bluuebunny/crossref_metadata_2025 huggingface dataset:
Two‐dimensional analysis of proteins phosphorylated in E. coli cells
Authors: Desmarquets G. , Cortay J.C. , Cozzone A.J.
Published: July 2002

Protein phosphorylation
Bacterial protein kinase
Two‐dimensional analysis
Cytoplasmic phosphoprotein
Ribosome
Escherichia coli
C‐terminal amidation of neuropeptides
Authors: Gomez Sophie , di Bello Carlo , Hung Lam Than , Genet Roger , Morgat Jean-Louis , Fromageot Pierre , Cohen Paul
Published: July 2002

Biosynthesis of the C‐terminal carboxamide group of peptide hormones was studied using comparatively pGlu‐His‐Pro‐Gly and Glu‐His‐Pro‐Gly‐Lys‐Arg as putative precursors of the tripeptide, thyroliberin (TRH). Rat hypothalamus granules were found to contain an amide group forming activity which converts both peptide substrates into TRH. Comparison of the rate of conversion of the two substrates indicated that the C‐terminal dibasic extension favored a 10‐fold increase in the production of amidated peptide. It is suggested that this type of structure may be present in the putative biosynthetic precursor of TRH and that it may provide a better substrate for the enzyme(s) involved in C‐terminal amidation.
Revised assignments for the β‐, γ‐ and δ‐subunits of the acetylcholine receptor structural model
Authors: Kosower Edward M.
Published: July 2002

Revised assignments for the bilayer helices of the β‐,γ‐ and δ‐subunits of the acetylcholine receptor are presented. A new feature of the model is extensive charge matching between the polar groups of the ion channel elements of different subunits.
Hydrocortisone inhibits prostaglandin production but not arachidonic acid release from cultured macrophages
Authors: Wood John N. , Coote Paul R. , Rhodes John
Published: July 2002

We have investigated the action of hydrocortisosone on arachidonic acid mobilisation in cultures of mouse peritoneal macrophages, mouse L929 cells and the mouse macrophage‐like cell line RAW264. Hydrocortisone inhibits both arachidonic acid release and prostaglandin production by L929 cells. However, prostaglandin production by macrophages or RAW264 cells is inhibited with a concomitant stimulation rather than inhibition of arachidonic acid release. These data suggest that hydrocortisone acts at the level of phospholipase activity in fibroblasts but at a later stage of prostanoid production in macrophages.
A colicin A fragment containing the receptor binding domain can be directed to the periplasmic space in E. coli through gene fusion
Authors: Crozel Veronica , Lazdunski Claude , Lloubes Roland , Cavard Danielle
Published: July 2002

The central region of the colicin A polypeptide chain has been fused to the N‐terminal part of β‐lactamase through genetic recombination. This region comprising amino acid residues 70–335 confers on the hybrid protein the ability to protect sensitive cells from the lethal action of colicin A. Although colicin A belongs to the cytoplasmic compartment of E. coli, export of the hybrid protein to the periplasmic space was promoted by the signal peptide of β‐lactamase.
Internal mobility in a double‐stranded B DNA hexamer and undecamer
Authors: Clore G.Marius , Gronenborn Angela M.
Published: July 2002

The internal mobility of the deoxyribose H2'‐H2' ' and base C(H5)‐C(H6) and T(CH3)‐T(H6) vectors has been investigated by means of time‐dependent nuclear Overhauser enhancement (NOE) measurements in a B DNA hexamer and undecamer. Cross‐relaxation rates between these proton pairs are determined from the initial slopes of the time development of the NOEs, and, as the interproton distances between these proton pairs are fixed, apparent correlation times for the 3 interproton vectors are calculated from the cross‐relaxation rate data. It is shown that there is little residue to residue variation in the crossrelaxation rates of the interproton vectors within each oligonucleotide, that the mean apparent correlation times of the C(H5)‐C(H6) and T(CH3)‐T(H6) vectors are approximately equal and significantly greater than that of the H2'‐H2' ' vectors, and that the data for the H2'‐H2' ' vectors of both oligonucleotides and the C(H5)‐C(H6) and T(CH3)‐T(H6) vectors of the undecamer cannot be accounted for by isotropic tumbling alone. The data are analysed in terms of a two motion model with isotropic tumbling and a single internal motion. The relaxation time of the internal motion at 23 °C is ≲ 1 ns for the H2'‐H2' ' vectors of both oligonucleotides and ≲3 ns for the C(H5)‐C(H6) and T(CH3)‐T(H6) vectors of the undecamer. In the case of the H2'‐H2' ' vectors, however, the amplitude of the internal motion is found to be too large to be compatible with the known stereochemistry of DNA. This finding can only be explained by invoking additional degrees of internal freedom with a larger number of internal motions of small amplitude of the type deduced from the analysis of crystallographic thermal factors [(1984) J. Mol. Biol. 173, 361‐388].
Calcium, calmodulin‐dependent protein phosphorylation in Neurospora crassa
Authors: van Tuinen Diederik , Ortega Perez Ruben , Marme Dieter , Turian Gilbert
Published: July 2002

A calcium, calmodulin‐dependent protein kinase activity has been partially purified by calmodulin‐Sepharose affinity chromatography from the soluble fraction of Neurospora crassa. The phosphorylated peptide has an apparent molecular mass on SDS‐polyacrylamide gel of 47 kDa. The apparent half maximal phosphorylation is obtained after 1.5 min at 30° C in the presence of calcium and calmodulin. The apparent half maximal activation of the phosphorylation is obtained at 1 μM calcium, and 0.1 or 0.2 μM calmodulin from bovine brain or Neurospora, respectively. The 32P incorporation is enhanced about 10‐fold by calmodulin.
Presence of tropomyosin in adrenal chromaffin cells and its association with chromaffin granule membranes
Authors: Burgoyne Robert D. , Norman Kathyrn-Marie
Published: July 2002

The presence of tropomyosins in adrenal chromaffin cells was demonstrated by immunoperoxidase staining of cultured chromaffin cells by an anti‐tropomyosin antibody. In immunoblotting the antibody labelled polypeptides of M
r 39000, 36500 and 31000 in 100000 × g supernatants and heat‐stable fractions of adrenal medulla. The adrenal medullary tropomyosins did not comigrate with smooth muscle tropomyosins. The 39‐kDa and 31‐kDa polypeptides comigrated with polypeptides labelled by anti‐tropomyosin in brain supernatants. Chromaffin granule membranes possessed a 39‐kDa polypeptide that was labelled by anti‐tropomyosin.
Human mitochondrial aldehyde dehydrogenase inhibition by diethyldithiocarbamic acid methanethiol mixed disulfide: a derivative of disulfiram
Authors: MacKerell Alexander D. , Vallari Robert C. , Pietruszko Regina
Published: July 2002

A derivative and possible physiological metabolite of disulfiram, diethyldithiocarbamic acid methanethiol mixed disulfide, is shown here for the first time to inactivate the mitochondrial low‐K
m, isozyme of human aldehyde dehydrogenase (EC 1.2.1.3). By comparing inactivating effects of diethyldithiocarbamic acid mixed disulfides with thiols of increasing chain length evidence is provided that steric hindrance is the reason for lack of inhibition of the mitochondrial enzyme by disulfiram in vitro. Since methanethiol is a normal metabolite [(1983) Annu. Rev. Biochem. 52, 187–222] the results also suggest a mechanism by which aldehyde dehydrogenase is inhibited by disulfiram and diethyldithiocarbamic acid in vivo.
Retarded decline in poly‐ADPR content and poly‐ADPR synthetase activity in chicken dystrophic muscle
Authors: Yoshikawa Akira , Masaki Tomoh
Published: July 2002

The activity of poly‐ADPR synthetase declines just after hatching in normal chick muscle nuclei. However, in dystrophic chick muscle nuclei it decreases 5 weeks after hatching, A delayed decrease in the amount of poly‐ADPR is also observed in dystrophic chick muscle nuclei. These observations suggest that dystrophic muscle follows an abnormal developmental program.
1H‐NMR spectroscopy on elongation factor Tu from Escherichia coli
Authors: Kalbitzer H.R. , Leberman R. , Wittinghofer A.
Published: July 2002

1H‐NMR
Protein
EF‐Tu
Perdeuteration
Relationship between Na+‐dependent cytoplasmic pH homeostasis and Na+‐dependent flagellar rotation and amino acid transport in alkalophilic Bacillus
Authors: Sugiyama Shigeru , Matsukura Hiroshi , Imae Yasuo
Published: July 2002

The cytoplasmic pH homeostasis of alkalophilic Bacillus strains required the presence of Na+ in the medium, and Li+ was found to be equivalently substitutable for Na+. Flagellar rotation and amino acid transport of these bacteria also required Na+ but Li+ was not substitutable for Na+. Na+ concentration of about 1 mM was enough for the cytoplasmic pH homeostasis, while more than 10 mM Na+ was required for the full activities of flagellar rotation and amino acid transport. The addition of 150 mM ethanolamine to the cells at pH 9.6 disrupted the pH homeostasis and increased the cytoplasmic pH close to the external pH. Under this condition, however, flagellar rotation and amino acid transport were not so much affected. Thus, it is clear that flagellar rotation and amino acid transport themselves require the presence of Na+ in the medium, independent of the Na+‐dependent cytoplasmic pH homeostasis.
Magnesium‐dependent conformational changes of membrane proteins are related to the Mg2+ ‐dependent ATPase activity in cardiac sarcolemma
Authors: Vrbjar N. , Soos J. , Ziegelhöffer A.
Published: July 2002

Magnesium‐induced enzymatic and structural changes of membrane‐bound proteins in rat heart sarcolemma have been investigated. In the absence of ATP, increasing concentrations of magnesium within the range 0.1–10.0 mM gradually lowered the α‐helix content of sarcolemmal proteins. The same magnesium concentrations stepwise activated the Mg2+‐dependent ATPase in the presence of ATP. Mathematical and graphical analysis of the data yielded a quantitative relationship between magnesium‐induced stimulation of the Mg2+‐dependent ATPase activity and diminution of the α‐helix content of membrane proteins in cardiac sarcolemma.
A temperature dependent transition in the Pribnow box of the trp promoter
Authors: Lefevre Jean-François , Lane Andrew N. , Jardetzky Oleg
Published: July 2002

Proton NMR spectra of the trp operator‐promoter (sequence CGTACTAGTT.AACTAGTACG) show selective changes in chemical shift and relaxation rates over the range of temperature 0–45°C for the non‐exchangeable protons of A11 and A12 only. These bases are in the centre of the Pribnow box. The changes imply that at least three conformational states become significantly populated in this range of temperature, and probably involve a change in the propellor twists of A11 and A12 for one transition, and changes in the helical twist and local pitch for the other. As (1) mutations in the Pribnow box that destroy the TAA sequence impair promoter activity, and (2) the abortive initiation assay for RNA polymerase shows a transition near 20°C, we propose that the observed conformational transitions in the trp promoter are an essential feature of good promoters.
Glutathione transferases in primary rat hepatomas: the isolation of a form with GSH peroxidase activity
Authors: Meyer David J. , Beale Denis , Tan K.Hong , Coles Brian , Ketterer Brian
Published: July 2002

A previously uncharacterized glutathione (GSH) transferase which is not apparent in normal liver, accounts for at least 25% of the soluble GSH transferase content of primary hepatomas induced by feeding N,N‐dimethyl‐4‐aminoazobenzene. This enzyme is readily isolated, has an isoelectric point of 6.8, is composed of two identical subunits of apparent M
r 26 000 and has GSH transferase activity towards a number of substrates including benzo(a)pyrene‐7,8‐diol‐9,10‐oxide. It is unusual in that it has GSH peroxidase activity towards fatty acid hydroperoxides but not towards the model substrates, cumene hydroperoxide and t‐butyl hydroperoxide. It has been shown by tryptic peptide analysis to be distinct from GSH transferases composed of subunits 1, 2, 3,4 or 6 and has been designated GSH transferase 7‐7.
Rapid purification of calcium‐activated protease by calcium‐dependent hydrophobic‐interaction chromatography
Authors: Gopalakrishna Rayudu , Head James F.
Published: July 2002

Both low Ca2+‐ and high Ca2+‐requiring forms of Ca2+‐activated protease (calpains I and II) were found to bind to phenyl‐Sepharose in a calcium‐dependent manner, suggesting that both enzymes expose a hydrophobic surface region in the presence of Ca2+. Inclusion of leupeptin in column buffers prevented the loss of activity during hydrophobic‐interaction and substrate‐affinity chromatography. Under these conditions calpain II (high calcium‐requiring form) was rapidly purified from bovine brain and rabbit skeletal muscle using successive phenyl‐Sepharose and casein‐Sepharose columns.
Nonhistone proteins of the transcriptionally active chromatin fraction of Physarum polycephalum, associated with nucleosome linker DNA instead of histone H1
Authors: Czupryn Marta , Solnica Lilianna , Toczko Kazimierz
Published: July 2002

Physarum polycephalum
Nonhistone protein
Histone H1
Transcriptionally active chromatin
Lipid requirements for reconstitution of the delipidated β‐adrenergic receptor and the regulatory protein
Authors: Kirilovsky Jorge , Steiner-Mordoch Sonia , Selinger Zvi , Schramm Michael
Published: July 2002

Delipidation
Reconstitution
β‐Adrenergic receptor
Guanyl nucleotide binding protein
Regulatory protein
Lipid requirement
Expression of Semliki Forest virus capsid protein from SV40 recombinant virus
Authors: Jalanko Anu
Published: July 2002

Here, the proteolytic processing of the Semliki Forest virus (SFV) capsid protein was studied in the absence of other viral functions. Two different fragments of the SFV messenger cDNA, coding for capsid protein and 174 and 38 extra amino acids from the envelope proteins, respectively, were cloned in the late region of the SV40 viral DNA. Cells infected with the SV40 recombinant virus stocks were analyzed for the production of SFV capsid mRNA and polypeptide. Immunofluorescence staining of the infected cells indicated that the produced SFV capsid protein accumulated mainly in the nucleus. Polyacrylamide gel electrophoresis of the immunoprecipitated SFV capsid proteins showed that both recombinants yielded a labelled band equivalent in size to the SFV capsid protein. Thus the proteolytic processing takes place even under conditions where the capsid protein is the only virus‐specified protein synthesized.
A tetrameric inhibitor of insect α‐amylase from barley
Authors: Sanchez-Monge Rosa , Gomez Luis , Garcia-Olmedo Francisco , Salcedo Gabriel
Published: July 2002

A tetrameric inhibitor that is active against α‐amylase from the larvae of the insect Tenebrio molitor, but inactive against the enzyme from human saliva and against the endogenous one, has been described in barley endosperm. The subunits of the inhibitor have been identified as the previously characterized proteins CMa, CMb and CMd, of which only CMa was inhibitory by itself.
Gene structure of calcium‐dependent protease retains the ancestral organization of the calcium‐binding protein gene
Authors: Emori Yasufumi , Ohno Shigeo , Tobita Masako , Suzuki Koichi
Published: July 2002

The gene structure of calcium‐dependent protease (Ca2+‐protease) was determined. It comprises at least 21 exons, and these were assigned to the 4 functional domains of the protease. The protease domain does not show clear correlation between exons and functional units, but the calmodulin‐like calcium‐binding domain shows strong correlation. Each of the 4 consecutive calcium‐binding regions in the C‐terminal part of Ca2+‐protease is encoded by one exon. This gene structure supports the idea that the 4 calcium‐binding regions of calcium‐binding proteins such as calmodulin arose by 2 steps of gene duplication.
Protein kinase C negatively modulated by phorbol ester
Authors: Inagaki Masaki , Hagiwara Masatoshi , Saitoh Masahiro , Hidaka Hiroyoshi
Published: July 2002

Pretreatment of protein kinase C with 12‐O‐tetradecanoylphorbol‐13‐acetate (TPA) and phospholipid resulted in complete inhibition of ATP/phosphotransferase activity, irreversibly. The inactivation by TPA required the phospholipid, and TPA alone did not cause inactivation. Ca2+ and diacylglycerol mimicked TPA. This action of TPA was not general for all protein kinases as it did not accelerate the inactivation of the catalytic subunit of cAMP‐dependent protein kinase by phospholipid. The addition of MgATP to the reaction mixture completely protected protein kinase C from being inactivated by TPA, in the presence of phospholipid. The nucleotide‐binding site of the enzyme was probably influenced by the binding of TPA and phospholipid.
FAD and FMN flavoproteins participate in the sodium‐transport respiratory chain NADH:quinone reductase of a marine bacterium, Vibrio alginolyticus
Authors: Hayashi Maki , Unemoto Tsutomu
Published: July 2002

The sodium‐transport respiratory chain NADH:quinone reductase of a marine bacterium, Vibrio alginolyti‐cus, is composed of three protein subunits, α,β and γ. The β‐subunit contains FAD as a prosthetic group and corresponds to NADH dehydrogenase, which catalyses the reduction of ubiquinone to ubisemiquinone. In addition to β, subunits α. and γ are essential for the quinone reductase, which catalyses the reduction of ubiquinone to ubiquinol. The α‐subunit contains FMN and the reaction catalysed by subunit α is related to the coupling site of the sodium pump in the quinone reductase.
Relationship between the 43 kDa chlorophyll‐protein of PS II and the rapidly metabolized 32 kDa QB protein
Authors: Machold O.
Published: July 2002

The chlorophyll‐proteins of L‐[35S]methionine‐labelled thylakoid membranes isolated from cycloheximide‐treated Vicia faba plants were electrophoretically separated and correlated to the rapidly metabolized QB protein. Additionally, the chlorophyll a‐protein band with the highest specific radioactivity was excised and subjected to denaturing re‐electrophoresis. Based on these experiments it is presumed that the native QB protein is associated with pigments and represents a protein moiety of the 43 kDa chlorophyll a‐protein (Chl a‐P3) of PS II. This is reconcilable with the hypothesis that the QB protein carries the reaction center of PS II as deduced from its homology to the L and M subunits of the bacterial reaction center complex.
Solution structure of d‐GAATTCGAATTC by 2D NMR
Authors: Hosur R.V. , Ravikumar M. , Chary K.V.R. , Sheth Anu , Govil Girjesh , Zu-Kun Tan , Miles H.Todd
Published: July 2002

A new approach based on the correlated spectroscopy (COSY) in 2D NMR has been described for determination of sugar geometries in oligonucleotides. Under the usual low resolution conditions employed in COSY, the intensities of cross peaks depend on the magnitudes of coupling constants. There are five vicinal coupling constants in a deoxyribose ring which are sensitive to the sugar geometry. The presence, absence and rough comparison of relative intensities of COSY cross peaks arising from such coupling constants enable one to fix the sugar conformation to a fair degree of precision. The methodology has been applied to d‐GAATTCGAATTC. It is observed that ten out of the twelve nucleotide units in this sequence exhibit a rare Ol′‐endo geometry. The EcoRI cleavage sites (between G and A) in the dodecanucleotide show an interesting variation in the conformation with the two sugars attached to the Gs acquiring a geometry between C2′‐endo and C4′‐endo.
Modulation of the interaction between chemotactic cAMP‐receptor and N‐protein by cAMP‐dependent kinase in Dictyosteliumdiscoideum membranes
Authors: Ludérus M.E.E. , van der Meer R.F. , van Driel R.
Published: July 2002

Dictyostelium discoideum cells contain kinetically distinguishable surface cAMP receptors. Both GTP and GDP lower the receptor affinity by inducing conversion of slowly dissociating sites to fast dissociating sites, presumably by binding to a N‐protein [(1985) Mol. Cell. Biochem. 67, 119‐124, and (1986) Biochemistry 25, 1314‐1320]. In this paper we show that treatment of isolated membranes with cAMP‐dependent protein kinase abolished the GTP‐induced receptor transition, but not the one induced by GDP. The effect of GTP on the receptor kinetics could be restored by treatment of the membranes with alkaline phosphatase. These results indicate that in D. discoideum membranes phosphorylation of a signal‐transduction component reversibly abolishes the interaction of the cAMP receptor with the NGTP complex, but not that with the NSDP complex.
Changes in the expression of cell surface sialoglycoproteins during transition of human monocytes into macrophages
Authors: Heino Jyrki , Larjava Hannu , Penttinen Risto
Published: July 2002

Cell surface sialoglycoproteins of human mononuclear phagocytes in different maturation stages were labelled by the periodate/borohydride method and separated by SDS‐polyacrylamide gel electrophoresis. The main surface glycoproteins of peripheral blood monocytes had molecular masses of 115 and 95 kDa. During in vitro transition into adherent macrophages, the monocyte‐characteristic surface glycoproteins disappeared. Most of the changes in the surface glycoprotein pattern occurred during the first 24 h and after 96 h the changes were completed. The major sialoglycoproteins of the macrophage cell surface had molecular masses of 130 and 55 kDa. The macrophage cell surface showed further changes when cultured in the presence of synovial fluid (10%). These results may reflect the in vivo maturation of monocytes into tissue macrophages. In synovium, tissue‐derived factors may also take part in differentiatíon.
Formation of lipoxin B by the pure reticulocyte lipoxygenase
Authors: Kühn H. , Wiesner R. , Alder L. , Schewe T. , Stender H.
Published: July 2002

The pure reticulocyte lipoxygenase converts 5,15‐DiHETE via a lipoxygenase reaction to 5,14,15‐trihy‐droxy‐6, 8,10,12‐eicosatetraenoic acid (a lipoxin B isomer) as shown by GC/MS analysis of its trimethylsilyl ether. With arachidonic acid, 15‐HETE and 15‐HETE methyl ester this lipoxin B isomer was also formed. The results presented here indicate that pure mammalian lipoxygenases are able to form lipoxins via sequential multiple oxygenation of arachidonic acid or its hydroxy derivatives.
Further evidence for a role of sulfhydryls in the thioredoxin dependent activation of corn NADP‐malate dehydrogenase
Authors: Jacquot Jean-Pierre , Decottignies Paulette
Published: July 2002

The kinetics of activation and reduction of the corn leaf NADP‐malate dehydrogenase reveal that plant thioredoxins play a dual role in the activation of this enzyme in the presence of dithiothreitol (e.g. reductive and conformational). A mutant thioredoxin of E. coli in which Cys32 and Cys35 are replaced by an Ala and a Ser residue, respectively, was tested in the activation of corn NADP‐malate dehydrogenase either in the presence of DTT or in a light reconstituted system. While native E. coli or plant thioredoxinm strongly activated the NADP‐malate dehydrogenase, the mutant thioredoxin was unable to promote any activation of the enzyme. At the same time the mutant thioredoxin stimulated the activity of T7 DNA polymerase. These results clearly confirm that the thioredoxin requirement observed in the activation of NADP‐malate dehydrogenase is primarily needed for redox reactions and not for structural/conformational effects as is the case for the T7 DNA polymerase assay.
Inhibition of phorbol ester stimulated superoxide production by 1‐oleoyl‐2‐acetyl‐sn‐glycerol (OAG); fact or artefact?
Authors: Bonser R.W. , Dawson J. , Thompson N.T. , Hodson H.F. , Garland L.G.
Published: July 2002

OAG‐stimulated superoxide (O−
2) production by HL‐60 granulocytes showed enantiomeric specificity but reached a maximum of only 5% of that produced by either phorbol myristate acetate (PMA) or phorbol dibutyrate (PDBu). At 10–100 μM, OAG displaced specifically‐bound [3H]PDBu from intact HL‐60 cells by only 25%, suggesting limited cell penetration. OAG (10–100 μM) also inhibited PDBu‐stimulated O2 production by 25%; this inhibition was enantiomerically specific. However, at a lower concentration (3 μM), both enantiomers of OAG fully blocked O−
2 production stimulated by PMA (0.5 μM). This inhibition is probably artefactual, due to the hydrophobic PMA physically associating with OAG in the extracellular fluid.
N‐terminal amino acid sequences of the heavy and light chains of chicken liver cathepsin L
Authors: Wada Kenji , Tanabe Tadashi
Published: July 2002

Avian cathepsin L (EC 3.4.22.15) was first purified from chicken liver. The enzyme was composed of two polypeptides with M
r values of 28 000 (heavy chain) and 5000 (light chain). The two polypeptide chains of the enzyme were separated by gel filtration after cleavage of disulfide bonds. The N‐terminal amino acid sequences of the heavy and light chains were APRSVDWREKGYVTPVKDQGQCGSCWAFSTTGALEGQ and GKKYWIVKNSWGEKWGDKGYIYMAKDRKNHCGIATAASYP, respectively. These sequences show high homologies with those of the N‐terminal and C‐terminal portions of papain, respectively. The residues of the surrounding area of cysteine‐25 in the heavy chain were quite similar to those in the region around the active‐site cysteine of papain.
The effect of iron and agar on production of hydrogen peroxide by stimulated and activated mouse peritoneal macrophages
Authors: Thompson H.Lorraine , Stevenson Joan , Brock J.H.
Published: July 2002

The effect of iron on H2O2 production by mouse peritoneal macrophages exposed to opsonised zymosan has been investigated. Macrophages elicited with thioglycollate broth produced less H2O2 than macrophages activated by Corynebacterium parvum, and levels were not affected by prior incubation of the cells with 0.1 mM iron nitrilotriacetate. However, preincubation with the iron chelator desferrioxamine (1 mM) reduced H2O2 production by both types of macrophages. Incubation of macrophages with agar, a component of thioglycollate broth, also reduced H2O2 production, particularly by C. parvum‐activated macrophages. The results indicate that although iron appears to be necessary for H2O2 production by macrophages, the low level of production by thioglycollate‐elicited macrophages is not due to an inadequate level of metabolically utilisable iron, but may be a result of prior ingestion of agar present in the broth.
Chemical modification of the brown fat mitochondrial uncoupling protein with tetranitromethane
Authors: Rial Eduardo , Nicholls David G.
Published: July 2002

Tetranitromethane reacts with the uncoupling protein of intact brown fat mitochondria. The chloride permeability in the absence of the inhibitory nucleotide GDP is not affected, but the affinity with which GDP binds is decreased, and the coupling between binding of nucleotide and inhibition of chloride permeation is broken.
The amino acid sequence of the aliphatic amidase from Pseudomonas aeruginosa
Authors: Ambler R.P. , Auffret A.D. , Clarke P.H.
Published: July 2002

Amino acid sequence studies show that the aliphatic amidase (EC 3.5.1.4) from Pseudomonas aeruginosa PAC142 consists of a single polypeptide chain of 346 residues, giving an M
r of 38400. The evidence from the amino acid studies is in complete agreement with that deduced from the DNA sequence of the amiE gene. Studies of the protein from Pseudomonas putida A87 show that it differs from the Ps. aeruginosa protein by about 30 amino acid substitutions. It now becomes possible to relate changes in the enzyme which result in altered specificity to structural changes in the protein.
Sequence conservation in the 3′‐untranslated regions of neurone‐specific enolase, lymphokine and protooncogene mRNAs
Authors: Day I.N.M. , Allsopp M.T.E.P. , Moore D.C.McN. , Thompson R.J.
Published: July 2002

The C‐terminal protein‐coding and the entire 3′‐untranslated regions of a cDNA corresponding to human neurone‐specific enolase mRNA have been sequenced. The 3′‐untranslated region is 892 bases long and shows a high degree of homology with the 3′‐untranslated region of rat neurone‐specific enolase mRNA. This sequence conservation is not seen in non‐neuronal enolase mRNAs. Features of the conserved sequence include an A‐rich region approx. 250 bases from the stop codon at a point corresponding to the polyadenylation signal site in non‐neuronal enolase mRNA, and a repeating ATTT sequence. This unusual motif in eukaryotic mRNAs has previously been reported in the 3′‐untranslated regions of lymphokine and protooncogene mRNAs.
Cross‐reconstitution of isolated F1‐ATPase from potato tuber mitochondria with F1‐depleted beef heart and yeast submitochondrial particles
Authors: Norling B. , Hamasur B. , Glaser E.
Published: July 2002

Cross‐reconstitution of isolated potato mitochondrial F1‐ATPase with F1‐depleted beef heart and yeast submitochondrial particles is reported. Potato F1 binds to the heterologous membrane and confers oligomycin sensitivity on the ATPase activity of the reconstituted system. Binding of F1 is promoted by the presence of Mg2+ with the maximal stimulatory efrect at 20 mM. Mg2+ increase the sensitivity to oligomycin of the reconstituted system consisting of potato F1 and yeast membranes, however, they do not influence oligomycin sensitivity of potato F1 and beef heart membranes.
The efficiency of dNTP complex formation with human placenta DNA polymerase α as demonstrated by affinity modification
Authors: Doronin S.V. , Lavrik O.I. , Nevinsky G.A. , Podust V.N.
Published: July 2002

The interaction of deoxyribonucleoside 5′‐mono‐, di‐ and triphosphates with human placenta DNA polymerase α was examined. Dissociation constants of enzyme complex formation with dNMP, dNDP and dNTP were determined from the data on enzyme affinity modification by imidazolide of dTMP. The basic role of the primary template‐primer interaction with the enzyme in dNTP complex formation is shown. The template‐dependent nucleotide interaction does not occur in the case of dNMP and dNDP in comparison with dNTP. The significant contribution of the γ‐phosphate of dNTP in this process is demonstrated.
PA‐II, the L‐fucose and D‐mannose binding lectin of Pseudomonas aeruginosa stimulates human peripheral lymphocytes and murine splenocytes
Authors: Avichezer Dody , Gilboa-Garber Nechama
Published: July 2002

Pseudomonas aeruginosa lectin PA‐II agglutinates human peripheral lymphocytes and stimulates mitogenesis (predominantly in T cells), like the plant lectins PHA and Con A. Murine splenocytes are also agglutinated and stimulated by PA‐II as by Con A. Sialidase treatment of the human and murine cells enhances their agglutination and augments the stimulation of human lymphocytes at low PA‐II concentrations. The PA‐II agglutinating and mitogenic effects are specifically inhibited by L‐fucose. The bacterial source and the specificity of PA‐II for L‐fucose are both rare features among the hitherto described mitogenic lectins. However, since this lectin also binds mannose, a mannose‐bearing receptor might be involved in its mitogenicity.
α‐h‐ANP is the only form of circulating ANP in humans
Authors: Theiss G. , John A. , Morich F. , Neuser D. , Schröder W. , Stasch J.-P. , Wohlfeil S.
Published: July 2002

It is therefore concluded that α‐h‐ANP is the only form of ANP circulating in human plasma.
Epidermal growth factor (EGF), tumor promoter 12‐O‐tetradecanoylphorbol 13‐acetate (TPA) and calcium ionophore A23187 increase cytoplasmic free calcium and stimulate arachidonic acid release and PGE2/6‐keto PGF1α production in cultured porcine thyroid cells
Authors: Takasu Nobuyuki , Yamada Takashi , Shimizu Yoshifusa
Published: July 2002

Epidermal growth factor (EGF), 12‐O‐tetradecanoylphorbol 13‐acetate (TPA) and calcium ionophore A23187 increase cytoplasmic free calcium ([Ca2+]i) and stimulate arachidonic acid release and production of PGE2 and 6‐keto PGF1α an end metabolite of PGI2, in cultured porcine thyroid cells. Addition of EGF, TPA or A23187 to the cells loaded with fura‐2, a fluorescent Ca2+ indicator, causes an immediate increase in [Ca2+]i which is the earliest event after mitogen stimulation. This [Ca2+]i response occurs immediately, reaching a maximum within several seconds. EGF, TPA and A23187 stimulate arachidonic acid release and PGE2 and 6‐keto PGF1α production; the maximum effects are obtained after 2–4 h incubation. EGF, TPA and A23187 increase [Ca2+]i and then stimulate arachidonic acid release and PG production.
Characterization of domain borders and of a naturally occurring major fragment of staphylococcal α‐toxin
Authors: Blomqvist Lennart , Bergman Tomas , Thelestam Monica , Jörnvall Hans
Published: July 2002

A naturally occurring staphylococcal α‐toxin fragment with an apparent membrane‐binding capacity but without toxic activities is shown to be derived from the C‐terminal half of the intact polypeptide chain by cleavage between position 134 and 135 in the parent molecule. The resulting N‐terminus is slightly ragged with a fragment start not only at position 135 but also at the adjacent position 136. Another naturally occurring fragment starts at position 9, derived from an original cleavage between position 8 and 9 in the parent molecule. Analysis of non‐purified fragment mixtures confirmed these positions and established that only one further region, at positions 71–72, is partly sensitive to proteolysis under natural conditions. Trypsin treatment has limited effects on the native toxin molecule, giving essentially only two initial cleavages with resultant large fragments. One of these cleavages is at the peptide bond between position 131 and 132, thus only three residues away from the position of the major naturally occurring cleavage. The other bond sensitive to trypsin is between position 8 and 9, thus identically positioned to the cleavage occurring naturally. Together, all the cleavages define a region in a central segment of the polypeptide chain that has all the properties of an inter‐domain segment. The C‐terminal half appears to constitute a membrane‐binding domain, and the N‐terminal half a structure needed for full biological activity, functionally subdividing the parent polypeptide chain.
Protonmotive‐force‐driven leucine uptake in yeast plasma membrane vesicles
Authors: Opekarová Miroslava , Driessen Arnold J.M. , Konings Wil N.
Published: July 2002

Plasma membranes prepared from Saccharomyces cerevisiae by the concanavalin A method were fused with liposomes by a freeze‐thaw sonication method. A ΔΨ of −109 mV was generated in the sealed vesicles by a valinomycin‐mediated potassium diffusion potential. This component of protonmotive force was able to drive a transient uptake of leucine.
Monoclonal antibodies against the 1,4‐dihydropyridine receptor associated with voltage‐sensitive Ca2+ channels detect similar polypeptides from a variety of tissues and species
Authors: Norman R.I. , Burgess A.J. , Allen E. , Harrison T.M.
Published: July 2002

Four monoclonal antibodies have been raised against voltage‐sensitive Ca2+ channel dihydropyridine receptors from rabbit skeletal muscle. When tested by immunoblot assay of denatured transverse tubule membranes in reducing polyacrylamide gels, each recognised a single polypeptide of Mr
~ 140000 that co‐migrated with the large glycoprotein subunit of the purified receptor. In blots of nonreducing gels, a larger protein of Mr
~ 170000 was seen and three of the antibodies recognised additional components at Mr
~ 310000 and ~ 330000. Crossreactive material of similar molecular mass was also seen in rabbit heart and brain, and in the skeletal muscle of other species.
Dexamethasone‐induced reduction of phospholipase D activity in the rat Possible role of lipocortin
Authors: Kobayashi Mutsuhiro , Bansal Vinay S. , Singh Indrapal , Kanfer Julian N.
Published: July 2002

Subcutaneous injection of dexamethasone resulted in a reduction of demonstrable phospholipase D activity of rat brain and liver microsomes. Partially purified rat lung lipocortin inhibited the activity of both microsomal and partially purified rat brain phospholipase D. These results show that phospholipase D activity is suppressed by dexamethasone and one of the possible mechanisms of inhibition may be a phospholipase inhibitory protein, lipocortin.
Early cortical plate specific glycoprotein in a marsupial species belongs to the same family as fetuin and α2HS glycoprotein
Authors: Jones S.E. , Dziegielewska K.M. , Saunders N.R. , Christie D.L. , Sueiras-Diaz J. , Szelke M.
Published: July 2002

Two related glycoproteins, fetuin in species of the order Artiodactyla (cattle, sheep, pig) and α2HS glycoprotein in the human [(1987) Cell Tissue Res. 248, 33–41] have a very specific distribution in the developing brain. We have isolated and determined the first 15 N‐terminal residues of a similarly distributed glycoprotein in the developing brain of the tammar wallaby (Macropus eugenii). The degree of homology is the same between wallaby glycoprotein and α2HS glycoprotein as between fetuin and α2HS glycoprotein (46%). Antibodies made to synthetic peptides of fetuin were used to identify the wallaby glycoprotein. A polyclonal antibody to the purified glycoprotein was used for immunocytochemical identification of brain cells positive for this protein.
Human recombinant interleukin‐1α‐mediated stimulation of procollagenase production and suppression of biosynthesis of tissue inhibitor of metalloproteinases in rabbit uterine cervical fibroblasts
Authors: Ito Akira , Goshowaki Hideko , Sato Takashi , Mori Yo , Yamashita Kyoko , Hayakawa Taro , Nagase Hideaki
Published: July 2002

Influence of human recombinant interleukin‐1 (hrIL‐1) on collagen metabolism was investigated with rabbit uterine cervical fibroblasts. Enzyme‐linked immunosorbent assays for collagenase and tissue inhibitor of metalloproteinases (TIMP) indicated that hrIL‐1 participates in both stimulation of procollagenase production and suppression of TIMP synthesis by uterine cervical cells. IL‐1 did not modulate collagen synthesis. In addition, the sensitivity to IL‐1 of uterine cervix from ovariectomized rabbits was augmented by estradiol‐17β treatment. Thus it is proposed that IL‐1 accelerates collagenolysis in the cervical tissue and its effect on uterine cervix is hormonally regulated.
Neurohypophysial hormones of the 1‐month‐old bovine fetus: Absence of vasotocin during mammal development
Authors: Rouille Y. , Levy B. , Chauvet M.T. , Chauvet J. , Acher R.
Published: July 2002

The neurohypophysial hormones of the 1‐month‐old bovine fetus have been identified by their positions in ion‐exchange chromatography and their retention times in high‐pressure reverse‐phase partition chromatography. Arginine vasopressin and oxytocin have been recognized. The molar ratio vasopressin/oxytocin in neurohypophysis is about 6 in the 1‐month‐old fetus compared with 4 in the 3‐month‐old fetus, 2.7 in the 7‐month‐old fetus and 1 in the adult. Vasotocin is virtually absent even in the early fetus (less than 0.1% of arginine vasopressin). The occurrence of a vasotocin gene expressed in the fetus but silent in the adult appears unlikely.
Human T cell specific proteinase (HuTSP) is encoded by the T cell and natural killer cell specific human Hanukah factor (HuHF) gene
Authors: Fruth Uli , Eckerskorn Christoph , Lottspeich Friedrich , Kramer Michael D. , Prester Marlot , Simon Markus M.
Published: July 2002

HuTSP, a serine proteinase which is specifically associated with activated T lymphocytes, was purified to homogeneity and characterized. N‐terminal amino acid sequencing revealed identity with a sequence predicted from the human Hanukah factor (HF) gene, which was isolated from a human T cell cDNA library. The dimeric structure of HuTSP, together with its extensive sequence homologies with the murine T cell specific proteinase, MTSP‐1, suggests phylogenetic conservation of this serine proteinase family.
Assay of phosphotyrosyl protein phosphatase using synthetic peptide 1142–1153 of the insulin receptor
Authors: King Martin J. , Sale Graham J.
Published: July 2002

Synthetic peptide 1142–1153 of the insulin receptor was phosphorylated on tyrosine by the insulin receptor and found to be a potent substrate for dephosphorylation by rat liver particulate and soluble phosphotyrosyl protein phosphatases. Apparent K
m values were 5 μM. V
m values (nmol phosphate removed/min per mg protein) were 0.62 (particulate) and 0.2 (soluble). This corresponds to 80% of total activity being membrane‐associated, indicating that membrane‐bound phosphatases are important receptor phosphatases. The phosphatase activities were distinct from acid and alkaline phosphatase. In conclusion peptide 1142–1153 provides a useful tool for the further study and characterization of phosphotyrosyl protein phosphatases.
Total synthesis of the cystatin α gene and its expression in E. coli
Authors: Katunuma Nobuhiko , Yamato Masayuki , Kominami Eiki , Ike Yoshimasa
Published: July 2002

A gene encoding cystatin α has been chemically synthesized, cloned and expressed in E. coli. The gene of 318 base pairs was assembled by enzymatic ligation of 19 oligonucleotides and cloned into a pBR322‐derived expression plasmid down stream of the tac promoter. The expression product of the synthetic gene has been purified by Sephadex G‐50 column chromatography and shown to have the same properties as those of the authentic protein isolated from rat epidermis.
The relationship between activity and the axial g=2.06 EPR signal induced by CO in the periplasmic (Fe) hydrogenase from Desulfovibrio vulgaris
Authors: Patil D.S. , He S.H. , Der Vartanian D.V. , Le Gall J. , Huynh B.H. , Peck H.D.
Published: July 2002

The effect of exposure to carbon monoxide on the activity of the (Fe) hydrogenase from Desulfovibrio vulgaris has been determined. Concentrations of carbon monoxide which completely inhibit hydrogenase activity and induce formation of the axial g=2.06 EPR signal up to 0.8 spin/molecule do not cause irreversible inhibition of the (Fe) hydrogenase.
Secretion of a new growth factor, smooth muscle cell derived growth factor, distinct from platelet derived growth factor by cultured rabbit aortic smooth muscle cells
Authors: Morisaki Nobuhiro , Kanzaki Tetsuto , Koshikawa Tomoko , Saito Yasushi , Yoshida Sho
Published: July 2002

In attempts to determine the mechanism of proliferation of arterial smooth muscle cells (SMC) in intimal atheromatous lesions, autocrine secretion of growth factors by SMC has recently received much attention. Here we report a new growth factor named smooth muscle cell derived growth factor (SDGF). Cultured rabbit medial SMC secreted SDGF for 1 week during their incubation in serum‐free media only after at least 4 passages. SDGF differed from platelet derived growth factor (PDGF) physicochemically, immunologically, and biologically. The properties of SDGF also seemed different from those of other known growth factors that stimulate the proliferation of mesenchymal cells.
A 5 kb intergenic region containing oril in the mitochondrial DNA of Saccharomyces cerevisiae is dispensable for expression of the respiratory phenotype
Authors: Piškur Jure
Published: July 2002

A mutant mitochondrial DNA of Saccharomyces cerevisiae strain R 0.54 has been shown to have a 5 kb deletion which removes the oril sequence and its surrounding intergenic sequences between the pro tRNA and 15 S rRNA genes. The deleted DNA has been sequenced and is found to be flanked by two 11 bp direct repeats. The deletion mutant displays a normal phenotype in non‐fermentable media. This implies that the intergenic sequence is not required for expression of the respiratory phenotype.
The mitochondrial ATP synthase inhibitor protein binds near the C‐terminus of the F1 β‐subunit
Authors: Jackson Philip J. , Harris David A.
Published: July 2002

The specific, mitochondrial ATP synthase protein (IF1) was covalently cross‐linked to its binding site on the catalytic sector of the enzyme (F1‐ATPase). The cross‐linked complex was selectively cleaved, leaving IF1 intact to facilitate the subsequent purification of the F1 fragment to which IF1 was cross‐linked. This fragment was identified by sequence analysis as comprising residues 394–459 on the F1 β‐subunit, near the C‐terminus. This finding is discussed in the light of secondary structure predictions for both IF1 and the F1 β‐subunit, and sequence homologies between mitochondrial and other ATP synthases.
Post‐translational modification of the β‐subunit of the human fibronectin receptor
Authors: Jaspers Martine , de Strooper Bart , Spaepen Marijke , van Leuven Fred , David Guido , van den Berghe Herman , Cassiman Jean-Jacques
Published: July 2002

Monoclonal antibody DH12, directed against the β‐subunit of the fibronectin receptor recognizes a doublet of proteins (100 and 110 kDa) in Western blots of solubilized whole fibroblasts. Pulse‐chase experiments with [35S]methionine in human skin fibroblasts suggested that the two proteins might be metabolically related as precursor (100 kDa) and product (110 kDa). Endo H digestion and [3H]fucose labeling suggested that maturation converted the high‐mannose oligosaccharides (100 kDa) to the endoglycosidase H resistant complex type (110 kDa). This was supported by N‐glycanase digestion and by chemical deglycosylation which showed a single polypeptide. Surface iodination of intact cells labeled only the presumed mature β‐subunit.
A unique mRNA species for a regulatory subunit of cAMP‐dependent protein kinase is specifically induced in haploid germ cells
Authors: Øyen Ole , Scott John D. , Cadd Gary G. , McKnight G.Stanley , Krebs Edwin G. , Hansson Vidar , Jahnsen Tore
Published: July 2002

Cyclic AMP (cAMP) and its action by way of cAMP‐dependent protein kinase is important for sperm motility. Previous studies on germ cells have demonstrated a selective decrease in the amount of type I cAMP‐dependent protein kinase during spermatid development, and that type II was the major form present in elongating spermatids and in mature sperm. This would indicate activation of a gene in haploid germ cells, encoding a regulatory subunit of type II protein kinase. However, haploid expression of such a gene has so far not been shown. In the present study we demonstrate high‐levelled expression of a unique mRNA species for a specific regulatory subunit of type II cAMP‐dependent protein kinase at late stages of spermatogenesis, i.e. during spermatid elongation.
Crystallization of cytochrome P‐450scc from bovine adrenocortical mitochondria
Authors: Iwamoto Yoshiki , Tsubaki Motonari , Hiwatashi Atsuo , Ichikawa Yoshiyuki
Published: July 2002

Cytochrome P‐450scc (P‐450scc), a cholesterol side‐chain cleavage enzyme from bovine adrenocortical mitochondria, has been crystallized for the first time. Upon removal of glycerol from the solution of the native enzyme complexed with pyridoxal 5′‐phosphate (PLP) by microdialysis against distilled water, reddish and planar crystals appeared. The crystals of native P‐450scc were also obtained by the same procedure. We identified the crystals as the P‐450scc‐PLP complex or native P‐450scc by absorption spectroscopy and SDS‐polyacrylamide gel electrophoresis, and characterized them under a polarization microscope.
Calcium uptake by tonoplast and plasma membrane vesicles from spinach leaves
Authors: Malatialy L. , Greppin H. , Penel C.
Published: July 2002

Tonoplast‐ and plasma membrane‐enriched fractions were simultaneously prepared from the same crude membrane suspension from spinach leaves by free‐flow electrophoresis. They were used for in vitro ATP‐dependent Ca2+ ‐uptake studies. The plasma membrane‐enriched fraction accumulated Ca2+ at a rather slow rate by a mechanism which was inhibited by orthovanadate, insensitive to nitrate and activated by calmodulin. The tonoplast‐enriched fraction accumulated Ca2+ at a high rate for 5 min, then the accumulated Ca2+ progressively leaked out of the vesicles. The tonoplast transport system was partly inhibited by nitrate, but also by vanadate, completely abolished by the protonophore CCCP and activated by calmodulin and the Ca2+ antagonist verapamil.
Correlation among the proton charges and molecular masses of myosin subunits
Authors: Béchet Jean-Jacques , d'Albis Anne
Published: July 2002

The molecular masses and isoelectric points of myosin light and heavy chains were calculated from their known primary sequences and their respective distribution in a two‐dimensional graph is displayed. Implications for the electrophoretic study of myosin subunits are inferred from this analysis.