bluuebunny/crossref_metadata_2025 huggingface dataset:
Quantification of DNA‐dependent RNA polymerase subunits and initiation factor(s) by antibody‐linked polymerase assays
Authors: Lerbs-Mache S.
Published: July 2002

The antibody‐linked polymerase assay is a method which allows one to assign RNA polymerase activity to SDS‐denatured polypeptides on nitrocellulose membranes using antibodies which were raised against only partially purified polymerase preparations. Here we show that with this method not only enzyme subunits but also initiation factor(s) can be determined in crude homogenates. Moreover the determination is quantitative. Therefore changes in the amount of individual polymerase subunits and factor(s) can be visualized within different crude homogenates.
The Finnish type of the LDL receptor gene mutation: Molecular characterization of the deleted gene and the corresponding mRNA
Authors: Aalto-Setälä Katriina
Published: July 2002

In one third of Finnish patients with the heterozygous form of familial hypercholesterolemia the disease is due to a gross deletion at the 3′‐end of the LDL receptor gene. The present study demonstrates that an 8‐kb deletion completely eliminates exons 16 and 17 and a part of exon 18. Cloning and partial sequencing of a DNA fragment from the mutated allele indicated that the 5′‐boundary of the deletion lies within intron 15 while the 3′‐breakpoint is located at nucleotide 3390 in exon 18. RNA blot hybridization studies revealed that the mutated allele encodes a truncated 4.2 kb mRNA (normal, 5.3 kb). This type of mutation has not been reported in other ethnic groups.
Effects of t‐butyl‐4‐hydroxyanisole and other phenolic antioxidants on tumoral cells and Trypanosoma parasites
Authors: Ferreira J. , Coloma L. , Fones E. , Letelier M.E. , Repetto Y. , Morello A. , Aldunate J.
Published: July 2002

The antioxidant food additives 2(3)‐tert‐butyl‐4‐hydroxyanisole (BHA), 2,6‐di(tert‐butyl)‐p‐cresol (BHT) and the methyl and propyl esters of gallic acid inhibited Trypanosoma cruzi culture growth and oxygen consumption. The I
50 values for growth and oxygen uptake with BHA were 0.284 and 0.400 and for BHT 0.083 and 0.235 mM, respectively. Moreover, BHA inhibited the respiration of several tumor cells, as well as of the procyclic and bloodstream trypomastigote forms of T. brucei brucei, with I
50 in the range 0.29–0.52 mM. Inhibition of the parasites' oxygen uptake by BHA was not of the pure Michaelis‐Menten type, but may be of a mixed form. It is postulated that these compounds are inhibitors because they resemble ubiquinone.
An antiserum to the C‐terminus of the Alzheimer amyloid precursor recognizes a soluble 70 kDa protein
Authors: Simpson James , Bladon Christine M. , Yates Celia M. , Harmar Anthony J.
Published: July 2002

A rabbit antiserum to the C‐terminus of the putative brain amyloid precursor was used to probe Western blots of tissue proteins separated by SDS‐PAGE. The antiserum specifically labelled a protein of approx. 70 kDa in the Tris buffer‐soluble fraction of brain samples from rat, Alzheimer subjects, cases of young and old Down's syndrome, and age‐matched controls. The 70 kDa protein was present in low concentrations in human liver and kidney, and was undetectable in human skeletal muscle. The 70 kDa protein may be a metabolite of the amyloid precursor.
Differential sensitivity to pertussis toxin of 3T3 cells transformed with different oncogenes
Authors: Rey I. , Suarez H. , Lavelle F. , Tocque B.
Published: July 2002

Pertussis toxin (PT), which blocks the activity of several G‐proteins, has been found to exert a marked inhibitory effect on the DNA synthesis induced in 3T3 cells by serum or growth factors. 3T3 cells transformed with human c‐ras oncogenes (Ha‐ras, Ki‐ras, N‐ras) or with src, an oncogene coding for a protein kinase, have lost sensitivity to growth control by PT, even though substrates for PT can still be ADP‐ribosylated in vivo. In contrast, 3T3 cells transformed with the SV40 virus behave like normal untransformed cells with respect to the ability of PT to decrease their growth rate. Oncogenes can thus likely be classified either as ‘responders’ or ‘non‐responders’ to PT.
Cloning and sequencing of a Clostridium perfringens sialidase gene
Authors: Roggentin Peter , Rothe Bernd , Lottspeich Friedrich , Schauer Roland
Published: July 2002

Escherichia coli was transformed with pUC vectors containing Sau3A restriction fragments (RF) of Clostridium perfringens DNA. Two clones expressed sialidase activity when assayed with the fluorogenic substrate 4‐methylumbelliferyl‐α‐D‐N‐acetylneuraminic acid. A synthetic oligonucleotide representing the N‐terminus of the expressed enzyme hybridized with the clostridial insert and with a corresponding 2.1 kb Sau3A RF of the C. perfringens genome. The insert reduced to 1.4 kb, which still encoded active sialidase, has been sequenced. The structural gene encodes 382 amino acids representing an M
r of 42 770. A hydrophobic leader sequence is absent. Upstream from the initiation codon ATG, a GA‐rich region is found and considered as the Shine‐Dalgarno sequence. Homology with the N‐terminus of the Vibrio cholerae sialidase gene and with viral sialidase sequences was not found.
Mechanistic studies on the phosphorylation of photoexcited rhodopsin
Authors: Fowles Charles , Sharma Ram , Akhtar M.
Published: July 2002

The mechanism of the photophosphorylation of rhodopsin was studied using several synthetic peptides corresponding to the sequence of the phosphorylation domain. It was found that the decapeptide (residues 339–348) was effectively phosphorylated by rhodopsin kinase only when incubation was performed in the presence of both rhodopsin and light. These results are interpreted to suggest that in the dark‐adapted state rhodopsin kinase exists in an inactive conformation and that this is converted into a catalytically competent form only after interaction with metarhodopsin II (Rho*)
tRNAPhe deprived of 3′‐terminal adenosyl residue does not stimulate adenosine aminoacylation catalysed by phenylalanyl‐tRNA synthetase from Escherichia coli
Authors: Boutorin A.S. , Gordienko V.A. , Lavrik O.I. , Moor N.A.
Published: July 2002

Phenylalanyl‐tRNA synthetase from Escherichia coli does not catalyze the [14C]phenylalanyl residue transfer from phenylalanyl‐adenylate to adenosine either in the presence or absence of homologous tRNAPhe and tRNAPhe
−A. When the reaction mixture contained dithiothreitol, radioactive substance was detected having a mobility on HPLC column close to that of aminoacyladenosine. The amount of this product depended on the concentration of dithiothreitol in the mixture. Phenylalanyl residue was suggested to undergo transfer from aminoacyladenylate to dithiothreitol molecule.
The effect of methanol and temperature on the kinetics of refolding of ribonuclease A
Authors: Fink Anthony L. , Anderson William D. , Hattersley James E. , Lustig Brooke S.
Published: July 2002

Unfolded ribonuclease A consists of 20% fast refolding (UF) and 80% slow refolding material (US). The latter consists of at least two different forms which refold at different rates. We have used absorbance and fluorescence spectrophotometry to compare the kinetics of refolding in aqueous and aqueous‐methanol solutions. At 1°C and pH 3.0, the addition of increasing concentrations of methanol (to 50%, v/v) had negligible effect on the rates and amplitudes of the slow refolding US states. The effect of temperature on the two slow phases of refolding was determined in 35 and 50% methanol. From Arrhenius plots the energies of activation were found to be in the vicinity of 20 kcal/mol for both processes. The results suggest that both slow phases correspond to proline isomerization, and that the presence of methanol does not significantly perturb the overall refolding process. It is possible that the faster of the slow refolding phases corresponds to the isomerization of a proline residue which is trans in the folded native state but which undergoes extensive isomerization to the cis conformation in the unfolded state.
Isolation and primary structure of the gene encoding fructose‐1,6‐bisphosphatase from Saccharomyces cerevisiae
Authors: Entian Karl-Dieter , Vogel Rudi F. , Rose Matthias , Hofmann Lucia , Mecke Dieter
Published: July 2002

The gene encoding Saccharomyces cerevisiae fructose‐1,6‐bisphosphatase (FBP1) was isolated. Constructed fbpi::HIS3 null mutants were unable to grow with ethanol, and growth was restored after transformation with the cloned fbp gene. The gene codes for a protein of 347 amino acid residues with an M
r of 38131. Homology with the pig kidney cortex and the sheep liver enzyme is 47.7% and 46.6%, respectively, within a central core of 328 amino acid residues. The cloned promoter size was 318 bp and allowed only low level expression of the gene. This indicates a positive activation site (UAS) upstream of the cloned DNA fragment.
An example of substrate channeling between co‐immobilized enzymes Coupled activity of myosin ATPase and creatine kinase bound to frog heart myofilaments
Authors: Arrio-Dupont Martine
Published: July 2002

In myofilaments obtained by Triton X‐100 lysis of frog heart cells in high ionic strength medium, the activity of bound creatine kinase cannot be detected by a coupled enzymatic assay. ATP is channelized toward myosin ATPase, through the unstirred layer near myofilaments and cannot diffuse into the bulk solution. Model systems based upon the coupled kinetics of enzymes co‐immobilized on the same surface may explain this behaviour. This may also account for why myofilament‐bound creatine kinase is more efficient than free enzyme in the cytosol for the physiological recycling of ADP into ATP.
Analysis of intrasugar interproton NOESY cross‐peaks as an aid to determine sugar geometries in DNA fragments
Authors: Chary K.V.R. , Modi Sandeep
Published: July 2002

A systematic analysis of the conformation of deoxyribofuranose rings in DNA fragments has been described using two‐dimensional nuclear Overhauser effect spectroscopy (2D NOESY). The approach is based on the interpretation of the intrasugar proton‐proton distances which can be estimated using a low‐mixing‐time pure‐absorption mode ω1,‐scaled NOESY spectrum. The experimental distances are compared with the theoretical values calculated as a function of pseudorotation phase angle (P) describing the sugar geometries. The approach can be used as a complementary aid to J couplings for establishing sugar conformations in individual nucleotide units of DNA fragments. Using this strategy on d‐ACATCGATGT, we observed that individual nucleotides exhibit O4′‐ endo sugar pucker. The results rule out possibilities of the existence of a fast equilibrium (on the NMR time scale) between C2′‐ endo (or S‐domain) and C3′‐ endo (or N‐domain) sugar puckers.
The binding of eosin‐labeled subunit δ to the isolated chloroplast ATPase, CF1, as revealed by rotational diffusion in solution
Authors: Wagner Richard , Apley Enno C. , Engelbrecht Siegfried , Junge Wolfgang
Published: July 2002

We investigated the binding of subunit δ to solubilized chloroplast ATPase. Purified δ was covalently labeled with eosin 5‐isothiocyanate and its rotational correlation time was determined by a photoselection technique as a function of added CF1 (containing δ) and of CF1(−δ) (lacking δ). In aqueous buffer the rotational correlation time of labeled δ was 33 ns. This is compatible with a rather elongated shape with the dimensions 2b = 100 Å/2a = 28 Å. Binding of δ to CF1 decreased the rotational correlation time about 10‐fold. The result was a biphasic decay of the laser flash‐induced absorption anisotropy which was analyzed to yield the proportion of δ (bound to CF1) relative to δ (free). CF1(−δ), which completely lacked the δ‐subunit, bound one δ (mol/mol) with high affinity (K
d ≈ 100 nM) and at least another δ with about 20‐fold lower affinity. The δ‐containing CF1, revealed only the low‐affinity site(s) for δ. This was compatible with a 1:1 stoichiometry of δ in isolated CF1.
Immunological studies on the respiratory burst oxidase of pig blood neutrophils
Authors: Fukuhara Yukiko , Ise Yayoi , Kakinuma Katsuko
Published: July 2002

Recently, a flavin enzyme (pI 5.0), that is probably responsible for superoxide (O−
2)‐generating oxidase activity, was separated by isoelectric focusing‐polyacrylamide gel electrophoresis (IEF—PAGE) from neutrophil membranes in our laboratory [(1987) J. Biol. Chem. 262, 12316–12322]. In the present work, we performed immunological studies on this enzyme derived from pig blood neutrophils. The enzyme extract obtained on IEF—PAGE was injected into guinea pigs to raise antibodies. IgG antibody against the pI 5.0 protein inhibited maximally 54% of the O−
2‐generating activity of the membrane‐solubilized oxidase, whereas the normal serum IgG was not inhibitory at all. Our results further confirmed that the enzyme (pI 5.0) is one of the component(s) of the O−
2‐generating system. The enzyme gave rise to a band corresponding to a major protein of 72±4 kDa on both non‐denaturing and SDS—PAGE. Immunoblotting after SDS—PAGE demonstrated labelling of peptides of 70–72, 28–32 and 16–18 kDa.
Primary structure of protein B from Pseudomonas putida, member of a new class of 2Fe‐2S ferredoxins
Authors: Morrice Nicholas , Geary Philip , Cammack Richard , Harris Alan , Beg Fatima , Aitken Alastair
Published: July 2002

The primary structure of the 2Fe‐2S ferredoxin (protein B) from the benzene dioxygenase system of Pseudomonas putida strain NCIB 12190 was determined by gas‐phase sequencing of the protein and its fragments. Fast atom bombardment mass spectrometry indicated a molecular mass of 11860 Da. The sequence contained five cysteine residues, four of which would be required to coordinate the iron‐sulphur cluster. The amino acid sequence determined in the present study is compared to that of a protein deduced from the DNA sequence from another strain of Pseudomonas putida. Little sequence homology was observed when protein B was compared to 2Fe‐2S ferredoxins from plant and cyanobacterial sources. The novel sequence determined here suggests a new class of ferredoxin, which is consistent with the observed mid‐point redox potential being significantly less negative ( −155 mV) than those of the 2Fe‐2S ferredoxins involved in photosynthesis (−310 to −455 mV)
Differentiation of tryptic enzymes based on enantiomeric specificity at the deacylation step
Authors: Yamada Hiroyuki , Tanizawa Kazutaka , Kanaoka Yuichi
Published: July 2002

The chiral specificity of tryptic enzymes at their deacylation step has been determined for the first time by virtue of ‘inverse substrates’ carrying optically active acyl groups. Differentiation of tryptic enzymes was also successful with these substrates. The stability of acyl‐thrombin is substantially higher than those of trypsin and plasmin when the (S)‐dihydro‐coumarilyl group is applied. This is in contrast to the result with its (R)‐antipode in which all three enzymes are not differentiated. The use of chiral p‐amidinophenyl esters is proposed as a versatile methodology for the design of specific inhibitors capable of discriminating among tryptic enzymes.
Glycoprotein nature of D2 dopamine receptors
Authors: Grigoriadis Dimitri E. , Niznik Hyman B. , Jarvie Keith R. , Seeman Philip
Published: July 2002

The glycoprotein nature of the ligand binding subunit of photoaffinity‐labeled striatal D2 receptors was investigated. Upon photolysis, [125I]N‐azidophenethylspiperone covalently incorporated into a major band of M
r, 94000 with an appropriate pharmacological profile for D2 receptors as assessed by autoradiography following SDS‐polyacrylamide gel electrophoresis. The exoglycosidase, neuraminidase, altered the electrophoretic mobility of the 94 kDa labeled band to 54 kDa with a slight modification in the binding affinity of [3H]spiperone. Endoglycosidase treatment (glycopeptidase‐F) produced a further increase in the mobility of the 94 kDa peptide to ∼43 kDa. A smaller specifically photolabeled D2 receptor peptide of 34 kDa does not contain terminal sialic acid but is an N‐linked glycoprotein as assessed by lectin affinity chromatography and susceptibility to digestion by glycopeptidase‐F to a peptide of ∼23 kDa.
Increased protein synthesis response to insulin in fibroblasts treated with the diacylglycerol kinase inhibitor R59022
Authors: Hesketh John E. , McKenzie Norma , Campbell Gillian P.
Published: July 2002

Insulin stimulated protein synthesis in quiescent 3T3 fibroblasts. This effect of the hormone was greater in the presence of the diacylglycerol kinase inhibitor R59022 (10−5M) over a range of insulin concentrations from 1μU to 1 mU/ml; R59022 increased the sensitivity of cells to insulin. The amount of radioactive diacylglycerol recovered from cells prelabelled with [3H]glycerol was increased transiently in response to insulin; the response was larger and prolonged in cells given the kinase inhibitor. The results (i) support the hypothesis that diacylglycerol production is part of the signal pathway by which insulin stimulates protein synthesis and (ii) suggest that inhibition of diacylglycerol breakdown leads to increased sensitivity to the hormone.
Complete cDNA sequence of a Dictyostelium ubiquitin with a carboxy‐terminal tail and identification of the protein using an anti‐peptide antibody
Authors: Müller-Taubenberger A. , Westphal M. , Jaeger E. , Noegel A. , Gerisch G.
Published: July 2002

The complete sequence of a Dictyostelium discoideum cDNA is presented that codes for monoubiquitin extended at its C‐terminus by a 52 amino acid tail. The sequence of both the ubiquitin portion and the tail is highly homologous to the one of Saccharomyces cerevisiae and to a partial mouse sequence. The highly basic tail sequence contains a putative metal and nucleic acid‐binding motif. The gene encoding the 0.6 kb mRNA of the C‐terminally extended ubiquitin is represented only once in the haploid genome. The 0.6 kb mRNA as well as its translation product, a 15 kDa protein, is expressed in exponentially growing cells and remains present for at least 5 h of development. Using antibodies against a synthetic peptide that corresponds to the C‐terminal amino acid sequence, a 15 kDa protein containing the extension was also detected in yeast.
Phosphorylation of brush border myosin at threonine on its 20 kDa light chains by a calmodulin‐independent kinase activates its ATPase
Authors: Borysenko Christopher , Rieker James P. , Swanljung-Collins Helena , Montibeller Judith , Collins Jimmy H.
Published: July 2002

A calmodulin‐independent kinase isolated from chicken intestinal brush border phosphorylates brush border myosin mainly at an apparently single threonine on its 20 kDa light chains. Phosphorylation to 1.9 mol phosphate/mol myosin activated the myosin actin‐activated ATPase about 12‐fold, to about 100 nmol/min per mg. Brush border myosin ATPase can thus be activated by phosphorylation either at threonine, by calmodulin‐independent kinase, or at serine, by calmodulin‐dependent myosin light chain kinase, as previously shown [(1987) FEBS Lett. 223, 262–266].
1H‐NMR investigation of the interaction between RNase T1 and a novel substrate analog, 2′‐deoxy‐2′‐fluoroguanylyl‐(3′–5′)uridine
Authors: Shibata Yasuyuki , Shimada Ichio , Ikehara Morio , Miyazawa Tatsuo , Inagaki Fuyuhiko
Published: July 2002

The interaction between RNase T1 and a non‐hydrolysable substrate analog, 2′‐deoxy‐2′‐fluoroguanylyl‐(3′–5′)uridine (GfpU), was investigated using 1H‐NMR spectroscopy. In the complex, the Gfp portion takes the syn form around the glycosidic bond and the 3′‐endo form for the ribose moiety, similar to those found in 3′‐GMP and 2′‐deoxy‐2′‐fluoroguanosine 3′‐monophosphate (Gfp). However, in contrast to the cases of these two inhibitors, the complex formation with GfpU at pH 6.0 was found to shift the His‐40 C2 proton resonance of RNase T1 to high field as much as 1 ppm. At pH 6.0, this histidine residue appears to be unprotonated in the complex, but is protonated in the free enzyme (pK
a of His‐40 being 7.9). His‐40, rather than Glu‐58, is probably involved in the catalytic mechanism as a Lewis base, supporting the recent results from site‐directed mutagenesis.
Identification of proteins of the 40 S ribosomal subunit involved in interaction with initiation factor eIF‐2 in the quaternary initiation complex by means of monospecific antibodies
Authors: Bommer Ulrich-Axel , Stahl Joachim , Henske Annemarie , Lutsch Gudrun , Bielka Heinz
Published: July 2002

Monospecific polyclonal antibodies against seven proteins of the 40 S subunit of rat liver ribosomes were used to identify ribosomal proteins involved in interaction with initiation factor eIF‐2 in the quaternary initiation complex [eIF‐2 × GMPPCP × [3H]Met‐tRNAf × 40 S ribosomal subunit]. Dimeric immune complexes of 40 S subunits mediated by antibodies against ribosomal proteins S3a, S13/16, S19 and S24 were found to be unable to bind the ternary initiation complex [eIF‐2 × GMPPCP × [3H]Met‐tRNAf]. In contrast, 40 S dimers mediated by antibodies against proteins S2, S3 and S17 were found to bind the ternary complex. Therefore, from the ribosomal proteins tested, only proteins S3a, S13/16, S19 and S24 are concluded to be involved in eIF‐2 binding to the 40 S subunit.
Effects of female sex hormones on the activity of serum hyaluronidase
Authors: Khoja Samir M.
Published: July 2002

The activities of serum hyaluronidase from female rats were measured during pregnancy. In pregnant female rats, the activity of serum hyaluronidase was found to increase initially and to fall to a minimum by the last day of the gestation period, but the activity of the enzyme increased after delivery and was similar to normals at 21 days post‐partum. The activity of hyaluronidase obtained from bovine leucocytes was significantly increased when leucocytes were incubated with various concentrations of progesterone. It is suggested that female sex hormones affect lysosomal membranes making them lyse more readily and hence release hyaluronidase.
Influence of probe structure on unique (regiospecific) cleavage of RNA by RNase H
Authors: Metelev V.G. , Zayakina G.V. , Ryabushenko I.L. , Krynetskaya N.F. , Romanova E.A. , Oretskaya T.S. , Shabarova Z.A.
Published: July 2002

Chimeric oligo(ribo‐deoxyribo)nucleotides with an internucleotide pyrophosphate bond are novel probes for regiospecific hydrolysis of RNA by RNase H. It has been shown that the use of d(TGTGTAT)ppGCCAU leads to unique hydrolysis of the TMV RNA fragment pAAUGGCAUACAC between C10 and A11.
Fusion proteins with both insecticidal and neomycin phosphotransferase II activity
Authors: Höfte Herman , Buyssens Saskia , Vaeck Mark , Leemans Jan
Published: July 2002

Hybrid proteins consisting of N‐terminal fragments of increasing length of a Bacillus thuringiensis insecticidal protein (Bt2) fused to neomycin phosphotransferase II (NPTII) were produced in Escherichia coli. The minimum fragment required for insect toxicity is comprised of the first 607 amino acids of Bt2. Fusion proteins not containing this minimum fragment were non‐toxic. The NPTII activity of the different non‐toxic hybrid proteins varied considerably but was not correlated with the length of the Bt2 fragment. Fusion proteins including the minimum toxic fragment of Bt2 exhibited insecticidal and NPTII activity comparable to that of the individual proteins. This was largely independent of the fusion point within Bt2. Our data suggest that the conformation of the Bt2 polypeptide exerts an important influence on the enzymatic activity of the fused NPTII protein. The combination of insecticidal activity and a dominant selectable trait into one protein offers important advantages for the generation of insect resistant transgenic plants.
Regulation of interleukin‐6 receptor expression in human monocytes and hepatocytes
Authors: Bauer Joachim , Lengyel Gabriella , Bauer Tilman Martin , Acs George , Gerok Wolfgang
Published: July 2002

Human blood monocytes normally express the interleukin‐6 receptor. Treatment of cultured monocytes with endotoxin, interleukin‐1β, or interleukin‐6 results in a decrease of interleukin‐6 receptor mRNA levels. Glucocorticoids also cause a drop in monocytic interleukin‐6 receptor mRNA levels. We also found interleukin‐6 receptor expression in cultured human hepatocytes, but in contrast to monocytes, where interleukin‐6 receptor mRNA is repressed by the ligand and by interleukin‐1, treatment of hepatocytes with interleukin‐6 or interleukin‐1 resulted in increased interleukin‐6 receptor mRNA levels. Induction of interleukin‐6 receptor mRNA in hepatocytes was less pronounced when glucocorticoids were omitted from the culture medium. We conclude that during noninflammatory homeostasis, blood monocytes are involved in binding of trace amounts of circulating interleukin‐6. During inflammatory events, the main tissue target of interleukin‐6 may be changed from the monocytic population not only to activated B‐cells, but also to the hepatocytes.
Synthesis of a (desSer1 Ile29 Leu89) chicken cystatin gene, expression in E. coli as fusion protein and its isolation
Authors: Auerswald Ennes A. , Genenger Gabriele , Assfalg-Machleidt Irmgard , Kos Janko , Bode Wolfram
Published: July 2002

A synthetic gene coding for the cysteine proteinase inhibitor (desSer 1 Ile29 Leu89) chicken cystatin was cloned and expressed in E. coli. The gene was assembled from 12 oligonucleotides and inserted into vector pUC 8. Expression as fusion protein was performed in a temperature‐inducible E. coli system. The expression product was synthesized as 20% of total E. coli protein. The fusion protein was purified, the chicken cystatin homologue was split off with CNBr and the N‐terminal sequence confirmed up to position 37. The properties of the purified material correspond to those of natural chicken cystatin. The recombinant cystatin variant binds anti‐chicken cystatin IgG, is inhibitorily active and displays K
i values with papain and with cathepsin B similar to those determined for natural chicken cystatin.
The domain structure of the inhibitor subunit of human inter‐α‐trypsin inhibitor reflects the exon structure of its gene
Authors: Vetr Helga , Kögler Manuela , Gebhard Wolfgang
Published: July 2002

The gene coding for the inhibitor subunit of the human plasma protein complex inter‐α‐trypsin inhibitor has been cloned. The exon structure of the gene corresponds with the organization of the protein in two distinct inhibitor domains. The exons coding for the inhibitor domains each comprise the complete information for a typical Kunitz‐type proteinase inhibitor structure. In contrast to bovine aprotinin, the mature inhibitor protein contains amino acid residues flanking the inhibitor domains on both sides. These amino acid residues are encoded by additional exons.
Reductive chain separation of botulinum A toxin — a prerequisite to its inhibitory action on exocytosis in chromaffin cells
Authors: Stecher B. , Gratzl M. , Ahnert-Hilger G.
Published: July 2002

Cleavage of the disulfide bond linking the heavy and the light chains of tetanus toxin is necessary for its inhibitory action on exocytotic release of catecholamines from permeabilized chromaffin cells [(1989) FEBS Lett. 242, 245–248; (1989) J. Neurochem., in press]. The related botulinum A toxin also consists of a heavy and a light chain linked by a disulfide bond. The actions of both neurotoxins on exocytosis were presently compared using streptolysin O‐permeabilized bovine adrenal chromaffin cells. Botulinum A toxin inhibited Ca2+‐stimulated catecholamine release from these cells. Addition of dithiothreitol lowered the effective doses to values below 5 nM. Under the same conditions, the effective doses of tetanus toxin were decreased by a factor of five. This indicates that the interchain SS bond of botulinum A toxin must also be split before the neurotoxin can exert its effect on exocytosis.
Discrepancy between the intestinal lactase enzymatic activity and mRNA accumulation in sucklings and adults Effect of starvation and thyroxine treatment
Authors: Freund J.-N. , Duluc I. , Raul F.
Published: July 2002

The accumulation profile of intestinal lactase mRNA was investigated in suckling and adult rats and pigs. We found no correlation between the lactase enzymatic activity and the accumulation of the messenger at both developmental stages. Modulation of lactase activity by starvation or thyroxine treatment had no effect on lactase mRNA accumulation in the rat intestine. These results confirm that thyroxine modulates lactase expression essentially at the post‐transcriptional level.
The effect of butylated hydroxytoluene, an inhibitor of lipid peroxidation, on the calcium‐induced uncoupling of rat liver mitochondria
Authors: Novgorodov Sergei A. , Gogvadze Vladimir G. , Medvedev Boris I. , Zinchenko Valeryi P.
Published: July 2002

The accumulation of Ca2+ by rat liver mitochondria in the presence of Pi results in spontaneous activation of respiration, accompanied by progressive loss of the accumulated cation. The lipid peroxidation inhibitor, butylated hydroxytoluene, completely prevents and reverses the loss of accumulated Ca2+ and restores respiration to the state 4 level, but exerts no effect on the rate of Ca2+ accumulation and respiration in the presence of the uncoupler. The strong inhibition by butylated hydroxytoluene of ruthenium red‐insensitive Ca2+ efflux has also been observed. No correlation between the BHT‐sensitive Ca2+ loss and the formation of malonic dialdehyde in mitochondria has been found. The data obtained suggest that the Ca2+‐induced uncoupling of mitochondria is mainly due to the appearance of electrogenic ion fluxes that are controlled by the initial steps of lipid peroxidation.
The structure and properties of horse muscle acylphosphatase in solution Mobility of antigenic and active site regions
Authors: Saudek Vladimir , Williams Robert J.P. , Ramponi Giampietro
Published: July 2002

The solution structure of acylphosphatase determined by proton nuclear magnetic resonance spectroscopy is described. The results allow us to discuss the fold of the protein (101 amino acids), to correlate the exposure and the mobility of the backbone with the antigenicity, and to locate the active site.
Identification of the major tRNAPhe binding domain in the tetrameric structure of cytoplasmic phenylalanyl‐tRNA synthetase from baker's yeast
Authors: Fasiolo F. , Sanni A. , Potier S. , Ebel J.P. , Boulanger Y.
Published: July 2002

Native cytoplasmic phenylalanyl‐tRNA synthetase from baker's yeast is a tetramer of the α2β2 type. On mild tryptic cleavage it gives rise to a modified ∡2β′2 form that has lost the tRNAPhe binding capacity but is still able to activate phenylalanine. In this paper are presented data concerning peptides released by this limited proteolytic conversion as well as those arising from exhaustive tryptic digestion of the truncated β′ subunit. Each purified peptide was unambiguously assigned to a unique stretch of the β subunit amino acid sequence that was recently determined via gene cloning and DNA sequencing. Together with earlier results from affinity labelling studies the present data show that the Lys 172—Ile 173 bond is the unique target of trypsin under mild conditions and that the N‐terminal domain of each β subunit (residues 1–172) contains the major tRNAPhe binding sites.
cDNA cloning and nucleotide sequence of rat muscle‐specific enolase (ββ enolase)
Authors: Ohshima Yuka , Mitsui Hiromi , Takayama Yoichi , Kushiya Etsuko , Sakimura Kenji , Takahashi Yasuo
Published: July 2002

The nucleotide sequence of rat muscle‐specific enolase cDNA was determined by sequencing three cDNA clones encoding this enolase isozyme. The nearly full‐length cDNA consists of 13‐bp 5′‐ and 84‐bp 3′‐noncoding regions and a poly(A) tail in addition to a 1302‐bp coding region encoding a polypeptide composed of 434 amino acid residues. The deduced primary structure of this enolase isozyme is about 80% similar to those determined previously for rat neuron‐specific and non‐neuronal enolase isozymes. Southern blot analysis suggested strongly the existence of a single copy of the muscle‐specific enolase gene per haploid genome. The mRNA for this enolase isozyme was detected in rat skeletal muscle on day 1 after birth and its level increased rapidly during 10–30 days after birth without any change in its size (1500 bases).
Preparation and characterization of a ruthenium‐labeled Rhus stellacyanin
Authors: Farver Ole , Pecht Israel
Published: July 2002

Reaction of Cu(II)‐stellacyanin with excess (NH3)5Ru(H2O)2+ yields a protein to which (NH3)5Ru(III) is coordinated. The binding stoichiometry of Ru:Cu is (2.1 ± 0.1):1 as shown by ICP atomic emission measurements. In the 1H‐NMR spectrum of the Ru(III)‐labeled protein, the signals from the C‐2H and C‐4H protons of His‐32 and His‐100 are absent. The ESR spectra of the native oxidized Cu(II)‐stellacyanin and of the Ru(III)‐labeled protein are identical in spite of the (NH3)5Ru(His)3+ ions being a low‐spin d5 system. These data are interpreted as being the result of binding of two RU(III) ions to the modified stellacyanin at two proximal imidazole side‐chains which are within a separation distance that allows for their electronic coupling.
Human platelets release α‐6‐L‐fucosyltransferase upon activation
Authors: Antoniewicz J. , Bykowska K. , Zdebska E. , Kościelak J.
Published: July 2002

Previously we have shown that human platelets release α‐6‐L‐fucosyltransferase (EC 2.4.1.68) during coagulation of blood [(1987) Glycoconjugate J. 4, 43–49]. Here we report that agonists which induce platelet aggregation bring about release of the enzyme. In quantitative terms the release of α‐6‐L‐fucosyltransferase by washed, aggregated platelets was very similar to that occurring during blood coagulation.
A peptidyl α‐amidation activity in chromaffin granules of bovine adrenal medulla
Authors: Bastiaensen Erik , De Potter Werner
Published: July 2002

Peptidyl α‐amidation activity in bovine adrenal medulla has been localized in chromaffin granules by density gradient centrifugation. The activity was found to be both soluble and membrane‐associated. Both enzymatic activities were stimulated by the addition of Cu2+ and ascorbate. The pH maximum for α‐amidation in the chromaffin granules in pH 8.0–8.5. By gel filtration, the soluble enzyme activity appeared as a protein of approx. 40 kDa. It is suggested that this enzyme is involved in the carboxyl‐terminal amidation of metorphamide, amidorphin and neuropeptide Y.
Family of human Na+,K+‐ATPase genes Structure of the putative regulatory region of the α+‐gene
Authors: Sverdlov E.D. , Bessarab D.A. , Malyshev I.V. , Petrukhin K.E. , Smirnov Yu.V. , Ushkaryov Yu.A. , Monastyrskaya G.S. , Broude N.E. , Modyanov N.N.
Published: July 2002

The primary structure of the putative regulatory region of a gene of the Na+,K+‐ATPase multigene family in the human genome has been determined. This region includes the first exon with all of the untranslatable sequence of mRNA and a dozen nucleotides, coding for the first four amino acids of the hypothetic precursor of the α+‐subunit. The entire region comprises over 1400 bp. The possible role of specific nucleotide blocks within this region in comparison with other genes is discussed.
Expression of human 5‐lipoxygenase cDNA in Escherichia coli
Authors: Noguchi Masato , Matsumoto Takashi , Nakamura Motonao , Noma Masana
Published: July 2002

A cDNA for human 5‐lipoxygenase (5LO) was inserted into the vector pKC (constructed from pKK223‐3 by replacing its replication origin with that of pUC18) and expressed in Escherichia coli. The enzyme expressed was purified to homogeneity from the cellular soluble fraction. The purified enzyme showed both 5LO and leukotriene A4 synthase activities, which were stimulated by Ca2+ and ATP. Its molecular mass (78 kDa) and NH2‐terminal sequence were identical with those of 5LO purified from human leukocytes. The availability of the expression system will facilitate further studies on its regulation and the reaction mechanism of the enzyme.
Characterization of γ‐crystallins from eye lenses of shark: closer structural similarity to mammalian than other piscine γ‐crystallins?
Authors: Chiou S.-H.
Published: July 2002

Lens crystalline were isolated and characterized from sharks of the cartilaginous fishes. Four crystallin fractions corresponding to α‐,⨿H‐,βL‐ and γ‐crystallins, similar to those of mammalian crystallins, were obtained. The native molecular masses and subunit structures of these purified fractions were analyzed by gel permeation chromatography, SDS gel electrophoresis and isolectric focusing, revealing the typical subunit compositions with various extents of heterogeneity in each orthologous crystallin class. Amino acid and N‐terminal sequence analyses corroborate the identification and classification of crystallin classes based on electrophoresis. Unexpectedly, it was found that the amino acid composition and N‐terminal sequence of shark γ‐crystallin are more closely related to those of bovine than carp γ‐crystallin. This finding may have some bearing on the divergence and specification of γ‐crystallins between the phylogenetic lines of mammals and fishes.
N‐Nitroso‐β‐lactams as β‐lactamase inhibitor
Authors: Tanizawa Kazutaka , Santoh Keiko , Kanaoka Yuichi
Published: July 2002

N‐Nitroso‐β‐phenyl‐β‐lactam has been found to be a specific inhibitor of β‐lactamase. N‐Nitroso‐α‐phenyl‐β‐lactam, by contrast, was virtually ineffective although a transient inhibition of short duration was observed. The acyl enzyme derived from the β‐phenyl isomer is presumably involved in a cross‐linking reaction, whereas that from the α‐phenyl isomer was quenched by spontaneous hydrolysis without formation of a covalent bond. No inhibitory effect of the β‐phenyl isomer on chymotrypsin has been observed.
Transport of CMP‐N‐glycoloylneuraminic acid into mouse liver Golgi vesicles
Authors: Lepers Anne , Shaw Lee , Cacan René , Schauer Roland , Montreuil Jean , Verbert André
Published: July 2002

CMP‐Neu5Gc has been shown to be transported into mouse liver Golgi vesicles by a specific carrier the characteristics of which were investigated in detail. In the system employed, CMP‐Neu5Gc enters the Golgi vesicles within 2 min; transport was saturable with high concentrations of the sugar‐nucleotide and was dependent on temperature. A kinetic analysis gave an apparent K
m of 1.3 μM and a maximal transport velocity of 335 pmol/mg protein per min. Almost identical values were obtained with CMP‐Neu5Ac, under the same incubation conditions. Furthermore, the uptake of CMP‐Neu5Gc was inhibited by CMP‐Neu5Ac, a substrate analogue. Conversely, the uptake of CMP‐Neu5Ac was inhibited by CMP‐Neu5Gc to the same extent, leading to the conclusion that the transport of CMP‐Neu5Ac and CMP‐Neu5Gc is mediated by the same carrier molecule. This transport system for CMP‐Neu5Gc involves both CMP and CMP‐Neu5Gc since intravesicular CMP induced the entry of external CMP‐Neu5Gc.
Electron transfer reactions in photosystem I following vitamin K1 depletion by ultraviolet irradiation
Authors: Biggins John , Tanguay Normand A. , Frank Harry A.
Published: July 2002

Photosystem I preparations were irradiated with UV to destroy vitamin K1 in situ. The depletion of vitamin K1 resulted in inactivation of NADP+ photoreduction and introduction of a ∼220 ms component in the flash generated P700+ re‐reduction at room temperature. The photoreduction of the terminal FeS centers FA and FB in control and vitamin K1‐depleted preparations at 7 K were comparable. The data confirm that vitamin K1 is functionally implicated in primary electron transfer reactions in PS I at physiological temperature, and that the anomalous results at cryogenic temperature may be explicable in terms of a by‐pass of the vitamin K1 acceptor site or heterogeneity introduced into the photosystem by quinone removal.
Regulation of colominic acid biosynthesis by temperature: Role of cytidine 5′‐monophosphate N‐acetylneuraminic acid synthetase
Authors: González-Clemente Camino , Luengo José M. , Rodríguez-Aparicio Leandro B. , Reglero Angel
Published: July 2002

Synthesis of colominic acid in Escherichia coli K‐235 is strictly regulated by temperature. Evidence for the role of cytidine 5′‐monophospho‐N‐acetylneuraminic acid (CMP‐Neu5Ac) synthetase in this regulation was obtained by measuring its level in E. coli grown at 20 and 37°C. No activity was found in E. coli grown at 20°C. CMP‐Neu5Ac started to be quickly synthesized when bacteria grown at 20°C were transferred to 37°C and was halted when cells grown at 37°C were transferred to 20°C. These findings suggest that temperature regulates the synthesis of this enzyme and therefore the concentration of CMP‐Neu5Ac necessary for the biosynthesis of colominic acid.
Effect of starvation and insulin in vivo on the activity of the pyruvate dehydrogenase complex in rat skeletal muscles
Authors: Denyer Gareth S. , Lam David , Cooney Gregory J. , Caterson Ian D.
Published: July 2002

The in vivo responses of pyruvate dehydrogenase (PDH) complex to starvation and insulin was assessed in heart, diaphragm and red quadriceps muscle. PDH complex activity was decreased by starvation (3.4–10.2‐fold), the magnitude of change depending on muscle type. Insulin increased PDH activity in all muscle types. In fed rats, this effect was relatively small (1.25–1.29‐fold). In starved rats there were effects in heart (4.3‐fold) and red quadriceps (1.7‐fold) but no effect in diaphragm. These results demonstrate that PDH complex in different groups of muscle has different insulin sensitivity (particularly in tissues from starved animals).
Patterns of nucleotide substitutions and implications for the immunological diversity of human immunodeficiency virus
Authors: Shimizu Nobuaki , Okamoto Takashi , Moriyama Etsuko N. , Takeuchi Yasuhiro , Gojobori Takashi , Hoshino Hiroo
Published: July 2002

Human immunodeficiency virus (HIV) exhibits immunological hypervariability, which has been an obstacle to successful production of effective anti‐HIV vaccines. In this study, we estimated patterns of nucleotide and amino acid substitutions in the env gene of HIVs, with the aim of finding characteristics of the mechanism which generates the immunological diversity of the env protein of HIVs. We found that nucleotide changes between A and G are predominant compared to those between other nucleotides. Since this feature is consistent with the pattern of nucleotide substitutions of other retroviral genes but is quite different from those of most eukaryotic genes, a high rate of nucleotide substitution between A and G appears to be specific for retroviruses including HIVs. We discuss the biological relationship between this biased substitution and the mechanism generating hypervariability of epitopes on the env protein of HIVs.
A class of non‐selective cation channels in human fibroblasts
Authors: Galietta Luis J.V. , Mastrocola Teresa , Nobile Mario
Published: July 2002

Non‐selective cation channels were detected in membrane patches of cultured human fibroblasts. The channels had a unitary conductance which ranged from 14 to 25 pS in symmetrical 130 mM NACl and were permeable to both sodium and potassium ions. Open channel probability was dependent either on the membrane potential and the Ca2+ concentration on the intracellular side of the membrane. High Ca2+ concentrations in the millimolar range were needed to keep the channel active.
Calcium ion binding by thermitase
Authors: Briedigkeit Lutz , Frömmel Cornelius
Published: July 2002

Thermitase is a thermostable member of the subtilisin protease family. It was found that this enzyme binds very strongly one calcium ion. This metal ion cannot be removed without denaturation by any known method. A second binding site for calcium ions can be depleted partially by gel filtration, similar to the strong binding site of subtilisin Carlsberg. In both enzymes the calcium ion bound to this site can be removed completely using Ca2+ chelating reagent. In thermitase and subtilisin Carlsberg, depleting calcium ions from any binding site results in a significant decrease of stability against autolysis.
Inositol trisphosphate‐stimulated calcium release from Acer microsomal fractions involves the uptake of potassium
Authors: Canut H. , Carrasco A. , Graziana A. , Boudet A.M. , Ranjeva R.
Published: July 2002

Microsome fractions from Acer cell‐suspension cultures imported calcium from the incubation medium in an ATP‐dependent manner. Intravesicular calcium was released on addition of inositol trisphosphate. Outward calcium transport was associated with inward potassium transport that was inhibited by a potassium channel blocker but not by calcium channel blockers. Therefore, there is a link between inositol trisphosphate‐controlled events and the potassium channel in plants.
The 34 kDa mitochondrial protein, phosphorylation of which is inhibited by vanadate, is the α‐subunit of succinyl‐CoA synthetase
Authors: Křivánek Jiří , Nováková Ludmila
Published: July 2002

Our previous characterization of the 34 500 Da protein, which occurs exclusively in mitochondria and the phosphorylation of which is inhibited rather than stimulated by vanadate, suggested that it might be the α‐subunit of succinyl‐CoA synthetase (SCS). In the present communication we show that in a commercial preparation of SCS there is only one band (34 500 Da) which is phosphorylated under endogenous phosphorylation conditions in vitro. The relative molecular weight of this band, sensitivity of its phosphorylation to the inhibitory action of both vanadate and vanadyl as well as dose response curves are identical to those described for the mitochondrial ‘34 kDa’ protein. This is the first demonstration of an inhibitory effect of vanadium ions on the autophosphorylation of the α‐subunit of SCS.
Studies on the reactive site of the cystatin superfamily using recombinant cystatin A mutants
Authors: Nikawa Takeshi , Towatari Takae , Ike Yoshimasa , Katunuma Nobuhiko
Published: July 2002

For study of the inhibition mechanism of the cystatin superfamily, cystatin A artificial mutants were obtained in which a well‐conserved QVVAG region in the cystatin superfamily was changed to KVVAG or QVTAG and these mutants were then expressed in E. coli. For this, genes with these sequences were synthesized enzymatically from 11 oligodeoxy‐nucleotides and expressed under the tac promoter gene of the E. coli plasmids. The products expressed were then purified on Sephadex G‐50 and HPLC DEAE‐5PW columns. The substitutions in cystatin A were confirmed by the amino acid compositions, N‐terminal amino acid sequences and elution positions on ion‐exchange chromatography of the products. The K
i values of these products for the cysteine proteinases, papain and cathepsins B, H and L, were determined in comparison with those of wild type recombinant cystatin A. Results showed that the cystatin A mutants had similar inhibitory activities to those of wild type recombinant cystatin A. Namely replacement of amino acids in the QVVAG sequence of cystatin A did not significantly affect the inhibitory activities on these proteinases. The results suggest that the QVVAG region is less important than the N‐terminal region of cystatin for inhibitory activities on cysteine proteinases.
Effect of subtransition in 1,2‐dipalmitoylphosphatidylcholine model membrane on the spin probe motion
Authors: Gollová J. , Balgavý P.
Published: July 2002

The effect of subgel → gel phase transition (subtransition) on conventional electron spin resonance spectra of cholestane, fatty acid and alkylammonium‐type spin probes has been studied in aqueous 1,2‐dipalmitoyl‐sn‐phosphatidylcholine dispersions. The cooperative onset of the cholestane spin probe rotation about its long axis, with an effective correlation time of 2–3 ns, has been detected at a temperature coinciding with the calorimetric substransition, indicating onset of the host lipid rotational motion. The lipid rotation results in dissolution of the spin probe clusters in the host lipid. In the gel phase, the lateral distribution of impurity molecules is more isotropic than in the subgel phase.