bluuebunny/crossref_metadata_2025 huggingface dataset:
Absence of 7‐acetyl taxol binding to unassembled brain tubulin
Authors: Takoudju Martin , Wright Michel , Chenu Jacques , Guéritte-Voegelein Françoise , Guénard Daniel
Published: July 2002

The effect of taxol on microtubule proteins at 0°C is controversial. In order to determine if taxol is unable to bind to unassembled tubulin, as has been hypothesized, the binding of [3H]acetyl taxol has been studied using equilibrium microdialysis. Ac‐taxol bound to microtubules at 37°C and the binding remained stable when the temperature was lowered to 0°C. Ac‐taxol bound also at 0°C to microtubules stabilized with rhazinilam. In contrast, there was no binding of Ac‐taxol to unassembled tubulin, either free tubulin at 0°C or tubulin, complexed with several microtubule poisons, at 0 and 37°C.
Determination of the H+ /ATP ratio of the H+ transport‐coupled reversible chloroplast ATPase reaction by equilibrium studies
Authors: Strotmann Heinrich , Lohse Detlev
Published: July 2002

Fluorescence quenching of 9‐aminoacridine was followed to ascertain the state of light‐induced transthylakoidal ΔH+ at energetic equilibrium with a subsequently added mixture of ATP, ADP and phosphate. In the measured range, the logarithm of relative fluorescence quenching at equilibrium is a linear function of the imposed phosphate potential. The slope of the line is shown by mathematical deduction to be equal to the ATP/H+ stoichiometry of the H+‐coupled reversible ATPase reaction. Determination of the stoichiometry by this method neither relies on the standard phosphate potential nor on the internal thylakoid volume. The results confirm that during ATP synthesis or hydrolysis 3 H+ per ATP are translocated through the CF0‐CF1 complex.
In vivo NMR studies of the alga Dunaliella salina embedded in beads
Authors: Oren-Shamir M. , Avron M. , Degani H.
Published: July 2002

To facilitate in vivo NMR studies of microorganisms under stable environmental conditions a technique was developed in which algae are embedded in Ca‐alginate beads and continuously perfused during the measurements. Dunaliella salina was shown to grow, multiply and recover from osmotic shocks while embedded within the beads. Using this technique the detailed kinetics of glycerol metabolism of Dunaliella salina following an osmotic shock, were investigated.
Characterization of a peripheral‐type benzodiazepine‐binding site in the mitochondria of Chinese hamster ovary cells
Authors: Riond J. , Vita N. , Le Fur G. , Ferrara P.
Published: July 2002

The isoquinoline carboxamide derivative [3H]PK11195, a ligand for the peripheral‐type benzodiazepine (BZD) receptor, binds to Chinese hamster ovary (CHO) cell mitochondria in a specific and saturable manner. Scatchard analysis showed the presence of a single‐binding site with an apparent dissociation constant (K
d) of 12.0 ± 1.0 nM and a maximal binding capacity of 23.0 ± 2.0 pmol/mg protein. The pharmacological characterization of this CHO BZD‐binding site, based on the displacement of [3H]PK11195 by several drugs of known binding specificity, indicated that it is of the peripheral‐type. The photoaffinity probe [3H]PK14105, a nitrophenyl derivative of [3H]PK11195, specifically labeled a 17 kDa CHO mitochondrial protein. This 17 kDa protein was purified from digitonin‐solubilized mitochondria by gel‐filtration chromatography and two reverse‐phase HPLC steps. The purified material migrated as a single band on silver stained or autoradiographed SDS‐polyacrylamide gels, and had an amino acid composition corresponding to a 17 kDa protein rich in Leu, Val, Ala, Gly, and Pro. Analysis of the amino‐terminal sequence of the purified 17 kDa protein revealed a blocked amino‐terminus.
Activation of the purified erythrocyte plasma membrane Ca2+‐ATPase by organic solvents
Authors: Benaim Gustavo , de Meis Leopoldo
Published: July 2002

In this report it is shown that organic solvents mimic the stimulatory effects of calmodulin and acidic phospholipids on the erythrocyte plasma membrane Ca2+‐ATPase. The solvents used were dimethyl sulfoxide (20%, v/v), glycerol (20%, v/v), ethylene glycol (20%, v/v) and polyethylene glycol (M
r, 6000–8000) (10%, w/v). These solvents increased both the affinity for Ca2+ and the turnover number of the enzyme. The increase in Ca2+ affinity is additive to that achieved with calmodulin. The calcium cooperativity observed in the presence of calmodulin disappears after the addition of dimethyl sulfoxide to the medium. The present data support the proposal that activation of the erythrocyte plasma membrane Ca2+‐ATPase is promoted by hydrophobic interactions along the enzyme molecule.
The role of charge neutralization and cooperative binding of linker histone in. the higher‐order structure of chromatin
Authors: Watanabe Fumiyuki
Published: July 2002

The binding mode and stoichiometry of interaction between soluble rat liver chromatin and histone H1 (H1) were studied. H1 binding to chromatin is cooperative. Chromatin accepts 3.6 molecules of H1/nucleosome at 0 M salt, close to the required ratio for neutralization of 90% of the charges on the phosphate groups of chromatin (4.0 H1 molecules/nucleosome). The proposal is put forward that critical charge neutralization (90%) has a significant influence on the irregular appearance of chromatin.
Phosphorylation of NADPH‐cytochrome c reductase in guinea pig peritoneal macrophages stimulated with phorbol myristate acetate
Authors: Tamoto Koichi , Hazeki Kaoru , Nochi Hiromi , Mori Yoki , Koyama Jiro
Published: July 2002

The plasma membrane‐bound NADPH‐cytochrome c reductase of guinea pig macrophages (Mφ) was found to be phosphorylated when [32P]phosphate‐labeled cells were stimulated with 12‐phorbol 13‐myristate acetate (PMA). The time course of phosphorylation was parallel to that of O−
2‐generating activity elicited. These results suggest that the reductase participates as a flavoprotein in activation of the respiratory burst NADPH oxidase, when phosphorylated.
Evidence for two closely related isozymes of arylamine N‐acetyltransferase in human liver
Authors: Grant Denis M. , Lottspeich Friedrich , Meyer Urs A.
Published: July 2002

Acetyl CoA‐dependent arylamine N‐acetyltransferase (EC 2.3.1.5) is the target of a genetic polymorphism in the metabolism of drugs and carcinogens. N‐Acetyltransferase was purified 1000‐fold from cytosol of human liver and its identity was verified by amino acid sequence homology of two of its tryptic peptides with published rabbit and chicken N‐acetyltransferase sequences. Enzyme activity correlated with the presence of two proteins, NAT‐1 and NAT‐2, with indistinguishable molecular masses (31 kDa). NAT‐1 and NAT‐2 could be separated by anion‐exchange chromatography and were functionally distinguished by their different apparent affinities for the acceptor amine sulfamethazine (SMZ). Antibodies raised against NAT‐1 were able to recognize both isozymes on Western blots.
The cytosolic G‐protein α‐subunit in human neutrophils responds to treatment with guanine nucleotides and magnesium
Authors: Rudolph Uwe , Schultz Günter , Rosenthal Walter
Published: July 2002

The hydrodynamic properties of the Gi2 α‐subunit in human neutrophil cytosol were analyzed. The s
20,w value was 3.3 S at 30°C and 4.1 S at 4°C. Reconstitution of the cytosolic α‐subunit with βγ‐complex purified from porcine brain resulted in an s
20,w value of 4.0 S at 30°C and 4.2 S at 4°C. Treatment of cytosol with G‐protein‐activating agents, GTPγS and MgCl2, decreased the s
20,w value to 2.4 S at 30°C and 2.9 S at 4°C. Our results indicate that the cytosolic α‐subunit of neutrophils is an inactive monomeric species at 30°C capable of interacting with G‐protein βγ‐complex and responsive to treatment with activating agents leading to the two activated forms, α* at 4°C and α** at 30°C.
Selective cleavage of glycyl bonds by papaya proteinase IV
Authors: Buttle David J. , Ritonja Anka , Pearl Laurence H. , Turk Vito , Barrett Alan J.
Published: July 2002

The specificity of papaya proteinase IV (PPIV) has been examined with small substrates and a protein. With both classes of substrate, the enzyme shows a marked selectivity for cleaving glycyl bonds. Boc‐Ala‐Ala‐Gly‐NHPhNO2 is a convenient substrate for routine assays that discriminate well against chymopapain, the most common contaminant of PPIV. Sixteen cleavage points in β‐trypsin were identified, of which 13 are glycyl bonds. Tentative suggestions are made as to the reasons for lack of cleavage of some other glycyl bonds. The structure of PPIV has been modelled on that of papain, and we suggest that the replacement of the highly conserved residues Gly‐65 and Gly‐23 by arginine and glutamic acid, respectively, can account for the specificity of PPIV.
Comparative properties of human α‐1‐proteinase inhibitor glycosylation variants
Authors: Guzdek Amalia , Potempa Jan , Dubin Adam , Travis James
Published: July 2002

Variant forms of human α‐1‐proteinase inhibitor (α‐1‐PI), obtained by the treatment of human Hep G2 cells with specific inhibitors of glycosylation were tested for both inhibitory activity and heat stability. All were found to have the same second‐order association rate with human neutrophil elastase, indicating a lack of importance of the carbohydrate moiety. In contrast, incompletely glycosylated forms of α‐1‐PI were found to be heat sensitive relative to the mature protein, suggesting a role for carbohydrate in protein stabilization.
Diet‐ and hormone‐induced reversal of the carbamoylphosphate synthetase mRNA gradient in the rat liver lobulus
Authors: Moorman Antoon F.M. , de Boer Piet A.J. , Charles Robert , Lamers Wouter H.
Published: July 2002

A hybridocytochemical analysis of adult liver from normal control and from hormonally and dietary‐treated rats was carried out, using radioactively‐labelled probes for the mRNAs of glutamine synthetase (GS), carbamoylphosphate synthetase (CPS) and phosphoenolpyruvate carboxykinase (PEPCK). In line with previous findings, GS mRNA is exclusively expressed in a small pericentral compartment, CPS mRNA exclusively in a contiguous large periportal compartment and PEPCK mRNA across the entire porto‐central distance. The density of labelling in CPS and PEPCK mRNA‐positive hepatocytes decreases in a porto‐central direction. Starvation resulted in a reversal of the gradient of CPS mRNA within its periportal compartment; glucose refeeding counteracted this effect. Livers of glucocorticosteroid‐treated, starved or diabetic rats also revealed a reversal of the normal gradient of CPS mRNA, but now across the entire porto‐central distance. The patterns of expression of GS and PEPCK mRNA remained essentially unchanged, notwithstanding substantial changes in the levels of expression. It is concluded that blood‐borne factors constitute the major determinants for the expression patterns of CPS mRNA within the context of the architecture of the liver lobulus.
Hormone glycosylation required for lutropin receptor recognition in sheep testis
Authors: Sairam M.R. , Bhargavi G.N. , Yarney T.A.
Published: July 2002

The high affinity binding sites for ovine pituitary lutropin (oLH) present in DLS‐1 sheep testis recognized only the fully glycosylated ovine or bovine hormone (bLH) in receptor binding assays using 125I‐labeled oLH. Chemically deglycosylated (DG‐) oLH or bLH which were fully active with other lutropin receptors (rat/pig) were completely inert in the DLS‐1 receptor assay. In the same membranes, the FSH (follitropin) receptor reacted well with both glycosylated FSH and DG‐oFSH. In recombination studies, lutropin formed by glycosylated native α‐ and β‐subunits of the hormone was fully active but when one of the subunits was in the deglycosylated form, receptor binding activity was greatly reduced. The presence of glycosylated α‐subunit in the recombined hormone gave rise to 5 × more activity than DG‐α + β. All these preparations were fully active in the rat/pig receptor assays for LH. These results demonstrate that lutropin hormone glycosylation is essential for optimum receptor recognition in the sheep testis, further emphasizing the importance of correct glycosylation in oLHα hormone function.
Autolysis of isolated adipocytes by endogenously produced fatty acids
Authors: Strålfors Peter
Published: July 2002

Isolated rat adipocytes, in which lipolysis was maximally stimulated by isoproterenol, continued to produce fatty acids in excess of the high‐affinity binding capacity of the fatty acid acceptor bovine serum albumin in the incubation medium. At an average of 17 mol fatty acids/mol serum albumin, there was a burst of lactate dehydrogenase activity recovered in the incubation medium, indicating cell lysis. It is concluded that endogenously produced fatty acids will cause autolysis of adipocytes in vitro. Actively lipolysing adipocytes were more fragile than resting cells, since increasing amounts of lactate dehydrogenase activity was recovered in the medium during active lipolysis.
Time and mode of synthesis of the sexual inducer glycoprotein of Volvox carteri
Authors: Balshüsemann Dirk , Jaenicke Lothar
Published: July 2002

The synthesis of the sex‐inducing glycoprotein of Volvox carteri was investigated. A highly specific antiserum directed against pheromone deglycos ylated with trifluoromethane sulphonic acid was used to detect inducer protein in maturing cultures of the male strain IPS‐22. Immunoblot analysi showed that inducer synthesis is restricted to a very late phase of sperm development, a few hours before the sperm cells die. The pheromonc is co‐translationally glycosylated to yield a well‐defined pattern of isoinducers.
Phosphotyrosine‐containing proteins in Dictyostelium discoideum
Authors: Schweiger Anton , Mihalache Oana , Muhr Angelika , Adrian Iris
Published: July 2002

Phosphotyrosine‐containing proteins in Dictyostelium discoideum were detected by immunoblot analysis and inununoprecipitation using a monoclonal anti‐phosphotyrosine antibody. The iodinated antibody recognized on bots a cluster of 205–220 kDa polypeptides and bands of 107 and 60 kDa. The 107 and 60 kDa polypeptides and, in addition, a 82 kDa one became phosphorylated on tyrosine when the immunoprecipitate was incubated with [γ‐32p]ATP. In preparations from differentiating cells the intensity of the label was increased in the 60 kDa band and decreased in the 107 and 205–220 kDa bands.
Purification and characterization of mouse DNA polymerase α devoid of primase activity
Authors: Takada-Takayama Ritsuko , Suzuki Masashi , Enomoto Takemi , Hanaoka Fumio , Ui Michio
Published: July 2002

A simple method was developed for the isolation of primase‐free DNA polymerase‐α from the DNA polymerase‐α‐primase complex of mouse FM3A cells. The polymerase was separated from primase submits by chromatography on a single‐stranded DNA‐cellulose column in the presence of 50% etylene glycol. The primase‐free DNA polymerase‐α contained two polypeptides with molecular masses of 180000 and 68000. Analysis of the DNA products with poly(dA)‐oligo(dT)10 as template‐primer revealed that both primase‐free DNA polymerase‐α and the DNA polymerase‐α‐primase complex predominantly synthesized short DNA with less than 30 nucleotides, but that the DNA polymerase‐α‐primase complex also synthesized some longer DNA with more than 300–400 nucleotides.
In vivo and in vitro mutants of FNR the anaerobic transcriptional regulator of E.coli
Authors: Sharrocks Andrew D. , Green Jeffrey , Guest John R.
Published: July 2002

FNR regulates the expression of target genes in response to anaerobiosis. It resembles the catabolite gene activator or cAMP‐receptor protein (CRP) except for the presence of an N‐terminal cysteine cluster, which may form a redox‐sensing iron‐binding site. Site‐directed mutagenesis has shown that 3 of the 4 cysteine residues in the N‐terminal cluster (Cys‐20, ‐23 and ‐29, but not Cys‐16) and the only other cysteine residue (Cys‐122), are essential for the normal activation and repression of PNR‐dependent promoters. Deletion of residues Pro‐3‐Arg‐9 (inclusive) had no effect, but FNR was inactivated by a frameshift extending through the C‐terminal DNA‐binding domain. Four independent in vivo mutants contained identical Gly‐96→Asp substitutions, which may inactivate FNR by distorting a sharp turn between β‐strands in the predicted structure.
Antibiotics ‐ cloning of biosynthetic pathways
Authors: Kleinkauf Horst , von Döhren Hans
Published: July 2002

Biosynthetic pathways leading to antibiotics have often been found to be clustered, and new organizational forms of multifunctional enzymes have been discovered. Such polyenzymes accomplish the synthesis of complex metabolites such as peptides or polyketides by a sequence of enzymatic reactions. So, reactions leading to the tripeptide precursor of β‐lactam antibiotics. ACV, or to the cycloundecapeptide cyclosporine have been fused into single polypeptide chain synthetases, respectively. In certain isofunctional sites restricted similarities have been detected.
Characterization of rat brain NCAM mRNA using DNA oligonucleotide probes
Authors: Andersson Anna-Maria , Gaardsvoll Henrik , Giladi Eliezer , Dahl Britta , Bock Elisabeth
Published: July 2002

A number of different isoforms of the neural cell adhesion molecule (NCAM) have been identified. The difference between these is due to alternative splicing of a single NCAM gene. In rat brain NCAM mRNAs with sizes of 7.4, 6.7, 5.2, 4.3 and 2.9 kb have been reported. We have synthesized six DNA oligonucleotides, that hybridize to different exons in the NCAM gene. Furthermore we have constructed three oligonucleotides, that exclusively hybridize to mRNAs lacking certain exons, by letting them consist of sequences adjacent to both sides of the splice sites. By means of these probes we have characterized the five NCAM mRNAs in rat brain.
Phytochrome in lower plants Detection and partial sequence of a phytochrome gene in the moss Ceratodon purpureus using the polymerase chain reaction
Authors: Thümmler Fritz , Beetz Axel , Rüdiger Wolfhart
Published: July 2002

The polymerase chain reaction was carried out with primers hybridizing to conserved regions of the phytochrome genes. With DNA from the moss Ceratodon purpureus 5 overlapping fragments were obtained resulting in a continuous nucleotide sequence of 1474 bp. The deduced amino acid sequence showed homology of around 60% with all known phytochrome sequences. The sequences contained a conserved chromophore attachment site. In light‐grown Ceratodon protonemata the phytochrome mRNA with the size of about 4.5 kb was detected.
Formylmethanofuran: Tetrahydromethanopterin formyltransferase from Methanosarcina barkeri Identification of N5‐formyltetrahydromethanopterin as the product
Authors: Breitung J. , Thauer R.K.
Published: July 2002

Formylmethanofuran: tetrahydromethanopterin formyltransferase was purified from methanol grown Methanosarcina barkeri to apparent homogeneity and characterized with respect to its molecular and kinetic properties. The enzyme was found to be very similar to the formyltransferase from H2/CO2 grown Methanobacterium thermoautotrophicum. It also catalyzed the formation of N
5‐formyltetrahydromethanopterin rather than of N
10‐formyltetrahydromethanopterin from formylmethanofuran and tetrahydromethanopterin.
Saturation of the processing of newly synthesized rRNA in isolated brain mitochondria
Authors: Enriquez J.A. , López-Pérez M.J. , Montoya J.
Published: July 2002

Isolated rat brain mitochondria, when incubated in the presence of (α‐32P)UTP in an appropriate incubation buffer, in which the energy requirements are provided by exogenous ADP in the presence of an oxidizable substrate, are able to support mitochondrial DNA transcription and RNA processing in a way faithfully resembling the in vivo process. Furthermore, we have strikingly found a saturation of the synthesis of mature 16 S and 12 S rRNA under conditions in which their RNA precursor as well as the mature mRNAs continue being synthesized. This suggests that synthesis of mature rRNAs could be regulated at the level of processing of their precursor rather than at the level of transcription.
The influence of heptane‐1,2,3‐triol on the size and shape of LDAO micelles Implications for the crystallisation of membrane proteins
Authors: Timmins Peter A. , Hauk Jürgen , Wacker Thomas , Welte Wolfram
Published: July 2002

The presence of small amphiphiles has been found to be necessary in the crystallization of several membrane‐protein surfactant complexes. It has been suggested that the role of the small amphiphile may be to reduce the size of the surfactant belt around the protein, making the formation of crystals easier. Thus far it was not known if this would involve changes in micellar size in general or whether the small amphiphile would merely replace LDAO during crystal growth. In the present study we have used small angle neutron scattering to study mixed micelles of lauryldimethyl amine oxide (LDAO; hydrogenated and deuterated) and heptane‐1,2,3‐triol (HP). Our results show that with increasing overall HP concentrations mixed LDAO/HP micelles of decreasing mass and radius are formed. The composition of these micelles has been determined. HP thus may decrease the size of the surfactant belt around a protein before crystallisation by insertion into a host micelle. As HP is a ‘small amphiphile’ compared to the surfactants used for solubilization of membrane proteins, the curvature of the host micelle will be increased by its insertion.
Characterization of a novel zinc binding site of protein kinase C inhibitor‐1
Authors: Mozier Ned M. , Walsh Michael P. , Pearson James D.
Published: July 2002

The zinc‐binding properties of an endogenous protein inhibitor of protein kinase C was studied. Equilibrium gel penetration revealed that 1 mol of this protein binds 0.97 mol of zinc with a dissociation constant of 4.3 μM. The site of zinc‐binding. MVVNEGSDGGQSVYHVHLHVLGGR, was indentified by a multi‐step process consisting of tryptic digestion, fragment isolation, transfer to nitrocellulose, and hybridization with 65ZnCl2. Binding of 65ZnCl2 to selected synthetic fragments further localized the site of interaction to the sequence QSVYHVHLHVL. This region contains 3 closely positioned histidine residues and represent a novel zinc‐binding site.
Inactivation of human cystatin C and kininogen by human cathepsin D
Authors: Lenarčič Brigita , Krašovec Marta , Ritonja Anka , Olafsson Isleifur , Turk Vito
Published: July 2002

A papain inhibitor or 22 kDa was isolated from human placenta and shown to be identical to residues Cys246‐Leu373 of the third domain of human kininogen. This kininogen domain and recombinant human cystatin C were inactivated by peptide bond cleavages at hydrophobic amino acid residues due to the action of cathepsin D. These results further support the proposed role cathepsin D in the regulation of cysteine proteinase activity.
Cloning and sequence analysis of brain cDNA encoding a Xenopus D2 dopamine receptor
Authors: Martens Gerard J.M. , Molhuizen Henri O.F. , Gröneveld Diet , Roubos Eric W.
Published: July 2002

A D2 dopamine receptor pharmacologically different from the mammalian D2 receptor has previously been characterized in the amphibian Xenopus laevis. Here we report the cloning of a Xenopus D2 receptor which revealed about 75% amino acid sequence identity with its mammalian counterpart and the presence of an additional 33 amino acid sequence in the 3rd cytoplasmic loop instead of the additional 29 residues in the large form of the mammalian D2 receptor, All 7 predicted transmembrane domains are highly conserved between the Xenopus and mammalian D2 receptors, as are the 1st and 2nd intracellular loop, the 1st and 3rd extracellular loop and the carboxy‐terminal portion of the receptors. The amino‐terminal portion, the 2nd extracellular loop and the middle portion of the 3rd intracellular loop of these receptors, however, differ considerably, Knowledge of the locations of these regions of conservation and divergence within the D2 receptors or Xenopus and mammals will help to delineate portions of the receptor molecule that are functionally important. Interestingly, the 5‐untranslated region of the Xenopus D2 receptor mRNA contains 4 small open reading frames which may affect translational efficiency.
Luminescence of peptide‐bound terbium ions Determination of binding constants
Authors: Dadlez Michal , Góral Jakub , Bierzyński Andrzej
Published: July 2002

Luminescence of Tb3+ ions bound to a calmodulin fragments has been studied. It is shown that during their lifetime excited ions dissociate from the peptide. If concentration of free peptide is high enough they can be coordinated again. As a consequence, observed terbium luminescence lifetime and intensity depends not only on binding equilibrium, but also on concentration or free peptide molecules. In such a system terbium binding constant cannot be correctly determined by simple steady‐state measurements of luminescence intensities. Instead, terbium luminescence decay curves measured at various peptide concentrations must be analysed. Such an analysis has been made for a fragment of the IIIrd calcium binding domain of rat testis calmodulin. Rate constant or terbium association and the equilibrium binding constant corresponding to the best fit of theoretical functions to experimental points have been determined.
Structure and organization of the gene encoding rat S‐adenosylmethionine decarboxylase
Authors: Pulkka Anitta , Ihalainen Ritva , Aatsinki Jyrki , Pajunen Antti
Published: July 2002

The gene for S‐adenosylmethionine decarboxylase (AdoMetDC) was isolated from a rat genomic library using AdoMetDC cDNA as a probe. Nucleotide sequence analysis shows that the rat AdoMetDC gene consists of 8 exons which encode a protein identical to that inferred by a rat AdoMetDC cDNA sequence. The exons range in length from 43 to 1964 base pairs spanning 15672 bases of chromosomal DNA. All of the exon/intronjunctions were found to conform to the consensus splice donor and acceptor sequences, Exon 8 corresponds to the 3′ noncoding region of the 2 species of AdoMetDC mRNA which are formed by alternative utilization of 2 polyadenylation signals separated from each other by 1272 nucleotides. The transcription initiation site was located by SI nuclease protection and by primer extension analysis, −325 nucleotides upstream of the translation initiation codon. The promoter region of the rat AdoMetDC gene contains a TATA box at −29 base pairs. No typical GC or CAAT boxes are located in the promoter, but six GC boxes and several putative binding sites for both tissue‐specific and non‐specific transcription factors are found in the proximal part of intron I. Southern blot analyses reveal a complex hybridization pattern suggesting that there are multiple copies of the AdoMetDC gene in the rat genome.
Cysteine reactivity in sorbitol and aldehyde dehydrogenases Differences towards the pattern in alcohol dehydrogenase
Authors: Johansson Jan , Fleetwood Louise , Jörnvall Hans
Published: July 2002

In sorbitol dehydrogenase only one cysteine residue, Cys‐43, is reactive in both anionic buffer (phosphate) and zinc‐liganding buffer (imidazole) upon carboxymethylation. This is in contrast to the situation in the structurally related liver alcohol dehydrogenase, with either of two alternative Cys residues being reactive, and is compatible with differences in zinc‐binding and active site relationships between these two metalloenzymes. Unrelated aldehyde dehydrogenase, upon carboxamidomethylation, shows a third pattern, now less well defined but confirming the presence of a thiol function of Cys‐302 close to the active site.
The thyroid hormone response element is required for activation of the growth hormone gene promoter by nicotinamide analogs
Authors: Sánchez-Pacheco Aurora , Aranda Ana
Published: July 2002

N′‐Methylnicotinamide and nicotinamide, which decreased in vitro ADP‐ribosylation of nuclear proteins and/or cellular NAD+ content, selectively increased the basal expression of the rat growth hormone (GH) gene promoter and its response to triiodothyronine (T3). This increase was not found when the thyroid hormone response element (TRE) was deleted from the promoter. Transfection with an expression vector for the T3 receptor inhibited basal activity of the TRE‐containing promoter and repressed the stimulatory effect of N′‐methylnicotinamide. The addition of hormone relieved this inhibition and enhanced transcription above levels found in the absence of the transfected receptors. These results suggest a modulatory role of ADP‐ribosylation in hormonal regulation of gene expression.
Precise mapping and molecular characterization of the MFT1 gene involved in import of a fusion protein into mitochondria in Saccharomyces cerevisiae
Authors: Ito Masaki , Yasui Akira , Komamine Atsushi
Published: July 2002

Garrett et al. [Mol. Gen. Genet. 225 (1991) 483‐491] recently reported that an Atp2‐lacZ fusion protein was transported into mitochondria in yeast, thus identifying the MFT1 (mitochondrial fusion targeting) gene as a genomic fragment which complements a mutation (mftI) that failed in targeting a fusion protein into mitochondria [4]. They mapped this gene to the ORF, which we have independently identified as a gene homologous to the cyc07 gene, which is expressed specifically in the S phase during the plant cell cycle. We have mapped the MFT1 gene precisely and found that this gene should correspond to the neighboring ORF, rather than the ORF they identified.
Expression of a Drosophila GABA receptor in a baculovirus insect cell system
Authors: Lee Hwa-Jung , Rocheleau Thomas , Zhang Hai-Guang , Jackson Meyer B. , ffrench-Constant Richard H.
Published: July 2002

Recombinant baculoviruses containing two alternative splice forms of the Drosophila Rdl GABA receptor gene were constructed. Spodoptera frugiperda (Sf21) cells infected with either splice form expressed a transcript of expected size (2.5 kb). Western blotting of cell membrane extracts and immunoprecipitation experiments with an anti‐Rdl antiserum recognized a protein of the expected size of ~65 kDa. Whole cell patch clamp analysis of cells infected with either splice form revealed functional expression of GABA gated chloride ion channels which were blocked by application of 1 μM picrotoxinin. Following replacement of alanine 302 with a serine, a mutation associated with resistance to picrotoxinin and cyclodiene insecticides, mutant channels showed similar levels of insensitivity to picrotoxinin (~ 100‐fold) as those observed in recordings from cultured Drosophila neurons. The significance of the expression of an insect GABA receptor in an insect cell line and the similarity of the results from these functional expression studies to recordings from cultured neurons is discussed.
Cooperative face‐to‐face and edge‐to‐face aromatic interactions of tryptophan indole ring with N7‐quarternized guanine and neutral cytosine bases
Authors: Ishida Toshimasa , Tarui Mariko , In Yasuko , Ogiyama Michiyo , Doi Mitsunobu , Inoue Masatoshi
Published: July 2002

In order to investigate the effect of cytosine base upon the stacking interaction of N7‐quarternized guanine base with tryptophan indole ring, the X‐ray crystal structure of a 1:1 complex of model compounds 1 and 2 was carried out. Contrary to the expectation of the interaction of both molecules in aqueous solution, the crystal structure showed the first example of the simultaneous recognition of the Trp indole ring by guanine and cytosine bases by the coupling of the face‐to‐face and edge‐to‐face aromatic interactions, respectively.
cDNA structure and in situ localization of the Aplysia californica pro‐hormone convertase PC2
Authors: Ouimet Tanja , Mammarbachi Aida , Cloutier Thérèse , Seidah Nabil G. , Castellucci Vincent F.
Published: July 2002

The complete cDNA structure of the Aplysia californica pro‐protein and pro‐hormone convertase PC2 (aPC2) was obtained from a cDNA library of the nervous system. The deduced amino acid sequence revealed that aPC2 exhibits an 85%, 61% and 62% sequence identity to the Lymnaea stagnalis, Xenopus laevis and mouse PC2 homologues, respectively. The deduced primary sequence suggested a protein of 653 amino acids which includes a 27‐ and 88‐amino acid signal peptide and pro‐segment. The signal peptide and the C‐terminal segments are the least conserved regions. On Northern blots of nervous system we detected a transcript of 6.8 kb. The in situ hybridization histochemistry on the abdominal ganglion revealed intense labeling of the bag cells. Large peptidergic cells and clusters of sensory and motor neurons also contained high levels of aPC2 mRNA.
Enzymatic catalysis as a process controlled by protein conformational relaxation
Authors: Kurzyński Michał
Published: July 2002

Great progress in studies of protein dynamics in the past decade propels an essential alteration in our understanding of the enzymatic catalysis phenomenon. A careful analysis of assumptions made by the hitherto used conventional theory of chemical reactions shows that neither of them is in fact satisfied. One of the reasons is the presence of a slow interconformational dynamics within the protein native state. In consequence, the simple classical statement “enzymes accelerate reactions by decreasing the free energy of activation” represents only half of the truth. Enzymatic reactions actually proceed through ‘gates’ of relatively low free energy but it is not the process of activated gate crossing that limits the reaction rate, but the process of generally non‐activated gate opening, controlled by the conformational relaxation. Possible consequences of this fact are pointed out.
Monensin and forskolin inhibit the transcription rate of sucrase‐isomaltase but not the stability of its mRNA in Caco‐2 cells
Authors: Chantret I. , Lacasa M. , Chevalier G. , Swallow D. , Rousset M.
Published: July 2002

Treatment of Caco‐2 cells with forskolin (25 μM) or monensin (1 μM) has previously been shown to cause a marked decrease in the level of sucrase‐isomaltase (SI) mRNA, without any effect on the expression of dipeptidylpeptidase IV (DPP‐IV). In the present work, we report that there is no significant difference in the stability of SI mRNA between control and treated cells. On the other hand, we demonstrate a decrease in the transcription rate of SI mRNA which is sufficient to account for the decrease in the steady‐state level of SI mRNA both in forskolin‐ and monensin‐treated Caco‐2 cells.
Stabilization of xylanase by random mutagenesis
Authors: Arase Akemi , Yomo Tetsuya , Urabe Itaru , Hata Yasuo , Katsube Yukiteru , Okada Hirosuke
Published: July 2002

Four heat‐resistant mutants of xylanase (N56, N102, N104 and F1) were obtained by random mutagenesis. The mutant genes had the following amino acid changes: N56, Ser‐26 to Trp, Gly‐38 to Asp and Thr‐126 to Ser; N102, Gly‐38 to Asp; N104, Gly‐38 to Ser and Arg‐48 to Lys; F1, Ser‐12 to Cys. Kinetic studies showed that N104 is stabilized by an increase in the activation enthalpy, while the other mutants are stabilized by a decrease in the activation entropy.
Thermodynamic considerations on the formation of reactive species from hypochlorite, superoxide and nitrogen monoxide Could nitrosyl chloride be produced by neutrophils and macrophages?
Authors: Koppenol W.H.
Published: July 2002

Hypohalous acids are poor one‐electron oxidizing agents, such that reactions with hydrogen peroxide to yield radical species are not feasible. However, the oxidation of superoxide by hypohalous acids can be a source of hydroxyl or haline radicals. The oxidation of nitrogen monoxide by hypochlorous acid is favourable, but in all likelihood cannot compete with the diffusion‐controlled reaction with superoxide to yield peroxynitrite. The reaction of the latter with hypochlorous acid may lead to nitrosyl chloride, a strongly oxidizing agent [E°′(NOCl/NO•, Cl) = 1.0 V] that is capable of nitrosylating organic compounds and thereby generating mutagens or promutagens.
Interaction of F‐actin with phosphate analogues studied by differential scanning calorimetry
Authors: Nikolaeva Olga P. , Dedova Irina V. , Khvorova Irina S. , Levitsky Dmitrii I.
Published: July 2002

The thermal unfolding of F‐actin and the changes induced in it by the binding of phosphate analogues were studied by differential scanning calorimetry. It is shown that the conformation of actin is drastically altered by interaction with beryllium fluoride or aluminium fluoride, while the effects of vanadate and phosphate are negligible. The effect of beryllium fluoride on the F‐actin structure, as reflected in a significant increase of the actin thermal stability, is much more pronounced in the presence of Mg2+ than in the case of F‐actin polymerized by KCl or LiCl in the absence of Mg2+. It is concluded that differential scanning calorimetry is a very convenient method for probing the conformational changes in F‐actin caused by the interaction with phosphate analogues.
Monoclonal antibodies distinctively recognizing the subtypes of inositol 1,4,5‐trisphosphate receptor: Application to the studies on inflammatory cells
Authors: Sugiyama Tomoyasu , Furuya Akiko , Monkawa Toshiaki , Yamamoto-Hino Miki , Satoh Souichiro , Ohmori Kenji , Miyawaki Atsushi , Hanai Nobuo , Mikoshiba Katsuhiko , Hasegawa Mamoru
Published: July 2002

Monoclonal antibodies were raised that specifically recognize the COOH‐terminal sequences and the loop sequences between the fifth and the sixth transmembrane spanning regions of human inositol 1,4,5‐trisphosphate receptor (IP3R) type 1, 2 and 3. Western blot analysis using Jurkat cells, mouse cerebellum, COS‐7 expressing IP3R type 3 cDNA showed that those monoclonal antibodies reacted specifically with each of these three IP3R subtypes and that they do not cross‐react. These antibodies could be used for the specific immunoprecipitation of IP3Rs. Using these monoclonal antibodies, the expression profiles of IP3R‐subtype proteins were found to be different among inflammatory cells such as macrophages, polymorphonuclear cells, mast cells, eosinophils, splenocytes, thymocytes and megakaryocytic cells. Usually, more than one type of IP3R were expressed in a cell simultaneously. The observation of CMK cells under immunofluorescence confocal microscopy revealed that IP3R type 1 and type 2 are located at different subcellular fractions.
Adding a positive charge at residue 46 of Drosophila alcohol dehydrogenase increases cofactor specificity for NADP+
Authors: Chen Zhuo , Tsigelny Igor , Lee William R. , Baker Michael E. , Chang Simon H.
Published: July 2002

We previously reported that the D39N mutant of Drosophila alcohol dehydrogenase (ADH), in which Asp‐39 is replaced with asparagine, has a 60‐fold increase in affinity for NADP+ and a 1.5‐fold increase in k
cat compared to wild‐type ADH [Chen et al. (1991) Eur. J. Biochem. 202, 263–267] and proposed that this part of ADH is close to the 2′‐phosphate on the ribose moiety of NADP+. Here we report the effect of replacing Ala‐46 with an argine residue, an A46R mutant, on binding of NADP+ to ADH and its catalytic efficiency with the NADP+ cofactor, and a modeling of the three‐dimensional structure of the NAD+‐binding region of ADH. The A46R mutant has a 2.5‐fold lower K
m(app)NADP+
and a 3‐fold higher k
cat with NADP+ compared to wild‐type ADH; binding of NAD+ to the mutant was unchanged and k
cat with NAD+ was lowered by about 30%. For the A46R mutant, the ratio of k
cat/K
m of NAD+ to NADP+ is 85, over ten‐fold lower than that for wild‐type ADH. Our model of the 3D structure of the NAD+‐binding region of ADH shows that Ala‐46 is over 10 Å from the ribose moiety of NAD+, which would suggest that there is little interaction between this residue and NAD+ and explain why its mutation to arginine has little effect on NAD+ binding. However, the positive charge at residue 46 can neutralize some of the coulombic repulsion between Asp‐39 and the 2′‐phosphate on the ribose moiety of NADP+, which would increase its affinity for the A46R mutant. We also constructed a double mutant, D39N/A46R mutant, which we find has a 30‐fold lower K
m(app)NADP+
and 8‐fold higher k
cat with NADP+ as a cofactor compared to wild‐type ADH; binding of NAD+ to this double mutant was lowered by 5‐fold and k
cat was increased by 1.5‐fold. As a result, k
cat/K
m for the double mutant was the same for NAD+ and NADP+. The principle effect of the two mutations in ADH is to alter its affinity for the nucleotide cofactor; k
cat decreases slightly in A46R with NAD+ and remains unchanged or increases in the other mutants.
Identification of annexin 33 kDa in cultured cells as a binding protein of influenza viruses
Authors: Otto Matthias , Günther Anna , Fan Hua , Rick Oliver , Huang Richard T.C.
Published: July 2002

The binding of three influenza A and one influenza B virus strains to proteins of three continuously cultured cell lines was studied using protein overlay and immunostaining methods. The results obtained indicated the presence of both sialic acid‐dependent and ‐independent binding of the virus strains; virus binding to proteins in the molecular mass range from about 40 to 103 kDa was dependent on sialic acid, whereas binding to the 33 kDa protein was independent of sialic acid. The 33 kDa binding protein was identified as annexin, a widely distributed non‐glycosylated calcium‐dependent phospholipid‐binding protein.
Organization and chromosomal localization of the gene for the human bombesin receptor subtype expressed in pregnant uterus
Authors: Gorbulev Valentin , Akhundova Aida , Grzeschik K.-H. , Fahrenholz Falk
Published: July 2002

The gene encoding the human homologue of the guinea pig uterine bombesin receptor [(1992) Eur. J. Biochem. 208,405] was isolated from a genomic lambda library by the PCR/homology screening approach. The gene spans more than 4 kb and consists of 3 exons and 2 introns. The deduced amino acid sequence shows about 86% identity to that of guinea pig bombesin receptor. This subtype of bombesin receptor is expressed in the pregnant uterus and in two human tumour cell lines, T47D (ductal breast carcinoma) and A431 (epidermal carcinoma). PCR analysis of genomic DNA from human‐mouse cell hybrids allows the cloned gene to be localized to the region q26–q28 on chromosome X.
Cloning and sequence analysis of cDNA for a human homolog of eubacterial ATP‐dependent Lon proteases
Authors: Amerik Alexander Yu. , Petukhova Galina V. , Grigorenko Vitaly G. , Lykov Igor P. , Yarovoi Serge V. , Lipkin Valery M. , Gorbalenya Alexander E.
Published: July 2002

Overlapping cDNA clones containing mRNA for a putative Lon protease (LonHS) were isolated from cDNA libraries prepared from human brain poly(A)+ RNA. The determined nucleotide sequence contains a 2814‐bp open reading frame with two potential initiation codons (positions 62–64 and 338–340). The 5'‐terminal 337‐nucleotide fragment of LonHS mRNA is highly enriched with G and C nucleotides and could direct synthesis of the LonHS N‐terminal domain. More likely this region promotes initiation of protein synthesis from the second AUG codon in a cap‐independent manner. The amino acid sequence initiated at the second AUG codon includes 845 residues, over 30% of which are identical to those of eubacterial Lon proteases. Residues of the ‘A’ and ‘B’ motifs of NTP‐binding pattern and a plausible catalytic serine residue are conserved in LonHS. Northern blot analysis revealed LonHS mRNA in lung, duodenum, liver and heart, but not in thymus cells.
Low density lipoprotein oxidizability by copper correlates to its initial ubiquinol‐10 and polyunsaturated fatty acid content
Authors: Kontush Anatol , Hubner Christoph , Finckh Barbara , Kohlschutter Alfried , Beisiegel Ulrike
Published: July 2002

At an early stage of oxidation induced by Cu2+, the rate of oxidative modification of human low density lipoprotein (LDL) from healthy donors correlated negatively to its ubiquinol‐10 (r = −0.58, P < 0.01) and positively to its polyunsaturated fatty acid (PUFA) (r = 0.53, P < 0.05) content. The PUFA/ubiquinol‐10 ratio was the best predictor of LDL susceptibility to oxidation (r = 0.68, P < 0.01). No significant correlation between LDL oxidizability and its α‐tocopherol content was found at any oxidation stage. It is suggested that ubiquinol‐10 plays a central role in the early protection of LDL PUFAs against Cu2+‐induced oxidation whereas α‐tocopherol posesses both pro‐ and antioxidant activity.
Molecular cloning, nucleotide sequence and expression of a Sulfolobus solfataricus gene encoding a class II fumarase
Authors: Colombo Sonia , Grisa Margareth , Tortora Paolo , Vanoni Marco
Published: July 2002

Fumarase catalyzes the interconversion of l‐malate and fumarate. A Sulfolobus solfataricus fumarase gene (fumC) was cloned and sequenced. Typical archaebacterial regulatory sites were identified in the region flanking the fumC open reading frame. The fumC gene encodes a protein of 438 amino acids (47,899 Da) which shows several significant similarities with class II fumarases from both eubacterial and eukariotic sources as well as with aspartases. S. solfataricus fumarase expressed in Escherichia coli retains enzymatic activity and its thermostability is comparable to that of S. solfataricus purified enzyme despite a 11 amino acid C‐terminal deletion.
Expression of rat chymotrypsinogen in yeast: a study on the structural and functional significance of the chymotrypsinogen propeptide
Authors: Venekei István , Gráf László , Rutter William J.
Published: July 2002

The role of the propeptide sequence and a disulfide bridge between sites 1 and 122 in chymotrypsin has been examined by comparing enzyme activities of wild‐type and mutant enzymes. The kinetic constants of mutants devoid of the Cys1‐Cys122 disulfide‐linked propeptide show that this linkage is not important either for activity or substrate specificity. However this linkage appears to be the major factor in keeping the zymogen stable against non‐specific activation. A comparison of zymogen stabilities showed that the trypsinogen propeptide is ten times more effective than the chymotrypsinogen propeptide in preventing non‐specific zymogen activation during heterologous expression and secretion from yeast. This feature can also be transferred in trans to chymotrypsinogen; i.e. the chymotrypsin trypsin propeptide chimera forms a stable zymogen.
Crystallization and preliminary X‐ray diffraction analysis of boar seminal plasma spermadhesin PSP‐I/PSP‐II, a heterodimer of two CUB domains
Authors: Romero Antonio , Varela Paloma F , Sanz Libia , Töpfer-Petersen Edda , Calvete Juan J
Published: July 2002

Boar spermadhesin PSP‐I/PSP‐II (M
r 29 000–30 000), a non‐covalent heterodimer of two CUB domains, was crystallized in two crystal forms. Complete diffraction data sets for hexagonal (space group P61,522) and trigonal (space group P31,221) crystals have been collected up to 2.9 and 2.5 Å resolution, respectively. Cell constants of the hexagonal and trigonal crystal forms are , , and , , respectively. The calculated packing parameters (V
m) are 2.8 and 3.2 Å3/Da for the hexagonal and trigonal crystal forms, respectively, indicating that, in both cases, the asymmetric unit is constituted by one PSP‐I/PSP‐II heterodimer. This paper reports the first crystals of a protein built up by a CUB domain architecture.
Cloning and characterisation of the human adenosine A3 receptor gene
Authors: Murrison E.M. , Goodson S.J. , Edbrooke M.R. , Harris C.A.
Published: July 2002

We have cloned the gene for the human adenosine A3 receptor and report characterisation of its intron/exon structure and upstream untranslated region. The open reading frame is interrupted by a single intron of approximately 2.2 kb, within the coding sequence for the second cytoplasmic loop. Sequence analysis of the upstream region reveals no TATA box but the transcriptional start site has been mapped to a common nucleotide in three tissues by 5′‐RACE and RT‐PCR analysis. Northern blotting, 5′‐RACE PCR and analysis of upstream sequences, have provided no evidence for the occurrence of further introns in the upstream untranslated sequence or of transcriptional regulation by alternative splicing in this region.