bluuebunny/crossref_metadata_2025 huggingface dataset:
Regulation of fructose 2,6‐bisphosphate levels in cold‐acclimated brown adipose tissue of rat
Authors: Sobrino F. , Gualberto A. , Pintado E.
Published: July 2002

The effects of cold exposure and T4, administration on fructose 2,6‐bisphosphate levels, phosphofructokinase‐2 and pyruvate kinase activities were examined in rat brown adipose tissue. Cold adaptation (14 days) gave rise to a 2‐fold increase in the amount of fructose 2,6‐bisphosphate and phosphofructokinase‐2 activity, and increased the pyruvate kinase activity 4‐fold. If, in addition, the cold‐acclimated rats were treated with T4, these parameters were again significantly enhanced. The effect on phosphofructokinase‐2 was on the V
max, without modification of the K
m (for both fructose 6‐phosphate and ATP) of the enzyme. In the hypothyroid state, however, the activity of pyruvate kinase remains unchanged. These data support previous observations on stimulation of glycolytic flux during cold adaptation in brown adipose tissue, and a permissive role of thyroid hormones in the process.
Trapping the fast‐refolding state of ribonuclease A at subzero temperatures
Authors: Fink Anthony L. , Anderson William D. , Antonino Lisa
Published: July 2002

Unfolded ribonuclease A consists of a mixture of fast‐ and slow‐refolding species. It is generally accepted that the slow‐refolding states arise from isomerization of proline residues. We show that unfolding at subzero temperatures may be used to trap the fast‐refolding species Uf, since the rate of proline isomerization slows down at a much faster rate than protein unfolding. The unfolding was carried out in 5 M guanidine hydrochloride; at −15°C the protein unfolding process is complete within 30 s and under these conditions there is < 1.5% proline isomerization. By using ribonuclease in which Tyr‐115 was nitrated it was possible to rule out significant isomerization of Pro‐114 in the observed slow‐unfolding step.
Block of light responses of salamander rods by pertussis toxin and reversal by nicotinamide
Authors: Falk G. , Shiells R.A.
Published: July 2002

Transducin is the substrate for a pertussis toxin‐catalyzed ADP‐ribosylation in isolated retinal rod disk membranes [(1984) J. Biol. Chem. 259, 23–26]. The effects of the toxin on the light responses of intact dark‐adapted rods were studied. Applied close to a rod outer segment in a retinal slice, pertussis toxin depolarized the rod by a few millivolts and produced a long‐lasting depression of light responses, effects which depended on penetration of toxin into rods. Nicotinamide, an inhibitor of ADP‐ribosylation, not only blocked the action of the toxin, but also reversed the effects once established. The action of nicotinamide itself on rods indicates the presence of endogenous ADP‐ribosyltransferases which may constitute a control system modulating phototransduction. Inhibition of phospholipase C by neomycin had only transient effects indicating that the cGMP, rather than a phosphoinositide, pathway is primary in vertebrate phototransduction. Rapid reversal of pertussis toxin action suggests possible clinical applications of nicotinamide or congeners to the treatment of disease caused by ADP‐ribosylating bacterial toxins.
The binding of in vitro synthesized adenovirus DNA binding protein to single‐stranded DNA is stimulated by zinc ions
Authors: Vos Hans L. , van der Lee Frédérique M. , Sussenbach John S.
Published: July 2002

We have synthesized wild type DNA binding protein (DBP) of adenovirus type 5 (Ad5) and several truncated forms of this protein by a combination of in vitro transcription and translation. The proteins obtained were tested for binding to a single‐stranded DNA‐cellulose column. It could be shown that for binding of in vitro synthesized wild type DBP, it is necessary to add zinc ions to the in vitro translation system. Binding studies with the truncated proteins revealed that deletion of the carboxyl‐terminal 46 amino acids abolishes DNA binding.
A cytoplasmic‐membrane protein repressible by ammonium in Synechococcus R2: altered expression in nitrate‐assimilation mutants
Authors: Madueño Francisco , Vega-Palas Miguel A. , Flores Enrique , Herrero Antonia
Published: July 2002

A protein of ∼48 kDa was present in the cytoplasmic membrane of cells of Synechococcus R2 grown on nitrate or incubated in the absence of combined nitrogen, but not in ammonium‐grown cells. The level of this polypeptide was diminished in mutant strains that also exhibit reduced levels of other components of the nitrate‐reductive system. On the other hand, a mutant constitutive for nitrate uptake also synthesized constitutively the 48 kDa protein.
Monoclonal antibodies which differentiate high‐ and low‐affinity binding sites of interleukin‐2
Authors: Takemoto Hiroshi , Murai Yasuo , Ide Misao
Published: July 2002

Five monoclonal antibodies (L15, L20, L23, L34, and L61) against human recombinant interleukin‐2 were tested for their effects on the interleukin‐2 bioactivity and binding. Four of these monoclonal antibodies, L15, L20, L34, and L61, which had neutralizing activity, completely blocked interleukin‐2 binding to the high‐affinity receptor. On the other hand, L23, which had a very weak neutralizing activity, blocked interleukin‐2 binding to the low‐affinity receptor. These results suggest that there are at least two distinct binding sites on the interleukin‐2 molecule; those for the high‐affinity receptor and those for the low‐affinity receptor. These monoclonal antibodies should be useful tools in the study of the interaction between interleukin‐2 and interleukin‐2 receptor.
Allosteric inhibition of Dictyostelium discoideum fructose‐1,6‐bisphosphatase by fructose 2,6‐bisphosphate
Authors: Andrés Vicente , García-Salguero Leticia , Gómez María-Esther , Aragón Juan J.
Published: July 2002

It has been found that the inhibition of Dictyostelium discoideum fructose‐1,6‐bisphosphatase by fructose 2,6‐P2 greatly diminished when the pH was raised to the range 8.5–9.5, which resulted in a marked decrease of the affinity for the inhibitor with no change in the Km for the substrate. This provides evidence for the involvement of an allosteric site for fructose 2,6‐P2. Moreover, the fact that excess substrate inhibition also decreased at the pH values for minimal fructose 2,6‐P2 inhibition, and was essentially abolished in the presence of fructose 2,6‐P2, strongly suggests that this inhibition takes place by binding of fructose 1,6‐P2 as a weak analogue of the physiological effector fructose 2,6‐P2.
Simple graphical methods for use with complex ligand‐binding and enzyme mechanisms
Authors: Crabbe M.James C.
Published: July 2002

Simple graphical plots in distribution‐free space are shown to provide estimates of mechanism and kinetic parameters for complex steady‐state processes. The methods were tested using simulated 1:1, 1:2, 2:2, 2:3 and 3:3 functions, and real data from polyol dehydrogenase, NADPH oxidation in the presence of DL‐glyceraldehyde, and NADPH oxidation in the presence of an NADPH‐binding protein. Estimates of mechanism and parameters agreed closely with those found from non‐linear regression by computer.
Use of electrofocusing for the analysis and purification of turkey erythrocytes β1‐adrenoceptors
Authors: Marbach Irit , Levitzki Alexander
Published: July 2002

We show that following one cycle of alprenolol affinity chromatography of turkey erythrocyte β1,‐adrenoceptors, electrofocusing on polyacrylamide gels in digitonin, followed by electroelution, results in complete receptor purification. The overall yield from the electrofocusing‐electroelution step of turkey erythrocyte β‐adrenoceptor is 75 ± 3 %. In addition, we are able to demonstrate that receptor‐binding assays can be performed directly on the polyacrylamide gel, using 125I‐cyanopindolol. This method can be employed for minute quantities of receptor which is an advantage when one wishes to characterize rapidly the β‐adrenoceptor in its native state from tissues that may be available only in limited amounts. We also report, for comparison, on the behavior of the turkey erythrocyte β1,‐adrenoceptor on immobiline polyacrylamide gels and the ability to purify only partially the receptor on these gels.
Sequences both 5′ and 3′ to the transcription initiation site contribute to the ability of a mouse H‐2 gene to respond to type I interferon
Authors: Pascucci Anna , Pannuti Antonio , Mantia Girolama La , Lania Luigi
Published: July 2002

To investigate the cis‐acting DNA elements that are involved in the regulation of class I major histocompatibility complex genes by interferon, several promoter fragments of the H‐2Kk gene were linked to the reporter chloramphenicol acetyl transferase (CAT) gene, and the CAT expression was analyzed in stable transfected cell lines. The functional activities of progressive deletions of the 5′‐flanking region of the H‐2Kk gene linked to the CAT gene have allowed us to define a discrete cis‐acting DNA region necessary for interferon‐mediated stimulation. Moreover, the H‐2Kk gene transcribed by the nonregulated SV40 early promoter was also found to be under interferon regulation. Thus interferon enhancement of the H‐2Kk gene expression appears to be mediated by two cis‐acting elements, one located in the 5′‐flanking region and the other by sequences downstream from the transcription initiation site.
The binding of ligands to the 55 kDa component of the interleukin‐2 receptor triggers increased turnover of phosphate bound to an 85 kDa protein
Authors: Mire-Sluis Anthony R. , Hoffbrand A.Victor , Wickremasinghe R.Gitendra
Published: July 2002

The cell‐surface receptor for interleukin‐2 (IL‐2) consists of two unlinked polypeptides of 55 and 75 kDa (p55, p75). The monoclonal antibody antiTac binds to p55 alone. We show here that the binding of either IL‐2 or antiTac to the surface of T lymphocytes triggered the generation of cAMP. Reagents which activate adenyl cyclase by stimulation of its guanine nucleotide‐binding protein (Gs) also stimulated increases in cAMP. All of the above reagents, and cAMP itself, stimulated the turnover of phosphate residues bound to serine and threonine residues of an 85 kDa protein. The data provide evidence that the binding of ligands to the p55 component of the IL‐2 receptor generates a biochemical signal by the stimulation of adenyl cyclase via Gs, and that the consequent generation of cAMP and activation of cAMP‐dependent protein kinase modulates the turnover of p85‐bound phosphate groups.
Nucleotide residues of tRNA, directly interacting with proteins within the complex of the 30 S subunit of E. coli ribosome with poly(U) and NAcPhe‐tRNAPhe
Authors: Abdurashidova G.G. , Tsvetkova E.A. , Budowsky E.I.
Published: July 2002

With the aid of photoinduced tRNA‐protein cross‐linking, nucleotide residues A21, U45 and U60 were shown to interact directly with proteins S5, S7 and S9 respectively, in the complex of the 30 S subunit of E. coli ribosome with poly(U) and NAcPhe‐tRNAPhe. These nucleotide residues are located in the central part of the L‐form in the tertiary structure of RNA.
The effects of oxygen free radicals on the carbohydrate moiety of IgG
Authors: Griffiths H.R. , Lunec J.
Published: July 2002

The CH2‐linked glycoform of rheumatoid IgG is abnormal in having a reduced galactose content. This has been postulated to be a synthetic defect due to a decrease in the level of rheumatoid B cell galactosyltransferase. However, more recent work has indicated that agalactosylation may be common to chronic inflammatory diseases. In this work we have investigated the effect of oxygen free radicals (OFRs), which are generated by activated phagocytic cells at inflammatory sites, on the carbohydrate moiety of IgG. Radiolytically generated peroxy (ROO•) and hydroxyl radicals (OH•) but not superoxide anion radicals (O—2
−) were found to destroy galactose on IgG. After OH• attack, this was associated with an increase in the availability of N‐acetylglucosamine, possibly due to its presence as a terminal residue. These results suggest that the agalactosylation associated with chronic inflammation may not only be synthetic in nature, but may also be a consequence of post‐synthetic degradation by OFRs.
A growth factor for hepatocytes is inactivated by sub‐lethal γ‐irradiation in vivo or in vitro
Authors: Skouteris George G. , Ord Margery G. , Stocken Lloyd A.
Published: July 2002

Serum from partially hepatectomised rats promoted DNA synthesis in primary adult rat hepatocyte cultures. If the rats had been exposed to sub‐lethal γ‐irradiation immediately following operation or if their serum, collected at 3 h, was exposed to irradiation in vitro, the growth‐promoting activity was destroyed. Prostaglandin E2 also stimulated DNA synthesis in the cultures; if PGE2 was irradiated in serum from intact or partially hepatectomised rats its growth‐promoting activity was markedly diminished.
Accessibility of histone H1° and its structural domains to antibody binding in mononucleosomes
Authors: Banchev Todor , Srebreva Ljuba , Zlatanova Jordanka
Published: July 2002

This work is devoted to the study of the immunoreactivity of histone H1° and its major structural domains in mononucleosomes. Three types of antibody populations were used: (i) anti‐H1° which reacted with antigenic determinants situated along the whole polypeptide chain; (ii) anti‐GH5 which recognized epitopes located in the globular region; and (iii) anti‐C‐tail antibodies reacting specifically with fragment 99–193 of the protein molecule. The anti‐GH5 antibodies gave a weak reaction, the C‐tail‐specific antibodies reacted relatively strongly and the antiserum to the intact molecule showed an intermediate level of reactivity. The relative intensifies of the immunoreaction could be interpreted as reflecting the exposure of the antigenic determinants of the individual protein domains in the monosome particle.
Two populations of the high‐potential form of cytochrome b‐559 in chloroplasts treated with 2‐(3‐chloro‐4‐trifluoromethyl)amino‐3,5‐dinitrothiophene (Ant 2p)
Authors: Barabás Klára , Garab Győző
Published: July 2002

Two populations of the high‐potential form of cytochrome b‐559 (cyt b‐559HP) can be distinguished in chloroplasts. In the presence of ≥ 1 μM Ant 2p [2‐(3‐chloro‐4‐trifluoromethyl)anilino‐3,5‐dinitrothiophene] about one‐half of the cyt b‐559 is autooxidized in the dark. This fraction of cyt b‐559HP undergoes rapid (t

≈2 ms) photoreduction followed by reoxidation in the dark with t

100–200 ms. Photoreduction can be inhibited by DCMU [3‐(3,4‐dichlorophenyl)‐1,1‐dimethylurea] and UHDBT [5‐(n‐undecyl)‐6‐hydroxy‐4,7‐dioxobezothiazole] but not by DBMIB (2,5‐dibromo‐3‐methyl‐6‐isopropyl‐p‐benzoquinine), which suggests the involvement of QB or the plastoquinone pool. Another fraction of cyt b‐559HP undergoes slow (t

≥ 1 s), irreversible photooxidation in the presence of ≥ 0.1 μM Ant 2p. The presently available data suggest that the heterogeneity of cyt b‐559HP originates from PS IIα centers.
Structure of the rat liver ribosome 40 S subunit: Freeze‐drying and high‐resolution shadow casting
Authors: Vasiliev V.D. , Selivanova O.M. , Lutsch G. , Westermann P. , Bielka H.
Published: July 2002

Small ribosomal subunits from rat liver have been studied by electron microscopy using freeze‐drying and high‐resolution shadow casting. The absolute hand of the asymmetric subunit has been determined and its three‐dimensional model with a ‘right’ location of the side protuberance has been constructed. The results evidence that pro‐ and eukaryotic ribosomes have a unique and principally similar structural organization.
Conservation of local electric fields in the evolution of Cu,Zn superoxide dismutase
Authors: Desideri Alessandro , Falconi Mattia , Parisi Valerio , Rotilio Giuseppe
Published: July 2002

The trend of the electric field and the value of the electric field flux, sensed by the superoxide substrate in the proximity of the active site, were found to be constant in three highly homologous Cu,Zn superoxide dismutases from ox, pig and sheep, which display large differences in net protein charge and distribution of electrically charged surface residues but very similar catalytic rate constants. The spatial relationship of charges on the protein surface apparently has been conserved during the evolution of this enzyme to create electrostatic facilitation of catalysis.
Butylmalonate is a transition state analogue for aminocylase I
Authors: Röhm Klaus-Heinrich
Published: July 2002

Butylmalonate (butyl propanedioic acid) is a slow‐binding inhibitor of porcine renal aminoacylase I (EC 3.5.1.14), causing transients of activity with half‐times of more than 10 min. At 25°C and pH 7.0, the dissociation rate of the complex is approximately 6 × 10−4 s−1, while the rate constant of complex formation is in the order of 20 M−1·s−1. In good agreement with these data, steady‐state kinetics yield an estimated inhibition constant around 100 μM. Molecular mechanics calculations showed that conformation and charge distribution of butylmalonate are strikingly similar to those of the putative transition state of aminoacylase catalysis.
Diacylglycerol breakdown in plasma membrane of rat intestinal epithelial cells Effect of E. coli heat‐stable toxin
Authors: Banik Nupur Dey , Ganguly Uma
Published: July 2002

Rat intestinal epithelial cells were isolated and the activity of the enzyme diacylglycerol lipase (DG lipase, EC 3.1.1.3) was investigated. When cells were treated with Escherichia coli heat‐stable toxin (ST) liberation of endogenous glycerol and fatty acids was observed. The enzyme responsible for this effect could be demonstrated to be a DG lipase by using specific substrates. It was found that the activity of DG lipase was increased 5–6‐fold with the substrates diolein and 1,2‐dioleyl‐rac‐glycerol and triolein being neutral lipid insensitive to DG lipase. ST had no direct effect on the DG lipase. The enzyme DG lipase was activated via a chain reaction due to the hydrolysis of phosphatidylinositol (PI) by the enzyme PI‐specific phospholipase C stimulated by ST.
Modulation by somatostatin of nerve‐mediated activation of glycogenolysis in the perfused rat liver
Authors: Beckh Karlheinz , Ehlenz Klaus , Arnold Rudolf
Published: July 2002

Perivascular nerve stimulation of rat livers perfused in situ with erythrocyte‐free Krebs‐Henseleit buffer at constant pressure in a non‐recirculating system resulted in an increase of glucose and lactate production and in a decrease of portal flow. Infusion of somatostatin in different concentrations (2 × 10−7, 10−8, 10−9 mol·l−1) reduced the nerve‐mediated activation of glucose release maximally to 66%. There was only a slight effect on the lactate output, the nerve‐mediated reduction of portal flow was unaltered. In controls, somatostatin alone had no effect on the metabolic and hemodynamic parameters. In order to differentiate between a presynaptic and postsynaptic mechanism, the noradrenaline overflow was calculated. The unaltered release of the neurotransmitter in the presence or absence of somatostatin excluded a presynaptic mechanism. To mimic the nerve effects on the carbohydrate metabolism and on the hemodynamics, noradrenaline (2 × 10−7 mol·l−1) was infused instead of the nerve stimulation over a period of 5 min. Somatostatin did not change the endocrine effects of the catecholamine under these conditions. The nerve‐dependent effect of somatostatin suggests that other neurotransmitters (e.g. VIP) or mediators (e.g. prostanoids) may be influenced by somatostatin.
Bovine lens 23, 21 and 19 kDa intrinsic membrane proteins have an identical amino‐terminal amino acid sequence
Authors: Rao Gadiparthi N. , Gutekunst Karen A. , Church Robert L.
Published: July 2002

We have isolated bovine lens intrinsic membrane proteins (MP) having molecular masses of 19, 21 and 23 kDa. Limited amino acid sequence analysis of the amino‐terminal portion of each of these polypeptides revealed a 100% homology in sequence for the number of residues determined (20 amino acids). Northern blot analysis of bovine lens mRNA using a labeled antisense oligonucleotide probe common to the amino acid sequence of these three peptides revealed a single band having an apparent molecular size of 0.8 kb. Taken together, these findings suggest a genetic commonality between these polypeptides.
Translational inhibition by eIF‐2‐phospholipid complex in mammalian cell‐free systems
Authors: Pelaez Fernando , de Haro Cesar
Published: July 2002

The polypeptide chain initiation factor 2 (eIF‐2) binds phospholipid (PL) and becomes a potent inhibitor of translation in hemin‐supplemented reticulocyte lysates [De Haro et al. (1986) Proc. Natl. Acad. Sci. USA 83, 6711–6715]. This binding is independent of calcium ions and seems to be specific for phosphatidylinositol or phosphatidylserine; phosphatidic and arachidonic acids are inactive. Like α‐subunit‐phosphorylated eIF‐2, eIF‐2·PL traps GEF in a non‐dissociable eIF‐2·PL·GEF complex whereby GEF is no longer able to recycle. Initiation is inhibited when no free GEF is available. Translational inhibition by eIF‐2·PL is rescued by equimolar amounts of eIF‐2·GEF. On the basis of this stoichiometry, we have estimated that reticulocyte lysates contain about 60 pmol of GEF/ml (60 nM) eIF‐2·PL also inhibits translation in cell‐free mouse liver extracts and this inhibition is prevented by reticulocyte eIF‐2·GEF suggesting that GEF also functions in liver. However, the eIF‐2·PL complex does not affect translation in such non‐mammalian eukaryotic systems as wheat germ and Drosophila embryos.
Glutathione transferase in the free‐living nematode Panagrellus redivivus
Authors: Papadopoulos A. , Brophy P.M. , Crowley P. , Ferguson M. , Barrett J.
Published: July 2002

The free‐living nematode Panagrellus redivivus was found to have relatively high cytosolic glutathione transferase activity. Chromatofocusing indicated that at least four GSH transferase forms were present in the nematode cytosol. An endogenous molecular factor interfered with the binding of the cytosolic GSH transferases to a glutathione affinity matrix and binding only occurred after a partial purification step. The major resolved GSH transferase form B was a basic enzyme that showed no biochemical homology to the three mammalian multigene GSH transferase families and appeared to interact with plant phenols, possible natural substrates. A minor GSH transferase form AT showed a biochemical relationship to the mammalian α family including catalytic activity with a model lipid hydroperoxide substrate.
Covalent peptide transfer to cell membrane proteins (peptidyl transferase)
Authors: Najjar Victor A. , Bump Nancy J.
Published: July 2002

HL60 cells, rabbit peritoneal granulocytes or membrane preparations of these cells incorporate radioactivity when reacted with the radioactive peptide tuftsin [3H Pro3]‐Thr‐Lys‐Pro‐Arg. The radioactivity which is not diminished by repeated treatments with TCA and NaOH, is covalently bound to a membrane acceptor protein of 100 kDa. The peptide is not displaced by large concentrations of its constituent amino acids. The acceptor protein is resolved into one labeled peak by gel filtration on Sephadex G‐200, Sephacryl S‐300 and by SDS‐PAGE. Digestion by trypsin and chymotrypsin results in the production of smaller fragments.
Adenosine formation by isolated rat kidney mitochondria
Authors: Henke Wolfgang , Ziegler Mathias , Dubiel Wolfgang , Jung Klaus
Published: July 2002

Isolated rat kidney mitochondria are able to generate extraordinary amounts of adenosine. About one‐third of the adenosine formed only results from the degradation of adenine nucleotides. Pyridine nucleotides may contribute to adenosine formation. Nevertheless, there must be an additional, as yet unidentified, acid‐insoluble compound in mitochondria which is able to form a significant portion of adenosine.
Interactions of formylmethionyl‐leucyl‐phenylalanine, adenosine, and phosphodiesterase inhibitors in human monocytes Effects on superoxide release, inositol phosphates and cAMP
Authors: Elliott Keith R.F. , Leonard Edward J.
Published: July 2002

Cessation of the fMLF‐induced burst of human monocyte superoxide release was associated with a rise in cAMP. This was not due to inhibition of phophodiesterase (PDE), the major form of which was the PDE IV isozyme. The action of burst inhibitors did not correlate with cAMP levels: Rolipram, a PDe IV inhibitor, increased cAMP 6‐fold, with minimal effects on the burst; whereas theophylline increased cAMP less than 2‐fold but decreased the burst to less than half. Although theophylline and the adenylate cyclase activator, adenosine, inhibited fMLF‐induced superoxide release, they did not inhibit production of inositol phosphates. Thus, these studies on inhibition of superoxide release implicated neither cAMP nor inositol phosphates.
Uptake and modification of dietary polyprenols and dolichols in rat liver
Authors: Jakobsson Åsa , Swiezewska Ewa , Chojnacki Tadeusz , Dallner Gustav
Published: July 2002

Short and long dolichols and polyprenols in free form or esterified with fatty acids were incorporated into liposomes and administered to rats through a gastric tube. The free alcohols were taken up by the liver to different extents. While uptake in other organs was less, it also involved the fatty acid esters. The use of systems other than liposomes did not increase the efficiency of uptake. Most of the administered lipids were recovered in the lysosomes. Exogenous dolichols and polyprenols were both partly esterified in the liver and, to some extent, also phosphorylated; a portion of the polyprenols was also α‐saturated. These results indicate that various polyisoprenes are taken up, to a small extent, from the diet by tissues under normal conditions and in liver these dietary lipids undergo terminal modifications.
The gene for human thioredoxin maps on the short arm of chromosome 3 at bands 3p11‐p12
Authors: Lafage-Pochitaloff-Huvalé M. , Shaw A. , Dessarps F. , Mannoni P. , Fradelizi D. , Wollman E.E.
Published: July 2002

Thioredoxin, a ubiquitous enzyme possessing an oxidoreductase activity, has recently been cloned in human. Using in situ chromosomal hybridization with a human thioredoxin cDNA probe, we have precisely localized the thioredoxin gene on chromosome 3 at bands 3p11‐p12.
Fatty acylation of yeast glycoproteins proceeds independently of N‐linked glycosylation
Authors: Appukuttan P.S. , Wu Henry C.
Published: July 2002

The relationship between protein glycosylation and fatty acylation of glycoproteins was studied in the wild‐type and asparagine‐linked glycosylation‐deficient mutants (alg1 and alg2) of Saccharomyces cerevisiae. At the non‐permissive temperature (37°C), both mutant cells exhibited increased incorporation of [3H]palmitate into five polypeptides based on SDS‐PAGE. In contrast, the wild‐type yeast cells contained [3H]palmitate‐labeled polypeptides of higher molecular weights, which were converted to the bands seen in the mutant cells upon treatment of the cell extract with endoglycosidase H prior to SDS‐PAGE. In addition, labeling of the wild‐type yeast cells with [3H]palmitate in the presence of tunicamycin revealed the incorporation of [3H]palmitate into the same five bands as found in the alg1 and alg2 mutants at the non‐permissive temperature without tunicamycin. These results indicate that fatty acylation of glycoproteins proceeds independently of protein N‐glycosylation in yeast cells.
Purification of a DNA methyltransferase from Bacillus natto B3364
Authors: Kim Ella L. , Maliuta Staniclav S.
Published: July 2002

An S‐adenosyl‐L‐methionine:DNA‐methyltransferase, termed M · BnaI, was purified from Bacillus natto B3364 strain by successive column chromatography. The molecular weight determined by gel filtration was 37 kDa for M · BnaI. Analysis of methyltransferase by sodium dodecyl sulfate—polyacrylamide gel electrophoresis showed correspondence of the M · BnaI activity with one protein band at a molecular weight of 35 kDa. Sequencing of pUC19 DNA methylated with M · BnaI showed the cytosine‐5 methylation in the BnaI recognition sequence GGAT↓CC at the position indicated by the arrow.
Thrombin‐induced platelet aggregation is affected by external Na+ independently of the Na+/H+ exchange
Authors: Agam G. , Argaman A. , Livne A.
Published: July 2002

Thrombin affects blood platelets by activation of Na+/H+ exchange and induction of aggregation, but the relationship between these effects is under debate. The present study attempts to clarify whether the activation of the exchanger activity is required for platelet aggregation. In apparent support of such a requirement, thrombin‐induced aggregation is higher in Na+ medium than in N‐methylglucamine+ medium and is inhibited by sphingosine, an inhibitor of protein kinase C known to regulate the Na+/H+ exchanger. However, the inhibition of aggregation by sphingosine occurs in both Na+‐containing and Na+‐free media, the aggregation is identical in Na+ and K+‐containing media, and is not inhibited by 5‐N‐(3‐aminophenyl)amiloride, at a concentration 10‐fold higher than its K
i for platelet Na+/H+ exchange. Furthermore, at low concentration (0.005 U/ml) thrombin induces aggregation but does not activate the exchange. It is concluded that the activation of Na+/H+ exchange is not required for thrombin‐induced platelet aggregation and that the apparent augmentation of aggregation by Na+ is due to an inhibitory effect of N‐methylglucamine+.
Light‐scattering changes in the bacteriorhodopsin photocycle
Authors: Drachev L.A. , Kaulen A.D. , Zorina V.V.
Published: July 2002

Light‐scattering changes were found to accompany the bacteriorhodopsin photocycle in a suspension of purple membranes. A light‐scattering signal comprises three phases, connected with (i) bR → M transition, (ii) transition of M to P intermediate (other names: N,R350 intermediate), (iii) P → bR transition. Light‐scattering changes seem to result from changes in the purple membrane shape in response to the bacteriorhodopsin conformational changes during the photocycle.
Reversible reorganization of the chlorophyll‐protein complexes of photosystem II in cyanobacterium cells in the dark ag
Authors: Lebedev N.N. , Van Ni Chan , Krasnovsky A.A.
Published: July 2002

A new emission band at 673 nm was detected in the low‐temperature fluorescence spectrum of dark‐adapted cyanobacteria Gloeotrichia raciborski. The excitation spectrum of this band was close to the absorbance of the isolated reaction centre of photosystem II. The relative intensities of the bands of chlorophyll and pheophytin in this spectrum showed the relative concentrations of these pigments to be about 3:1. The intensity of the band increased with darkness (half‐time about 2 h). Under illumination the band rapidly disappeared (half‐time about 60 s). The appearance of a 673 nm band in the dark and its disappearance in the light were accompanied by a decrease, and, respectively, an increase in the fluorescence of the PS II band at 697 nm.
Clones containing variant forms of complete human rRNA genes characterization and sequence of their transcription initiation region
Authors: Renalier Marie-Hélène , Joseph Nicole , Bachellerie Jean-Pierre
Published: July 2002

In order to assess the extent of structural heterogeneity among ribosomal genes in the human genome, several cosmid clones, each containing an entire transcription unit, have been isolated and analyzed. Five cloned genes were recognized as structurally different from each other to some extent, either within the transcription unit or in its immediate vicinity. The sequence of a 1.2 kb region encompassing the transcription start site has been determined for the five cloned genes. Point differences among the genes are observed at five nucleotide positions within the analyzed portion of the 5′ external transcribed spacer of the ribosomal gene. Moreover, upstream from the transcription start site, a unique difference is observed with the absence from the three genes of a 19‐nucleotide long stretch which is present in the two other variant gene forms.
Topological analysis of the amino‐terminal region of lactose permease using the Escherichia coli outer membrane protein, OmpA, as a marker
Authors: MacIntyre Sheila , Eschbach Marie-Luise , Schwarz Heinz , Ehring Ruth
Published: July 2002

LacY‐ompA fusions, encoding the N‐terminal 50, 71 or 143 residues of lactose permease, were constructed. The observed orientation of the OmpA part of each hybrid protein with respect to the plasma membrane supports current models of the N‐terminus of Lac permease. Hybrids possessing the entire mature OmpA were very stable; those with only a part thereof were much less stable. Due to their in vivo stability and accessibility to antibody it is proposed that such hybrids may represent potential models to investigate the assembly pathway of lactose permease.
Reconstitution of cyc− S49 membranes by in vitro translated Gsα
Authors: Journot L. , Bockaert J. , Audigier Y.
Published: July 2002

After ADP‐ribosylation by cholera toxin which promotes dissociation of the subunits, the α‐subunit of Gs(Gsα) remained strongly associated with plasma membranes of wild‐type S49 cells, since its interaction with the membrane was insensitive to 1 M KCl. Its association with the membrane was partially disrupted by 6 M urea and totally abolished by treatment with alkali at pH ≥ 11.5. In vitro translated Gsα could interact with plasma membranes from the cyc− mutant of S49 cells as revealed by its cosedimentation with the membrane fraction and incubation of reconstituted membranes with GTPγS did not alter anchorage of Gsα. The characteristics of the association of in vitro translated Gsα with cyc− membranes after GTPγS treatment, i.e. sensitivity to 1 M KCl, 6 M urea and alkali treatment, were very similar to those described for the ADP‐ribosylated form in wild‐type membranes. Restoration of the coupling between the adrenergic receptor and adenylate cyclase further confirmed the vectorial reconstitution of cyc− membranes by in vitro translated α‐subunit of Gs.
Epitope mapping of the monoclonal antibody FAC2 on the apoprotein of CPa‐1 in photosystem II
Authors: Frankel Laurie K. , Bricker Terry M.
Published: July 2002

Using a combination of cyanogen bromide cleavage and endoproteinase digestion we have shown that the putative epitope for the monoclonal antibody FAC2 lies in the region 360Pro‐391Ser on the apoprotein of CPa‐1. This region lies entirely within the large extrinsic loop of this protein. We have shown previously that the epitope of FAC2 becomes exposed in oxygen‐evolving membranes upon treatment with alkaline Tris which releases all four of the manganese associated with the oxygen‐evolving site of photosystem II. The epitope is not exposed, however, after CaCl2 treatment and exposure to low concentrations of chloride, conditions which lead to the release of two of the four manganeses associated with the oxygen‐evolving site [(1987) Arch. Biochem. Biophys. 256, 295–301]. These results suggest that, upon release of the chloride‐insensitive manganese from photosystem II membranes, a conformational change occurs which leads to the exposure of 360Pro‐391Ser on CPa‐1 to the monoclonal antibody FAC2.
Activation of protein kinase C alters the interaction of α2‐adrenoceptors and the inhibitory GTP‐binding protein (Gi) in human platelets
Authors: García-Sáinz J.Adolfo , Gutiérrez-Venegas Gloria
Published: July 2002

The effect of 12‐tetradecanoyl phorbol 13‐acetate (TPA) on the hormonal modulation of adenylate cyclase was studied. The effect of epinephrine (α2‐adrenergic action) was markedly diminished in membranes from TPA‐treated platelets as compared to the controls. Interestingly, the inhibitory effect of guanylyl imido diphosphate (Gpp(NH)p) was not altered. Neither the number of α2‐adrenoceptors nor their affinity for [3H]yohimbine were affected by the treatment with TPA. In control platelets, 77% of the receptors were in a high‐affinity state for epinephrine and 22% in a low‐affinity state; Gpp(NH)p shifted the receptor affinity towards the low‐affinity conformation. In membranes from TPA‐treated platelets, the receptors were in the low‐affinity state and no further decrease in affinity was induced by Gpp(NH)p. Our data suggest that activation of protein kinase C in platelets blocks the hormonal inhibition of adenylate cyclase by interfering with the receptor‐Gi interaction.
The F1‐ATPase of vibrio alginolyticus
Authors: Dmitriev O.Yu. , Grinkevich V.A. , Skulachev V.P.
Published: July 2002

The F1‐type ATPase has been isolated from membrane preparations of marine alkalotolerant bacterium. Vibrio alginolyticus. The enzyme was found to consist of two major subunits of 55 and 58 kDa and at least two minor components (38 and 23 kDa). Amino acid sequences of N‐terminal regions of the major subunits revealed close homology with those of E.coli H+ ‐ATPase and of Propionigenium modestum Na+‐ATPase.
Mechanism of the ATP‐dependent phosphatidylserine synthesis in liver subcellular fractions
Authors: Barańska Jolanta
Published: July 2002

Phosphatidylserine; Base‐exchange reaction; ATPase, Ca2+,Mg2+‐; (Endoplasmic reticulum)
In vitro nucleosome assembly with plant histones
Authors: Hofmann Dieter , Zentgraf Hanswalter , Kahl Günter
Published: July 2002

Nucleosome; DNA‐histone interaction; Nucleosome reconstitution; Core histone; (Plant)
Caffeine releases oscillating opening of calcium‐dependent potassium channels in the alga Eremosphaera viridis
Authors: Förster Birgit , Thaler Manfred , Urbach Wolfgang , Simonis Wilhelm
Published: July 2002

Caffeine; K+ channel, Ca2+‐dependent; Oscillating transient potential; (Eremosphaera viridis)
Serum proteins as mediators of hemin efflux from red cell membranes: specificity of hemopexin
Authors: Solar I. , Muller-Eberhard U. , Shaklai N.
Published: July 2002

Hemin; Hemopexin; Albumin; Erythrocyte membrane
Kinetic studies on the prenyl chain elongation by undecaprenyl diphosphate synthase with artificial substrate homologues
Authors: Ohnuma S. , Koyama T. , Ogura K.
Published: July 2002

Undecaprenyl diphosphate synthase; Prenyltransferase; Prenyl chain elongation; Polyprenyl diphosphate; Artificial substrate; Substrate homologue
Spatial and temporal expression pattern of retinoic acid receptor genes during mouse bone development
Authors: Noji Sumihare , Yamaai Tomoichiro , Koyama Eiki , Nohno Tsutomu , Taniguchi Shigehiko
Published: July 2002

Retinoic acid receptor gene; Hybridization, in situ; Bone formation; (Mouse, Forelimb)
Defined monoclonal antibodies to Escherichia coli β‐galactosidase as a tool for characterisation of recombinant expression products
Authors: Bröker Michael , Harthus Hans-Peter
Published: July 2002

Galactosidase, β‐; Monoclonal antibody; Recombinant expression product; Epitope mapping; (E. coli)
Hypomorphic C4B∗ 15 variant of the fourth component of complement
Authors: Christenson Marie J. , LaRosa Thomas , Jung Mark , Bias Wilma B. , McLean Robert H.
Published: July 2002

We report a rare ‘hypomorphic’ C4 allotype detected during routine screening in controls for the Rogers:1 epitope. C4B∗15 was distinguished by having only faint staining when using polyclonal anti‐C4 antibody on agarose inimunoelectrophoresis (e.g. hypomorphic), having relatively weak hemolytic activity but being strongly reactive with monoclonal antibody to Rodgers 1. TaqI restriction fragment length polymorphism (RFLP) demonstrated that C4B∗ 15 segregated with 7 kb and 5.4 kb C4 gene fragments and with the haplotype HLA‐A2,C‐, B50,BW6,DR7,DQ2,DR52,S07C2(1,15). The 5.4‐kb fragment was more intense than the 7.0‐kb fragment, suggesting duplication of the 5.4‐kb fragment. This hypomorphic C4 allotype (genotype frequency = 0.0088) has diminished expression of C4 epitopes commonly recognized by polyclonal anti‐C4 and may be missed by standard phenotyping methods.
Is a four‐state model sufficient to describe actomyosin ATPase?
Authors: Tesi Chiara , Barman Thomas , Travers Franck
Published: July 2002

Four‐ [(1984), J. Biol. Chem. 259, 11908] and six‐ [(1985) Science 227, 999] state models have been proposed for actomyosin ATPase. A key experiment in deciding between these is whether or not there is a transient Pi burst at high actin. In the first, the cleavage and release of products rates are similar and the Pi burst is low; in the second, there are additional product complexes and the Pi burst is large. We reinvestigated the problem by carrying out burst experiments under the conditions in [(1985) Science 227, 999]. Since we find that the Pi burst at high actin is low, we conclude that the four‐state model is sufficient to describe actomyosin ATPase.
Conformational effects of nucleotide exchange in ras p21 proteins as studied by fluorescence spectroscopy
Authors: Skelly Jane V. , Suter David A. , Kuroda Reiko , Neidle Stephen , Hancock John F. , Drake Alex
Published: July 2002

The intrinsic fluorescence properties of the oncogene protein p21N‐ras, p21H‐ras and one of its transforming mutants, p21N‐ras (Va1112), have been investigated. A mutant containing a single tryptophan at position 28 in p21H‐ras (Trp28) has been specifically engineered to provide a probe of protein conformation on nucleotide binding. The proteins produced essentially similar circular dichroism spectra typical of alpha/beta proteins. A decrease in the intensity of the fluorescence emission spectrum due to tyrosine occurred on GDP/GTP nucleotide exchange in the native and mutant proteins. Selective excitation of the single tryptophan in p21 produced a decrease in fluorescence intensity which was accompanied by a blue shift in the wavelength of maximum emission on nucleotide exchange. A reduction in the residual Mg2+ ion concentration enhanced this effect.
A second consensus sequence of ATP‐requiring proteins resides in the 21‐kDa C‐terminal segment of myosin subfragment 1
Authors: Burke Morris , Rajasekharan Kallikat N. , Maruta Shinsaku , Ikebe Mitsuo
Published: July 2002

Previous comparisons of sequence homologies of ATP‐requiring enzymes have defined three consensus sequences which appear to be involved in the binding of the nucleotide. One of these was identified in the N‐terminal 27‐kDa segment of the myosin heavy chain but the other two sequences have not hitherto been located in myosin. The present paper proposes that one of these other two consensus sequences is in the 21‐kDa C‐terminal portion of S1 and that it may contribute to the ATP binding domain.
Reversal by EGTA of the enhanced secretory responsiveness of mast cells due to treatment with ouabain
Authors: Johansen T. , Knudsen T. , Bertelsen H.
Published: July 2002

The effect of EGTA on the enhancement by ouabain of compound ‐induced secretion from mast cells was compared with the effect on the Na+‐K+ pump activity. The time‐dependent secretory enhancement by ouabain was blocked by addition of EGTA to the cell suspension concomitantly with the addition of ouabain, and EGTA caused a large increase in the pump activity. Addition of 10 μM EGTA to ouabain‐treated cells stopped but did not reverse the enhancement. The experiments show that the effect of ouabain was due to changes in a calcium pool utilized in compound ‐induced secretion following changes in the Na+,K+ pump activity.
Molecular weight analysis of isopenicillin N synthase by electrospray mass spectrometry
Authors: Aplin R.T. , Baldwin J.E. , Fujishima Y. , Schofield C.J. , Green B.N. , Jarvis S.A.
Published: July 2002

The use of electrospray mass spectrometry as a tool in analytical biochemistry was illustrated by determination of the molecular weights of wildtype and recombinant isopenicillin N synthase (IPNS). The molecular weight of recombinant IPNS produced using an expression system which generated soluble protein was found to be between 38 364 and 38 376 Da, ca 60 mass units higher than that of the wildtype material, consistent with the presence of an additional N‐terminal glycine in the former. Observed molecular weights were all ca 70 Da higher than that calculated from sequence information, consistent with the complexion of a partially hydrated iron atom to the enzyme during analysis.
Membrane lateral pressure as a modulator of glycerol‐3‐phosphate dehydrogenase activity
Authors: Amler Evžen , Jasińska Renata , Drahota Zdeněk , Zborowski Józef
Published: July 2002

Michaelis‐Menten kinetics of glycerol‐3‐phosphate dehydrogenase activity in proteoliposomes from brown adipose tissue mitochondria with exogenously added phospholipids or cholesterol was measured. It was shown that changes in membrane lipid composition affected the membrane lateral pressure and therefore modulated the enzyme activity, namely V
max value. Contrarily, changes in surface charge caused by minute amounts of phosphatidylserine or charged organic substances influenced only the apparent K
m value. The role of bulk phospholipids in regulation of glycerol‐3‐phosphate dehydrogenase is discussed.
GABAB receptors couple to G proteins Go, G∗o and G·i1 but not to Gi2
Authors: Morishita Rika , Kato Kanefusa , Asano Tomiko
Published: July 2002

We studied the selectivity of GABAB receptors for coupling to G proteins by testing the ability of various purified G proteins to increase GABA binding to N‐ethylmaleimide (NEM)‐treated membranes of bovine brain. The addition of Go, G∗
o or gi1 to NEM‐treated membranes increased GABA binding in a dose‐dependent manner. However, the addition of Gi2 did not elicit a marked increase in GABA binding. When α subunits of G proteins were mixed with various brain βγ subunit complexes composed of different γ subunits, and they were added to the NEM‐treated membranes, Gi2 with any βγ subunits hardly increased GABA binding. On the other hand, Go with any βγ subunits caused a marked increase, though Go with a small γ subunit was more effective than that with a large γ subunit. These data suggest that the selective coupling of the G proteins to GABAB receptors is determined by the α subunit.
Identification and structural analysis of a ribosomal RNA gene promoter from Thiobacillus ferrooxidans
Authors: Takamiya Monica , Salazar Oriana , Vargas Danilo , Jedlicki Eugenia , Orellana Omar
Published: July 2002

The 5'‐terminus of a rRNA operon (rrnT2) from Thiobacillus ferrooxidans was characterized. The rRNA promoters from this microorganism were identified by means of a functional assay in Escherichia coli. DNA sequencing of the promoter region, upstream the 16 S rRNA gene, showed the presence of a consensus sequence for bacterial ribosomal promoters. Other features such as a ‘discriminator’ sequence, antiterminator elements and an upstream hexanucleotide common to several rRNA operons were also found. Two other putative transcription promoters were also identified.
Importance of the G27‐A43 mismatch at the anticodon stem of Escherichia coli tRNAThr2
Authors: Komine Yuriko , Inokuchi Hachiro
Published: July 2002

The tRNAThr2 isoacceptor of E. coli has a G‐A mismatch at positions 27–43. When the anticodon of this tRNA was converted to an amber anticodon (CUA), this tRNA showed suppressor activity in E. coli. Moreover, introduction of the base pair (G‐C or U‐A) at positions 27–43 of this suppressor tRNA reduced its suppressor activity. These results indicate that the G27‐A43 mismatch is necessary for full function of tRNAThr2.
Phosphorylation and activation of p40 tyrosine kinase by casein kinase‐1
Authors: Vila Jordi , Payne D.Michael , Zioncheck Thomas F. , Harrison Marietta L. , Itarte Emilio , Weber Michael J.
Published: July 2002

Because examination of regulatory trans‐phosphorylations can help elucidate the cellular functions of tyrosyi protein kinases, we have investigated the effects of phosphorylation by casein kinase‐1 on the activity of the p40 tyrosyi protein kinase. We find that casein kinase‐1 can phosphorylate the p40 tyrosyl kinase on serine and threonine residues, in part on a unique tryptic peptide. The phosphorylation induces a substantial increase in the tyrosyl protein kinase activity of p40, in contrast to most instances in which serine/threonine phosphorylation inhibits activity of tyrosyl protein kinases. These findings raise the possibility that p40 might be part of a protein phosphorylation network in which casein kinase‐1 participates.
The new model of DNA structures
Authors: Zhang J. , Zhang H.Y.
Published: July 2002

The new model of the DNA dynamic state is proposed in terms of Watson‐Crick (WC) and Corey‐Pauling‐Kortum (CPK) models by the modification of the cis ladder conformation. The model may rationally explain the DNA replication, transcription, renaturation, hybridization, premelting and breach etc.