated following calcium‐loading of erythrocytes cannot be solely explained by the action of a polyphosphoinositol‐lipid‐specific phospholipase C with subsequent phosphorylation of diacylglycerol to phosphatidic acid. Our data demonstrate that calcium‐loading of intact erythrocytes, but not of red cell ghost membranes, causes a small but significant decrease in the relative amount of phosphatidylcholine (PtdCho). In order to identify the mechanisms responsible for calcium‐mediated hydrolysis of PtdCho, we encapsulated Ptd[Me‐14C]Cho‐containing rat liver microsomes into erythrocytes and studied the generation of [Me‐14C]choline and phospho[Me‐14C]choline. We found that choline was the only detectable 14C‐labeled product. Furthermore, incubation of erythrocytes with calcium under hypotonic conditions and in the presence of [14C]PtdCho vesicles and ethanol resulted in the formation of [14C]phosphatidylethanol. Together, these results suggest that the loss of PtdCho during calcium‐loading of human erythrocytes is caused by a previously unrecognized PtdCho‐hydrolyzing phospholipase D, resulting in direct generation of phosphatidic acid. Analysis of the molecular species composition of PtdCho, phosphatidic acid, and diradylglycerol, confirm the simultaneous actions of PtdCho‐hydrolyzing and polyphosphoinositol‐lipid‐hydrolyzing phospholipases in calcium‐loaded human erythrocytes.
Expression of the Shigella dysenteriae type‐1 lipopolysaccharide repeating unit in Escherichia coli K12/Shigella dysenteriae type‐1 hybrids
Authors: FÄLT Inger C. , SCHWEDA Elke K. H. , WEINTRAUB Andrej , STURM Sabine , TIMMIS Kenneth N. , LINDBERG Alf A.
Published: March 2005

The structures of the polysaccharide part of lipopolysaccharides isolated from eight Escherichia coli K12/Shigella dysenteriae type 1 hybrids have been determined using sugar and methylation analysis plus 1H‐ and 13C‐nuclear magnetic resonance spectroscopy. The hybrids express parts of the S. dysenteriae type 1 O‐antigen tetrasaccharide repeating unit because of the presence of pSS3, a plasmid expressing an α‐galactosyl:lipopolysaccharide transferase and pSS9, a pBR322 plasmid expressing S. dysenteriae type 1 rfb genes. The various classes of hybrids are the result of transposon Tn 1000 insertions in pSS9 inactivating different rfb genes. The following structural elements were found.

E. coli K12 (pSS3) and E. coli K12 (pSS3, pSS9‐6; a class I hybrid); α‐d‐Galp(1→3)β‐d‐GlcpNAc(1→. Class IV hybrids: E. coli K12 (pSS3, pSS9‐36); (pSS3, pSS9‐107) and (pSS3, pSS9‐114); α‐l‐Rhap(1→2)α‐d‐Galp(1→3)β‐d‐GlcpNAc(1→. Class V hybrids: E. coli K12 (pSS3, pSS9‐78) and (pSS3, pSS9‐111); α‐l‐Rhap(1→3)α‐l‐Rhap(1→2)α‐d‐Galp(1→3)β‐d‐GlcpNAc(1→.

The structural sequences are identical to those found in the lipopolysaccharide from native S. dysenteriae type 1. In the hybrid strains, the terminal non‐reducing GlcNAc residue of the E. coli K12 core is fully substituted by S. dysenteriae type 1 repeating units, or parts thereof.
Regulation of elongation factor‐2 by multisite phosphorylation
Authors: REDPATH Nicholas T. , PRICE Nigel T. , SEVERINOV Konstantin V. , PROUD Christopher G.
Published: March 2005

We have studied the phosphorylation of protein synthesis elongation factor eEF‐2, the effects of phosphorylation on its activity and dephosphorylation of phosphorylated eEF‐2 by protein phosphatases‐2A and ‐2C. Extensive analysis of phosphopeptides generated from eEF‐2 phosphorylated in vitro, by subsequent digestion with CNBr and trypsin indicated that Thr56 and Thr58 are the only residues significantly phosphorylated, consistent with our earlier report. They are also the only two residues to be significantly phosphorylated in reticulocyte lysates: in this system mono‐phosphorylated eEF‐2 corresponded only to phosphorylation of Thr56, no factor phosphorylated at only Thr58 being detected. Phosphorylation of Thr56 and Thr58 was found to be an ordered process, modification of Thr56 preceding, and apparently being required for, phosphorylation of Thr58. This presumably explains why the only species of mono‐phosphorylated eEF‐2 detected are phosphorylated at Thr56.

The eEF‐2 kinase could phosphorylate a synthetic peptide based on residues 49–60 of eEF‐2 (RAGETRFTDTRK), albeit only at a very low rate, and with a very high Km, compared to eEF‐2 itself. The kinase phosphorylated the residues corresponding to Thr56 and Thr58, apparently in a random manner, but not Thr53.

In the light of the existence of two phosphorylation sites in eEF‐2, the relationship between phosphorylation and activity was investigated. Activity was measured in the poly(U)‐directed synthesis of polyphenylalanine, where both the bis‐ and mono‐phosphorylated (mono at Thr56) forms of the factor were found to be completely inactive. Indeed, the phosphorylated species appeared to be able to impair the activity of non‐phosphorylated eEF‐2 in this system. Experiments using reticulocyte lysates also indicated that both phosphorylated forms of eEF‐2 were inactive in the translation of physiological templates, but no evidence for dominant inhibition by these species was obtained.

Protein phosphatases‐2A and ‐2C (PP‐2A and PP‐2C) can each efficiently dephosphorylate phosphorylated eEF‐2. While bis‐phosphorylated eEF‐2 was a better substrate for PP‐2A than mono‐phosphorylated factor (phosphorlated at Thr56), the converse was true for PP‐2C. This seemed to be due, at least in part, to the inhibition of dephosphorylation of Thr56 by PP‐2C by the presence of phosphate on Thr58. Nevertheless, PP‐2C exhibited a preference for dephosphorylation of Thr56 in bis‐phosphorylated eEF‐2, while PP‐2A showed no such preference. These findings are discussed in terms of current knowledge of the specificity of these two protein phosphatases.
Are there different water requirements in different steps of a catalytic cycle?
Authors: BARRABIN Héctor , SCOFANO H. M. , de GÓMEZ‐PUYOU M. Tuena , GÓMEZ‐PUYOU A.
Published: March 2005

The Ca2+‐ATPase from sarcoplasmic reticulum was transferred in an active form to a low‐water system composed of toluene, phospholipids, and Triton X‐100 (TPT). The Ca2+‐ATPase activity in the TPT system with 4.0% water (by vol.) was about 50% of the activity observed in all‐aqueous mixtures. Phosphate formation was linear with time up to 20% of ATP hydrolysis and, as expected from an enzyme‐catalysed reaction, activity was linear with protein concentration. No ATPase activity was detected in the presence of 3 mM EGTA, indicating that the enzyme retained its Ca2+ dependence in the TPT system. A hyperbolic response to ATP concentration was observed with a Km of 0.15 mM. There was no detectable ATPase activity at water concentrations below 1.5% (by vol.). With 2.0% water, activity became detectable and increased as the water content was progressively raised to 7.0% (by vol.). Higher amounts of water produced unstable emulsions. Enzyme phosphorylation by ATP and dephosphorylation took place in the TPT system. The velocities of both enzyme phosphorylation and dephosphorylation increased with increments in the water content. The enzyme could also be phosphorylated in the TPT system by inorganic phosphate. However, in comparison to ATP, phosphorylation by phosphate took place with significantly lower amounts of water. It is suggested that at low amounts of water, the enzyme is in a relatively rigid conformation and, as the water content is increased, the ATPase acquires more flexibility and, hence, the capacity to carry out catalysis at higher rates. Nevertheless, the release of conformational constraints of the catalytic site of the E2 conformer takes place at water concentrations much lower than those needed for the expression of catalytic activity by the E1 conformer.
The action of Trypanosoma cruzi trans‐sialidase on glycolipids and glycoproteins
Authors: FERRERO‐GARCÍA Miguel Angel , TROMBETTA Sergio E. , SÁNCHEZ Daniel O. , REGLERO Angel , FRASCH Alberto C. C. , PARODI Armando J.
Published: March 2005

Addition of sialic acid residues in the human pathogen Trypanosoma cruzi glycoconjugates is mediated by a trans‐sialidase and not by a CMP‐sialic acid:glycoconjugate sialyltransferase. Incubation of trans‐sialidase with N‐[galactose‐14C]acetyllactosamine and O‐linked oligosaccharides, N‐linked glycopeptides (both obtained from fetuin) or sialyllactose showed that the last three compounds were donors of sialic acid residues to the first one. Moreover, N‐ and O‐linked oligosaccharides in asialofetuin and asialomucin, respectively, served as acceptors of sialic acid units. Gangliosides GM3, GD1a and GT1b but not GM2, GM1a GD1b donated sialic acid units to N‐acetyllactos amine when incubated with trans‐sialidase. This showed that only sialic acid units bound to terminal galactosyl residues were transferred. GM1a and GD1b, and GD1b to GT1b when incubated with the appropriate donor. The fact that asialo‐GM1a was converted to a ganglioside migrating as GD1a on thin‐layer chromatography suggested that sialic acid units may be transferred to internal galactosyl residues, although once linked to those residues they can not be further transferred to other glycoconjugates. Sialic acid residues linked α2,3‐ but not α2,6‐ or α2,8‐ were transferred by the trans‐sialidase. Methyl β‐galactoside but not methyl α‐galactoside served as acceptor of sialic acid units, thus suggesting that terminal α‐linked galactosyl units in T. cruzi and mammalian glycoproteins are not sialylated by the enzyme. As the trans‐sialidase employed in these experiments has been shown to be located on the external surface of the parasite and to be shed to the medium, the relatively broad specificity shown by the enzyme with respect to protein‐ and lipid‐linked oligosaccharides strongly suggests that infection by T. cruzi might alter the sialic acid distribution in glycoproteins and glycolipids of the mammalian host.
The rat mitochondrial 3‐hydroxy‐3‐methylglutaryl‐coenzyme‐A‐synthase gene contains elements that mediate its multihormonal regulation and tissue specificity
Authors: GIL‐GÓMEZ Gabriel , AYTÉ José , HEGARDT Fausto G.
Published: March 2005

Mitochondrial 3‐hydroxy‐3‐methylglutaryl‐coenzyme‐A (HMG‐CoA) synthase, a liver‐specific enzyme, is a constituent of the HMG‐CoA cycle responsible for ketone‐body synthesis.

We report the isolation and characterization of genomic clones that encompass the gene for rat mitochondrial HMG‐CoA synthase. The gene spans at least 24 kbp and contains ten exons and nine introns. The 5′ flanking region of the gene has also been cloned and characterized. Exon 1 contains the untranslated sequence of the transcript, extending downstream to enclose the coding region for the putative mitochondrial‐targeting signal (35 amino acids). The 1149‐bp proximal region of the transcription start point permits transcription of a reporter gene in transfected hepatoma cells but not in an extrahepatic cell line, confirming the function of the promoter. A truncated construct of 142 bp is still able to promote transcription in hepatoma cells, suggesting the presence of liver‐specific enhancer elements in the proximal promoter region. The 5′ flanking region contains typical promoter elements, including a TATA box and several putative recognition sequences for transcription factors involved in controlling both basal‐level and hormone‐modulated transcription rates. Furthermore, the presence in the mitochondrial HMG‐CoA‐synthase promoter of cis‐elements, responsible for the multihormonal regulation of transcription, is supported by transient transfection experiments.
Nucleotide binding and GTP hydrolysis by the 21‐kDa product of the c‐H‐ras gene as monitored by proton‐NMR spectroscopy
Authors: LÖW Andreas , SPRINZL Mathias , LIMMER Stefan
Published: March 2005

Proton‐NMR signals in the downfield region (below ∼ 10 ppm) have been shown to provide a useful spectroscopic window to monitor the binding of guanine nucleotides to the active site of GTP/GDP‐binding proteins via H‐bonds, as specified here by the 21‐kDa product of the c‐H‐ras gene (p21). The time/course of the intensity change of certain peaks upon addition of GTP to nucleotide‐free p21 corresponds to the GTP hydrolysis rate as determined by HPLC. Though there are fewer potential H‐bond acceptors in the GDP‐bound protein than in the GTP complex, more downfield peaks are found in the former complex, suggesting tighter binding of GDP. Moreover, inspection of the downfield proton‐NMR spectra permits rapid detection of subtle changes of the active site induced by complexation with slowly hydrolyzing GTP analogues resulting from mutations of the amino acid sequence, especially in the phosphate binding loop. Our studies strongly suggest that no major conformational change of the phosphate‐binding region occurs upon nucleotide complexation that precedes the catalytic step. Besides, it is suspected that the Ser17 hydroxyl group is involved in nucleotide binding and GTP hydrolysis.
A 1H‐NMR study of the transcription factor 1 from Bacillus subtilis phage SPO1 by selective 2H‐labeling
Authors: REISMAN Joseph M. , HSU Victor L. , JARIEL‐ENCONTRE Isabelle , LECOU Christian , SAYRE Michael H. , KEARNS David R. , PARELLO Joseph
Published: March 2005

1H‐NMR experiments have been performed on transcription factor 1 (TF1) encoded by Bacillus subtilis phage SPO1. To study this 22‐kDa homodimeric DNA‐binding protein, a selective 2H‐labeling strategy has been employed. Complete sequence‐specific assignments of all the resonances from the five aromatic residues were determined by a modified standard sequential‐assignment procedure. The reduced contribution of spin diffusion upon the long‐mixing‐time nuclear‐Overhauser‐enhancement spectroscopy for the selectively 2H‐labeled variants, as opposed to the fully 1H‐containing protein, has allowed for the identification of the spin system and of the long‐range dipolar contacts between Phe28 and Phe47 protons in the protein core and between Phe61 and Phe97 protons. The latter suggests an interaction between the proposed ß‐ribbon DNA‐binding arm and the carboxy terminus of the paired monomer. A previously proposed TF1 structural model [Geiduschek, E. P., Schneider, G. J. & Sayre, M. H. (1990) J. Struct. Biol. 104, 84–90)] has been modified using constrained‐energy‐minimization calculations incorporating the experimentally determined set of aromatic‐to‐aromatic contacts. This new model has been analyzed with regard to the relative mobility and the relative solvent accessibility of the aromatic residues which have been measured by the nonselective T1 relaxation times of the aromatic resonances for the fully 1H‐containing protein and the relaxation time enhancements upon selective 2H‐labeling, respectively.
Phosphorylatio of solubilised dark‐adapted rhodopsin
Authors: DEAN Kevin R. , AKHTAR Muhammad
Published: March 2005

A protocol for the separation of phosphorhodopsin from phospho‐opsin has been developed. The method takes advantage of the finding that, while 0.5%N,N‐dimethyldodecylamine‐N‐oxide completely solubilises membrane‐embedded phosphorhodopsin, at this concentration of detergent, phospho‐opsin is only sparingly soluble. Phosphorhodopsin solubilised in this manner may be freed from contaminant phospho‐opsin by chromatography on hydroxyapatite. Using this method, the rhodopsin‐kinase‐catalysed phosphorylation of photoexcited rhodopsin and native rhodopsin was studied in rod outer‐segment membranes at different levels of bleaching. Prior to analysis of the phosphorylation mixture, the phosphorylated form of photoexcited rhodopsin was converted into phospho‐opsin by treatment with NH2OH. It was found that, while at a 5% bleach level the amount of phosphorhodopsin produced was 15% that of phospho‐opsin, at 60% bleaching the phosphorhodopsin was less than 1% of phospho‐opsin. The phosphorylation reaction under different bleaching conditions was also studied in a completely soluble system (using 2% dodecyl maltoside) and the pattern of phosphate incorporation into rhodopsin versus opsin was identical to that in the membrane system. We have previously proposed that rhodopsin kinase normally exists in an inactive form and is only activated following interaction with photoexcited rhodopsin. The present work strengthens this conclusion and also shows that, following activation, the kinase preferentially phosphorylates photoexcited rhodopsin but can also act upon unbleached rhodopsin. Two possible mechanisms for the activation of the kinase are considered. From the distribution of phosphorhodopsin and phospho‐opsin at different bleaching levels, the relatives rates of the phosphorylation of photoexcited rhodopsin (kR*) and rhodopsin (kR) were calculated. KR*lkR values for the membrane system of 71 ± 20 and, for the solubilised system, of 80 ± 19 were obtained. The algebraic equation used to obtain these values highlights the fact that the ratio of the concentrations of the two substrates, photoexcited rhodopsin and rhodopsin, in a sample, determines the final distribution of phosphate between bleached and unbleached rhodopsin. This conclusion may contribute to the understanding of ‘high‐gain’ phosphorylation observed previously.
Isolation and characterization of a cDNA clone encoding a Pleurodeles lectin
Authors: TIFFOCHE Christophe , CHESNEL Amand , JEGO Patrick , LE PENNEC Jean‐Paul
Published: March 2005

A cDNA encoding a lectin secreted by the oviduct of Pleurodeles Waltl has been isolated and sequenced. The cDNA was identified by comparing the N‐terminal amino‐acid sequence of the purified P. Waltl lectin polypeptides with the amino‐acid sequence deduced from the cDNA. The two chains of the mature protein can be encoded within a unique mRNA. Two mRNA were also found in the oviduct extracts. However, they probably result from differential polyadenylation events. The mRNA are strictly localized in the anterior part of the oviduct and increase after estradiol stimulation, two characteristics which have been previously demonstrated for the protein. P. Waltl is known to possess a very high DNA content (∼2×1010bp) but the aforementioned results and Southern‐blot experiments suggest a unique or a least a very low gene‐copy number for this protein.

The amino‐acid sequence of the P. Waltl lectin deduced from the cDNA sequence shows similarities with the C‐type carbohydrate‐recognition domains of animal lectins as defined by Drickamer [Drickamer, K. (1988) J. Biol. Chem. 263, 9557–9560]. Although it is regulated by estradiol, the P. Waltl lectin amino‐acid sequence shows a higher similarity with animal lectins involved in the defence of the organism than with those involved in reproduction and development.
Conformational characterization by circular‐dichroism spectroscopy of various fragments and analogs of calcitonin‐gene‐related peptide
Authors: MIMEAULT Murielle , ST‐PIERRE Serge , FOURNIER Alain
Published: March 2005

A conformational study by circular‐dichroism spectroscopy of calcitonin‐gene‐related peptide (CGRP) and related fragments and analogs was carried out in structure‐promoting solvent mixtures. The structural characterization of rat CGRPα and the two isoforms of human CGRP, α and β, revealed that these peptides possess very similar conformational features. The far‐ultraviolet circular‐dichroism spectra, in pure water, of human CGRPα, (hCGRPα), [Acm‐Cys2,7]hCGRPα, various fragments and analogs indicated that these peptides exhibited predominantly a random‐coil conformation. The addition of increasing concentrations of 1,1,1,3,3,3‐hexafluoro‐2‐propanol to the peptide solutions resulted in a transition from a random‐coil conformation to a stabilized α‐helical structure. The substantial loss of helical content measured with [Acm‐Cys2,7]hCGRPα, [Acm‐Cys2,7]hCGRP‐(1–24)‐CONH2 and hCGRP‐(8–37), compared to hCGRPα, suggested that the N‐terminal disulfide bridge of hCGRPα is essential for adopting a highly stabilized α‐helical conformation. Moreover, the lower helical content of hCGRP‐(8–37), as compared to [Acm‐Cys2,7]hCGRPα, as well as spectroscopic results measured with various fragments and analogs of hCGRP‐(8–37) revealed that N‐terminal residues found in the peptide segment 1–12 are important for the full conservation of the amphiphilic α‐helix. In addition, the similar α‐helical content of hCGRP‐(8–37) and hCGRP‐(8–18) indicated that the C‐terminal segment 19–37 is not essential for the stabilization of the α‐helix structure.
Isomeric sn‐1‐octadecenyl and sn‐2‐octadecenyl analogues of lysophosphatidylcholine as substrates for acylation and desaturation by plant microsomal membranes
Authors: SPEERLING Petra , HEINZ Ernst
Published: March 2005

To provide supporting and independent evidence for lipid‐linked desaturation of acyl groups in plant microsomal membranes, ether‐analogous substrates were synthesized and used for in‐vitro desaturation studies. The substrates included 1‐O‐(9‐cis‐octadecenyl)‐sn‐glycero‐3‐phosphocholine and 2‐O‐(9‐cis‐octadecenyl)‐sn‐glycero‐3‐phosphocholine as well as labelled 1‐O‐(9‐cis‐[9,10‐3H2]octadecenyl)‐sn‐glycero‐3‐phosphocholine. In experiments with microsomal membranes from developing fruits of sunflower, it was shown that both isomeric alkenyl ether phospholipids were acylated with [14C]oleoyl‐CoA and [14C]palmitoyl‐CoA. In the presence of O2 and NADH, the oleoyl groups incorporated into both compounds, i.e. at the sn‐1 and sn‐2 positions of the glycerol back‐bone of the substrate, were desaturated to linoleoyl residues in similar proportions. Under the same conditions, an additional double bond, but not an enol‐ether double bond, was introduced into the ether‐linked side chain of acylated 1‐O‐(9‐cis‐[9,10‐3H2]octadecenyl)‐sn‐glycero‐3‐phosphocholine. This represents the first demonstration of this type of desaturation with an alkenyl ether phospholipid and confirms previous conclusions that plants introduce second and further double bonds into lipid‐linked acyl groups.
cDNA cloning and mitochondrial import of the β‐subunit of the human electron‐transfer flavoprotein
Authors: FINOCCHIARO Gaetano , COLOMBO Irma , GARAVAGLIA Barbara , GELLERA Cinzia , VALDAMERI Giulio , GARBUGLIO Nicoletta , DIDONATO Stefano
Published: March 2005

We have isolated a cDNA clone which encodes the entire β‐subunit of human electron‐transferring flavoprotein (ETF) by screening an expression library from human liver using polyclonal antibodies against porcine ETF. This cDNA encodes a protein of 255 amino‐acid residues with a predicted molecular mass of 27 877 Da which shows a high degree of similarity with partial amino acid sequences obtained from both rat liver and Paracoccus denitrificansβ‐ETF. Northern‐blot analysis shows that the human β‐ETF mRNA is approximately 1 kb in size and is abundant in liver, heart and skeletal muscle.

Incubation with intact mitochondria indicates that the cDNA‐encoded β‐ETF polypeptide contains the information necessary to reach the mitochondrial matrix. These data are in agreement with previous experiments suggesting that β‐ETF, unlike the majority of nuclear‐encoded mitochondrial matrix proteins, does not have a cleavable leader peptide. Furthermore, when valinomycin is added to the incubation mixture, the import is abolished, thus demonstrating that it is an energy‐dependent process.

Interestingly, the sequence analysis of β‐ETF protein identifies a 26.3% identity with the Fix A gene product of the nitrogen‐fixing bacterium Azorhizobium caulinodans.
Coordinate changes in the expression of troponin subunit and myosin heavy‐chain isoforms during fast‐to‐slow transition of low‐frequency‐stimulated rabbit muscle
Authors: LEEUW Thomas , PETTE Dirk
Published: March 2005

The purpose of this study was to follow the time course of changes in the expression of myosin heavy chain (HC) and troponin (Tn) subunit isoforms during fast‐to‐slow transition as induced in rabbit fast‐twitch muscle by low‐frequency stimulation. The evaluation of changes in the relative concentrations of myosin and troponin subunit isoforms were supplemented by measurements of relative protein synthesis rates using an in situ labeling technique. Changes in the amounts of mRNA encoding fast troponin C (TnC) were followed by Northern blot analysis, those for fast and slow troponin I (TnI) by in vitro translation of total RNA. The various fast myosin heavy chain (HC) and fast troponin T (TnT) isoforms were exchanged sequentially. Myosin HCIId which is the predominant fast isoform in rabbit tibialis anterior, was exchanged with HCIIa and, finally, the latter was replaced by the slow myosin HCI. The replacement of HCIId by HCIIa was accompanied by an exchange of TnT1f and TnT2f with TnT3f. The expression of HCI was accompanied by an exchange of TnT3f with the slow TnT isoforms, TnT1s and TnT2s. The changes in the relative concentrations of the TnT isoforms were preceded by similar changes of their relative synthesis rates. Pronounced decreases in the fast TnI and TnC isoforms occurred only with prolonged stimulation and were preceded by changes of the specific mRNAs and decreases in relative synthesis rates. The parallel time courses of the sequential transitions in both the myosin heavy chain and troponin T isoforms suggest the existence of coordinate programs of expression serving specific functional requirements.
Inhibitor‐2 functions like a chaperone to fold three expressed isoforms of mammalian protein phosphatase‐1 into a conformation with the specificity and regulatory properties of the native enzyme
Authors: ALESSI Dario R. , STREET Alasdair J. , COHEN Philip , COHEN Patricia T. W.
Published: March 2005

Three isoforms of mammalian protein phosphatase‐1 (PP1α, PP1β and PP1γ) were expressed in Escherichia coli and purified to near homogeneity. The activities of all isoforms towards phosphorylase, phosphorylase kinase and myosin and their sensitivities to inhibitor‐2 were similar to the native PP1 catalytic subunit (PP1C) isolated from vertebrate tissues. Like PP1C, they each formed a complex with the glycogen‐targetting(G) subunit which directs PP1C to glycogen particles in skeletal muscle. However, other properties differed strikingly from native PP1C. The expressed isoforms were 100–600‐fold less sensitive to inhibitor‐1, 3–5‐fold less sensitive to okadaic acid, 5–100‐fold less sensitive to microcystin‐LR and approximately 20‐fold more active in dephosphorylating histone H1 than native PP1C. Although PP1γ (like PP1C) was active in the absence of Mn2+, expressed PP1α and PP1β were completely dependent on Mn2+ for activity. PP1β, like PP1C, interacted with the myofibrillar‐targetting(M) complexes from skeletal‐muscle and smooth‐muscle producing species with enhanced myosin‐phosphatase activity, whereas expressed PP1α and PP1γ did not.

The expressed isoforms of PP1 combined with inhibitor‐2 to form an inactive complex (PP1I) that could be reactivated by the glycogen‐synthase‐kinase‐3(GSK3)‐catalysed phosphorylation of inhibitor‐2. This procedure transformed the properties of all three expressed isoforms to those of native PP1C. Their sensitivities to inhibitor‐1, okadaic acid and microcystin‐LR were increased greatly, their histone‐phosphatase activities decreased and the activities of PP1α and PP1β became independent of Mn2+. Furthermore PP1α and PP1γ now interacted with the M complexes in a similar manner to PP1β and PP1C. Conversely, incubation of native PP1C with 50 mM NaF caused conversion to a Mn2+‐dependent form with properties similar to those of the expressed isozymes.

The G subunit from skeletal muscle or the M complex from smooth muscle could displace PP1C from activated PP1I, but not inactive PP1I, to form G‐subunit/PP1C and M‐complex/PP1C heterodimeric complexes. Inhibitor‐2 was also found to be essential for the reactivation of PP1C from 6 M guanidinium chloride in the absence of Mn2+. Taken together, the results suggest that inhibitor‐2 is critical for the correct folding of nascent PP1C polypeptides, that its function is similar to that of a molecular chaperone and that it acts as a cytosolic reservoir of PP1C molecules which can be directed to the required subcellular locations following the synthesis of specific targetting subunits.
γ‐Butyrobetaine hydroxylase
Authors: RÜETSCHI Ulla , NORDIN Ingalill , ODELHÖG Birgit , JÖRNVALL Hans , LINDSTEDT Sven
Published: March 2005

γ‐Butyrobetaine hydroxylase is a 2‐oxoglutarate‐dependent dioxygenase that catalyzes the hydroxylation of γ‐butyrobetaine to carnitine, the last step in the biosynthesis of carnitine from lysine. The primary structure of the enzyme from Pseudomonas sp. AK1 has been determined. Sequence analysis of the intact protein and of peptides from essentially three different digests established the presence of a peptide chain containing 383 residues, and an N‐terminal truncated form of 382 residues. The two chains have molecular masses of 43 321 Da and 43 207 Da, respectively, and are identical except for the presence or absence of an N‐terminal asparagine residue; the shorter form starts with an alanine residue. In preparations of the dimeric protein, the two chains occur in an approximate ratio of 1:1. There are nine cysteine residues and 13 histidine residues, i. e. amino acids which have been postulated as ligands for iron binding. In spite of functional similarities, there appears to be no clear sequence similarities with any of the other mammalian 2‐oxoglutarate‐dependent dioxygenases so far characterized.
A cold‐inducibleBombyxgene encoding a protein similar to mammalian sorbitol dehydrogenase
Authors: NIIMI Teruyuki , YAMASHITA Okitsugu , YAGINUMA Toshinobu
Published: March 2005

To facilitate the study of the induction of sorbitol dehydrogenase by acclimation to 5 °C in diapause eggs of the silkworm,Bombyx mori, two cDNA libraries from eggs and larval fat bodies were screened with anti‐(sorbitol dehydrogenase) serum, and a positive cDNA was cloned from the fat‐body cDNA library. 1039 nucleotides determined from the cDNA corresponded to a protein‐coding region consisting of 346 amino acids. The missing regions (containing two amino acids at the 5′ end and a stop codon at the 3′ end) were supplemented with the genome sequence. The deduced amino‐acid sequence had 45–47% identity with mammalian sorbitol dehydrogenases. The results led us to conclude that the cDNA for aBombyxhomolog of mammalian sorbitol dehydrogenase was isolated, which was designated as BmSDH. Analyses of Northern hybridization and reverse transcription/polymerase chain reaction showed that the transcript of BmSDH occurred after chilling for 40–50 days when the diapause eggs were exposed to 5 & deg;C from two days after oviposition to break the diapause. The changing pattern in the amount of BmSDH transcript was well correlated with those in the activity of sorbitol dehydrogenase and the amount of the enzyme protein in diapause eggs. Further, the transcript of BmSDH was localized in yolk cells. The results indicate that the yolk nuclei‐dependent gene expression of BmSDH is induced by acclimation to 5 °C in diapause eggs.
A comparative laser‐flash absorption spectroscopy study of Anabaena PCC 7119 plastocyanin and cytochrome c6 photooxidation by photosystem I particles
Authors: MEDINA Milagros , DÍAZ Antonio , HERVÁS Manuel , NAVARRO José A. , GÓMEZ‐MORENO Carlos , DE LA ROSA Miguel A. , TOLLIN Gordon
Published: March 2005

Laser‐flash absorption spectroscopy has been used to investigate the kinetics of electron transfer from reduced cytochrome c6 and plastocyanin, isolated from Anabaena PCC 7119, to oxidized P700 in photosystem‐I particles isolated from the same cyanobacterium and from spinach. For all metalloproteins and photosystems, the observed rate constant has a non‐linear protein‐concentration dependence, thus suggesting complex formation preceding electron transfer. Plastocyanin and cytochrome c6 have similar association constants for complex formation with spinach photosystem I, but the copper protein exhibits a higher intracomplex‐electron‐transfer rate constant (twofold). With Anabaena photosystem I, the two redox proteins are more effective with respect to both complex formation (5–10‐fold) and electron transfer (1.5–4‐fold) than with the spinach photosystem. In all cases, the observed rate constants for electron‐transfer monotonically decrease with increasing NaCl or MgCl2 concentration. This is interpreted in terms of the involvement of attractive electrostatic interactions, which result in the initial collision complex having the most productive orientation for the electron transfer process, without a requirement for further reorientation. The magnitude of the response to MgCl2 suggests the occurrence of specific ion effects as well. In the absence of added salts, the reduction rate of oxidized P700 increases with pH from approximately 6 to 8, but decreases slightly at pH 8.5.
Stoichiometry and redox behaviour of metals in cytochrome‐c oxidase
Authors: STEFFENS Guy C. M. , SOULIMANE Tewfik , WOLFF Gerhard , BUSE Gerhard
Published: March 2005

The early observation of extra copper in preparations of cytochrome‐c oxidase has recently lead to a renewed interest in its stoichiometry and possible redox function. In various, pure preparations (heme A contents close to the theoretical value of 9.79 nmol/mg protein for the 13‐subunit bovine enzyme) protein‐related metal stoichiometries of 3 Cu, 2 Fe, 1 Zn, 1 Mg/monomer with Mr 204266 were determined.

Despite the presence of five potential redox metal ions, reductive and reoxidative titrations indicate the presence of only four one‐electron‐accepting/donating species in the ligand‐free enzyme.

Participation of two copper ions in a binuclear copper site acting as a one‐electron acceptor may explain both the observed copper stoichiometry and the redox behaviour. The homology of the C‐terminal sequence of subunit II with one of the copper‐binding sites in nitrous‐oxide reductases provides possible ligands for complexing two copper ions in a binuclear center.