bluuebunny/crossref_metadata_2025 huggingface dataset:
Inhibition of cyclic AMP efflux by insect pheromones and fatty acids
Authors: Kanter Joan R. , Hagey Lee R. , Brunton Laurence L.
Published: July 2002

Avian erythrocytes export cyclic AMP by a means that prostaglandins A1 and A2, but not other eicosanoids, inhibit (EC50 ≈ 45 nM). Several insect pheromones and the fatty acyl components of common membrane phospholipids also inhibit cyclic AMP efflux (EC50 ≈ 3O μM). The presence of at least one double bond in the acyl chain enhances the effect. Unlike PGA, fatty acids probably do not act via formation of a glutathione adduct but very likely by altering membrane fluidity. Inhibition of cyclic AMP export provides a mechanism by which products of phospholipid metabolism can influence the cyclic AMP signaling pathway.
A novel extracellular subtilisin inhibitor produced by a Streptomyces sp.
Authors: Kourteva Yordanka , Boteva Raina
Published: July 2002

The amino acid composition and inhibitory properties of a protein (SI‐1–72) isolated from the culture medium of a Streptomyces sp. have been investigated. SI‐1–72 appears to be a monomer protein of molecular mass about 13 100 Da and amino acid composition which differs from that of the inhibitors of the Streptomyces subtilisin inhibitor (SSI) family. Furthermore, it was found to exhibit novel specificity: strong inhibitory effect against microbial alkaline proteinases, moderate effect towards chymotrypsin and elastase, and no inhibition of the other serine proteinases, as well as of the cysteine, aspartate and metallo‐proteinases.
Modification of Bacillus subtilis elongation factor Tu by N‐tosyl‐L phenylalanyl chloromethane abolishes its ability to interact with the 3′‐terminal polynucleotide structure but not with the acyl bond in aminoacyl‐tRNA
Authors: Jonák Jiří , Karas Karel
Published: July 2002

Modification of B. subtilis EF‐TU by N‐tosyl‐L‐phenylalanyl chloromethane destroyed its ability to promote protein synthesis and resulted in selective dissociation of the two binding activities of the protein for aminoacyl‐tRNA. The modified EF‐Tu was completely ineffective in the protection of the 3′‐terminal CCA structure of tRNA against pancreatic ribonuclease, while remaining almost fully active in the protection of the ester bond between the 3′‐terminal adenosine and the amino acid residue in aminoacyl‐tRNA.
A nuclear protein associated with actively transcribed nucleosomes exhibits Zn2+‐binding activity
Authors: Liew C.C. , Chen H.Y.
Published: July 2002

The presence of the zinc‐finger motif in nuclear protein structure often confers specific DNA‐binding capabilities and, consequently, may implicate such proteins as being involved in gene regulation. We measured the zinc‐binding capacity of nuclear proteins by using a zinc (Zn2+) blotting technique. This assay revealed that some non‐histone proteins are selectively associated with this metal ion. Of particular interest, a nuclear protein, B2 (M
r 68000; pI 7.3), associated with actively transcribed nucleosomes and possibly involved with premessenger splicing, exhibits Zn2+‐binding activity. Moreover, this activity can be achieved with as little as 2 μg of nuclear protein and the zinc‐binding capacity is, on average, 2 ions per molecule of the protein B2.
Role of supernumerary subunits in mitochondrial cytochrome c oxidase
Authors: Planques Y. , Capitanio N. , Capitanio G. , De Nitto E. , Villani G. , Papa S.
Published: July 2002

Role of supernumerary subunits of bovine heart cytochrome c oxidase has been investigated by examining the influence on the enzymatic activity of their removal by Chromatographic procedures or controlled digestion by trypsin. Is has been shown that partial proteolytic cleavage of subunit IV results in depression of respiratory activity and of redox‐linked proton translocation. Selective removal by gel‐filtration of subunit VIb has no significant influence on the redox and protonmotive activity of the oxidase.
Tryptophan‐130 is the most reactive tryptophan residue in rabbit skeletal myosin subfragment‐1
Authors: Peyser Y.Michael , Muhlrad Andras , Werber Moshe M.
Published: July 2002

Rabbit skeletal muscle myosin subfragment‐1 (S‐1) was reacted with dimethyl(2‐hydroxy‐5‐nitrobenzyl)sulfonium bromide (DHNBS) resulting in modification of 0.8 tryptophan residues per S‐1. In order to assign the most reactive tryptophan of the 5 S‐1 tryptophans, antibodies were raised in rabbits against bovine serum albumin modified with DHNBS. The antibodies reacted with the 27 kDa tryptic fragment of DHNBS‐treated S‐1, indicating that the reactive tryptophan resides on this domain. The 27 kDa fragment was isolated from DHNBS‐treated S‐1 and was further cleaved at a single cysteine residue by 2‐nitro‐5‐thiocyanobenzoic acid. This cleavage resulted in two peptides, each of them containing one tryptophan. The antibodies reacted with the smaller peptide consisting of residues 122–204. The only tryptophan residing on this peptide is Trp130, and this is therefore the most reactive tryptophan of S‐1.
Structure of the gene encoding concanavalin A from Canavalia gladiata and its expression in Escherichia coli cells
Authors: Yamauchi Daisuke , Minamikawa Takao
Published: July 2002

We have cloned and sequenced the gene encoding concanavalin A (Con A) from Canavalia gladiata. The sequence covers the whole transcribed region as well as the 5'‐ and 3'‐untranscribed sequences. The coding sequence lacks introns. Con A expressed in Escherichia coli cells was purified by Sephadex G‐50 affinity chromatography. The precursor of Con A expressed in E. coli undergoes a peptide cleavage and ligation in the same way as that synthesized during seed maturation.
Comparison of mitochondrial cationic channels in wild‐type and porin‐deficient mutant yeast
Authors: Fèvre Florence , Chich Jean-François , Lauquin Guy J.M. , Henry Jean-Pierre , Thieffry Michel
Published: July 2002

Bilayers were formed at the tip of microelectrodes from a suspension of proteoliposomes derived from wild‐type and porin‐deficient mutant yeast mitochondria. In both preparations, identical cationic channels of large conductance were recorded. This result rules out any relationship between this channel and the outer membrane voltage‐dependent anion channel, the activity of which is carried by porin. The ionic selectivity and the voltage‐dependence of the yeast cationic channel suggest that it is related to that recently described in mammalian mitochondria. This hypothesis is further supported by the fact that both channels are blocked by a mitochondrial addressing peptide.
Lack of inhibition by colicin M suggests bactoprenol independence of MDO biosynthesis
Authors: Harkness Robin E. , Fiedler Waltraud , Braun Volkmar
Published: July 2002

Biosynthesis of membrane‐derived oligosaccharides (MDO), located in the periplasmic space of Escherichia coli, was not inhibited by colicin M, an inhibitor of bactoprenyl phosphate regeneration. This result suggests that bactoprenol does not serve as a lipid carrier of MDO oligosaccharides across the cytoplasmic membrane.
Competitive binding of the troponin T‐specific pool of caldesmon antibodies and tropomyosin to skeletal troponin T and smooth muscle caldesmon
Authors: Birukov Konstantin G. , Shirinsky Vladimir P. , Vorotnikov Alexander V. , Gusev Nikolai B.
Published: July 2002

The fraction of polyclonal caldesmon antibodies cross‐reacting with rabbit skeletal troponin T are shown to compete with smooth muscle tropomyosin for caldesmon and troponin T, as revealed by ELISA method. The epitope recognized by these antibodies was also found in M
r 77 kDa non‐muscle caldesmon. These results provide functional confirmation for the suggestion that the regions of amino acid sequence homology in caldesmon isoforms and troponin T belong to the tropomyosin binding sites.
McrI: a novel class‐II restriction endonuclease from Micrococcus cryophilus recognizing 5'‐CGRY/CG‐3'
Authors: Brensing-Küppers Jutta , Reischl Udo , Schmitz Gudrun S. , Kaluza Klaus , Jarsch Michael , Kessler Christoph
Published: July 2002

A new class‐II restriction endonuclease, McrI, with a novel sequence specificity as isolated from the Gram‐positive eubacterium Micrococcus cryophilus. McrI recognizes the palindromic hexanucleotide sequence The novel enzyme in the presence of Mg2+‐ions cleaves specifically both strands as indicated by the arrows. The staggered cuts generate 3'‐protruding ends with single‐stranded 5'‐RY‐3' dinucleotide extensions. The McrI recognition sequence was deduced from mapping data on DNAs of bacteriophages φX174RF and M13mp18RF characterized by one and four cleavage sites, respectively. The cut positions within both strands of the recognition sequence were determined in sequencing experiments by analyzing hydrolysis of phosphodiester bonds within a Polylinker region of M13mp18RF DNA containing an additional McrI recognition site including treatment with T4 DNA polymerase. The novel enzyme may be a useful tool for cloning experiments by completion of the enzymes EclXI (5'‐C/GGCCG‐3'), NotI (5'‐GC/GGCCGC‐3'), PvuI (5'‐CGAT/CG‐3') as well as EaeI (5'‐Y/GGCCR‐3') and XhoII (5'‐Y/GATCR‐3') characterized by partly identical sequence specificities.
Amino acid sequence of pheromone‐inducible surface protein in Enterococcus faecalis, that is encoded on the conjugative plasmid pPD1
Authors: Nakayama Jiro , Nagasawa Hiromich , Isogai Akira , Clewell Don B. , Suzuki Akinori
Published: July 2002

The major pheromone‐inducible protein, PD78, believed to contribute to bacterial conjugation, was purified from Enterococcus (formerly Streptococcus) faecalis cells containing the plasmid pPD1. A cloned EcoRI‐Bg/II 3.6‐kbp fragment of the plasmid pAM351 (pPD1::Tn916) contained an open reading frame corresponding to 467 amino acid residues representing PD78. In a central region of the deduced protein, there is a repeated sequence of X‐X‐Pro that is repeated 15 times. This is analogous to the Gln‐Gln‐Pro repeat in the C‐terminal region of TraD product encoded on the R 100 plasmid in Escherichia coli.
Release of the component of Streptococcus faecalis Na+‐ATPase from the membranes
Authors: Kakinuma Yoshimi , Igarashi Kazuei
Published: July 2002

The Na+‐stimulated ATPase activity of Streptococcus faecalis was lost by washing the membranes with ethylenediaminetetraacetic acid (EDTA). ATPase activities of both the EDTA extract and the stripped membranes did not show any stimulation by Na+ ions. However, the Na+‐stimulated ATPase was readily reconstituted by an incubation of these fractions combined. It was only reconstituted from the fractions prepared under the condition that the Na+‐ATPase is amplified, and not from those boiled or digested by trypsin. Thus, the component of Na+‐ATPase of this organism is capable of being released from the membranes.
Monoclonal antibody evidence for structural similarities between the central rod regions of actinin and dystrophin
Authors: Man Nguyen thi , Ellis J.M. , Ginjaar I.B. , van Paassen M.M.B. , van Ommen G.-J.B. , Moorman A.F.M. , Cartwright A.J. , Morris G.E.
Published: July 2002

A monoclonal antibody, MANDYS141, binds to both dystrophin and actinin on Western blots (SDS‐denatured), but only to actinin in frozen sections of human muscle (native conformation). It differs from a polyclonal cross‐reacting antiserum in that it binds to several muscle isoforms of actinin (smooth, fast and slow) from man, mouse and chicken and recognises a quite different part of the proposed triple‐helical region of dystrophin (amino acids 1750–2248). The results suggest that structural homologies between actinin and dystrophin occur more than once in their central helical regions and provide experimental support for an actinin‐like central rod model for dystrophin.
Synthesis and characterization of an N‐terminal‐specific 125I‐photoaffinity derivative of μ‐Conotoxin GIIIA which binds to the voltage‐dependent sodium channel
Authors: Becker Stefan , Liebe Reinhardt , Gordon Robert D.
Published: July 2002

An N‐terminal, iodinatable photoaffinity derivative of μ‐Conotoxin GIIIA, 4‐Azido‐salicylyl‐μ‐Conotoxin GIIIA (CTXASA), was synthesized by solid phase peptide synthesis. The binding of 125I‐CTXASA to the voltage dependent sodium channel from electroplax of Electrophorus electricus was specific, as demonstrated by saturation binding experiments. Using autoradiography, 125I‐CTXASA labeled a protein with a molecular mass of 260 kDa, consistent with the apparent molecular mass of the sodium channel. This labeling could be suppressed by excess of tetrodotoxin and μ‐Conotoxin GIIIA.
Evidence that the tightly bound magnesium in tubulin is associated with the N‐site GTP
Authors: Osei Anthony A. , Everett Grover W. , Himes Richard H.
Published: July 2002

In an attempt to determine whether the tightly bound Mg2+ found in purified tubulin in associated with the N‐site GTP or the E‐site GDP or GTP, we removed the E‐site nucleotide by several means: (i) alkaline phosphatase treatment; (ii) displacement using excess GMPPCP; and (iii) polymerizing tubulin in the presence of alkaline phosphatase and non‐hydrolyzable analogues. The Mg2+ content remained equal to about 1 tubulin under conditions where denaturation did not occur. Moreover, the Mg/GTP ratio always remained equal to 1. These results indicate that the Mg2+ is associated with the N‐site GTP.
Synthesis of 8‐N3‐[2'‐32P]NADP(H), a new photoaffinity label for NADP(H)‐specific enzymes
Authors: Hartog A.F. , Berden J.A.
Published: July 2002

The synthesis of the photoaffinity label 8‐N3‐[2'‐32P]NADP+ is described. The synthesis consists of the following steps: first 8‐N3‐AMP is synthesized from AMP or ATP and then the 8‐N3‐AMP is coupled with NMN, using DCCD as coupling reagent. Finally, the formed 8‐N3‐NAD+ is phosphorylated with [γ‐32P]ATP, using NAD‐kinase. The formed product is purified and analysed and is shown to react well with NADP‐specific enzymes and not to react with NAD‐specific enzymes. Additionally, it is proposed that in the NAD‐kinase reaction 2 mol ATP are used for the formation of 1 mol NADP: one molecule of ATP is hydrolyzed to ADP and phosphate while the other delivers the phosphate for the phosphorylation of NAD+.
The in vivo and in vitro oxidation of molecular form 1 of biliverdin reductase to molecular form 3 by diamide
Authors: Tomaro María Luján , Frydman Judith , Frydman Rosalía B.
Published: July 2002

Administration of phenylhydrazine to rats converted molecular form 1 of the liver biliverdin reductase into its disulfide bridged dimer (molecular form 3). This oxidative dimerization was shown not to be mediated by the NAD+‐dependent dehydrogenase [(1984) Biochem. Biophys. Res. Commun. 121, 249‐254]. Administration of diamide produced the same conversion. Although hepatic levels of GSH also decreased, no mixed disulfides of the reductase and GSH could be detected. Administration of the antioxidants allopurinol and α‐tocopherol together with the diamide did not affect this conversion of molecular forms produced by the latter. The diamide also oxidized molecular form 1 of biliverdin reductase in vitro and molecular form 3 was formed. The chemical oxidation took place at a high rate and was partially inhibited by GSH but not by cysteine.
Inactivation of Ca2+‐, Na+K+‐, and H+K+‐ATPases with a carbodiimide derivative of ATP
Authors: Murphy Alexander J.
Published: July 2002

The αmma;P adduct of ATP with 1‐ethyl‐3‐(3‐dimethylaminopropyl)carbodiimide (ATP‐EDC) was synthesized and incubated with the Ca‐ATPase of sarcoplasmic reticulum with the result that time‐dependent complete loss of the enzyme's activity occurred. The inactivation required calcium and magnesium while ATP had a protective effect. ATP‐EDC incubation with the NaK‐ATPase and HK‐ATPase produced partial (> 50%) inactivation, but had no effect on myosin Sl, pyruvate kinase and hexokinase, suggesting that this ATP analog is a specific inactivator of the so‐called ’quo;P‐type’ ATPases.
Phosphorylation of rat liver inorganic pyrophosphatase by ATP in the absence and in the presence of protein kinase
Authors: Vener Alexander V. , Smirnova Irma N. , Baykov Alexander A.
Published: July 2002

Cytoplasmic inorganic pyrophosphatase of rat liver can be phosphorylated by cAMP‐dependent protein kinase on a serine residue with a concomitant increase in enzymic activity. Phosphorylation is also observed in the absence of protein kinase, but in this case much higher concentrations of ATP are required and the stability characteristics of the phosphoenzyme resemble those of an acyi phosphate. Kinase‐free phosphorylation of the animal inorganic pyrophosphatase, unlike that of microbial pyrophosphatases, does not activate the enzyme. Pyrophosphatase may thus provide a new example of an enzyme whose evolution involves convergence of regulatory phosphorylation mechanisms.
The oral hypoglycemie agent, U‐56324, inhibits the activity of ATP‐sensitive potassium channels in cell‐free membrane patches from cultured mouse pancreatic B‐cells
Authors: Hopkins William F. , Fatherazi Sahba , Cook Daniel L.
Published: July 2002

U‐56324, a hypoglycemic agent derived from nicotinic acid, inhibited the activity of ATP‐sensitive potassium channels in excised patches from mouse pancreatic B‐cells. The effect of U‐56324 on channel activity was reversible and concentration‐dependent while it had no effect on single channel conductance. The positional isomer, U‐59588, which has relatively little hypoglycemic activity, had no effect on channel properties. U‐56324, at the same concentrations, had no effect on calcium‐activated potassium channels. The basis for the potentially antidiabetic properties of U‐56324 may therefore be due to direct and specific inhibition of ATP‐sensitive potassium channels.
Evidence for the ability of L10 ribosomal proteins of Salmonella typhimurium and Klebsiella pneumoniae to regulate rplJL gene expression in Escherichia coli
Authors: Paton E.B. , Woodmaska M.I. , Kroupskaya I.V. , Zhyvoloup A.N. , Matsuka G.Kh.
Published: July 2002

Genes rplJ, coding for ribosomal protein L10 of Salmonella typhimurium and Klebsiella pneumoniae, have been cloned on pUC plasmid. The resultant multicopy recombinant plasmids were detrimental for the growth of normal JM101
E. coli host cells and harmless for the mutant JF3029 host. This negative effect is the evidence for the ability of heterologous L10 proteins to regulate expression of rplJL genes in E. coli. Nucleotide sequence was determined completely for S. typhimurium rplJL ‘DNA portion and partially for rplJL’ genes of K. pneumoniae. According to the nucleotide sequence data obtained three amino acid substitutions differ L10 proteins of S. typhimurium and E. coli and the long range, providing for the coupled translations of L10 and L7/L12 cistrons in E. coli mRNA is also valid for S. typhimurium and K. pneumoniae.
Degradation of crystallins from a psoriatic patient undergoing PUVA therapy
Authors: e Melo Teresa Sa , de Castro José Cime , Ribeiro Luisa , Conte Joao , Lawrence David , Bazin Marc , Santus René
Published: July 2002

Comparative physico‐chemical and spectroscopic analyses were carried out in human lens proteins obtained from extracts of normal, senile and PUVA cataracts. Mass recovery analysis reveals a large protein concentration loss in the PUVA cataract relative to the normal lens and senile cataract. This protein loss parallels an increase in the degraded polypeptide chains. However, the tryptophan content (2.1 of 20 kDa protein subunit) and the apparent fluorescence quantum yield (ϕf = 0.056) of the tryptophan residues which are believed to be involved in the development of UV‐induced cataracts are unchanged after age‐related alterations and/or in vivo photochemistry associated with psoralen (8MOP) photosensitized reactions.
Molecular cloning, nucleotide sequence and expression of the gene encoding prepro‐polygalacturonasell of Aspergillus niger
Authors: Bussink Hendrik J.D. , Kester Harry C.M. , Visser Jaap
Published: July 2002

PolygalacturonaseII of Aspergillus niger was fragmented using CNBr and the NH2‐terminal fragment and another fragment were partially sequenced. The polygalacturonaseII (pgaII) gene was then isolated by using an oligonucleotide mixture based on the internal amino acid sequence as a probe. The nucleotide sequence of the pga II structural gene was determined. It was found that polygalacturonaseII is synthesized as a precursor having an NH2‐terminal prepro‐sequence of 27 amino acids. The cloned gene was used to construct polygalacturonaseII over‐producing A. niger strains. PolygalacturonaseII was isolated from one such strain and was determined to be correctly processed and to be fully active.
Neutron scattering study of the (γ‐B) catalytic domains of complement proteases Cl̄r and Cl̄s
Authors: Zaccaï Giuseppe , Aude Catherine A. , Thielens Nicole M. , Arlaud Gérard J.
Published: July 2002

The catalytic domains of Cl̄r and Cl̄s, comprising the C‐tenninal region of the A chain (γ), disulphide‐linked to the B chain, were obtained by limited proteolysis of the native proteases with chymotrypsin and plasmin, respectively, and studied by small angle neutron scattering. For CIs (γ‐B), a molar mass of 45 000 ± 5000 , and a relatively large radius of gyration (R
g) of 28 ± 1 Å were determined, excluding a single globular domain. The corresponding values for Cl̄r (γ‐B), (90,000 , R
g, = 34 ± 1 Å) are consistent with a dimer involving the loose packing of two (γ‐B) subunits. Various models of the dimer are discussed in the light of neutron scattering and other data.
Modulation of albumin‐like protein and lysozyme production by bovine tracheal gland serous cells
Authors: Jacquot Jacky , Dupuit Florence , Benali Rachid , Spilmont Christophe , Puchelle Edith
Published: July 2002

Bovine tracheal submucosal gland serous cells were cultured in medium supplemented with either 10% fetal calf serum or 2% Ultroser G, a commercial serum substitute for cell culture. The proteins synthesized and secreted into the culture medium during [35S]methionine pulse, chase and isoproterenol‐stimulated periods were analyzed. Marked differences in the patterns of secretory radiolabeled proteins with M
r values ranging from 15000 to 95000 were observed between pulse and chase media of cells cultured in fetal calf serum and Ultroser G. In the presence of Ultroser G, albumin‐like protein production was inhibited 95% as compared to cultures incubated with fetal calf serum. A bovine lysozyme‐type enzymatic activity was detected only in medium from stimulated cells cultured in Ultroser G. The results suggest that bovine tracheal serous cells synthesize different proteins according to the composition of culture medium and release certain proteins when adrenergically stimulated.
Comparison of the mode of action of ras p21 with those of protein kinases A and C in the stimulation of gene expression in NIH/3T3 cells
Authors: Oku Naohisa , Kaibuchi Kozo , Fukumoto Yasuo , Hori Yuichi , Fujioka Hiroyuki , Takai Yoshimi
Published: July 2002

To compare the mode of action of ras p21 with those of protein kinases A and C in the regulation of gene expression in NIH/3T3 cells, we investigated the transcriptional activity of various enhancer/promoters and enhancer motifs in the cells transfected with the c‐Ha‐ras
val12 complementary DNA (cDNA). The results indicate that the c‐Ha‐ras
val12 protein stimulates the enhancer/promoters of the c‐fos gene, the metallothionein IIA gene, the simian virus 40 (SV40) virus genome and the Rous sarcoma (RS) virus genome, and the serum‐response element and the 12‐O‐tetradecanoylphorbol‐13‐acetate (TPA)‐response element in a manner independent of protein kinases A and C in NIH/3T3 cells.
2‐Deoxy‐D‐glucose resistant yeast with altered sugar transport activity
Authors: Novak Srdjan , D'Amore Tony , Stewart Graham G.
Published: July 2002

The transport of glucose and maltose in Saccharomyces cerevisiae was observed to occur by both high and low affinity transport systems. A spontaneously isolated 2‐deoxy‐D‐glucose resistant mutant was observed to transport glucose and maltose only by the high affinity transport systems. Associated with this was an increase in the V
max values, indicating derepression of the high affinity transport systems. The low affinity transport systems could not be detected. This mutant will be important in examining the repression regulatory and sugar transport mechanisms in yeast.
Inactivation of vesicular stomatitis virus by photosensitization following incubation with a pyrene‐fatty acid
Authors: Rabia S.Abou , Fibach E. , Kotler M. , Gatt S.
Published: July 2002

Vesicular stomatitis virus (VSV) was incubated with pyrene dodecanoic acid (P12), a fluorescent derivative of a medium‐chain length fatty acid, and subjected to irradiation with a long wavelength ultra‐violet light source at 366 nm (UVA). This inactivated the virus, resulting in a drastic decrease of its infectivity. The virus inactivation was proportional to the concentration of the pyrene‐fatty acid, the length of exposure of the virus to P12 and the irradiation dose.
The competitive inhibition of Trichoderma reesei C30 cellobiohydrolase I by guanidine hydrochloride
Authors: Woodward Jonathan , Carmichael Jeffrey S. , Capps Katherine M. , Herrmann Paul C. , Lee Norman E.
Published: July 2002

TheP‐nitrophenylcellobiosidase (PNPCase) activity of Trichoderma reesei cellobiohydrolase I (CBH I) was competitively inhibited by concentrations of guanidine hydrochloride (Gdn HC1) that did not affect the tryptophan fluorescence of this enzyme. The K
m of CBH I, 3.6 mM, was increased to 45.4 mM in the presence of 0.14 M Gdn HCl, the concentration that was required to inhibit the enzyme by 50%. A similar concentration of lithium chloride and urea had little effect on the PNPCase activity of CBH I. Maximal inhibition was pH dependent, occurring in the range of pH 4.0 to 5.0, which is in the range for maximal activity. Analysis of the inhibition data indicated that 1.2 molecules of Gdn HCl combine reversibly with I molecule of CBH I. Other hydrolases and proteases were also inhibited by Gdn HCl. It is suggested that the inhibition of CBH I by Gdn HCl occurs as a result of the interaction between the positively charged guanidinium group of Gdn HCl and the carboxylate group of glutamic acid 126, postulated to be in the catalytic center of this enzyme.
Immobilized cytochrome P‐450LM2
Authors: Myasoedova Ksenia N. , Berndt Peter
Published: July 2002

Subunit interactions in the purified hexameric cytochrome P‐450LM2
have been studied using covalent binding of one of the 6 protomers to an insoluble matrix. High ionic strength, large‐scale pH changes, guanidine chloride and sodium cholate taken at membrane‐solubilizing concentrations, had no effect on the aggregation state of the immobilized hemoprotein. SDS caused a 6‐fold decrease in the amount of the bound cytochrome. Non‐ionic detergents (Emulgen 913, octylglucoside, Tritons) induced hexamer dissociation. In the presence of Emulgen 913 (> 0.2%), monomers and immobilized dimers were obtained as cytochrome P‐450 was studied in an aqueous medium and in the immobilized state, respectively. Immobilized dimers could be reconstituted to hexamers by treatment with an excess of solubilized monomers after removal of the detergent. In the presence of various phospholipids, which increased the immobilized cytochrome P‐450LM2
demethylase activity and induced characteristic spectral changes, no hexamer dissociation was shown. The data obtained are thus in agreement with the suggestion that hexameric arrangement is inherent in the cytochrome P‐450 when it is bound to the native membranes.
Bidirectional promoter activity of the 5′ flanking region of the mouse thymidine kinase gene
Authors: Weichslbraun Irene , Ogris Egon , Wintersberger Erhard
Published: July 2002

The 5′ flanking region of the gene coding for cytoplasmic thymidine kinase (TK) in the mouse (a total of 490 bp upstream of the initiation codon) was tested for promoter activity using the chloramphenicol acetyltransferase gene as reporter. It was found that the region can be devided into two parts, one of which carries promoter activity in the direction of TK, whereas the 5′‐half has promoter activity in the opossite direction. A fragment of 140 bp was sufficient for growth dependent promoter activity in the direction of TK, although about 100 bp further upstream, enhanced the activity. Expressions from the divergent promoter was independent of cell growth.
Fusion of negatively charged phospholipid vesicles by α‐latrotoxin
Authors: Lishko V.K , Terletskaya Ya.T , Trikash I.O
Published: July 2002

α‐Latrotoxin‐induced fusion of liposomes has been described using large unilamellar vesicles composed of phosphatidylcholine/phosphatidylethanolamine/cardiolipin at a molar ratio of 2:3:5. Vesicle fusion was monitored by terbium/dipicolinic acid assay as well as by fluorescence energy transfer measurement. The enhancement of the fusogenic effect of LTX by low concentrations (0.1–3 mM) of CaCl2 has been demonstrated. The efficiency of other divalent cations on the LTX fusogenic activity was shown to decrease in the sequence Ca > Cd > Sr > Mg > Ba. LTX‐induced fusion was accompanied by the increase of vesicle size measured by laser correlation spectroscopy. It is concluded that fusogenic action of LTX may be involved in its effect on synaptic apparatus.
Identification and primary structure of a calbindin 9K binding domain in the plasma membrane Ca2+ pump
Authors: James Peter , Vorherr Thomas , Thulin Eva , Forsen Sture , Carafoli Ernesto
Published: July 2002

Bovine calbindin 9K has been conjugated to a bifunctional, photoactivatable, cleavable and radioactive cross‐linker. It has been photolyzed in the presence of preparations of the purified crythrocyte Ca2+ pump, and shown to interact with it in the presence of Ca2+. The affinity of the interaction has been studied using the fluorescence enhancement of dansylated calbindin 9K incubated with the synthetic calmodulin binding domain of the pump. Two versions of the domain have been used, one corresponding to its full length (28 residues), one to about of it (20 residues). The affinity of the interaction was between 5 and 10 times lower than in the case of calmodulin.
N‐Methyl‐N‐(5‐carboxypentyl)‐1‐deoxynojirimycin, a new affinity ligand for the purification of trimming glucosidase I
Authors: Bause Ernst , Gross Achim , Schweden Jürgen
Published: July 2002

The paper describes the synthesis of a new type of affinity resin containing N‐methyl‐N(5‐carboxypentyl)‐1‐deoxynojirimycin as the ligand attached to AH‐Sepharose 4B, which allows the purification of trimming glucosidase 1 from a detergent extract of pig liver crude microsomes in one step and with high yield. The structure of the affinity ligand was designed on the basis of the observation that N, N‐dialkylated derivatives of 1‐deoxynojirimycin do strongly inhibit trimming glucosidase I, but not nonspecific aryl‐a‐glucosidases, including glucosidase II. The specific binding of glucosidase I eliminates the need of additional purification steps with their associated losses which were required with the previously synthesized N‐5(‐carboxypentyl)‐AH‐Sepharose 4B resin in order to achieve a homogenous enzyme preparation.
Transcriptional activity of the phosphoenolpyruvate carboxykinase gene decreases in regenerating rat liver
Authors: Milland Julie , Schreiber Gerhard
Published: July 2002

Rat cytosolic phosphoenolpyruvate carboxykinase (PEPCK) gene expression and enzyme activity in liver were studied in rats fasted for 12 hours before and after partial hepatectomy, sham operation or no operation. Transcriptional activity and mRNA levels decreased in regenerating liver compared to sham‐operated and unoperated controls. In contrast, PEPCK enzyme activity in regenerating liver was similar to that in livers of sham‐operated and unoperated controls. Since all the animals were fasted the decrease in transcription is probably caused by some factor other than insulin, the known repressor of PEPCK gene expression.
Epitope mapping of human thyroglobulin reveals a central immunodominant region
Authors: Malthièry Yves , Henry Mireille , Zanelli Eric
Published: July 2002

Thyroglobulin is the thyroid hormone precursor and the major antigen frequently involved in autoimmune diseases. The primary structure of human thyroglobulin is known but the spatial structure remains largely undetermined. By using fusion protein produced in prokaryotic system we have characterized seven short immunoreactive properties carrying at least one epitope. None of them includes hormonogenic sites, but five are concentrated in the central part of the monomeric molecule, which thus emerges as the major immunogenic region of this protein.
An upward shift of intracellular pH rather than the final absolute pH value is critical for controlling gap junction permeability in tumor promoter‐treated cells
Authors: Budunova Irina , Mittelman Leonid A. , Margolis Leonid B.
Published: July 2002

The tumor promoter TPA is found to inhibit gap junction permeability in monolayer cultures of hamster fibroblasts. This effect is associated with an increase in intracellular pH. Here we show that neither an increase in pH1 alone nor TPA treatment under conditions preventing pH1‐shift affect gap junction permeability. It is not the level of pH1 reached, but rather the pH1‐shift itself that is essential for the inhibition of gap junction permeability in the presence of TPA.
Two new components of 9 and 14 kDa from spinach photosystem I complex
Authors: Ikeuchi Masahiko , Inoue Yorinao
Published: July 2002

Two formerly‐uncharacterized subunits of 9 kDa and 14 kDa were found in spinach PSI complex. The 9 kDa subunit was released upon removal of antenna chlorophyll complex, whereas the 14 kDa subunit was tightly bound to the core complex. We determined the N‐terminal amino acid sequence of the 9 kDa, and an internal sequence of the 14 kDa subunit after protease treatment, since the N‐terminus of the latter protein was blocked. These partial sequences suggested that both subunits are new PSI components.
Nucleosomes occurring in protein‐free hybridoma cell culture Evidence for programmed cell death
Authors: Franěk František , Dolníková Jana
Published: July 2002

In addition to monoclonal immunoglobulin. two kinds of nucleoproteins. NP1 and NP2, were isolated from the supernatants of hybridoma cultures set up in a protein‐free medium. As shown by SDS‐electrophoresis the two nucleoproteins shared a set or proteins (apparent M
r 11000 to 15000). and differed in the DNA moiety (∼ 150 bp in NP1. ∼ 350 bp in NP2). The amino acid composition of the protein moiety confirmed the nucleosomal origin of NP1 and NP2. The findings support the view that in hybridoma cultures the cells undergo death by apoptosis, i.e. a programmed process characterized by initial fragmentation of chromatin.
7‐Tetrahydrobiopterin is an uncoupled cofactor for rat hepatic phenylalanine hydroxylase
Authors: Davis Michael D. , Kaufman Seymour
Published: July 2002

Rat hepatic phenylalanine hydroxylase requires both a tetrahydropterin cofactor and molecular oxygen to convert phenylalanine to tyrosine. During the physiological hydroxylation, a single mol of the natural cofactor, tetrahydrobiopterin, is oxidized for each mol of phenylalanine converted to tyrosine. Artificial conditions have been devised in which the oxidation of the tetrahydropterin is uncoupled from the hydroxylation of the aromatic amino acid substrate. Recently, an isomer of tetrahydrobiopterin, 7‐tetrahydrobiopterin, has been isolated from the urine of certain mildly hyperphenylalaninemic children. We report in this communication that 7‐tetrahydrobiopterin may be an inefficient cofactor for phenylalanine hydroxylase because, in vitro, the phenylalanine‐dependent oxidation of 7‐tetrahydrobiopterin is accompanied by the hydroxylation of the aromatic amino acid substrate only about 15% of the time, i.e. the enzymatic oxidation of 7‐tetrahydrobiopterin is about 85% uncoupled from the hydroxylation of the amino acid substrate.
A novel strategy for converting recombinant viral protein into high immunogenic antigen
Authors: Hinuma Shuji , Hazama Masatoshi , Mayumi Aki , Fujisawa Yukio
Published: July 2002

Interleukin 2 (IL‐2) is a lymphokine promoting immune response and therefore has been investigated as an immunological adjuvant. In order to enhance the immunogenicity of recombinant viral protein, herpes simplex virus type 1 (HSV‐1) glycoprotein D (gD), we gentically created a fusion protein consisting of gD and human IL‐2. The fusion protein, without any other adjuvants, induced high antibody responses and cell‐mediated immunity to HSV‐1 in mice. Mice immunized with the fusion protein were protected against HSV‐1 infection. The results indicate that IL‐2‐fusing can provide a means for converting a weak immunogenic protein into a high immunogenic antigen, and the strategy would be widely applicable to the other antigens for pathogens.
Primary structure of the cDNA 5′‐terminal region encoding the N‐terminal domain of the rabbit muscle α‐actinin subunit
Authors: Jokhadze G.G. , Oleinikov A.V. , Alakhov Yu.B. , Nadirashvili N.Sh. , Zaalishvili M.M.
Published: July 2002

A number of cDNA clones have been obtained in summary encoding the N‐terminal domain containing 286 amino acid residues of the rabbit skeletal muscle α‐actinin subunit. α‐Actinin cDNA clones were isolated from specific cDNA libraries using the primer extension method for synthesis of the first cDNA chain. A strong stop signal for AMV reverse transcriptase in the 5′‐terminal region of mRNA of α‐actinin was found. It seems there is a G+C rich region (93% G+C nucleotides) including a continuous sequence of 23 G and C nucleotides encoding 6 glycine residues.
Different superstructural features of the light responsive elements of the pea genes rbcS‐3A and rbcS‐3.6
Authors: Cacchione Stefano , Savino Maria , Tufillaro Annamaria
Published: July 2002

Analyses of the DNA sequences, named Light Responsive Elements (LREs), relevant in the photoactivation of the two pea genes rbcS‐3A and rbcS‐3.6, encoding the same protein but differently expressed, have been carried out, taking advantage of two complementary methods: a theoretical analysis, based on conformational energy calculations, and an experimental evaluation of LREs curvature, derived from gel electrophoretic mobilities of multimers of the LREs and of oligonucleotides corresponding to the three boxes in which they can be dissected. Theoretical and experimental analyses show that the curvature or the rbcS‐3A LRE is larger than that of rbcS‐3.6 LRE, and seems to be correlated with rbcS‐3A higher transcription efficiency.
Interference of Ha‐ras with inositol trisphosphate‐mediated Ca2+‐release
Authors: Maly Karl , Kiani Alexander , Oberhuber Hermann , Grunicke Hans
Published: July 2002

Expression of a transforming Ha‐ras by dexamethasone in NIH3T3 cells transfected with a glucocorticoid‐inducible Ha‐ras construct results in a rapid desensitization of the intracellular Ca2+‐mobilizing system to bombesin. This effect precedes the down‐modulation of inositol trisphosphate (IP3) formation by several hours and is, therefore, not explained by an uncoupling of phosphoinositidase C. It is demonstrated that expression of Ha‐ras attenuates the Ca2+‐release by IP3 in permeabilized cells. The IP3 concentration required for half‐maximal Ca2+‐release is doubled in Ha‐ras expressing cells. Maximal Ca2+‐release which is obtained with 2,μM IP3 in control cells requires 10μM IP3 in cells expressing Ha‐ras. The desensitization of the IP3 receptors coincides with the desensitization of the Ca2+‐mobilizing system to bombesin. The results indicate that the Ha‐ras mediated desensitization of the Ca2+‐releasing system to bombesin is — at least in part — caused by a decrease in the affinity of the IP3 receptor to inositol trisphosphate.
Effect of heat shock on acetylcholinesterase activity in chick muscle cultures
Authors: Eichler Jerry , Toker Lilly , Silman Israel
Published: July 2002

The effect of heat shock was studied on the acetylcholinesterase activity of chick muscle primary cultures. In cultures transferred from 37°C to 45°C, a sharp drop in activity was followed by rapid spontaneous recovery. The time of onset of recovery resembled the time needed for expression of heat shock proteins. In cultures exposed to heat shock at 45°C and allowed to recover at 37°C, reappearance of acetylcholinesterase activity did not involve de novo protein synthesis since it was not prevented by cycloheximide. Our data raise the possibility of a role for heat shock proteins as molecular chaperones in rescuing heat‐denaturing acetylcholinesterase.
NMR studies of [U‐13C]cyclosporin A bound to human cyclophilin B
Authors: Neri Placido , Gemmecker Gerd , Zydowsky Lynne D. , Walsh Christopher T. , Fesik Stephen W.
Published: July 2002

NMR data (1H and 13C chemical shifts, NOEs) on [[U‐13C]cyclosporin A bound to cyclophilin B were compared to previously published data on the [U‐13C]CsA/CyPA complex [Fesik et al., (1991) Biochemistry 30, 6574–6583]. Despite only 64% sequence identity between CyPA and CyPB, the conformation and active site environment of CsA when bound to CyPA and CyPB are nearly identical as judged by the similarity of the NMR data.
Identification of 4‐chlorobenzoyl‐coenzyme A as intermediate in the dehalogenation catalysed by 4‐chlorobenzoate dehalogenase from Pseudomonas sp. CBS3
Authors: Löffler Frank , Müller Rudolf
Published: July 2002

The intermediate in the reaction catalysed by 4‐chlorobenzoate dehalogenase from Pseudomonas sp. CBS3 was identified as 4‐chlorobenzoyl‐CoA. One component of 4‐chlorobenzoate debalogenase worked as a a 4‐chlorobenzoyl‐CoA ligase catalysing the formation of 4‐chlorobenzoyl‐CoA from 4‐chlorobenzoate, coenzyme A and ATP. This intermediate was detected spectrophotometrically and by HPLC. 4‐chlorobenzoyl‐CoA was the substrate for the dehalogenase component, which catalysed the conversion to 4‐hydroxybenzoate with concomitant release or coenzyme A.
The vegetalizing factor A member of the evolutionarily highly conserved activin family
Authors: Tiedemann Heinz , Lottspeich Friedrich , Davids Michael , Knöchel Sigrun , Hoppe Peter , Tiedemann Hildegard
Published: July 2002

The mesoderm and endoderm inducing vegetalizing factor was partially sequenced after BrCN cleavage. A sequence which is highly conserved in activin A near the C‐terminal end was identified. This shows that the factor belongs to the activin family. The activins are not confined to embryos and gonads, but widely distributed in other tissues like calf kidney and calf liver. Functional aspects are discussed.
The rate of catabolism of dATP to deoxyadenosine during the growth of different cell lines in vitro
Authors: Marcussen Mads , Klenow Hans
Published: July 2002

A method has been developed for the determination of the rate of formation or deoxyadenosine from dATP in cultured cell lines. The lowest rate was found in the T‐cell‐derived Molt cell line while it was about 70‐fold higher in Balb c/3T3 mouse fibroblasts. In the B‐cell‐derived Raji cells and in the murine sarcoma cell line SEWA it had intermediary values. It is concluded that in some cell types like the 3T3‐cells the catabolism of dATP to deoxyadenosine may have a significant regulatory eftect on the cellular content of dATP.
Amino acid sequence of the unique protamine from yellow perch
Authors: Chao Heman , Davies Peter L.
Published: July 2002

By the criteria of gel electrophoresis, ion‐exchange chromatography, and reverse‐phase HPLC, yellow perch prolamine behaves as a single component. This observation was confirmed by automated Edman degradation which gave a single unambiguous amino acid sequence PRRRRHAARPVRRRRRTRRSSRVHRRRRAVRRRR. Yellow perch prolamine has 34 amino acids, including 21 arginines. It has two histidines, neither of which interrupts an arginine tract. It is unusual among fish protamines in not having a serine or threonine N‐terminal to the second arginine tract, and is unique in not being a mixture of components.
Left‐handed topology of super‐secondary structure formed by aligned α‐helix and β‐hairpin
Authors: Kajava A.V.
Published: July 2002

A novel super‐secondary structure common for many non‐homological proteins is considered. This folding pattern, consisting of adjacent along the chain α‐helix and β‐hairpin, has an aligned packing. It is found that one of the two possible ‘mirror‐symmetrical’ topologies is observed in proteins. The α‐helix + β‐hairpin structures have a similar pattern of hydrophobic residues in their amino acid sequences. The remaining part of a molecule or a domain is almost always located on the same side of the considered folding pattern. These results can be used in the prediction of three‐dimensional protein structure and protein design.
Assembly and processing of subunit II (PsaD) precursor in the isolated photosystem‐I complex
Authors: Cohen Yuval , Nechushtai Rachel
Published: July 2002

The precursor of photosystem I (PSI) subunit II (pre‐subunit II) synthesized in vitro, was found to bind to the holo‐PSI complex, both within the thylakoids and outside, after detergent extraction of PSI from the membranes. Chloroplast stromal fraction added to the purified PSI complexes, containing the labeled pre‐subunit II, induced the processing of the precursor to the mature form. This implies that processing can occur within the isolated complex, after the integration of the precursor. The results presented suggest that certain aspects of biogenesis of membranal protein complexes can be studied in detergent‐extracted purified complexes.
Interaction of a synthetic peptide of the interferon α2 C‐terminal part with human blood leukocytes. II. Effect on protein tyrosine phosphorylation
Authors: Danilkovich A.V. , Kharitonenkov A.I. , Freze K.V. , Shevalier A.F. , Kolosova O.V. , Bulargina T.V. , Kirkin A.F. , Gusev M.V.
Published: July 2002

Tyrosine phosphorylation in human blood lymphocytes was studied as a function of stimulation with concanavalin A (ConA) and treatment of the cells with interferon α2 (IFNα2) and/or an IFN‐derived C‐terminal synthetic peptide 2438 (amino acid residues 124–138). Both IFNα2 and the peptide 2438 decreased the level of protein tyrosine phosphorylation in the ConA‐stimulated cells. In unstimulated cells, IFNα2 increased, and the peptide 2438 decreased the level of the tyrosine phosphorylation. A possible correlation of these effects with stimulation or cell proliferation is discussed.
An insect picornavirus may have genome organization similar to that of caliciviruses
Authors: Koonin Eugene V. , Gorbalenya Alexander E.
Published: July 2002

Computer‐assisted analysis of the amino acid sequence of the product encoded by the sequenced 3′ portion of the cricket paralysis virus (CrPV), an insect picornavirus, genome showed that this protein is homologous not to the RNA‐directed RNA polymerases, as originally suggested, but to the capsid proteins of mammalian picornaviruses. Alignment of the CrPV protein sequence with those of picornavirus and calicivirus capsid proteins demonstrated that the sequenced portion of the insect picornavirus genome encodes the C‐terminal part of VP3 and the entire VP1. Thus CrPV seems to have a genome organization distinct from that of other picornaviruses but closely resembling that of caliciviruses, with the capsid proteins encoded in the 3′ part of the genome. On the other hand, the tentative phylogenetic trees generated from the VP3 alignment revealed grouping of CrPV with hepatitis A virus, a true picornavirus, not with caliciviruses. Thus CrPV may be a picornavirus with a calicivirus‐like genome organization. Different options for CrPV genome expression are discussed.
Isolation and characterization of the MHC linked β‐type proteasome subunit MC13 cDNA
Authors: Frentzel Stefan , Gräf Uwe , Hammerling Günter J. , Kloetzel Peter-M.
Published: July 2002

We have cloned and analysed the second mouse MHC‐linked proteasome subunit, designated MC13, which appears to be homologous to the human RING10 proteasome protein. The isolated cDNA has an ORF encoding a protein of 276 amino acids with a molecular weight of ca. 30 kDa. Sequence: alignment reveals that the subunit MC13 and several other mammalian proteasome subunits are encoded by a second proteasome gene family. This second gene family encodes subunits of the β‐type, reveals striking sequence similarities with the β‐subunit of archaebacterial proteasomes and is related to, but distinct from, the genes encoding the so‐called α‐type subunits.