bluuebunny/crossref_metadata_2025 huggingface dataset:
A distinct 2,5‐di‐(tert‐butyl)‐1,4‐benzohydroquinone‐sensitive calcium store in bovine adrenal chromaffin cells
Authors: Robinson Iain M. , Burgoyne Robert D.
Published: July 2002

The fluorescent Ca2+ indicator Fura 2 was used to monitor Ca2+ release induced by the Ins(1,4,5)P3‐mobilizing agonist angiotensin II (Ag II), caffeine and 2,5‐di‐(tert‐butyl)‐1,4‐benzohydroquinone (tuBHQ), in intact bovine adrenal chromaffin cells. Under low external Ca2+ conditions, tuBHQ, Ag II and caffeine elicited Ca2+ rises, indicating Ca2+ release from internal stores. Prior addition of Ag II had no noticeable effect on the extent of release of Ca2+ induced by tuBHQ. Stimulation of the cells with tuBHQ before either Ag II or caffeine, similarly had no effect on Ca2+ released by these two agonists. It was concluded, therefore, that there is a third intracellular Ca2+ store in bovine adrenal chromaffin cells, distinct and non‐overlapping, from those sensitive to caffeine or Ins(1,4,5)P3‐Mobilizing agonists.
Activation of respiration and loss of thermodynamic control in hyperthyroidism Is it due to increased slipping in mitochondrial proton pumps?
Authors: Luvisetto Siro , Schmehl Ibolya , Conti Elena , Intravaia Elena , Azzone Giovanni Felice
Published: July 2002

T3 administration increases the extent of non‐linearity in the flow—force relationship between pump proton conductance and protonmotive force. The effect is present also at the ATPase proton pump. These effects are not accompanied by changes in passive proton conductance. Incubation of mitocondria at 45°C also causes an increased non‐linearity, accompanied by a partial increase of proton conductance. It appears that the increase of respiratory activity following T3 administration is due to loss of thermodynamic control within or at the proton pumps, an effect which might be attributed to increased slipping.
Hydrogen bonding effects on 31P NMR shielding in the pyrophosphate group of NADPH bound to L. casei dihydrofolate reductase
Authors: Gerothanassis I.P. , Birdsall B. , Feeney J.
Published: July 2002

A comparison of 31P NMR chemical shift data and X‐ray structural data [Filman, D.J., Bolin, J.T., Matthews, D.A. and Kraut, J. (1982) J. Biol. Chem. 257, 13663–13672] for complexes of NADPH with L. casei dihydrofolate reductase indicates that solvation effects play a major role in influencing the 31P shielding of the pyrophosphate nuclei whereas changes in P‐O‐C5‐H5' torsion angle have little effect.
Role of metal ions in the assembly and decay of the transcription initiation complex on tRNA gene in yeast extracts
Authors: Szafrański Przemysław , Smagowicz W.Jerzy
Published: July 2002

Studies on formation of the transcription initiation complex on the tRNATyr gene in yeast extracts with the use of Sarkosyl‐challenge assay proved that a single magnesium ion is indispensable for the complex assembly in the absence of nucleotides. Other cations do not substitute for Mg2+. The optimal KCl concentration for transcription of the gene by the assembled initiation complex was found to be twice as high as that for the assembly process and close to that observed previously for the TFIIIC (or tau) factor dissociation from the promoter. The preformed complex remained stable for several hours at room temperature and its decay was not influenced by ionic strength. The data seem to support the notion that the TFIIIC factor is used only for assembly of the initiation complex and is not necessarily involved in the subsequent steps of transcription,
Repression of the CaMV 35S promoter by the octopine synthase enhancer element
Authors: Gatz Christiane , Katzek Jens , Prat Salome , Heyer Arnd
Published: July 2002

A 16 base‐pair palindrome upstream of the Agrobacterium tumefaciens octopine synthase (ocs) gene functions as a positive regulatory element in plant cells (Ellis et al. (1987) EMBO J. 6, 3203–3208; Fromm et al. (1989) Plant Cell 1, 977–984). We have converted it into a negative element by locating two copies flanking the TATA‐box of the constitutively expressed CaMV 35S promoter. The reduced promoter activity is very likely due to sterical hindrance of the ocs binding protein (OCSTF) × ocs complex with the transcription initiation complex. We propose that this type of constructs can be used for the identification of recognition sites for DNA‐binding proteins which are labile in vitro as well as for determining the DNA‐binding activity of a trans‐acting factor in vivo.
Presence of phospholipase C in coated vesicles from bovine brain Dual regulatory effects of GTP‐analogs
Authors: Martín Mairena , Ros Manuel , González-Calero Gloria , Cubero Ana
Published: July 2002

Bovine brain coated vesicles display free calcium‐dependent phospholipase C activity. Gpp(NH)p and GTPγS inhibited phospholipase C at nanomolar concentrations. Increasing concentrations of Gpp(NH)p and GTPγS reversed the inhibitory effects and stimulated phospholipase C activity. Preincubation of coated vesicles with pertussis toxin blocked the poorly‐hydrolyzable GTP‐analogs' inhibitory effects on phospholipase C. These data indicate that guanine nucleotides exert a dual regulatory control of phospholipase C in coated vesicles and that the inhibitory pathway is mediated by a pertussis toxin‐sensitive G‐protein.
On the mechanism of oligomycin inhibition of Ca2+‐induced mitochondrial respiration
Authors: Krasinskaya Inna P. , Kudryashova Irina A. , Yaguzhinsky Lev S.
Published: July 2002

The addition of oligomycin in the presence of Ca2+ increased the ADP pool in mitochondrial suspension. It is suggested that oligomycin inhibition of Ca2+‐induced mitochondrial respiratory activation is the function of the increased endogenous ADP pool. Low ADP concentrations (5–20 μM) produce the same inhibitory effect as oligomycin. The increase of ADP levels in the presence of glucose plus hexokinase resulted in the inhibition of Ca2+‐induced respiration, while the addition of phosphoenol pyruvate plus pyruvate kinase followed by a reduction in ADP levels, reversed the oligomycin inhibitory effect. One of the essential stages of ADP accumulation in mitochondrial suspensions in the presence of oligomycin and Ca2+ is proposed to be the formation of ADP from AMP and ATP, effected by adenylate kinase.
Dimeric structure of H+‐translocating pyrophosphatase from pumpkin vacuolar membranes
Authors: Sato Masa H. , Maeshima Masayoshi , Ohsumi Yoshinori , Yoshida Masasuke
Published: July 2002

Vacuolar membrane H+‐translocating pyrophosphatase (H+‐PPase) was purified from pumpkin seedlings. Its enzymatic properties including molecular size of constituting polypeptide (75 kDa) were very similar to those of mung bean H+‐PPase [(1989) J. Biol. Chem. 264, 20068–20073]. The native, functional molecular size of the pumpkin H+‐PPase was estimated to be 135–139 kDa from gel permeation HPLC of the purified enzyme in the presence of detergent and from radiation inactivation of the enzyme in vacuolar membranes. It is concluded that native, functional pumpkin H+‐PPase, and also probably H+‐PPases from other plants, is a dimer of 75 kDa subunits.
The retarded rate of acid‐catalyzed solvolysis of glycoside bonds between reducing‐end glucose residue and ceramide in glycosphingolipids compared with that of glycoside bonds between hexopyranosides
Authors: Shimamura Michio , Oku Manami , Yamagata Tatsuya
Published: July 2002

Rates of acid‐catalyzed solvolysis of glycoside bonds in glycosphingolipids were compared to establish a basis for conducting saccharide analysis. Permethylated globotetraosylceramide and asialogangliotriaosylceramide as model compounds for methylation and sugar composition analysis, respectively, were solvolyzed under acidic conditions and the sugar components thus obtained were determined at specified times by gas liquid chromatography, after they had been derivatized. Reducing‐end glucose residues in both compounds were liberated more slowly than other sugar residues. Glycoside bonds between reducing‐end glucose and ceramide in glycosphingolipids would thus appear to be more resistant towards acid‐catalysed solvolysis than other glycoside bonds between hexopyranosides.
Overproduction, purification and crystallization of Bacillus cereus oligo‐1,6‐glucosidase
Authors: Watanabe Kunihiko , Kitamura Kazuhisa , Hata Yasuo , Katsube Yukiteru , Suzuki Yuzuru
Published: July 2002

The gene coding for oligo‐1,6‐glucosidase from Bacillus cereus ATCC7064 has been overexpressed in Escherichia coli MV1184 cells under the control of the lac promoter in the genetically engineered plasmid pBCE4‐2. Oligo‐1,6‐glucosidase was purified in large quantities and was crystallized at 25°C by using a hanging drop vapor diffusion method with 53% saturated ammonium sulfate. The crystals have the shape of hexagonal bipyramids and belong to the space group P62 or P64 with lattice constants of a=b=106.1 Å, c=120.0 Å and γ=120°.
Transcriptional regulation of murine NADP+‐dependent methylenetetrahydrofolate dehydrogenase‐cyclohydrolase‐synthetase
Authors: Peri Krishna G. , MacKenzie Robert E.
Published: July 2002

The cytosolic NADP+‐dependent methylenetetrahydrofolate dehydrogenase‐methenyltetrahydrofolate cyclohydrolase‐formyltetrahydrofolate synthetase is ubiquitously expressed in all mouse tissues and cell lines examined. Northern analyses of the RNA indicated that there is an extensive variation in the levels of mRNA in different tissues. However, the gene is refractory to induction by serum, phorbol esters or growth factors in cultured fibroblasts. The mRNA of the NADP′‐dependent trifunctional enzyme is stabilized post‐transcriptionally by insulin‐like growth factor‐1.
Histone H1 inhibits eukaryotic DNA topoisomerase I
Authors: Richter Arndt , Kapitza Marion
Published: July 2002

Histone H1 inhibits the catalytic activity of topoisomerase I in vitro. The relaxation activity of the enzyme is partially inhibited at a molar ratio of one histone H1 molecule per 40 base pairs (bp) of DNA and completely inhibited at a molar ratio of one histone H1 molecule per 10 base pairs of DNA. Increasing the amount of enzyme at a constant histone H1 to DNA ratio antagonizes the inhibition. This indicates that topoisomerase I and histone H1 compete for binding sites on the substrate DNA molecules. Consistent with this we show on the sequence level that histone H1 inhibits the cleavage reaction of topoisomerase I on linear DNA fragments.
A family of laminin‐related proteins controlling ectodermal differentiation in Drosophila
Authors: Patthy László
Published: July 2002

It is shown that the proteins encoded by the tumor suppressor fat gene, the neurogenic slit gene and crumbs gene of Drosophila contain domains homologous with modules identified previously in laminin A. These proteins of Drosophila have a number of features in common; they have large extracellular regions containing laminin A modules linked to epidermal growth factor‐like domains, and they are all involved in cell—cell interactions that are crucial for correct morphogenesis or ectodermal tissues (development of midline neuroepithelia, organization of epihelial tissues etc.) It is suggested that the laminin A‐type modules of these proteins play important roles in the interactions that control ectodermal differentiation.
Conservation of binding site specificity of three yeast DNA binding proteins
Authors: Pretorius G.H.J. , Muller H.E.
Published: July 2002

Sequence specific binding of protein extracts from 13 different yeast species to three oligonucleotide probes and two points mutants derived from Saccharomyces cerevisiae DNA binding proteins were tested using mobility shift assays. The probes were high affinity binding sites for GRF1/RAP1/ ABF1 and CP1/CPF1. Most yeasts in the genus Saccharomyces showed specific binding to all three probes and also displayed similar sequence requirements when challenged by molar excesses of mutant probes. The affinities for the probes varied amongst the other yeasts tested, but in general, CPF1 binding activity was the most widespread, while the other two were more limited.
High protein mobility in skinned rabbit muscle fibres observed by 1H NMR spectroscopy
Authors: Kalbitzer Hans Robert , Schrumpf Matthias , Wray John
Published: July 2002

1H NMR spectra of skinned rabbit muscle fibers show a group of relatively sharp resonance lines which presumably originate from highly mobile protein domains. Comparison with the spectrum of myosin subfragment I suggests that these signals may come at least partly from mobile regions of the myosin head. NMR could possibly be used to characterize the movements of crossbridges in force generation.
Probing of pore in the Chara gymnophylla K+ channel by blocking cations and by streaming potential measurements
Authors: Pottosin Igor I.
Published: July 2002

The current—voltage (I–V) relationship of single K+ channels present in the Chara gymnophylla droplet membrane was studied. The channel presumably contains large mouths at both pore ends which are sufficiently wide to accomodate TEA+ as evidenced by internal and external TEA+ blockade. The voltage dependence of blockade by external Cs+ and Na+ reveals the multi‐ion occupancy of the channel. The value of streaming potential (4.0 mV/osmol) measured in the Chara K+ channel indicates that the channel contains up to nine water molecules in the narrow region. It is concluded that the length of this region is around 28 Å.
Interactions of Bowringia mildbraedii agglutinin with complex‐ and hybrid‐type glycans
Authors: Chawla Davinder , Animashaun Theresa , Hughes R.Colin
Published: July 2002

Affinity chromatography on Bowringia mildbraedii agglutinin (BMA) Sopharose of glycopeptides confirmed a previous report using oligo‐saccharides (Animashaun, T. and Hughes, R.C. (1989) J. Biol. Chem. 264,4657–4663) that high affinity binding requires the sequence Manα1→2 Manα1→6 Manα1→6 Manβ1→4. However, moderate binding was still exhibited by structures lacking this sequence provided the oligosaccharide core sequence Manα1→3[Manα1→6]Manβ1→4GlcNAc was present. This moderate binding was not affected by substitution with N‐acetylgluco‐samine at C2 and C4, respectively, of the Manα1→3 and Manβ1→4 residues and BMA Sepharose should prove to be a useful tool for the isolation of bisected or non‐bisected hybrid‐type glycans.
Human centrosomal epitope is shared specifically with human lactate dehydrogenase‐B isozyme
Authors: Gosti F. , Li S.S.L. , Maunoury R. , Bornens M.
Published: July 2002

A rabbit serum (0013) used to identify pericentriolar proteins from isolated centrosomes (Gosti‐Testu, F., Marty, M.C., Berges, J., Maunoury, R. and Bornens, M. (1986) EMBO J. 5, 2545–2550) was shown also to react through the same epitope with several non‐centrosomal proteins including a major 36 kDa cytosolic antigen. This protein was identified to be human lactate dehydrogenase and the co‐distribution of 0013 epitope on the centrosomal protein and on lactate dehydrogenase (LDH) was shown to be specific for human cells (Gosti, F., Marty, M.C., Courvalin, J.C., Maunoury, R. and Bornens, M. (1987) Proc. Natl. Acad. Sci. USA 84, 1000–1004). Human hepatic cells constitute, so far, the only exception to this co‐distribution rule. By using this cell type which expresses only the LDH‐A4 isozyme, we demonstrate that 0013 epitope is specific for the human LDH‐B subunit, making serum 0013 the strongest anti‐LDH‐B available so far. The evolutionary and physiological significance of this situation is discussed.
Biochemical characterization of the p51 sub‐unit of human immunodeficiency virus reverse transcriptase in homo‐ and heterodimeric recombinant forms of the enzyme
Authors: Dirani-Diab Rim El , Andreola Marie-Line , Nevinsky Georgyi , Tharaud Danièle , Barr Philip J. , Litvak Simon , Tarrago-Litvak Laura
Published: July 2002

The biochemical properties of the p51 subunit of HIV‐1 reverse transcriptase (RT) were studied in order to understand its role in the heterodimeric form p66/p51 found in virions. A recombinant form of RT, p51/p51, expressed in yeast, was purified and characterized. The enzyme was affinity labeled using a 5′ modified oligonucleotide primer, covalently linked, that was further elongated in the presence of a radioactive dNTP precursor. We found that the p51 subunit was labeled in the p51/p51 form, thus reflecting its activity, while this subunit was catalytically silent in the heterodimer, since only the p66 subunit was labeled in the latter recombinant form. Processivity studies showed long‐sized products synthesized by p51/p51, as in the case of the other RT Forms. The effect of primer tRNALys on the p51/p51 activity showed a strong inhibitory effect in the absence of KCl, similar to that observed with the p66/p51 form, while the same p51/p51 enzyme was strongly stimulated by tRNALys, like RT p66/p66, when KCl was present in the incubation mixture.
Unimpaired effect of insulin on glucokinase gene expression in hepatocytes challenged with amylin
Authors: Nouspikel Thierry , Gjinovci Asllan , Li Senlin , Iynedjian Patrick B.
Published: July 2002

Amylin appears to interfere with the action of insulin in muscle and possibly in liver. We have attempted to detect a direct antagonism between amylin and insulin in cultured rat hepatocytes. The stimulation of glucokinase gene expression was used as a marker of insulin action. Amylin proved ineffective in suppressing subsequent accumulation of glucokinase mRNA in response to maximal or submaximal doses of insulin. When applied to cells already induced by prior incubation with insulin alone, amylin failed to reverse induction, in contrast to the effectiveness of glucagon under the same conditions. Thus, amylin is not a physiological antagonist of insulin in the control of hepatic glucokinase gene expression.
Two‐dimensional crystals of membrane‐bound gastric H,K‐ATPase
Authors: Hebert Hans , Xian Yijing , Hacksell Ingrid , Mårdh Sven
Published: July 2002

H,K‐ATPase protomer. Through stain‐deficient contact regions four adjacent protein units were connected forming a tetrameric structure.
Subcellular localisation and processing of non‐specific lipid transfer protein are not aberrant in Rhizomelic Chondrodysplasia Punctata fibroblasts
Authors: Heikoop Judith C. , Ossendorp Bernadette C. , Wanders Ronald J.A. , Wirtz Karel W.A. , Tager Joseph M.
Published: July 2002

The import into peroxisomes and maturation of peroxisomal 3‐oxoacyl‐CoA thiolase are impaired in patients with the Rhizomelic form of Chondrodysplasia Punctata (RCDP). Here we show by means of immunoblotting and subcellular fractionation that non‐specific lipid transfer protein (nsLTP), another peroxisomal protein synthesised as a larger precursor, is localised in peroxisomes and is present as the mature protein in RCDP fibroblasts. Thus the component of the import machinery defective in RCDP is not required for the import of nsLTP into peroxisomes.
Effects of pressure on glucose transport in human erythrocytes
Authors: Thorne S.D. , Hall A.C. , Lowe A.G.
Published: July 2002

The operation of the human red cell glucose transporter has been studied at normal and high hydrostatic pressure to identify the step(s) which involve a volume change. Pressure inhibited zero‐trans and equilibrium exchange influx to similar extents, by decreasing the V
max but not significantly changing the K
m. The B
max and K
d of specific [3H]cytochalasin B binding were unaffected by pressure indicating no change to the number or affinity of functional transporters at pressure. Passive glucose transport was inhibited by pressure in a manner consistent with permeation across the lipid bilayer. These data indicate that there is a major change in volume during the translocation step of the glucose transporter which is rate‐limiting for transport.
A short form of the tick‐borne encephalitis virus NS3 protein
Authors: Pugachev K.V. , Nomokonova N.Yu. , Morozova O.V. , Pletnev A.G.
Published: July 2002

Using monoclonal antibodies to the tick‐borne encephalitis virus (TBE) nonstructural protein NS3 two forms of this protein were revealed in TBE‐infected mammalian cells: a full‐length form (69 kDa) and a short form (49 kDa) which has not been observed before and was called NS3′. Recombinant plasmids were constructed and various fragments of the TBE NS3 gene were expressed in rabbit reticulocyte lysate. By analyzing immune precipitates of 35S‐labeled translation products, we could monitor and localize internal cleavage of NS3, due to which the NS3′ protein was generated.
Anhydrosubtilisin‐catalyzed peptide synthesis
Authors: Tanizawa Kazutaka , Sugimura Akihiro , Kanaoka Yuichi
Published: July 2002

Anhydrosubtilisin was found to be a good catalyst for the peptide synthesis though its hydrolytic activity was fully inhibited. Amino acid (peptide) p‐chlorophenyl ester (acyl donor) was coupled with amino acid (peptide) amide in the presence of the modified enzyme. The method did not afford by‐products resulting from the hydrolysis of the acyl donor. This is the most advantageous characteristic of the method, since the hydrolysis of the acyl donor is unavoidable for the methods using catalytically active proteases. Anhydrosubtilisin was further shown to be useful for the fragment condensation of peptides.
Effect of glucagon on insulin receptor phosphorylation in intact liver cells
Authors: Issad Tarik , Young Steven W. , Tavaré Jeremy M. , Denton Richard M.
Published: July 2002

Evidence is presented that incubation of rat liver cells with glucagon leads to an increase in the phosphorylation of specific serine residues within insulin receptors, particularly in the presence of insulin. However, no changes in either the tyrosine phosphorylation of the receptors or the tyrosine kinase activity towards a synthetic peptide substrate was detected.
Applying machine learning methods for finding significant amino acid properties in proteins
Authors: Selbig Joachim , Kaden Frieder , Koch Ina
Published: July 2002

There are several possibilities for definition and derivation of sequence patterns associated with structural motifs, in particular on the secondary structure level which may be used to predict these structure elements. Sequence patterns consist of a number of consecutive positions along the polypeptide chain from which a certain quantity is specified. One of the important factors in deriving sequence patterns in terms of amino acid properties is how to find the most characteristic properties to specify a certain position and thus to avoid redundant physical information. We have applied machine learning methods to select the most significant amino acid properties describing a structurally determined sequence position. Results are given for the beginning of α‐helices. These methods may link the gap between amino acid patterns and property patterns and thus are valuable to improve protein structure prediction.
Melanocyte stimulating hormone activation of tyrosinase in B16 mouse melanoma cells Evidence for a differential induction of two distinct isoenzymes
Authors: Valverde P. , García-Borrón J.C. , Martínez-Liarte J.H. , Solano F. , Lozano J.A.
Published: July 2002

Tyrosinase induction in murine malignant melanocytes by αMSH is well known, but its molecular basis has not been characterized. Treatment of B16 melanoma cells with theophylline or αMSH mediates a larger induction of tyrosine hydroxylase than of dopa oxidase activity in total cell extracts, and in the melanosomal and microsomal fractions. No evidence for the modulation of a tyrosinase effector was found. SDS‐PAGE and specific activity stain demonstrated two forms of tyrosinase, with different degrees of induction by theophylline. These results agree with the recent proposal that two tyrosinases, encoded by different genes, are present in murine melanocytes.
GTP‐binding proteins in luminal and basolateral membranes from pars convoluta and pars recta of rabbit kidney proximal tubule
Authors: Buhl Anne Mette , Sheikh M.Iqbal , Steensgaard Jens , Røigaard-Petersen Hans , Jacobsen Christian
Published: July 2002

The GTP‐binding proteins on luminal and basolateral membrane vesicles from outer cortex(pars convoluta) and outer medulla (pars recta) of rabbit proximal tubule have been examined. The membrane vesicles were highly purified, as ascertained by electron microscopy, by measurements of marker enzymes, and by investigating segmental‐specific transport systems. The [35S]GTPγS binding to vesicles, and to sodium cholate‐extracted proteins from vesicles, indicated that the total content of GTP‐binding proteins were equally distributed on pars convoluta, pars recta luminal and basolateral membranes. The membranes were ADP‐ribosylated with [32P]NAD+ in the presence of pertussis toxin and cholera toxin. Gel electrophoresis revealed, for all preparations, the presence of cholera toxin [32P]ADP‐ribosylated 42 and 45 kDa Gα, proteins, and pertussis toxin [32P]ADP‐ribosylated 41 kDa Gαil, 40 kDa Gα12 and 41 kDa Gα13 proteins. The 2D electrophoresis indicated that Go's were not present in luminal nor in basolateral membranes of pars convoluta or pars recta of rabbit proximal tubule.
Relationship between membrane lipid mobility and spectrin distribution in lymphocytes
Authors: Langner Marek , Repasky Elizabeth A. , Hui Sek-Wen
Published: July 2002

We have previously established that T and B lymphocytes in situ are remarkably heterogeneous with respect to the cytoskeletal protein spectrin. Since in erythrocytes spectrin is known to play an important role in the regulation of membrane fluidity, lipid organization and lateral mobility of membrane proteins, we have sought to determine if the heterogeneous patterns of spectrin distribution that we have observed are related to possible differences in membrane lipid organization in these various subsets. To this end, we have utilized a fluorescent pyrene‐labelled phospholipid as a probe orthe lipid lateral mobility and have examined two related T cell systems maintained in vitro, DO.11.10 cells and a spontaneously arising variant, DO.11.10V. In these (and other cloned in vitro systems) we have previously observed that the cells homogeneously express one of the kinds of spectrin distribution patterns observed in situ. Thus the uniformity of staining of these systems permits us to address whether the various patterns of spectrin distribution may be predictive of differences in membrane lipid properties. Here we show that in cells in which there is little or no spectrin at the plasma membrane (DO.11.10) that the lipids in the plasma membrane are considerably less mobile than in its related variant in which spectrin is diffusely distributed within the cell and at the plasma membrane. From this and previous results, we conclude that differences in the distribution of the cytoskeletal protein spectrin among lymphocytes may be a useful parameter in helping to predict the status of membrane lipid organization.
Distinct and specific GAP activities in rat pancreas on the yeast GTP‐binding proteins Ypt1 and Sec4
Authors: Jena B.P. , Brennwald P. , Garrett M.D. , Novick P. , Jamieson J.D.
Published: July 2002

Previous studies have demonstrated that Sec4, a 23.5 kDa guanine nucleotide‐binding protein of the ras superfamily is required for exocytosis in the budding yeast Saccharomyees cerevisae, Ypt1, another ras‐like 23 kDa guanine nucleotide‐binding protein in yeast has been found to be involved in ER‐Golgi transport. A mammalian homologue of Ypt1 called rabl has also been identified. Recent studies using purified Sec4 protein have identified a component of yeast lysate that specifically stimulates the hydrolysis of GTP bound to Sec4. In the present study, purified recombinant Sec4 and Ypt1 proteins expressed in E. coli have been used as substrates to determine if GTPase activating proteins (GAPs) directed toward these proteins are present in rat pancreas. Our studies showed that 65% of Sec4‐GAP activity was associated with the 150,000 × g pancreatic particulate fraction with approximately 35% being found in the cytosol. On the other hand, more than 95% of Ypt1‐GAP activity was found to associate with the particulate fraction. Sec4 and Ypt1 competition assays further demonstrated the specificity of the Sec4 and Ypt1 GAPs. The results from the present study suggest the presence of a distinct GAP in the pancreas that interacts with Sec4, and another that interacts with Ypt1.
p56lyn catalyzes a reversible autophosphorylation reaction and a nucleoside diphosphate kinase reaction
Authors: Litwin Craig M.E. , Gendreau Marthe , Wang Jerry H.
Published: July 2002

The reversible autophosphorylation of the pp60 family tyrosine kinase, p56lyn has been characterized by a simple procedure that involves the examination of the enzyme catalyzed radioisotope exchange between ATP and ADP. The equilibrium constant of the reaction was determined to be 3.31 and corresponded to a standard free energy of hydrolysis of the phosphotyrosine bond in p56lyn of −8.03 kcal/mol. GDP was capable of substituting for ADP as phosphate acceptor so that p56lyn displayed a nucleoside diphosphate kinase activity.
Carbohydrates in mammalian tryptophanyl‐tRNA synthetase
Authors: Kovaleva Galina K. , Zheltova Anna O. , Nikitushkina Tatyana V. , Egorov Tsezi A. , Musoljamov Aleksander Ch. , Kisselev Lev L.
Published: July 2002

Homogeneous preparations of bovine tryptophanyl‐tRNA synthetase (EC 6.1.1.2) contain monosaccharides (mannose, fucose, galactose, N‐acetylglucosamine) as revealed by liquid chromatography. Their content comprises 2.5–3.0% (w/w) of the enzyme composed of two subunits (60 kDa × 2). The same set of sugars was detected in clastase and CNBr‐generated fragments (with molecular masses of approx. 40 kDa and 30 kDa, respectively). It is concluded that bovine tryptophanyl‐tRNA synthetase, in addition to being a metallo‐ and phosphoprotein, is also a glycoprotein.
Synthesis of a large peptide mimicking the DNA binding properties of the sex determining protein, SRY
Authors: Poulat F. , Guichard G. , Gozé C. , Heitz F. , Calas B. , Berta P.
Published: July 2002

The sex determining protein, SRY, has been recently described as containing a DNA binding motif, also called the SRY box. This 80 amino acid box was synthesized using the continuous flow solid‐phase technique. The product was then purified and tested according to such diverse criteria as its intrinsic structure or its biological activity (DNA binding capacity), and compared to the full‐length protein. The data indicate that the peptide is relevant for the properties described so far for the protein.
Properties of Pseudomonas aeruginosa exotoxin A ionic channel incorporated in planar lipid bilayers
Authors: Gambale F. , Rauch G. , Belmonte G. , Menestrina G.
Published: July 2002

Acidic conditions induce the incorporation of Pseudomonas aeruginosa exotoxin A into phospholipid planar bilayers and the formation of pores permeable to electrolytes. Channel openings occur as single events, although they may occasionally cluster in bursts. In 100 mM KCl, the elementary single channel current amplitude is 3.1 pA (at a transmembrane voltage of 100 mV), the mean open time is 1.3 ms, while bursts may last for several seconds. Noise analysis gave results identical to single channel analysis. Voltage pulse protocols and continuous cycling voltage ramps showed that the toxin channel is voltage dependent, having a higher probability of being open at positive voltages.
cDNA cloning and sequencing of tobacco chloroplast ribosomal protein L12
Authors: Li Yuqing , Itadani Hiraku , Sugita Mamoru , Sugiura Masahiro
Published: July 2002

Tobacco chloroplast ribosomal protein L12 was isolated as a ssDNA‐cellulose‐binding protein from a chloroplast soluble protein fraction. Based on the N‐terminal amino acid sequence of chloroplast L12, a cDNA clone was isolated and characterized. The precursor protein deduced from the DNA sequence consists of a transit peptide of 53 amino acid residues and a mature L12 protein of 133 amino acid residues. The chloroplast L12 protein was synthesized with a reticulocyte lysate and subjected to nucleic acid‐binding assays. L12 synthesized in vitro does not bind to ssDNA, dsDNA nor ribonucleotide homopolymers, but it binds to cellulose matrix.
Identification of the atrial natriuretic factor‐RIC receptor subtype (B‐clone) in cultured rat aortic smooth muscle cells
Authors: Fèthière James , Graihle Régis , De Léan André
Published: July 2002

The present report demonstrates the presence in cultured rat aortic smooth muscle cells of a natriuretic factor receptor subtype with a specificity typical of the ANF‐RIC (B‐clone) receptor subtype. To prove the existence of this receptor subtype in this cell line we show that pCNP‐(82–103) is the most potent activator of the intrinsic guanylate cyclase activity, and that [125I]pCNP‐(82–103) binds to a specific receptor subtype which is insensitive to the ANF‐R2 specific ligand, C‐ANF. The investigation of its binding characteristics show the rank potency order of the natriuretic factors in competing for pCNP binding to be pCNP > pBNP > rANF. Furthermore it was possible to covalently photolable this receptor subtype with underivatized [125I]pCNP and show that it is composed of a single subunit of 130 kDa with very high specificity for pCNP.
Minimal functional size of porcine lung and testicular angiotensin‐converting enzymes deduced from radiation inactivation analysis Interaction of two highly homologous domains in somatic isoenzyme
Authors: Sakaguchi Hajime , Hirose Shigehisa , Kume Tamikazu , Hagiwara Hiromi
Published: July 2002

Domain structures of porcine lung and testicular angiotensin‐converting enzymes (ACE) were studied by radiation inactivation to test the hypothesis that lung ACE has two catalytic sites localized to discrete, structurally independent domains (the N‐ and C‐domains) of approximately equal size. The minimum functional sizes of lung and testicular ACE, calculated from the inactivation curves obtained, were 140 and 74 kDa, respectively. Since testicular ACE has been demonstrated to contain only the C domain, this result indicates that the two domains in lung ACE are not independent but are, in fact, structurally tightly linked.
Generation of Gla‐domainless FVIIa by cathepsin G‐mediated cleavage
Authors: Nicolaisen Else Marie , Petersen Lars Christian , Thim Lars , Jacobsen Jes Kristian , Christensen Mogens , Hedner Ulla
Published: July 2002

Cougulation factor VII contains ten λ‐carboxyglutamic acid residues in the N‐terminal region (Gla‐domain) which are essential for the hemostatic function of FVII. The present study shows that granulocyte cathepsin G degrades the Gla‐domain of FVIIa in vitro. Characterization of the truncated FVIIa by SDS‐PAGE and N‐terminal amino acid sequence analysis revealed that cleavage had occurred between Tyr‐44 and Ser‐45 and that further cleavage was only obtained on extensive cathepsin G exposure. Cleavage of vitamin K‐dependent coagulation factors by cathepsin G may play a role in vivo, and it offers a convenient way of obtaining proteins deprived of their Gla‐domain for functional and structural studies.
NMR study of self‐paired parallel duplex of d(AAAAACCCCC) in solution
Authors: Luo Jia , Sarma Mukti H. , Yuan Ren-da , Sarma Ramaswamy H.
Published: July 2002

The oligonucleotide d(A5C5) in solution forms a parallel self‐duplex at neutral and low pH values. H2O NMR spectra at pH 5.1 indicate the presence of five imino resonances at lower temperatures; and the structure is stable up to 60°C. These signals ran arise only from the hemiprotonated C+·C pairs [Westhof, E. and Sundaralingham, M. (1980) Biochemistry 77, 1852–1856; Westhof, E. and Sundaralingham, M. (1980) J. Mol. Biol. 142, 331–361] and constitute the first direct observation of C+·C hemiprotonated pairs in solution. The cross peaks from Hl's and more than five distinct AH8's in 500 MHz 1H 2D‐NOESY spectra indicate that there are two conformationally different and energetically similar A‐tracts. There is good qualitative agreement between NOESY data and two theoretically derived structures in which A‐tracts are reverse Watson‐Crick and reverse Hoogsteen base‐paired, respectively.
Subunit interactions of the Go protein
Authors: van der Voorn Loesje , Hengeveld Trudi M. , Ploegh Hidde L.
Published: July 2002

The monoclonal antibody, MONO, recognizes an epitope on the G protein αo‐subunit [van der Voorn et al., submitted] and readily immunoprecipitates heterotrimeric Go proteins from solubilized, crude bovine brain membranes, as well as from a purified bovine brain G protein preparation. Upon incubation of the immunoprecipitates with GTPγS, all βγ‐subunits are released from the αo‐subunit. Thus, binding of MONO to the Go protein does not appear to interfere with release of bound GDP, binding of GTPγS or GTPγS‐induced subunit dissociation. However, we have been unable to induce a similar dissociaton of Go using its physiological activator, GTP. Surprisingly, we did not observe any dissociation of Go (bound to MONO) upon dilution in a range from 500 to 5 nM. Since an apparent X
4 of αo‐GDP for binding βγ of 340–390 nM has been reported (1989) J. Biol. Chem. 264, 20688–20696] our results would suggest that binding of MONO to the αo‐subunit induces an increased affinity of αo‐GDP for βγ. Alternatively, these results could be explained if, under the conditions used, the K
4 of αo‐GDP for βγ were at least two orders of magnitude lower than estimated previously.
Ion channels from the Bacillus subtilis plasma membrane incorporated into planar lipid bilayers
Authors: Alcayaga Carmen , Venegas Rodrigo , Carrasco Alicia , Wolff Daniel
Published: July 2002

Fusion of Bacillus subtilis plasma membrane vesicles with planar lipid bilayers induced the appearance of discrete current fluctuations characteristic of ion channels. These channels showed a wide range of conductances and kinetic behaviors. In 300 mM KCl, their conductances ranged from a few hundreds of pS to more than 1 nS, and most of them exhibited several sub‐states. The channels poorly discriminated between small univalent anions and cations. Some of them showed voltage dependence and most of them presented a complex gating kinetics. The results are consistent with the hypothesis of the presence in the B. subtilis plasma membrane of pores composed of subunits that function cooperatively.
Effects of 1,3‐chelation induced by cis‐diamminedichloroplatinum(II) on the stability of DNA duplexes
Authors: Urata Hidehito , Tamura Masahiro , Urata Masayuki , Akagi Masao
Published: July 2002

Several 1,3‐intra‐strand cross‐linked decadeoxynucleotide duplexes, modified with cis‐diamminedichloroplatinum(II) (cis‐DDP), and their base substitution analogues at the complementary site to the intervening base of the coordination sites, were synthesized and measured for UV‐melting, profiles to determine melting temperature (T
m) values. The results indicated the thermal stability of the oligonucleotide duplexes containing Pt‐induced 1,3‐intra‐strand cross‐linking did not depend on the kind of intervening base of the coordination site but rather on its complementary base. These results may explain the mutagenicity or cis‐DDP from a chemical aspect.
Kinetics of the N intermediate and the two pathways of recovery of the ground‐state of bacteriorhodopsin
Authors: Tokaji Zsolt , Dancsházy Zsolt
Published: July 2002

Absorption kinetic measurements at alkaline pH, in which bacteriorhodopsin (BR) is pre‐excited by another flash, indicate that a part of the recovery of the BR ground‐state is faster than the decay of the N intermediate of the photocycle. This fact proves the existence of a parallel pathway in the late part of the BR photocycle (the decay of Ms into BR), which does not include the N intermediate. We demonstrate that the decay of the Mr intermediate does not lead to any direct recovery of the BR ground‐state, and that excitation of N does not form an M‐like intermediate. Mr decays directly into the N intermediate, and photoexcitation of N leads to the formation of a red‐shifted form, O*. The kinetics of this red‐shifted intermediate are also presented.
Stimulation of receptor‐coupled phospholipase A2 by interferon‐γ
Authors: Ponzoni Mirco , Montaldo Paolo G. , Cornaglia-Ferraris Paolo
Published: July 2002

The biomolecular mechanisms that mediate signal transduction by type 11 (γ) interferon (IFN) are poorly understood. IFN‐γ is a potent growth inhibitory cytokine also endowed with antiviral, immunomodulatory, and differentiating activities on various cell targets, including neural cells. IFN‐γ induced a rapid and transient activation of phospholipase A2 in LAN‐5, a human neuroblastoma cell line. A consequence of phospholipase A2 activation was the release of arachidonic acid and the generation of lysophospholipids from membrane phospholipids. Treatment of pre‐labeled LAN‐5 cells with a receptor‐saturating concentration of IFN‐γ led to a time‐dependent release of [3H]arachidonic acid into the culture media and generation or [32P]lysophosphatidylcholine. Pretreatment of cultures with the phospholipase A2 inhibitor, bromophenacyl bromide, markedly inhibited both [3H]arachidonic acid release and lysophosphatidylcholine production induced by IFN‐γ treatment. Pretreatment of LAN‐5 cells with nordihydroguaiaretic acid, a lipoxygenase inhibitor, or with indomethacin, a cyclooxygenase inhibitor, amplified the release of[3H]arachidonic acid and production of lysophosphatidylcholine induced by non‐saturating concentrations of IFN‐γ. In parallel, and with the same time‐dependent effect, a significant decrease in phosphatidylcholine labeling was observed in IFN‐γ‐treated cells, further indicating that a potential signal transduction mechanism of IFN‐γ is the hydrolysis of membrane phosphatidylcholine by phospholipase A2.
Expression, characterization and purification of soluble G‐protein βγ dimers composed of defined subunits in baculovirus‐infected insect cells
Authors: Dietrich Alexander , Meister Michael , Spicher Karsten , Schultz Günther , Camps Montserrat , Gierschik Peter
Published: July 2002

Recombinant β1γ2 dimers of signal‐transducing guanine nucleotide‐binding proteins (G‐proteins) carrying a mutation known to block isoprenylation of the γ2 subunit were expressed as a soluble protein in baculovirus‐infected insect cells. The soluble βγ dimer was analyzed by sucrose density gradient centrifugation and purified to near homogeneity in the absence of detergents. The sedimentation velocity studies gave an s
20,w value of 4.1 ± 0.4 S. The two subunits segregated as a dimer upon sucrose density gradient centrifugation and purification by sequential ion exchange and hydroxylapatite chromatography. The results show that baculovirus‐infected insect cells can be employed for high level production of pure G‐protein βγ dimers suitable for functional and structural characterization.
C‐type natriuretic peptide in bovine chromaffin cells The regulation of its biosynthesis and secretion
Authors: Babinski K. , Féthière J. , Roy M. , De Léan A. , Ong H.
Published: July 2002

We report here the regulation of the biosynthesis and the secretion of C‐type natriuretic peptide (CNP) in cultured bovine chromaffin cells. The combined treatment with protein kinase A and ‐C activators induced a 6‐fold increase of intracellular levels of CNP‐(1–103). The biosynthesized CNP‐(1–103) was co‐released with its mature forms, typically CNP‐(51–103), upon stimulation by nicotine or depolarizing agents. This confirms the neuropeptidic character of this third member of the natriuretic peptide family, which might act as a neuromodulator or neurotransmitter.
Measurement of relative amounts of phospho‐ and dephospho‐B‐50(GAP‐43) peptides by fast atom bombardment‐mass spectrometry
Authors: Di Luca M. , de Graan P.N.E. , De Angelis L. , Gispen W.H. , Cattabeni F.
Published: July 2002

The biological role of phosphoproteins depends upon their degree of phosphorylation in vivo. Methods currently available to measure the degree of phosphorylation of a protein involve indirect procedures to detect the 32P‐phosphate incorporation. We report here a direct method to measure relative amounts of phospho‐ and dephospho‐forms of peptides based upon a mass spectrometric technique. The intensities of the molecular ions corresponding to the two forms of the peptides are proportional to their relative amounts. This is demonstrated for a peptide fragment of the protein B‐5O(GAP‐43) and for kemptide, respectively substrates for protein kinases C and A, and demonstrates the applicability of fast atom bombardment‐mass spectrometry to quantitate peptides bearing post‐translational modifications.
Investigation of spatial relationships and energy transfer between complexes B800‐850 and B890‐RC from Chromatium minutissimum reconstituted into liposomes
Authors: Moskalenko Andrey A. , Toropygina Olga , Godik Valentina I. , Timpmann Kou , Freiberg Arvi
Published: July 2002

Spatial relationships between difterent pigment‐protein complexes in the membranes of the purple photosynthetic bacterium, Chromatium minutissimum, have been studied. The possibility of restoring the function of efficient excitation energy transfer from bacteriochlorephyll molecules to the reaction centers in the system of soybean liposomes, reconstituted with pigment‐protein complexes B800‐850 and B890‐RC from C. minutissimum, has been explored. The chemical cross‐linking method, together with stationary and picosecond spectrally resolved fluoresence measurements were employed. It has been shown that after the incorporation of the complexes into the liposame membranes conditions for directed excitation energy transfer from the light‐harvesting pigments to the reaction centers are created, which are less optimal, however, than those in the native state. Possible reasons are considered.
Protein synthesis in HL‐60 cells treated with DMSO and hypoxanthine
Authors: Tyobeka E.M. , Masemola A.M.
Published: July 2002

Short‐term treatment of the HL‐60 cells with DMSO and hypoxanthine, inducers of granulocytic differentiation, was reported to cause a rapid increase in protein synthesis. This effect was ascribed to the insertion of inosine in the wobble position of the tRNA anticodon and consequently increasing codon recognition potential. In this study we have re‐investigated the effects of DMSO and/or hypoxanthine on protein synthesis. In contrast to their findings we were unable to demonstrate stimulated protein synthesis in either short‐ or long‐term treatment with these agents. Polysome analysis under these conditions revealed that polysomes were disaggregated. Finally, the activity of tRNA‐hypoxanthine ribosyltransferase, an enzyme responsible for the insertion of inosine in the anticodon, was also relatively low. Under these circumstances, we propose that tRNA modification is not essential in the regulation of protein synthesis.
Manganese deficiency and transcriptional regulation of mitochondrial superoxide dismutase in hepatomas
Authors: Borrello Silvia , De Leo Maria Emilia , Wohlrab Hartmut , Galeotti Tommaso
Published: July 2002

The presumed involvement of the transition metals manganese and copper in the regulation of the expression of the Mn‐ and CuZn‐containing superoxide dismutase genes has been investigated in normal and neoplastic tissues of the rat. Two hepatomas of the Morris line have been employed, the slow growing, highly differentiated 9618A and the fast growing, poorly differentiated 3924A. The data obtained indicate a control at the pretranslational level of the Mn‐containing enzyme, presumably exerted by the manganese ion. The CuZn‐containing superoxide dismutase is also regulated pretranslationally in the normal tissues examined and in the hepatoma 3924A. However, there is no indication for the involvement of the copper ion, which in the liver is mostly located in the cytosol bound to CuZnSOD, in such regulation. The possible role of a reduced redox state, concomitant to the manganese deficiency in hepatoma tissues, in the down regulation of Mn‐containing superoxide dismutase is discussed.