bluuebunny/crossref_metadata_2025 huggingface dataset:
Nucleotide sequence of the promoter region of the citrate synthase gene (gltA) of Escherichia coli
Authors: Hull Elizabeth P. , Spencer Margaret E. , Wood David , Guest John R.
Published: July 2002

The gltA gene, specifying the citrate synthase (EC 4.1.3.7) of Escherichia coli, has been isolated and the nucleotide sequence of a 752 basepair segment containing the gltA promoter and encoding 96 amino‐ terminal residues of the protien has been defined using the dideoxy/M13 method. The results confirm the location and transcriptional polarity of the gltA gene and indicate that the gltA transcript may contain a long leader sequence of 302‐306 nucleotides upstream from the coding region.
Biosynthesis of pigment‐protein complex polypeptides in bacteriochlorophyll‐less mutant cells of Rhodopseudomonas capsulata YS
Authors: Dierstein Roland
Published: July 2002

Bacteriochlorophyll‐less mutant cells of Rhodopseudomonas capsulata YS were capable of synthesizing pigment—protein complex polypeptides under conditions permitting the formation of the photosynthetic apparatus in the wild type. Individual polypeptides were identified by immunoprecipitation. Pulse‐chase experiments revealed that the polypeptides did not accumulate in the membranes but underwent rapid degradation. The data suggest that bacteriochlorophyll is needed to stabilize the polypeptides of pigment—protein complexes.
Reversible helix‐coil transition of pig stomach myosin rod
Authors: Cross R.A. , Bardsley R.G. , Ledward D.A. , Small J.V. , Sobieszek A.
Published: July 2002

Studies using differential scanning calorimetry indicated that the isolated rod domain of the pig stomach myosin undergoes a single reversible helix‐coil transition with a temperature midpoint of 325 K at physiological pH and ionic strength. Similar behaviour was observed for both the isolated LMM and SF‐2 fragments of the rod. The results suggest that the smooth myosin rod has rather uniform structural stability along its length, and is thus unlikely to contain a specialised region which undergoes a substantial force‐generating helix‐coil transition under physiological conditions.
Proteins of the Bacillus stearothermophilus ribosome
Authors: White Stephen W. , Appelt Krzysztof , Dijk Jan , Wilson Keith S.
Published: July 2002

The structure of protein S5 from the small subunit of the Bacillus stearothermophilus ribosome is described to a resolution of 5 Å. The molecular boundary is visible and shows the protein to be essentially compact although slightly elongated in one dimension.
Increases in permeability of Escherichia coli outer membrane induced by polycations
Authors: Katsu Takashi , Yoshimura Shuichi , Fujita Yuzaburo
Published: July 2002

The action of polycations (such as polylysine and compound 48/80) on Escherichia coli was studied with use of Ca2+, K+, and TPP+ ion‐selective electrodes. Rapid efflux of Ca2+ was observed when a polycation was added in cell suspension. The polycation treatment promoted a drug‐inducing K+ release from the cytoplasmic membrane. TPP+ uptake was also increased by addition of a polycation. Without the polycation treatment, the uptake of TPP+ was largely suppressed due to a permeability barrier of the outer membrane. The results show that a polycation disrupted the permeability barrier of the outer membrane.
Newly synthesized proteins are degraded by an ATP‐stimulated proteolytic process in isolated pea chloroplasts
Authors: Malek Ladislav , Bogorad Lawrence , Ayers Arthur R. , Goldberg Alfred L.
Published: July 2002

Up to 22% of the [3H]leucine‐labeled proteins synthesized chloroplasts in the light was degraded during subsequent incubation for 20–40 min. The degradation of these radioactive proteins was more rapid in the light than in the dark and was at least 2‐fold greater in the presence of 5 mM ATP in light or darkness. Exogenous amino acids did not influence degradation rates, although they promoted protein synthesis. Overall, proteins from thylakoid and stromal fractions were degraded at comparable rates. Analysis by electrophoresis in denaturing polyacrylamide gels revealed that many proteins decreased in both fractions. Certain low molecular mass stromal proteins were lost almost completely during a 90 min incubation in the presence of ATP, while others were unaffected or decreased only slightly. Thus Chloroplasts, like eukaryotic and prokaryotic cells and mithochondria, contain an ATP‐stimulated proteolytic system.
The complete amino acid sequence of hirudin, a thrombin specific inhibitor
Authors: Dodt Johannes , Müller Hans-Peter , Seemüller Ursula , Chang Jui-Yoa
Published: July 2002

Color carboxymethylation of cysteine residues with a new chromophoric reagent dimethylaminoazobenzene iodoacetamide, was applied to the micro‐sequence analysis of hirudin, a thrombin specific inhibitor. Six cysteine residues of the reduced hirudin were detected as colored phenylthiohydantoin derivative and 3 tryptic peptides of hirudin (all containing cysteines) were isolated as colored peptide. The complete hirudin sequence, including 6 uncertain positions left in the previous report [Petersen T.E. (1976) in: Protides of the Biological Fluids; 23rd Colloquium, pp. 145, Pergamon Press, London] was established.
The isozyme pattern of glutathione S‐transferases in rat heart
Authors: Ishikawa Toshihisa , Sies Helmut
Published: July 2002

The isozyme pattern of glutathione S‐transferases of rat heart differs markedly from that of liver. Heart lacks the majority of basic isozymes recognized in rat liver, and isoelectric focusing of glutathione S‐transferases obtained from affinity chromatography on S‐hexylglutathione‐linked Sepharose 6B revealed that 3 major acidic isozymes with pI values of 6.8, 6.3 and 4.9 are present in heart, at least two of which were not found in liver. In addition, multiple bands were found in the pI range 5.0–5.5 for glutathione S‐transferase activity and protein staining.
Mutations in the regulatory subunit of soluble phosphofructokinase from yeast
Authors: Nadkarni Medha , Parmar Latika , Lobo Zita , Maitra P.K.
Published: July 2002

Mutant alleles of the gene PFK2 have been obtained that alter the sensitivity to ATP inhibition of the soluble yeast phosphofructokinase. One of the alleles makes the enzyme sensitive to micromolar concentrations of ATP. Intragenic revertants of PFK2 mutants confirm that the PFK2 gene determines not only the regulatory properties of the soluble enzyme but also the catalytic activity of particulate phosphofructokinase.
An electrogenic proton pump in plasma membranes from the cellular slime mould Dictyostelium discoideum
Authors: Strandmann Ralph Pogge-von , Kay Robert R. , Dufour Jean-Pierre
Published: July 2002

Plants and fungi possess an outwardly directed plasma membrane proton pump that may regulate intracellular pH. We provide the first demonstration that amoebae of the slime mould Dictyostelium discoideum also possess a similar proton pump. It can be assayed either as an ATPase activity in highly purified plasma membranes or as a proton pump, after solubilization and reconstruction into liposomes. The pump is inhibited by vanadate, diethylstilbestrol (DES) and miconazole but not by azide or ouabain. The proton pump described here may represent the target for the action of DES and miconazole, both of which have previously been shown to induce stalk cell formation during the in vitro development of Dictyostelium.
Spin label studies of sulfhydryl environment in plasma fibronectin
Authors: Lai Ching-San , Tooney Nancy M. , Ankel Else G.
Published: July 2002

Plasma fibronectin
Spin label
ESR
Sulfhydryl group
Isolation, properties and tissue distribution of rat glutathione transferase E
Authors: Meyer David J. , Christodoulides Lucia G. , Tan K.Hong , Ketterer Brian
Published: July 2002

Glutathione transferase E (5‐5)
Rat liver
Purification
Subunit size
Tissue distribution
Methylation of a middle repetitive DNA sequence class during differentiation in Friend erythroleukemia cells
Authors: Tolberg Mary Ellen , Smith Steven S.
Published: July 2002

We have examined the methylation patterns within middle repetitive sequences in Friend erythroleukemia cells. Mouse‐interspersed‐family‐1 (MIF‐1) and a group characterized by a 1350‐bp Eco‐Bam fragment cloned into pBR322 as plasmid pFS‐13, are both less modified in Friend cell DNA than in normal tissue DNA. The pattern of methylation present in pFS‐13 homologous sequences was found to be stable during cell division, i.e., somatically inherited, and stable during differentiation induced by HMBA.
An edeine resistant mRNA‐dependent protein synthesis system from a Saccharomyces cerevisiae mutant
Authors: Herrera Flor , Moreno Nancy , Martínez José Antonio
Published: July 2002

A cell‐free protein synthesizing system from a mutant of Saccharomyces cerevisiae translated exogenous mRNA in the presence of 2μM edeine, while a similar system from wild‐type strain was completely inhibited by the drug. The mutant ribosomes showed an affinity for [125I]edeine comparable to the wild‐type ribosomes, thereby suggesting that these macromolecules alone were not responsible for the edeine‐resistant capacity of the mutant.
Immunological identification of four different polypeptides in ‘subunit VII’ of mammalian cytochrome c oxidase
Authors: Kuhn-Nentwig Lucia , Kadenbach B.
Published: July 2002

Rat liver cytochrome c oxidase was separated by SDS‐gel electrophoresis into 13 polypeptide bands. Monospecific antisera against the isolated polypeptides VIIa, VIIb and VIIc were raised in rabbits. Cytochrome c oxidase was blotted on nitrocellulose and incubated with the antisera. The antisera reacted only with their corresponding polypeptides, indicating no immunological relationship between polypeptides VIla, VIIb and VIIc. The data also exclude that these polypeptides are proteolytic breakdown products of larger subunits.
Homology between the α and β subunits of chloroplast and bacterial proton‐translocating ATPases
Authors: Deno Hiroshi , Sugiura Masahiro
Published: July 2002

The α and β subunits of tobacco chloroplast proton‐translocating ATPase show 25% sequence homology. When these subunits from tobacco chloroplast and Escherichia coli are compared, 66 amino acid residues are identical and the majority of them are localized in 4 regions. Some nucleotide‐binding enzymes contain sequences homologous to the 4 regions, suggesting that these regions have common functions in catalysis.
Nuclear Overhauser effects and the assignment of the proton NMR spectra of proteins
Authors: Dobson Christopher M. , Howarth Martin A. , Redfield Christina
Published: July 2002

A systematic procedure is outlined to reveal assignments in the 1H NMR spectrum of a protein that are consistent with a limited set of nuclear Overhauser effect data and with specific assumptions. The results of applying this to a group of resonances in the spectrum of hen lysozyme, most of which were previously assigned by independent methods, are described. The number of possible assignment schemes obtained depends on the distance constraints chosen for the Overhauser effect data, and the investigation of this dependence is proposed as a method of assessing the confidence that can be placed in individual assignments obtained in this way.
Glycogen metabolism: a 13C‐NMR study on the isolated perfused rat heart
Authors: Lavanchy N. , Martin J. , Rossi A.
Published: July 2002

Glycogen synthesis from D‐[1‐13C]glucose was observed in the perfused rat heart by 13C‐NMR spectroscopy at 62.9 MHz. The glycogenogenesis was stimulated by pretreatment of the animals with isoprenaline. Whereas in hearts from control rats the incorporation of D‐[1‐13C]glucose into the glycogen remained below the detection threshold, 5 min proton‐decoupled 13C‐NMR spectra revealed, in hearts from treated rats, a significant labelling of the glycogen within the first minutes of the perfusion and a further linear increase of the glycogen resonance for up to 25 min. This model was used to monitor the appearance of 13C‐labelled lactate during ischemia.
1H‐NMR of met‐enkephalin in AOT reverse micelles
Authors: De Marco Antonio , Zetta Lucia , Menegatti Enea , Guarneri Mario
Published: July 2002

1H‐NMR spectra of Met‐enkephalin dissolved in AOT/isooctane reverse micelles are reported at various temperatures. The NH temperature coefficients are compared with those obtained for the same peptide in normal SDS micelles, in bulk water, and in DMSO. The results show that the opioid molecule undergoes the greatest folding in the reverse micellar system. Shift reagents selectively dissolved in the water pools or in the hydrophobic matrix affect the Phe‐4 or the Tyr‐1 aromatic resonances, respectively. This suggests that the phenylalanyl sidechain is spatially close to the carboxyl group at the C‐terminus, whereas the tyrosyl ring points towards the outer region of the AOT micelles.
Acetyl‐blocked N‐terminal structures of sorbitol and aldehyde dehydrogenases
Authors: Fairwell Thomas , Krutzsch Henry , Hempel John , Jeffery Jonathan , Jörnvall Hans
Published: July 2002

Two new dehydrogenase structures, the 354‐residue polypeptide chain of sorbitol dehydrogenase (from sheep liver) and the 500‐residue polypeptide chain of cytoplasmic aldehyde dehydrogenase (from human liver), have blocked N‐termini. The N‐terminal peptides were purified by reverse‐phase high‐performance liquid chromatography and submitted to mass spectrometry after derivatization. They were also analyzed by dipeptidyl carboxypeptidase digestion, utilizing gas chromatography‐mass spectometry for dipeptide identifications. Results are consistent and establish that sorbitol dehydrogenase has N‐terminal acetylalanine and aldehyde dehydrogenase N‐terminal acetylserine in amino acid sequences that are compatible with estimates from chemical analyses. The two N‐terminal residues found are typical of acetylated proteins in general, extend the group of known acetylated dehydrogenases, and show that these intracellular proteins are frequently N‐terminally acetylated.
A novel approach to the study of glycolipid receptors for viruses
Authors: Hansson Gunnar C. , Karlsson Karl-Anders , Larson Göran , Strömberg Nicklas , Thurin Jan , Örvell Claes , Norrby Erling
Published: July 2002

A method for the binding of virus to a silica gel thin‐layer chromatogram is presented. After development the chromatogram is overlayed with the 125I‐labelled virus and the bound virus is autoradiographed. Alternatively, the unlabelled virus may be detected after exposure to monoclonal antibody and labelled anti‐antibody. The Sendai virus strain used did not bind to brain gangliosides earlier proposed to be receptors, but bound to human erythrocyte gangliosides. This finding may be explained by the existence of Sendai virus variants with different receptor specificities.
Novel crystal forms of a proteolytic core of the single‐stranded DNA‐binding protein (SSB) from E. coli
Authors: Hilgenfeld R. , Saenger W. , Schomburg U. , Krauss G.
Published: July 2002

A proteolytic core of the Escherichia coli single‐stranded DNA‐binding protein (SSB) has been crystallized from phosphate buffer. Crystals suitable for X‐ray data collection display monoclinic space group C2 with α = 106.8, b = 62.3 c = 100.2 Å, β = 112° and contain one tetramer of proteolysis product SSB*‐A per asymmetric unit. Two other crystal forms have been obtained in the presence of the inhibitor diisopropylfluorophosphate.
Structural analysis of a new GC‐specific insertion element IS186
Authors: Chong Pele , Hui Ivy , Loo Tip , Gillam Shirley
Published: July 2002

A new insertion sequence, IS186, has been identified from Escherichia coli and sequenced. It contains 1336 base pairs with a terminal inverted repeat of 22 nucleotides. A long open reading frame, from an ATG codon at position 55 to a TAG termination codon at 1327, could code for a polypeptide of 424 amino acids. This element recognizes GC‐rich regions as target sites for insertion.
Inhibition of 5‐lipoxygenase by vitamin E
Authors: Reddanna Pallu , Krishna Rao M. , Channa Reddy C.
Published: July 2002

Purified 5‐lipoxygenase from potato tubers was inhibited strongly by vitamin E and its analogs. The inhibition by d‐α‐tocopherol was found to be irreversible and non‐competitive with respect to arachidonic acid. An IC50 of 5μM was calculated for d‐α‐tocopherol. The inhibition appears to be unrelated to its antioxidant function. Binding studies with 14C‐labelled d‐α‐tocopherol revealed that there is a strong interaction between vitamin E and 5‐lipoxygenase. Tryptic digestion and peptide mapping of 5‐lipoxygenase‐vitamin E complex indicate that vitamin E binds strongly to a single peptide. These studies suggest that cellular vitamin E levels may have profound influence on the formation of leukotrienes.
Membrane lectins on human monocytes
Authors: Roche Annie-Claude , Midoux Patrick , Bouchard Philippe , Monsigny Michel
Published: July 2002

Freshly isolated human monocytes, which do not contain cell‐surface mannose‐specific receptors, bind mannose 6‐phosphate and actively endocytose mannose 6‐phosphate‐bearing neoglycoproteins (6‐P‐Man‐F‐BSA). Three days after isolation, human monocytes endocytose very actively 6‐P‐Man‐F‐BSA as well as Man‐F‐BSA, and the endocytosed neoglycoproteins are rapidly degraded. These results were obtained in quantitative flow cytofluorometry by using a panel of fluoresceinylated sugar‐substituted serum albumins (neoglycoproteins). Thus, in contrast to mannose receptors which appear only after maturation, mannose 6‐phosphate receptors are already present on freshly isolated human monocytes.
Breakdown and synthesis of polyphosphoinositides in fMetLeuPhe‐stimulated neutrophils
Authors: Cockcroft S. , Barrowman M.M. , Gomperts B.D.
Published: July 2002

The interconversions of the inositol‐containing lipids (PI, PI‐P and PI‐P2) and their products (DG, inositol phosphates and PA) in human and rabbit neutrophils stimulated with fMetLeuPhe and PMA have been examined. PMA causes only the phosphorylation of PI to PI‐P whereas fMetLeuPhe causes phosphorylation of both PI and PI‐P yielding PI‐P2 and the hydrolysis of all three lipids. While the predominant reaction is breakdown of PI to PA catalysed by phospholipase D, approx. 2% of PI is converted to polyphosphoinositides and then broken down by the phospholipase C route yielding inositol phosphates and DG. The latter reaction occurs without detectable lag and is a function of receptor occupancy. The amount of inositol trisphosphate thus formed would be sufficient to release Ca2+ from intracellular stores.
Changes in activity and F1 content of mitochondrial H+‐ATPase in regenerating rat liver
Authors: Buckle Malcolm , Guerrieri Ferruccio , Papa Sergio
Published: July 2002

Submitochondrial particles prepared from rat liver during hepatic regeneration exhibit a depressed ATPase activity which is correlated with a decrease in F1 subunit content as shown by SDS‐PAGE. Use of an antibody directed against the F1 portion of the H+‐ATPase complex demonstrated that there is a definite decrease in the amount of β‐subunit of F1 in both Submitochondrial particles and mitochondria from rat liver 24 h after partial hepatectomy
Cyclic AMP independence of Escherichia coli protein phosphorylation
Authors: Dadssi M. , Cozzone A.J.
Published: July 2002

The effect of cyclic AMP on protein phosphorylation was analyzed comparatively in two strains of E.coli differing in their capacity to synthesize this nucleotide, one of them lacking the adenylate cyclase activity. The results obtained from both in vivo and in vitro experiments concurred in showing that the bacterial protein kinase activity is cAMP‐independent.
A one‐step purification of a plant lysozyme from in vitro cultures of Rubus hispidus
Authors: Bernasconi P. , Pilet P.E. , Joliés P.
Published: July 2002

A Rubus hispidus suspension culture, prepared from a callus, showed lysozyme and chitinase activities. A filtered growth medium was used as a lysozyme source. The enzyme was purified by a rapid one‐step purification technique.
Induction of histone H1° differs with different treatments among different cell lines
Authors: Hall Jeffrey M. , Davis Cheryl , Cole R.David
Published: July 2002

Histone H1°
Cell culture
Butyrate
DMSO
Serum withdrawal
Density inhibition
Phorbol ester and calcium act synergistically to enhance neurotransmitter release by brain neurons in culture
Authors: Zurgil Neomi , Zisapel Nava
Published: July 2002

Phorbol ester
Calcium
Brain neuron
Neurotransmitter
Changes in the glutathione status of plasma, liver and muscle following exhaustive exercise in rats
Authors: Lew Helen , Pyke Steven , Quintanilha Alexandre
Published: July 2002

Glutathione
Oxidative stress
Physical exercise
Sequence of the OXA2 β‐lactamase: comparison with other penicillin‐reactive enzymes
Authors: Dale J.W. , Godwin D. , Mossakowska D. , Stephenson P. , Wall S.
Published: July 2002

The nucleotide sequence of the unusual plasmid‐mediated OXA2 β‐lactamase is presented, and compared with other β‐lactamases. The OXA2 enzyme has similar features at the presumed active site, but no other significant regions ofhomology with other penicillin‐reactive enzymes. The active site homology may therefore represent convergent evolution of otherwise dissimilar genes.
Increased β‐aminoisobutyric acid in rat liver with 6‐azauracil and its enantiomer
Authors: Tamaki Nanaya , Fujimoto Shigeko , Mizutani Naomi , Mizota Chizuru
Published: July 2002

When 6‐azauracil was subcutaneously injected, β‐aminoisobutyric acid and β‐alanine contents were increased 22 and 61‐fold, respectively, in rat liver. Incorporation of [methyl‐14Cithymine into β‐aminoisobutyric acid was increased to 42‐fold by 6‐azauracil treatment. The absolute configuration of this amino acid was proved to be the (R)‐form by means of a gas‐chromatographic technique. 6‐Azauracil inhibited β‐alanine‐pyruvate aminotransferase activity with an I
50 of approx. 2.5 mM.
Phosphorylation of hormone‐sensitive lipase by cyclic GMP‐dependent protein kinase
Authors: Strålfors Peter , Belfrage Per
Published: July 2002

In intact rat adipocytes hormone‐sensitive lipase has been shown to be phosphorylated on serine residues in two different phosphorylation sites: a regulatory site phosphorylated by cyclic AMP‐dependent protein kinase and a basal site, which does not directly affect the enzyme activity, phosphorylated by cyclic AMP‐independent protein kinase(s) [(1984) Proc. Natl. Acad. Sci USA 81, 3317‐3321]. Cyclic GMP‐dependent protein kinase catalyzed the phosphorylation of the same two phosphorylation sites on the isolated enzyme, at serine residues. Both sites were phosphorylated at about the same rate, with the hormone‐sensitive lipase activity concomitantly enhanced.
Kinetic analysis of short‐term effects of α‐agonists on gluconeogenesis in isolated rat hepatocytes
Authors: Leverve X.M. , Groen A.K. , Verhoeven A.J. , Tager J.M.
Published: July 2002

Isolated hepatocytes from fasted rats were perifused with glycerol as gluconeogenic substrate. Stimulation of gluconeogenesis with phenylephrine (10−5M) as α‐adrenergic agonist consisted of two distinct phases. The first phase was a transient stimulation of gluconeogenesis and was accompanied by transient changes in cytosolic and mitochondrial redox state; this phase was abolished by the transaminase inhibitor aminooxyacetate. The second phase was a stable stimulation of less magnitude, without change in redox state and insensitive to addition of aminooxyacetate. It is concluded that the first phase is due to a transient enhancement of flux through the malate/aspartate shuttle and that the stable phase is probably due to a stimulation of mitochondrial glycerol‐3‐phosphate dehydrogenase and glycerol kinase.
Inhibition of hepatocyte plasma membrane Ca2+‐ATPase activity by menadione metabolism and its restoration by thiols
Authors: Nicotera Pierluigi , Moore Margo , Mirabelli Francesca , Bellomo Giorgio , Orrenius Sten
Published: July 2002

Incubation of isolated rat hepatocytes with cytotoxic concentrations of menadione resulted in inhibition of plasma membrane Ca2+‐ATPase activity. This could be restored by subsequent treatment with either dithiothreitol or reduced glutathione, suggesting that the inhibition by menadione was due to oxidation of sulfhydryl groups critical for Ca2+‐ATPase activity.
What family of ATPases does the vacuolar H+‐ATPase belong to?
Authors: Lichko L.P. , Okorokov L.A.
Published: July 2002

Polyacrylamide gel electrophoresis (PAGE) of partially purified ATPase from vacuoles of Saccharomyces carlsbergensis under non‐dissociating conditions revealed 3 bands with ATPase activity. Further PAGE in dissociating conditions showed the similarity in composition of these 3 ATPase preparations. They were assumed to contain the same vacuolar ATPase exhibiting, however, different electrophoretic mobility due to the formation of enzyme complexes with different proteins and phospholipids. The ATPase preparation with the highest electrophoretic mobility contained 6 subunits of 75, 62, 16, 14, 12 and 9 kDa. Inhibitors of vacuolar ATPase [14C]DCCD and [14]NEM bound to a 9 kDa polypeptide, while [14C]DES associated with a polypeptide of 75 kDa. A partially purified preparation of the vacuolar ATPase was not phosphorylated by [γ‐32P]ATP under conditions when plasma membrane ATPase formed a phosphorylated intermediate. Our results show that vacuolar H+‐ATPase consists of several polypeptides, does not form the phosphorylated intermediate, and evidently represents a new type of H+‐ATPase of yeast.
Mechanism of coupling periodate‐oxidized nucleosides to proteins
Authors: Senapathy Periannan , Ali Mir Ahmed , Jacob Mathai T.
Published: July 2002

Nucleoside‐antigen
Periodate oxidation
Schiff base
Protein cross‐linking
Polymerization
Antibody production
Interactions of the human red cell membrane tyrosine kinase with heparin
Authors: Boivin P. , Galand C. , Bertrand O.
Published: July 2002

Heparin interacts with protein kinases in various ways; the different patterns of behavior of heparin towards protein kinases contributes to the characterization of these enzymes. We studied the interactions between heparin and a new type of tyrosine kinase extracted from the normal human red cell membrane. We found that heparin inhibited kinase activity by competition with ATP. Furthermore the interaction of heparin with the red cell membrane tyrosine kinase allowed us to use heparin‐agarose chromatography as a step towards tyrosine kinase purification.
cDNA clones coding for the β‐subunit of human liver alcohol dehydrogenase have differently sized 3'‐non‐coding regions
Authors: Heden Lars-Olof , Höög Jan-Olov , Larsson Kerstin , Lake Mats , Lagerholm Eva , Holmgren Arne , Vallee Bert L. , Jörnvall Hans , von Bahr-Lindström Hedvig
Published: July 2002

Three different size classes of cDNA clones coding for the β1‐rsubunit of human alcohol dehydrogenase (ADH) were characterized from a human liver cDNA library. Clones were identified by hybridization with synthetic oligodeoxyribonucleotides. A total of 2530 nucleotides were determined, covering an ADH‐coding region of 1122 nucleotides, a preceding 72‐nucleotide segment and 3 types of 3'‐non‐coding region. The coding nucleotide sequence is in full agreement with the amino acid sequence of the β1subunit. Of 8 clones identified, 6 had a short, 213‐nucleotide 3'‐non‐coding region; 1 an intermediate, 590‐nucleotide 3'‐region; and 1 a long, 1330‐nucleotide 3'‐region. In addition, 2 unused polyadenylation signals were found. These results suggest that human liver β‐ADH mRNAs occur in several size classes, and that in addition to the consensus sequence AATAAA further signals are important for 3'‐end formation.
Sequence determination of a peptide fragment from electric eel acetylcholinesterase, involved in the binding of quaternary ammonium
Authors: Kieffer Brigitte , Goeldner Maurice , Hirth Christian , Aebersold Ruedi , Chang Jui-Yoa
Published: July 2002

Specific photoaffinity labelling of purified electric eel acetylcholinesterase by 3H‐labelled p‐(N,N‐dimethyl‐amino) benzenediazonium fluoroborate allows the identification of a labelled peptide fragment which is described as being involved in the binding of quaternary ammonium ions on this enzyme. Denaturation and proteolytic cleavage of the inactivated enzyme gave a mixture of peptide fragments. The purification of one labelled fragment, containing over 15% of the radioactivity incorporated in the enzyme, led to the following sequence: Gly‐Ser‐X‐Phe. The relatively low amount of this tetrapeptide did not allow us to determine the nature of the labelled residue X.
Limited proteolysis of the cellobiohydrolase I from Trichoderma reesei
Authors: Van Tilbeurgh Herman , Tomme Peter , Claeyssens Marc , Bhikhabhai Rama , Pettersson Göran
Published: July 2002

Limited proteolysis of the cellobiohydrolase I (CBH I, 65 kDa) from Trichoderma reesei by papain yields a core protein (56 kDa) which is fully active against small, soluble substrates such as the chromophoric glycosides derived from the cellodextrins and lactose. Activity against an insoluble substrate, such as Avicel, is however completely lost and concomitantly decreased adsorption onto this microcrystalline cellulose is observed. The peptide (10 kDa), initially split off during proteolysis, is identified as the heavily glycosylated carboxy‐terminal of the native CBH I. Depending on the experimental conditions the core protein is further nicked in between disulfide bonds, but its properties and stability do not appreciably differ from those of intact CBH I. These results lead to the proposal of a bifunctional organisation of the CBH I: one domain, corresponding to the carboxyterminal, acts as a binding site for insoluble cellulose and the other, localised in the core protein, contains the active (hydrolytic) site.
A calorimetric study of Ca2+ binding by the parvalbumin of the toad (Bufo): distinguishable binding sites in the molecule
Authors: Tanokura Masaru , Imaizumi Masamoto , Yamada Kazuhiro
Published: July 2002

Microcalorimetric titrations of the major isotype of parvalbumin (tPA) from toad (Bufo) skeletal muscle, with Ca2+ in the presence and absence of Mg2+ and with Mg2+ in the absence of Ca2+, have been carried out at 25°C and pH 7.O. The results indicate that the two binding sites in each molecule are distinguishable from each other for both Ca2+ binding and Mg2+ binding. Such a characteristic is distinctly different from those of other parvalbumins. The enthalpy changes determined are distinctly different from those of bullfrog parvalbumins on Ca2+ or Mg2+ binding, but are similar to those on Mg2+‐Ca2+ exchange. The results indicate that the reaction of Mg2+‐Ca2+ exchange is driven almost entirely by the large favorable enthalpy change.
The similarities and differences in structures between kringle 1 of prothrombin and kringle 4 of plasminogen
Authors: Williams R.J.P. , Esnouf P. , Lawrence M. , Cederholm-Williams S.A.
Published: July 2002

Kringle structure
NMB
Prothrombin
Plasminogen
β‐sheet
Lysine site
Protein mobility
Activation by phorbol esters of protein kinase C in MCF‐7 human breast cancer cells
Authors: Issandou Marc , Bayard Francis , Darbon Jean-Marie
Published: July 2002

Exposure of MCF‐7 human breast cancer cells to the phorbol ester 12‐O‐tetradecanoylphorbol‐13‐acetate (TPA) leads to the inhibition of cell proliferation. We investigate here the short‐term effects of TPA on subcellular distribution of protein kinase C, and on protein phosphorylation in cultured MCF‐7 cells. We report a rapid and dramatic decrease in cytosolic protein kinase C activity after TPA treatment. Only 30% of the enzymatic activity lost in the cytosol was recovered in the particulate fraction. These data suggest that subcellular translocation of protein kinase C is accompanied by a rapid down‐regulation of the enzyme (70%). Furthermore, TPA and other protein kinase C activators rapidly induce the phosphorylation of a 28 kDa protein in intact MCF‐7 cells. Phorbol esters devoid of tumor‐promoting activity are ineffective both for inducing these early biochemical events and for inhibiting cell proliferation.
Phospholipase C in rat liver plasma membranes
Authors: Melin Per-Martin , Sundler Roger , Jergil Bengt
Published: July 2002

Phospholipase C activity against phosphoinositides in isolated rat liver plasma membranes has been examined using exogenous substrates. The enzyme hydrolyzed phosphatidylinositol 4,5‐bisphosphate 30–40‐times faster than phosphatidylinositol 4‐monophosphate, while phosphatidylinositol was not a substrate. Maximum activity was observed with 1.1 mM phosphatidylinositol 4,5‐bisphosphate at pH 5.0. The enzyme was stimulated by micromolar concentrations of Ca2+. The GTP analogue guanylyl (β,γ‐methylene)diphosphonate enhanced phospholipase C activity at and above 0.3 μM Ca2+, but was inhibitory at 0.1 μM Ca2+. This supports the suggestion that plasma membrane phospholipase C is regulated by guanine nucleotide‐binding protein, but indicates a regulatory mechanism different from that of other enzymes regulated by such proteins.
N‐Methyl‐4‐phenylpyridine (MMP+ together with 6‐hydroxydopamine or dopamine stimulates Ca2+ release from mitochondria
Authors: Frei Balz , Richter Christoph
Published: July 2002

The nigrostriatal neurotoxin N‐methyl‐1,2,3,6‐tetrahydropyridine (MPTP) causes Parkinsonism in humans and laboratory animals. MPTP neurotoxicity is dependent on its oxidation to N‐methyl‐4‐phenylpyridine (MPP+). The mechanism by which MPP+ causes destruction of dopamine‐containing nigrostriatal cells is unknown. Here we show that MPP+ but not MPTP is taken up by energized mitochondria. MPP+ in the presence of dopamine and particularly of 6‐hydroxydopamine stimulates Ca2+ release from mitochondria. Ca2+ release is accompanied by hydrolysis of intramitochondrial pyridine nucleotides. Our findings suggest that the MPTP‐induced model of Parkinson's disease may be due to a disturbed Ca2+ homeostasis in dopamine neurons.
The iron‐sulfur composition of the active site of hydrogenase from Desulfovibrio vulgaris (Hildenborough) deduced from its subunit structure and total iron‐sulfur content
Authors: Hagen W.R. , van Berkel-Arts A. , Krüse-Wolters K.M. , Voordouw G. , Veeger C.
Published: July 2002

Hydrogenase
Iron‐sulfur cluster
(Desulfovibrio vulgaris)
Molecular cloning of human prostate specific antigen cDNA
Authors: Lundwall Åke , Lilja Hans
Published: July 2002

A λgt11 clone encoding prostate specific antigen has been isolated from a human prostate cDNA library. The cDNA insert of 1415 nucleotides hybridizes specifically to a prostate mRNA species of 1.5 kb. The nucleotide sequence codes for part of a signal peptide, a short propiece and the mature protein of 237 amino acid residues. The M
r for the non‐glycosylated protein was 26 089. One potential site for N‐linked carbohydrate attachment was identified. The primary structure shows extensive homology with proteases of the kallikrein family.
Influence of Mg2+ and Ca2+ bound to 1,5‐IAEDANS‐labeled phosphorylated and dephosphorylated heavy meromyosin complexed with F‐actin on polarized fluorescence of the fluorophore
Authors: Borovikov Yu.S. , Wrotek M. , Aksenova N.B. , Lebedeva N.N. , Kakol I.
Published: July 2002

Dephosphorylated and phosphorylated heavy meromyosin, fluorescently labeled with 1,5‐IAEDANS attached at the SH1 group, was introduced into myosin‐free ghost fibres and the polarized fluorescence of the bound label was measured. The results depended on whether the divalent cation binding sites on heavy meromyosin were saturated with Mg2+ or Ca2+. The calculated angles of absorption and emission dipoles and the amount of random fluorophores were significantly changed, indicating that the random mobility and orientation of the fluorophores of phosphorylated and dephosphorylated heavy meromyosin heads complexed with F‐actin in the ghost fibre depend on saturation of heavy meromyosin with Ca2+ or Mg2+. The presence of bound Ca2+ has an opposite effect on the polarized fluorescence of phosphorylated and dephosphorylated 1,5‐IAEDANS‐heavy meromyosin.
Two additional bacteriophage‐associated glycan hydrolases cleaving ketosidic bonds of 3‐deoxy‐D‐manno‐octulosonic acid in capsular polysaccharides of Escherichia coli
Authors: Altmann Friedrich , März Leopold , Stirm Stephan , Unger Frank M.
Published: July 2002

Two bacteriophages degrading 3‐deoxy‐D‐manno‐2‐octulosonic acid‐(KDO)‐containing capsules of Escherichia coli strains were identified. Using modifications of the thiobarbituric acid assay, it was shown that each phage contains a glycan hydrolase activity cleaving one type of ketosidic linkage of KDO. Thus, the enzyme from phage ψ95 catalyses the hydrolysis of β‐octulofuranosidonic linkages of the K95 glycan; and ψ1092, the α‐octulopyranosidonic linkages of the K? antigen of E. coli LP1092. No cross‐reactivity of the phage enzymes with other KDO‐containing capsular polysaccharides was observed.
Structure and order of the protein and carbohydrate domains of prothrombin fragment 1
Authors: Harlos K. , Boys C.W.G. , Holland S.K. , Esnouf M.P. , Blake C.C.F.
Published: July 2002

The three‐dimensional structure of prothrombin fragment 1 has been determined by X‐ray crystallography at 3.8 Å resolution. The fragment is composed of a number of structural units, some of which are ordered while others are disordered. The ordered part of the structure includes a compact kringle unit, a helical domain and a carbohydrate chain. The kringle structure is organized around a close pair of buried disulfide bridges. One of its carbohydrate chains, that attached to Asn 101, is fully ordered, but the carbohydrate chain attached to Asn 77 appears to be disordered. The calcium binding unit is composed of a disordered part containing all ten γ‐carboxyglutamic acid residues and an ordered part forming the helical domain. The highly conserved residues Phe 41, Trp 42 and Tyr 45, which form a hydrophobic cluster on the first helix, interact around a crystallographic two‐fold axis with the equivalent residues in another molecule to form a dimer in the crystal.
Vertical fluctuations of phospholipid acyl chains in bilayers
Authors: Wardlaw John R. , Sawyer William H. , Ghiggino Kenneth P.
Published: July 2002

The possibility of vertical displacement of acyl chains in lipid bilayers has been examined by quenching the fluorescence of 2‐(9‐anthroyloxy)palmitic acid with 5‐ and 16‐doxylstearates in dipalmitoylphosphatidyl‐choline unilamellar vesicles. Measurement of lifetime and steady‐state quenching showed that the dynamic component of quenching was independent of the transverse position of the quencher indicating that a quencher at the 16‐position could interact with a fluorophore at the 2‐position with high frequency. The differences in steady‐state quenching could be accounted for by the differences in the static component of quenching. The results provide further evidence for rapid vertical displacements of acyl chains in phospholipid bilayers.
Proteins of the Thermus thermophilus ribosome Purification of several individual proteins and crystallization of protein TL7
Authors: Sedelnikova S.E. , Agalarov S.C. , Garber M.B. , Yusupov M.M.
Published: July 2002

The procedure of selective removal of eight proteins from the 50 S ribosomal subunit of the extreme thermophilic bacterium Thermus thermophilus has been developed based on extraction at 60°C in the presence of 0.5 M or 1 M NH4Cl and 50% ethanol. CM‐Sepharose CL column chromatography of the protein mixture under non‐denaturing conditions yielded five proteins with a purity of 95% or higher. Crystals of one of these proteins, namely TL7 (probably an analog of L6 protein from the Escherichia coli ribosome) have been obtained using the ‘hanging drop’ method with ammonium sulphate as a precipitant.
Inositol 1,4,5‐trisphosphate and 5′‐GTP induce calcium release from different intracellular pools
Authors: Henne Volkmar , Piiper Albrecht , Söling Hans-Dieter
Published: July 2002

It has been shown recently by several groups that 5′‐GTP can release calcium from intracellular compartments independently from inositol 1,4,5‐trisphosphate (Ins(1,4,5)P3) by a mechanism which seems to be different from that used by Ins(1,4,5)P3. We report here for the first time that the 5′‐GTP‐sensitive and the Ins(1,4,5)P3‐sensitive calcium pools reside in different intracellular compartments.
The structure of mouse L1210 dihydrofolate reductase‐drug complexes and the construction of a model of human enzyme
Authors: Stammers D.K. , Champness J.N. , Beddell C.R. , Dann J.G. , Eliopoulos E. , Geddes A.J. , Ogg D. , North A.C.T.
Published: July 2002

The structure of mouse L1210 dihydrofolate reductase (DHFR) complexed with NADPH and trimethoprim has been refined at 2.0 Å resolution. The analogous complex with NADPH and methotrexate has been refined at 2.5 Å resolution. These structures reveal for the first time details of drug interactions with a mammalian DHFR, which are compared with those observed from previous X‐ray investigations of DHFR/inhibitor complexes. The refined L1210 structure has been used as the basis for the construction of a model of the human enzyme. There are only twenty‐one sequence differences between mouse L1210 and human DHFRs, and all but two of these are located close to the molecular surface: a strong indication that the active sites are essentially identical in these two mammalian enzymes.
Isolation of a thiol‐activated T‐kininogenase from the rat submandibular gland
Authors: Barlas Aydin , Gao Xiaoxing , Greenbaum Lowell M.
Published: July 2002

T‐kininogenase (T‐kgnase) activity has been investigated in tissues of the rat and submandibular glands of the rat, mouse and guinea pig. Both rat and mouse submandibular homogenates showed high T‐kgnase activity. The enzyme has been purified 360‐fold from rat submandibular gland homogenate supernatant fluid. The enzyme has an apparent molecular mass of 28 kDa and a pH optimum of 8.0 toward T‐kininogen. It cleaved T‐kininogen in catalytic quantities to release T‐kinin (Ile‐Ser‐bradykinin) and small quantities of bradykinin and an unknown kinin. The activity of the enzyme was increased 10‐fold in the presence of thiol groups (dithiothreitol) and inhibited by leupeptin (90%) and to a lesser extent by aprotinin (49%), TLCK (46%) and soybean trypsin inhibitor (27%). Pepstatin and PMSF did not inhibit the enzyme. Studies on substrate specificity, pH optimum and agents which inhibit T‐kgnase activity demonstrate that this enzyme is different from plasma and tissue kallikreins, cathepsin D, esterase A and esterase B (other known kininogenases). It is the first thiol‐activated kininogenase to be reported.
Heterogeneity of the amino acid sequence of phytochrome from etiolated oat seedlings
Authors: Grimm Rudolf , Lottspeich Friedrich , Rüdiger Wolfhart
Published: July 2002

Phytochrome from etiolated oat seedlings was digested with subtilisin. A 16 kDa fragment was isolated and investigated by microsequencing. Its amino‐terminal sequence SLPGGSMEV/ML revealed a heterogeneity (valine and methionine) at position 9. This proves expression of several phytochrome genes on the protein level. One can conclude from the ratio valine/methionine that the isophytochrome derived from AP5 (containing methionine) is a major gene product besides isophytochromes derived from genes AP4 and AP3 (containing valine).
Chemical synthesis and expression in E. coli of a human Val8‐ calcitonin gene by fusion to a synthetic human interferon‐γ gene
Authors: Ivanov I. , Gigova L. , Jay E.
Published: July 2002

A gene coding for human Val8‐calcitonin (Val8‐hCT) was synthesized by the solid‐phase phosphite approach and fused to a synthetic human immune interferon‐γ (IFN‐γ) gene. The IFN gene was previously shown to be expressed at a very high level in E. coli [(1986) Gene, in press] due to the control of a strong synthetic promoter and strong ribosome binding site. The cells harboring the fused gene produced 100‐150 μg per 1 of bacterial suspension of immunoreactive calcitonin in the form of hybrid IFN‐γ‐Val8‐hCT protein consisting of 140 amino acids. The Val8‐hCT can be released from this protein by CNBr treatment.
An integrated prediction of secondary, tertiary and quaternary structure of glucose dehydrogenase
Authors: Hönes J. , Jany K.-D. , Pfleiderer G. , Wagner A.F.V.
Published: July 2002

Based on homology of partial sequences, on physico‐chemical evidence and on studies using chemical modification, we came to the tentative conclusion that tetrameric glucose dehydrogenases from Bacillus megaterium and B. subtilis should have a structure closely related to that of lactate dehydrogenase. The overall homology of primary structures was found to be very low, however, and independent predictions of secondary structure produced a clearly different pattern of β‐strands and α‐helices. We nevertheless tried a manual prediction based on the hydrophobic nature of internal β‐sheet and on the amphiphilic character of external helices. This treatment led to the identification of analogues of all the β‐strands present in lactate dehydrogenase with the exception of βC. Six amphiphilic helices were identified corresponding to αB, αC, αD, α1F, α2F and α3G in lactate dehydrogenase. Conserved functional residues were found at analogous positions. The Q and R intersubunit contacts could be identified and partial proteolysis was found to occur on the outer surface of the tetramer. The structure was found to explain the better binding of NADP as compared to NAD+ and offered a rationalization of the role of the essential lysine at position 201.
Identification of nucleus‐encoded F0I protein of bovine heart mitochondrial H+‐ATPase as a functional part of the F0 moiety
Authors: Zanotti Franco , Guerrieri Ferruccio , Capozza Guiseppe , Houštěk Josef , Ronchi Severino , Papa Sergio
Published: July 2002

The F0I protein of apparent M
r 27000, previously characterized [(1988) Eur. J. Biochem. 173, 1–8] as a genuine component of bovine heart F0, has been sequenced and shown to be identical with the nucleus encoded 24668 Da protein characterized earlier [(1987) J. Mol. Biol. 197, 89–100]. It is directly shown by proteolytic cleavage and reconstitution experiments that this protein, denoted here as PVP from the single‐letter codes of the last three residues of the N‐terminus, is involved in proton conduction by F0 and in its sensitivity to oligomycin.
Seminal pepsinogen C is not identical with, but is very similar to gastric pepsinogen C
Authors: Szecsi P.B. , Koch C. , Foltmann B.
Published: July 2002

Human seminal pepsinogen C has been purified and compared with gastric pepsinogen C. The two zymogens cannot be distinguished by amino acid compositions and sequences of the first 28 N‐terminal amino acid residues are identical. Apparent immunological identity is observed with polyclonal antisera. Monoclonal antibodies toward seminal pepsinogen C have been produced. One is able to recognize a non‐carbohydrate antigenic determinant only present in seminal pepsinogen C.
Regulation of a rat lung protein tyrosine kinase activity by reversible phosphorylation/dephosphorylation
Authors: Srivastava Ashok K. , Chiasson Jean-Louis
Published: July 2002

Incubation of a partially purified protein tyrosine kinase from rat lung with Mg2+ and ATP resulted in about 10–15‐fold activation of the enzyme activity as judged by the phosphorylation of poly(Glu:Tyr,4:1), an exogenous substrate. The activation was time dependent and was associated with the phosphorylation of a single protein band of 50 kDa. Phosphoamino acid analysis of the phosphorylated protein indicated that tyrosine was the amino acid being phosphorylated. Upon gel filtration on a Sephacryl S‐200 column, the phosphorylated protein co‐eluted with protein tyrosine kinase and ATP‐binding activities, suggesting that all three activities are part of the same protein. In addition, pretreatment of the partially purified protein tyrosine kinase with alkaline phosphatase inhibited its enzyme activity which could be restored by reincubation with Mg2+ and ATP. These data suggest that a temporal relationship exists between the phosphorylation and the activation states of rat lung protein tyrosine kinase, and that the phospho‐ and dephospho‐ forms represent the active and inactive (or less active) forms, respectively, of the enzyme.