bluuebunny/crossref_metadata_2025 huggingface dataset:
Partial purification and determination of molecular weights of glyoxalases by filtration on dextran gel
Authors: Jerzykowski Tadeusz , Matuszewski Wojciech , Winter Romana
Published: July 2002

Glyoxalase I and glyoxalase II (EC. 4.4.1.5 and EC 3.1.2.6) were separated by gel filtration on Sephadex G‐75 and G‐100. This simple procedure permitted also the partial purification of glyoxalase II. The purification coefficient in a single run from supernatant from beef liver was about 1 : 30 compared with 1 : 15 after the fifth step of purification with classical methods.
Glycyl‐tRNA synthetase from rat liver: The role of tRNA in formation of glycylhydroxamates
Authors: Favorova O. , Kisselev L.
Published: July 2002

One glycyl‐tRNA synthetase from rat liver does not form glycylhydroxamates in the absence of tRNA. Glycyl‐hydroxamate accumulated during the incubation of enzyme preparation with glycine, ATP, tRNA and NH2OH is formed by non‐enzymatic hydroxylaminolysis of glycyl‐tRNA.
An initiation factor causing dissociation of E. coli ribosomes
Authors: Albrecht J. , Stap F. , Voorma H.O. , Van Knippenberg P.H. , Bosch L.
Published: July 2002

Purification of crude initiation factors, essential for polypeptide synthesis in cell‐free systems of E. coli, yielded a fraction DF which causes dissociation of 70 S ribosomes. Its stoichiometric action on 70 S ribosomes is antagonized by increasing Mg2+ concentrations but not by the addition of 30 S and 50 S subunits washed with high salt concentration. GTP did not stimulate this dissociating action. 2 μg of our most purified preparation caused 100% dissociation of 100 μg of 70 S ribosomes without added GTP. DF‐induced dissociation is a very rapid process at 37°C and is temperature‐dependent in the range of 0°–37°C. DF, which is thermolabile factor, is much less or not effective with complexed 70 S ribosomes bearing peptidyl‐tRNA and mRNA.
Participation du cytochrome P450 dans l'oxydation des alcanes chez Candida tropicalis
Authors: Gallo M. , Bertrand J.C. , Azoulay E.
Published: July 2002

Candida tropicalis strain 101 possesses a hydroxylase system when grown on tetradecane as the carbon source which is active towards hydrocarbons and fatty acids. This system including cytochrome P
450 and NADPH‐cytochrome c reductase has been localized exclusively in the microsomal fraction.
Inhibition competitive de la Phé:tRNA ligase des embryons de ble par l'acide aurine tricarboxylique
Authors: Carias J.R. , Julien R.
Published: July 2002

Aurin tricarboxylic acid (A.T.A.), an inhibitor of protein biosynthesis (initiation and elongation steps), acts also as a competitive inhibitor of phenylalanine, in the ATP‐PPi exchange and tRNAPhe aminoacylation reactions catalysed by cytoplasmic wheat germ phenylalanine:tRNA ligase.
Immunological determination of (1 → 3),(1 → 4)‐β‐D‐glucan endohydrolase development in germinating barley (Hordeum vulgare)
Authors: Stuart I.Montgomery , Fincher Geoffrey B.
Published: July 2002

Proteins in homogenates of germinating barley were separated by sodium dodecyl sulphate—polyacrylamide gel electrophoresis and, following electrophoretic transfer to nitrocellulose paper, the two (1 → 3), (1 → 4)‐β‐D‐glucan 4‐glucanohydrolases (EC 3.2.1.73) were individually quantitated in the 0–10 pmol range using crossreactive antibodies raised against either enzyme. The two enzymes develop in approximately equimolar proportions during the germination process.
A possible novel interaction between the 3′‐end of 18 S ribosomal RNA and the 5'‐leader sequence of many eukaryotic messenger RNAs
Authors: Sargan David R. , Gregory Stephen P. , Butterworth Peter H.W.
Published: July 2002

A novel interaction between the 5′‐untranslated region of eukaryotic messenger RNAs and non‐contiguous sequences in the 18 S ribosomal RNA is proposed. The small ribosomal RNA contains, at its 3′‐terminus, a heavily conserved hairpin structure. It is suggested that mRNA 5′‐leader sequence stabilises this structure by interacting with other conserved nucleotides which flank it. Sequences closely related to the required sequence (A—U—C—C—A—C—C) occur quite commonly in eukaryotic mRNAs and are often found immediately upstream from the AUG‐codon. This interaction may have a role in the events which lead up to the initiation of protein synthesis.
Distribution of calmodulin within wheat leaf cells
Authors: Muto Shoshi
Published: July 2002

Subcellular distribution of calmodulin within wheat leaf cells was measured by a enzymatic method (activation of calmodulin‐deficient NAD kinase from pea seedlings) or a radioimmunoassay. Most of cellular calmodulin was localized in cytosol fraction (89–93%), and remainders were in the mitochondrial (5–9%), the chloroplast (1–2%) and the microsomal (< 1%) fractions. Calmodulin concentration in the chloroplast was sufficient to saturate NAD kinase which was localized mostly in the chloroplast. Therefore, the light‐induced Ca2+ transport in intact wheat chloroplasts (Muto, S. FEBS Lett. (1982) 139, 250–254] may play an important role in regulation of chloroplast NAD kinase activity.
Replicon fusion in mouse cells is very sensitive to DNA damage induced by N‐methyl‐N′‐nitro‐N‐nitrosoguanidine
Authors: Wintersberger Erhard , Mudrak Ingrid
Published: July 2002

Incubation of mouse cells with N‐methyl‐N′‐nitro‐N‐nitrosoguanidine causes a strong inhibition of DNA replication the extent of which varies with the cell line used. Analysis of the products synthesized in drug‐treated cells indicates a particularly severe effect on the joining of replicons while other steps in DNA synthesis like initiation and chain elongation are much less affected. The data indicate that replicon fusion may be extremely sensitive to changes in the topology of DNA induced by the introduction of rare single‐strand breaks during repair of N‐methylated purines produced by incubation of cells with small amounts of the methylating agent
Methylation of elongation factor 1α in mouse 3T3B and 3T3BSV40 cells
Authors: Coppard N.J. , Clark B.F.C. , Cramer F.
Published: July 2002

Two‐dimensional gel electrophoretic (NEPHGE) analysis of proteins from mouse 3T3B and 3T3B/SV40 cells labelled with [methyl‐3H]methionine in the presence of cycloheximide have revealed that the elongation factor 1α (EF‐1α) in these cells is methylated and that the extent of methylation is higher in the SV40 transformed cell type. It is suggested that methylation may account for differences in growth properties for the different cell types.
Interaction between hysteretic regulation and redox modulation of glucose‐6‐phosphate dehydrogenase from Anacystic nidulans
Authors: Udvardy János , Juhász András , Farkas Gábor L.
Published: July 2002

The glucose‐6‐phosphate dehydrogenase (G6PDH) of cyanobacteria is a hysteretic enzyme which is also subject to redox modulation [FEBS Lett. 126 (1981) 85–88]. We have found that the hysteretic and redox properties of G6PDH exhibit specific interactions: (1) The hysteretic forms of G6PDH (‘hypoactive’ ⇌ ‘hyperactive’), obtained at pH 7.5 and 6.5, respectively, differ in their redox properties. The ‘hypoactive’ form is easily activated by oxidation whereas the ‘hyperactive’ form is easily deactivated by reduction. (2) At low G6P concentrations (1 mM) only the oxidized form of G6PDH has significant activity. An increase in G6P level diminishes the difference between the activity of oxidized and reduced G6PDH forms.
Identification of the proteins interacting with tRNA in rat liver small ribosomal subunits
Authors: Reboud A.M. , Dubost S. , Reboud J.P.
Published: July 2002

Irradiation at 254 nm of the rat liver 40 S ribosomal subunit—poly(U)—Phe[32P]tRNA complex induced a covalent linkage of tRNA with a limited number of ribosomal proteins. After RNA hydrolysis, S10 was found to be the protein most highly labeled by radioactive nucleotides. Some radioactivity was also associated with protein S6‐6a, S3a, S2 and S13–15.
Relaxed mutants of Escherichia coli RNA polymerase
Authors: Nene Vishvanath , Glass Robert E.
Published: July 2002

Unusual guanosine nucleotides synthesised during amino acid or energy source starvation are thought to be the effectors of the stringent response. In vitro experiments suggest that the magic spot compounds alter transcription specificity of RNA polymerase by binding to the enzyme. However, there is no good in vivo evidence for such an interaction. We define sites on the β‐subunit of RNA polymerase which, when altered, yield E. coli mutants apparently insensitive to the presence of ppGpp.
Pyrophosphate: fructose 6‐phosphate phosphotransferase in germinating castor bean seedlings
Authors: Kruger Nicholas J. , Kombrink Erich , Beevers Harry
Published: July 2002

The distribution of enzymes interconverting fructose 6‐phosphate and fructose 1,6‐bisphosphate has been studied in a range of tissues from castor bean seedlings. In each tissue the activity of PPi:fructose 6‐phosphate phosphotransferase was greater than phosphofructokinase and substantial compared with fructose 1,6‐bisphosphatase. PPi:fructose 6‐phosphate phosphotransferase in endosperm is apparently confined to the cytoplasm. The role of this latter enzyme in vivo is discussed.
The role of lipid peroxidation in the induction of cation transport in rat liver mitochondria
Authors: Marshansky V.N. , Novgorodov S.A. , Yaguzhinsky L.S.
Published: July 2002

Lipid peroxidation in mitochondria induced by Fe2+ in the presence of ascorbate or by cumene hydroperoxide in the presence of phosphate results in a drop of the membrane potential and in K+ efflux. The inhibitors of ATP‐synthetase (oligomycin and dicyclohexylcarbodiimide (DCCD)) are capable of preventing lipid peroxidation, stabilizing the membrane potential and inhibiting potassium efflux. The same effects are observed in the presence of ionol or α‐tocopherol. In contrast to antioxidant protection the effects of oligomycin and DCCD are reversed by the uncoupler (FCCP). The functional link between non‐enzymatic lipid peroxidation, proton conduction through Fo component of ATP‐synthetase and induced cation transport is suggested.
Mechanisms of auxin‐induced plant cell elongation
Authors: Brummer Benno , Parish Roger W.
Published: July 2002

An alternative to the wall acidification theory of auxin‐induced elongation growth is presented. A central effect of auxin is postulated to be cytoplasmic acidification, possibly via an increase in the levels of cytoplasmic Ca2+. Activation of the outwardly directed electrogenic proton pump resulting from acidification would lead to changes in Δψ and transmembrane ion gradients. We argue that these changes, rather than wall acidification, are involved in elongation growth.
Bacillus cereus 569/H β‐lactamase I: Cloning in Escherichia coli and signal sequence determination
Authors: Mézes Peter S.F. , Yang Yue Qin , Hussain Musaddeq , Oliver Lampen J.
Published: July 2002

The gene, penPC, for β‐lactamase I of Bacillus cereus 569/H has been cloned and its expression studied in Escherichia coli. The protein product from the in vitro translation of penPC was shown by gel electrophoresis to have an M
r of 36 000 which is larger than the in vivo products fround in B. cereus and E. coli. The DNA sequence of the signal region was determined. It revealed that the smallest known mature form present in B. cereus culture fluids is preceded by 45–48 amino acids in pre‐β‐lactamase I, considering that there are 3 initiation codons in the same reading frame, one or more of which might be initiating translation. Unlike the Bacillus licheniformis 749/C β‐lactamase, which has a membrane‐bound thioether lipoprotein form, the single Cys residue in the B. cereus β‐lactamase I signal sequence is unmodified and a single processed form of the enzyme is present in E. coli cells carrying pen PC
The translation of the messenger for the poly(A)‐binding protein‐associated with translated mRNA is suppressed
Authors: Akhayat Omar , Vincent Alain , Goldenberg Samuel , Person Anne , Scherrer Klaus
Published: July 2002

In vivo protein synthesis in duck erythroblasts was compared to in vitro translation of polyribosomal and free cytoplasmic mRNA. The in vivo study showed the absence of de novo synthesis of the M
r 73 000 poly(A)‐binding protein found associated with all polyribosomal mRNA. In vitro translation demonstrated that the mRNA for this protein is absent from the polyribosomal mRNA fraction but constitutes a medium frequency messenger among the repressed free mRNA. This result confirms the existence of a qualitative translational control in terminal differentiating duck erythroblasts leading eventually to the arrest of the protein synthesizing machinery.
Specific phosphorylation of a protein in calcium accumulating endoplasmic reticulum from rat parotid glands following stimulation by agonists involving cAMP as second messenger
Authors: Plewe Gerd , Jahn Reinhard , Immelmann Andreas , Bode Christa , Söling Hans-Dieter
Published: July 2002

Stimulation of secretion in exocrine cells by agonists involving cAMP as second messenger is associated with the phosphorylation of a specific membrane‐associated 22.4‐kDa protein (protein III) (Jahn). Here it is shown by subcellular fractionation of rat parotid gland lobules that protein III is associated with the endoplasmic reticulum. The submicrosomal fractions containing protein III, also contain the ATP‐dependent microsomal calcium pump activity. Protein III in microsomal subfractions can be phosphorylated in vitro with catalytic subunit from cAMP‐dependent protein kinase. Phosphorylated protein III contains exclusively P‐serine. Protein III can be removed from ER‐membranes with acid chloroform—methanol or Triton X‐114, but not by high salt wash indicating that it is tightly associated with the membranes. Protein III is smaller than phospholamban and, in contrast to phospholamban, resistant to heating in SDS. A relationship between phosphorylation of protein III and microsomal calcium sequestration is discussed.
An amino acid substitution that blocks the deacylation step in the enzyme mechanism of penicillin‐binding protein 5 of Escherichia coli
Authors: Broome-Smith Jenny , Spratt Brian G.
Published: July 2002

A mutant of Escherichia coli has been described that produces an altered form of penicillin‐binding protein 5 which still binds penicillin but is unable to catalyse the release of the bound penicilloyl moiety. We show that the mutation is caused by a single nucleotide transition that results in a change from glycine at residue 105 of the wild‐type sequence of penicillin‐binding protein 5 to aspartate in the mutant.
Hydrophobic labeling of the membrane binding domain of acetylcholinesterase from Torpedo marmorata
Authors: Stieger S. , Brodbeck U. , Reber B. , Brunner J.
Published: July 2002

Membrane‐bound acetylcholinesterase (AChE) from the electric organ of Torpedo marmorata was labeled with the hydrophobic photoactivatable reagent 3‐trifluoromethyl‐3‐(m‐[125I]iodophenyl)diazirine ([125I]TID). Labeling with [125I]TID was restricted to the membranous polypeptide segment of AChE as shown upon conversion of the amphiphilic form to the hydrophilic one by limited digestion with proteinase K. The labeled membranous segment, which has an M
r of approx. 3000 was isolated by gel filtration on Sephadex LH‐60 in ethanol/formic acid.
HMG‐like proteins of Physarum polycephalum: Association with the transcriptionally active chromatin
Authors: Czupryn Marta , Toczko Kazimierz
Published: July 2002

HMG‐like proteins were isolated from nuclei of the acellular slime mold Physarum polycephalum. These proteins represented 0.6 ± 0.2% of nuclear proteins and approx. 6% of DNA by weight. Polyacrylamide gel electrphoresis demonstrated the presence of 3 major proteins, designated HMG‐1P, HMG‐2P and HMG‐14/17P. None of these proteins comigrated with any of the calf thymus HMGs. Physarum HMG‐like proteins were found to be preferentially associated with transcriptionally active chromatin fraction, as well as being released from nuclei by light DNase I digestion. In contrast, the content of histone H1 was significantly lower in active than in inactive chromatin fraction.
A cross‐linking mechanism for the heparin‐enhanced antithrombin—thrombin reaction?
Authors: Chang Jui-Yoa
Published: July 2002

Emerging evidence suggests that a simple cross‐linking reaction through ionic interaction may be the primary cause of the heparin‐accelerated antithrombin inhibition of α‐thrombin.
Determination of the functional molecular size of vasopressin isoreceptors
Authors: Crause P. , Boer R. , Fahrenholz F.
Published: July 2002

The molecular size of vasopressin receptors in the intact membrane‐bound state was determined by radiation inactivation (target size analysis). For the V1 receptor in rat liver a molecular size of (76 ± 8) kDa was determined. For the V2, receptor in rat kidney and bovine kidney molecular sizes of (95 ± 4) and (108 ± 11) kDa were found. Statistical analysis gave evidence for size differences between rat liver and rat kidney receptors or differences between rat liver and bovine kidney receptors, but not between kidney receptors from different species. The results suggest that V1 and V2 receptors can be distinguished by functional properties as well as by their size.
Fluidizing effect of endogenous ubiquinone in bovine heart mitochondrial membranes
Authors: Fato Romana , Bertoli Enrico , Castelli Giovanna Parenti , Lenaz Giorgio
Published: July 2002

Extraction of endogenous ubiquinone from lyophilized beef heart mitochondria results in increases of both the order parameter of the spin label 5‐NS and of the rotational correlation time of 16‐NS; reconstitution with the pentane extract results in restoration of the original spectral parameters. On the other hand, addition of purified ubiquinone homologs restores the original spectra only in the case of 16‐NS, whereas the order parameter of 5‐NS is restored by addition of mixed phospholipids. The same amounts of ubiquinone homologs incorporated in mixed phospholipid vesicles induce much lower effects. It is suggested that ubiquinone in mitochondria is intercalated with the lipid chains of the membrane in such a way to perturb the fluidity of the hydrophobic core.
Solubilization and reconstitution of the Na+‐motive NADH oxidase activity from the marine bacterium Vibrio alginolyticus
Authors: Tokuda Hajime
Published: July 2002

The Na+‐motive NADH oxidase activity from Vibrio alginolyticus was extracted with octylglucoside and reconstituted into liposomes by dilution. On the addition of NADH, the reconstituted proteoliposomes generated Δψ (inside positive) and ΔpH (inside alkaline) in the presence of a proton conductor CCCP, and accumulated Na+ in the presence of valinomycin. These results indicate that the NADH oxidase activity, reconstituted in opposite orientation, leads to the generation of an eletrochemical potential of Na+ by the influx of Na+.
Glyceraldehyde, but not cyclic AMP‐stimulated insulin release is preceded by a rise in cytosolic free Ca2+
Authors: Wollheim Claes B. , Ullrich Susanne , Pozzan Tullio
Published: July 2002

The dynamics of changes in membrane potential, cytosolic free Ca2+ [Ca2+]i and immunoreactive insulin release were assessed in RINm5F cells. Membrane depolarization and a rise in [Ca2+]i preceded the stimulation of insulin release by D‐glyceraldehyde. Forskolin, which raised the cellular cyclic AMP levels, stimulated insulin release without changing membrane potential or [Ca2+]i. It is concluded that cyclic AMP acts on insulin release not by mobilizing Ca2+ but by another, as yet undefined, mechanism.
ADP‐ribosyltransferase from beef liver which ADP‐ribosylates elongation factor‐2
Authors: Iglewski Wallace J. , Lee Herbert , Muller Patrick
Published: July 2002

Fragment A of diphtheria toxin and Pseudomonas toxin A intoxicate cells by ADP‐ribosylating the diphthamide residue of elongation factor‐2 (EF‐2) resulting in an inhibition of protein synthesis [1–3]. A cellular enzyme from polyoma virus transformed baby hamster kidney (pyBHK) cells ADP‐ribosylates EF‐2 in an identical manner [4]. Here we describe a similar cellular enzyme from beef liver which transfers [adenosine‐14C]ADP‐ribose from NAD to EF‐2. The 14C‐label can be removed from the EF‐2 by snake venom phosphodiesterase as a soluble product which comigrates with AMP on TLC plates, indicating the 14C‐label is present on EF‐2 as monomeric units of ADP‐ribose. Furthermore, the forward transferase reaction catalyzed by the beef liver ADP‐ribosyltransferase is reversible by excess diphtheria toxin fragment A, with the formation of 14C‐labeled NAD, indicating that both transferases ADP‐ribosylate the same site on the diphthamide residue of EF‐2. Thus, beef liver and pyBHK mono(ADP‐ribosyl) transferases both modify the diphthamide residue of EF‐2, in a manner identical to diphtheria toxin fragment A and Pseudomonas toxin A. These results suggest the cellular enzyme is probably ubiquitous among eukaryotic cells.
Functional and structural analysis of acetylcholine receptor‐rich membranes after negative staining
Authors: Reinhardt Sigrid , Schmiady Hardi , Tesche Bernd , Hucho Ferdinand
Published: July 2002

Phosphotungstate (pH 7.4) used for negative staining of membranes from Torpedo electric tissue rich in acetylcholine receptor does not affect binding properties and cation permeability of the receptor and its ion channel. Uranyl salts, frequently used for negative staining, precipitate the receptor‐rich membranes making measurements of ligand binding and ion‐permeability regulation impossible. The gross ultrastructure in the two stains is not significantly different, but for future high‐resolution electron microscopy aiming at visualizing structural details of functional receptor molecules it is necessary to resort to a stain preserving native and active receptor. Uranyl salts are not applicable for this purpose. The electron micrographs obtained with phosphotungstate reveal two distinct structures in the receptor‐rich membrane: a closed ring (‘doughnut’) and an open ring (‘horseshoe’), with a ratio of abundance of about 3:2.
Isolation and characterization of human blood platelet gelsolin
Authors: Olomucki A. , Hue C. , Lefébure F. , Coué M.
Published: July 2002

A 90‐kDa protein‐actin stable complex was purified from blood platelets by a short and efficient procedure giving at the same time actin used in polymerization assays. 90‐kDa protein free of actin was prepared from the complex by 8 M ureau treatment and renaturation. By its molecular mass, immunological cross‐reactivity with macrophage gelsolin and its effect on G‐ and F‐actin the 90‐kDa protein appears as the platelet gelsolin.
Inhibition of testosterone biosynthesis by ethanol: relation to the pregnenolone‐to‐testosterone pathway
Authors: Eriksson C.J.P. , Widenius T.V. , Leinonen P. , Härkönei M. , Ylikahri R.H.
Published: July 2002

The concentrations of metabolites in the pregnenolone → testosterone pathway were determined in freezestopped testes in control rats and during ethanol intoxication (2 h after injection of 1.5 body wt). Ethanol lowered the mean testicular concentrations of testosterone (by 63–74%), androstenedione (49–81%), 17‐hydroxyprogesterone (60–76%), progesterone (29–67%) and pregnenolone (12–25%). 4‐Methylpyrazole had no effect on the ethanol‐induced changes. The present results reveal no inhibition at the 17‐hydroxyprogesterone → androstenedione → testosterone steps, but do not exclude inhibition before the step yielding pregnenolone and at the pregnenolone → progesterone → 17‐hydroxyprogesterone steps.
NMR studies on oligo‐deoxyribonucleotides containing N6‐methyladenine
Authors: Fazakerley G.Victor , Téoule Robert , Guy André , Guschlbauer Wilhelm
Published: July 2002

500 MHz 1H‐NMR and NOE measurements of d(GGATCC) and d(GGm6ATCC) show that both oligo‐nucleotides assume a B‐DNA conformation at low temperature. Around the melting temperature, however, the single and double strands of the N‐methylated form are in slow exchange on the NMR time scale. The preferred conformation of the adenine methyl group, cis to N1, retards base pairing and also destabilizes the double helix.
High tyrosine protein kinase activities in soluble and particulate fractions in bone marrow cells
Authors: Nakamura Shun-ichi , Takeuchi Fumito , Kondo Hiroki , Yamamura Hirohei
Published: July 2002

High tyrosine protein kinase activities were detected in soluble and particulate forms from bone marrow cells using synthetic peptide (Glu‐Asp‐Ala‐Glu‐Tyr‐Ala‐Ala‐Arg‐Arg‐Arg‐Gly) as a substrate. Total activity of tyrosine protein kinase was 2.4 times higher in the soluble fraction but the specific activity was slightly higher in the particulate one. Mg2+ or Mn2+ requirements of these two enzymes for maximal activity were quite different from each other. Physiological significance of these two forms of enzymes is briefly discussed.
Novel heparin‐activated protein kinase activity in rabbit skeletal muscle
Authors: Ahmad Zafeer , DePaoli-Roach Anna A. , Roach Peter J.
Published: July 2002

A heparin‐activated protein kinase has been identified in rabbit skeletal muscle. The enzyme, which had a native molecular mass of 70 kDa as judged by gel filtration, was stimulated 3‐ to 5‐fold by heparin, halfmaximally at 3 heparin. The stimulation by heparin was not reproduced by other polyanions such as polyaspartate and polyglutamate. The protein kinase was detected by its ability to phosphorylate glycogen synthase; it was ineffective in phosphorylating caseins, phosvitin, histone, or phosphorylase. Glycogen synthase was phosphorylated to a stoichiometry of 0.7–0.8 phosphates/subunit, exclusively at serine residues located in the COOH‐terminal CNBr‐fragment of the subunit, with a corresponding reduction in the ‐/+ glucose‐6P activity ratio from 0.96 to 0.43. The activity of the protein kinase was unaffected by the presence of Ca2+ and/or phospholipid, cyclic AMP or heat‐stable inhibitor protein of cyclic AMP‐dependent protein kinase. The enzyme was inhibited about 60% by the presence of glycogen, half‐maximal effect at 25 . The heparin‐activated protein kinase is clearly distinguishable from other known glycogen synthase kinases.
Heteromeric nature of glucocorticoid receptor
Authors: Gehring Ulrich , Arndt Holger
Published: July 2002

The wild‐type and a mutant receptor of S49.1 lymphoma cells have been shown by photoaffinity labelling to contain steroid‐binding polypeptides of M
r 94 000 and 40 000, respectively. We investigated the molybdate‐stabilized forms of these receptors and obtained M
r 325 000 and 285 000, respectively, by gel filtration and sedimentation analysis. Mild chymotrypsin treatment of the large wild‐type receptor resulted in a form of about M
r 290 000 which contained a steroid‐binding polypeptide of M
r 40 000. The data suggest that the high‐M
r forms of glucocorticoid receptors are heteromeric in nature and contain one steroid‐binding polypeptide per complex
Magnetic circular dichroism of DCPIP‐oxidised Desulfovibrio vulgaris hydrogenase
Authors: Stephens P.J. , Devlin F. , McKenna M.C. , Morgan T.V. , Czechowski M. , DerVartanian D.V. , Peck H.D. , LeGall J.
Published: July 2002

Ni‐free hydrogenases commonly exhibit a gav
> 2 EPR spectrum in their most oxidised level. This spectrum has been linked with the active site and generally attributed to an oxidised HIPIP‐type, [4Fe‐4S]3+ cluster. We report the low temperature magnetic circular dichroism (MCD) spectrum of 2,6‐dichlorophenolindo‐phenol (DCPIP)‐oxidised Desulfovibrio vulgaris hydrogenase (DvH2ase), which exhibits an axial gav
> 2 EPR spectrum. Paramagnetic MCD is observed, which however is shown to arise from an EPR‐silent, para‐magnetic species with S >
. No evidence of paramagnetic MCD arising from the gav
> 2 EPR‐detectable species is obtained. We conclude that: (1) DCPIP‐oxidised DvH2ase does not contain either a [4Fe‐4S]3+ or an oxidised 3Fe cluster; (ii) an EPR‐silent species with MCD characteristics somewhat similar to those of the oxidised ‘P’ clusters of the dye‐oxidized iron‐molybdenum protein of nitrogenase is present in DCPIP‐oxidised DvH2ase; (iii) the MCD and EPR of the gav
> 2 species exhibit characteristics in common with those of the gav
> 2 species produced by Fe(CN)6
3− oxidation of the [4Fe‐4S]2+ cluster of the 7Fe ferredoxin I of Azotobacter vinelandii [(7Fe)FdI). We suggest that the gav
> 2 EPR signal of this and other hydrogenases arises from a species chemically analogous to that observed in (7Fe)FdI.
Limited proteolysis of porcine pancreatic lipase
Authors: Bousset-Risso M. , Bonicel J. , Rovery M.
Published: July 2002

Mild chymotrypsin digestion of native lipase (449 amino acids) preferentially cleaved the Phe 335‐Ala 336 bond. On SDS‐gel electrophoresis, 3 major bands were observed: band 1 (52 kDa) representing native lipase, bands 2 and 3 (40 and 12 kDa) representing the two lipase fragments A and B. Fragment A does not retain lipase activity but maintains its ability to adsorb to interfaces. Fragment B was identified with the lipase C‐terminal region (336–449). It does not exhibit any activity towards tributyrylglycerol emulsions and any ability to adsorb to interfaces.
Study of the peptidasic site of cholinesterase: preliminary results
Authors: Chatonnet Arnaud , Masson Patrick
Published: July 2002

The peptidasic site of highly purified human plasma cholinesterase was investigated using active‐site‐directed inhibitors. Peptidase activity was assayed taking substance P as substrate. Inhibition by organophosphates indicated that the peptidasic site contained an active serine. The presence of essential histidine residues associated with serine was revealed by histidine modifications. Carboxyl group reagents showed that the active centre contained carboxyl groups in a non‐polar environment. The removal of sialic acids did not alter peptidase activity. The peptidasic site of cholinesterase shared many properties with serine proteases sites and esteratic sites of cholinesterases. In addition, with the peptidasic site, as well as the esteratic site, there was always the possibility of ‘aging’ when inhibited by DFP or soman.
Monoclonal antibodies block the bromelain‐mediated release of human placental alkaline phosphatase from cultured cancer cells
Authors: Jemmerson Ronald , Millan Jose Luis , Klier F.George , Fishman William H.
Published: July 2002

Certain monoclonal antibodies (mAbs) to human placental alkaline phosphatase (PLAP) block bromelain cleavage of a 2‐kDa segment from each of the two polypeptide chains of PLAP. These mAbs also prevent the release of PLAP from cultured cancer cell surfaces by bromelain. Such proteolysis‐blocking mAbs serve as tools to specifically modify the molecular topography of cell surfaces by protease treatment.
X‐ray studies on triclinic crystals of fatty acid binding protein
Authors: Pähler Arno , Maslowska Maria , Parge Hans E. , Schneider Michael , Steifa Manfred , Saenger Wolfram , Keuper Hermann J.K. , Spener Friedrich
Published: July 2002

Fatty acid binding protein (pI 7.0) from bovine liver cytosol was crystallized using polyethylene glycol 4000 and 6000 as precipitating agents. The crystals are triclinic, space group P1. One molecule of 14 kDa occupies the unit cell with constants a = 33.5 Å, b = 39.4 Å, c = 30.6 Å, α = 113.6°, β = 113.8°, γ = 88.8°. Crystal diffraction extends to at least 2.25 Å resolution and the crystals are stable in the X‐ray beam for more than 450 h. One native data set to 2.5 Å resolution has been collected.
The chloroplast 32 kDa protein is synthesized on thylakoid‐bound ribosomes in Chlamydomonas reinhardtii
Authors: Herrin David , Michaels Allan
Published: July 2002

A cloned cpDNA fragment containing a portion of the gene for the 32–36 kDa thylakoid protein of Chlamydomonas (polypeptide D‐1) was isolated. Hybridization probing of RNA from soluble and membrane fractions of Chlamydomonas showed that the mRNA for D‐1 is bound to thylakoid membranes. Run‐off translation of thylakoid‐bound polysomes (rough thylakoids) with [35S]‐methionine yields polypeptide D‐1 as the major product. Peptide mapping with S. aureus V‐8 protease of D‐1 synthesized (1) in vivo, (2) in vitro by rough thylakoids and (3) in the reticulocyte lysate directed by non‐polyadenylated RNA showed that D‐1 is synthesized as a precursor in the reticulocyte lysate but as the mature polypeptide by rough thylakoids.
Polymerization of G‐actin by caldesmon
Authors: Gała̧zkiewicz Barbara , Mossakowska Małgorzata , Osińska Hanna , Da̧browska Renata
Published: July 2002

Electron microscopy of negatively stained samples indicates that caldesmon induces polymerization of Gactin into filaments. Polymerization takes place in a very low ionic strength solution and is accompanied by an increase of intensity of fluorescence of G‐actin labelled with N‐(1‐pyrenyl)iodoacetamide. The effect of caldesmon is abolished by calmodulin in the presence of Ca2+
Expression in E. coli of finger‐domain lacking tissue‐type plasminogen activator with high fibrin affinity
Authors: Kagitani Hitoshi , Tagawa Michito , Hatanaka Kiyomi , Ikari Takaomi , Saito Akemi , Bando Hiroshi , Okada Kiyotaka , Matsuo Osamu
Published: July 2002

Tissue‐type plasminogen activator
Finger‐domain
Fibrin affinity
Plasmid E. coli tac promoter/operator
Detroit 562 cell
Light‐induced activation of the rod phosphodiesterase leads to a rapid transient increase of near‐infrared light scattering
Authors: Paul Kamps K.M. , Reichert Johannes , Hofmann Klaus Peter
Published: July 2002

The so‐called AT‐signal described here is a transient light‐induced increase of the near‐infrared scattering from isolated bovine rod outer segments (ROS). Freshly prepared ROS are permeabilized with 0.01% Triton X‐100 immediately before measurement in the presence of 1 mM GTP. The signal amplitude is saturated when ~ 2 rhodopsin molecules out of 30 000 are photo‐excited. The signal recovers rapidly ( ~ 90 s) and can be repeated in a succession of flashes. The AT‐signal can be prevented by pre‐activation of the phosphodiesterase (PDE) enzyme cascade at various levels: either at the level of G‐protein, using ALF4
− in darkness or GTPγS plus light; or at the level of the PDE catalytic unit, using protamine as an activator. The light sensitivity and kinetics of the AT‐signal are similar to published parameters of PDE activation. These data suggest that light‐induced activation of the PDE is the key reaction for the generation of the signal. On the other hand, blocking of the catalytic cGMP binding site by isobutylmethylxanthine only slightly affects the signal. We propose that the AT‐signal reflects a structural change linked to the transient removal of the PDE inhibitory subunit from the catalytic unit.
Glucuronidation of bile acids in human liver, intestine and kidney
Authors: Parquet M. , Pessah M. , Sacquet E. , Salvat C. , Raizman A. , Infante R.
Published: July 2002

The activities of UDP‐glucuronyl transferase(s) in homogenates and microsomal preparations of human liver, kidney and intestine were tested with hyodeoxycholic acid (HDC). The various kinetic parameters of the UDC‐glucuronidation were determined from time course experiments. In both liver and kidney preparations, HDC underwent a very active metabolic transformation: liver Km
= 78 μM, Vmax
= 3.3 nmol·min −1mg −1 protein; kidney Km
= 186 μM, Vmax
= 9.9 nmol·min −1.mg −1 protein. To our knowledge this is the first observation of both an extensive and comparable bile acid glucuronidation occurring in renal and hepatic tissues.
The dark (oxidized) form of the light‐activatable NADP‐malate dehydrogenase from pea chloroplasts is catalytically active in the presence of guanidine‐HCl
Authors: Scheibe R. , Fickenscher K.
Published: July 2002

The dark form of NADP‐malate dehydrogenase from pea leaves which has been shown to contain one disulfide bridge per subunit does not exhibit any catalytic activity in the absence of thiol reducing agents. Upon reduction of these disulfide bridges the enzyme becomes catalytically active. In this presentation, however, it is shown that the oxidized dark form of NADP‐malate dehydrogenase becames catalytically competent when assayed in the presence of 200–250 mM guanidine‐HCl. This guanidine‐dependent activity of the oxidized enzyme is characterized by higher apparent K
m values for the substratres as compared to the reduced enzyme, but is still specific for NADPH. Up to 25% of the V
maxOf the reduced enzyme was obtained for the oxidized guanidine‐activated NADP‐malate dehydrogenase. The results suggest that the reduction of the regulatory disulfide is not essential for catalytic activity.
Action of urokinase and trypsin on angiotensin, ACTH and the oxidized B chain of insulin
Authors: Yomtova V.M.
Published: July 2002

Trypsin
Urokinase specificity
HPLC enzyme kinetics
Angiotensin
ACTH
Oxidized insulin B chain
Identification of the 1H resonances of valine and leucine residues in dihydrofolate reductase by using a combination of selective deuteration and two‐dimensional correlation spectroscopy
Authors: Searle M.S. , Hammond S.J. , Birdsall B. , Roberts G.C.K. , Feeney J. , King R.W. , Griffiths D.V.
Published: July 2002

Lactobacillus casei dihydrofolate reductase (M
r, 18500) contains 16 valine and 14 leucine residues. By comparing the 2D COSY NMR spectra of normal and [γ‐2H6]valine enzyme we have been able to identify all 60 methyl resonances from these residues, and to connect the pairs arising from the same residue. This pairing of the methyl resonances was aided by the examination of the 2D RELAY spectrum which also allowed the CαH resonances (and hence the complete spin systems) of 14 of the valine residues to be identified. The combination of selective deuteration with 2D NMR techniques is shown to be a powerful general method for resolving 1H resonances in the complex spectra of proteins and for assigning them to amino‐acid type.
Cloning and DNA sequence of the 5'‐exonuclease gene of bacteriophage T5
Authors: Kaliman A.V. , Krutilina A.I. , Kryukov V.M. , Bayev A.A.
Published: July 2002

The nucleotide sequence of the BalI‐PstI fragment of T5 DNA, 1347 bp in length, coding for 5'‐exonuclease (D15 gene), has been determined. A coding region of the gene contains 873 bp and is preceded by a typical Shine‐Dalgarno sequence. The D15 gene belongs to a cluster, consisting of at least 3 genes, in which a termination codon of a preceding gene overlaps an initiation codon of the following one. The sequence contains an open reading frame for 291 amino acid residues. The molecular mass of the 5'‐exonuclease calculated from the predicted amino acid sequence is 33400 Da.
Complete nucleotide and deduced amino acid sequences of rat L‐type pyruvate kinase
Authors: Lone Yu-Chun , Simon Marie-Pierre , Kahn Axel , Marie Joëlle
Published: July 2002

Four overlapping cDNA clones for L‐type pyruvate kinase (PK‐L) were isolated from carbohydrate‐induced rat liver cDNA libraries. They contained all the coding sequence of the enzyme from the 7th codon and the entire 3'‐untranslated extension up to the poly(A) tail. The sequence of the first 7 codons and that of the 5'‐untranslated region were determined by primer extension. The analyzed PK‐L mRNA has 19 5'‐untranslated bases, 1629 coding bases and 1281 3'‐untranslated bases without the poly(A) tail; it corresponds to the heavier, 3.2 kb species of the L‐type mRNAs. The codons for the phosphorylatable site are, located at the 5'‐end of the messenger. The unusually long 3'‐untranslated extension contains a repetitive element complementary to the ‘brain‐specific’ identifier sequence described by Sutcliffe et al. [(1982) Proc. Natl. Acad. Sci. USA 79, 4942‐4946].
Maitotoxin stimulat es the formation of inositol phosphates in rat aortic myocytes
Authors: Berta Philippe , Sladeczek Fritz , Derancourt Jean , Durand Monique , Travo Pierre , Haiech Jacques
Published: July 2002

Maitotoxin is the most potent of the known marine toxins. The effect of maitotoxin on muscle contraction or hormone release was consistent with its action on the voltage‐sensitive channel. Indeed, calcium antagonists such as nifedipine or diltiazem were able to reverse the maitotoxin effects. Using smooth muscle cells, we have analysed the effects of maitotoxin on the inositol phosphate metabolism. Maitotoxin stimulates the inositol phosphate formation (5 ± 1.8‐fold in the presence of 10 mM LiCI). Moreover, this effect is not reversed, even partially by calcium antagonists, by α1 antagonists and is not mimicked by Ca2+ ionophores such as A23187 or calcium agonists such as Bay‐K 8644. The action of maitotoxin is further discussed in this paper.
The importance of individual nucleotides for the structure and function of rRNA molecules in E. coli
Authors: Meier N. , Göringer H.U. , Kleuvers B. , Scheibe U. , Eberle J. , Szymkowiak C. , Zacharias M. , Wagner R.
Published: July 2002

Methods of in vitro mutagenesis were employed to determine the importance of individual nucleotides within the ribosomal RNAs for the structure and function of E. coli ribosomes. A series of denned nucleotides in the genes for the 5 S and 16 S RNA were altered by transition and transversion mutations using either oligonucleotide‐directed or bisulfite‐catalyzed mutation procedures. Plasmids harbouring the mutated rRNA genes were expressed and the ribosomes containing such altered RNAs were investigated for impairments in RNA‐protein interaction assembly and mRNA‐coded tRNA binding.
The effect of the natural protein inhibitor on H+‐ATPase in hepatoma 22a mitochondria
Authors: Chernyak B.V. , Dukhovich V.F. , Khodjaev E.Yu.
Published: July 2002

The uncoupler‐induced inactivation of H+‐ATPase in hepatoma 22a and mouse liver mitochondria has been studied. The dependence of this process on ΔμH, and pH and ATP was established. The inactivated ATPase could be reactivated at alkaline pH values in the absence of ATP. These data indicate that the inactivation is apparently caused by the natural protein inhibitor. ATP‐ and pH‐dependent decrease of ATPase activity is also observed after Lubrol‐WX disruption of mitochondria. It can be proposed that practically all ATPase molecules in hepatoma mitochondria are in a catalytically active complex with the protein inhibitor. At low ΔμH this complex is inactivated via reversible pH‐dependent and irreversible ATP‐dependent rearrangements. The pH‐dependent rearrangement of the isolated protein inhibitor from hepatoma mitrochondria is also observed.
Biosynthesis and activity of DNA polymerase throughout the mitotic cycle of Physarum polycephalum
Authors: MacNicol A.M. , Banks G.R. , Cox R.A.
Published: July 2002

Measurements of DNA polymerase protein levels and polymerase activity through the naturally synchronous mitotic cycle of Physarum polycephalum show that active DNA polymerase‐α is synthesized throughout the G2 phase, in step with the profile of general protein biosynthesis. Three main components of P. polycephalum DNA polymerase of 200, 112 and 70 kDa were found to be immunologically related.
The effect of myosin light chain phosphorylation on the actin‐stimulated ATPase activity of myosin minifilaments
Authors: Levitsky Dmitri I. , Shuvalova Lyudmila A. , Poglazov Boris F.
Published: July 2002

It has been shown that in the absence of KCl, the actin‐stimulated Mg2+‐ATPase activity of rabbit skeletal myosin minifilaments with phosphorylated regulatory lights chains (LC2) exceeds 3–4‐fold that of myosin minifilaments with dephosphorylated LC2. Addition of KCl leads to a decrease in the difference between the two ATPase activities. LC2 phosphorylation considerably increases the rate of ATPase reaction and only slightly decreases the affinity of myosin minifilaments for F‐actin. It is suggested that the unusual effect of LC2 phosphorylation on the kinetic parameters of the actin‐stimulated ATPase reaction of myosin minifilaments can be accounted for by its influence on the interaction between myosin heads which results in the ordered self‐assembly of minifilaments.
Nebulin and titin expression in Duchenne muscular dystrophy appears normal
Authors: Fürst Dieter , Nave Rüdiger , Osborn Mary , Weber Klaus , Bardosi Attila , Archidiacono Nicoletta , Ferro Mariangela , Romano Valentino , Romeo Giovanni
Published: July 2002

Monoclonal antibodies which recognize different epitopes on either titin or nebulin show normal staining patterns on frozen sections of three muscle biopsies of Duchenne muscular dystrophy (DMD). Gel electrophoresis and immunoblotting performed on two of these muscle biopsies show the normal pattern of titin and nebulin polypeptides. Since the donor of one of these biopsies has a large deletion of the 5′‐region of the DMD gene, our results argue against the recent proposal that nebulin is the gene mutated in DMD.
Identification of Ca2+‐stimulated polyphosphoinositide phospholipase C in isolated plant plasma membranes
Authors: Melin Per-Martin , Sommarin Marianne , Sandelius Anna Stina , Jergil Bengt
Published: July 2002

A polyphosphoinositide phospholipase C has been identified in highly purified plasma membranes from shoots and roots of wheat seedlings. The enzyme preferentially hydrolysed phosphatidylinositol 4‐phosphate and phosphatidylinositol 4,5‐bisphosphate and had a different phosphoinositide substrate profile from soluble phospholipase C. The enzyme activity was lower in plasma membranes isolated from light‐grown shoots than from dark‐grown ones, whereas no differences in activity between plasma membranes from light‐ and dark‐grown roots were seen. Maximum activity of the membrane‐bound enzyme was observed around pH 6. It was activated by micromolar concentrations of Ca2+, but not by GTP or GTP analogues. The enzyme may participate in signal transduction over the plant plasma membrane.
Specific loss of LHCII phosphorylation in the Lemna mutant 1073 lacking the cytochrome b6/f complex
Authors: Gal Alma , Shahak Yosepha , Schuster Gadi , Ohad Itzhak
Published: July 2002

The thylakoid protein kinase(s) activity of Lemna perpusilla strain 6746 (wild type, WT) and the cytochrome (cyt) b
6/f‐less mutant 1073 was compared. Isolated thylakoids of both WT and mutant phosphorylated the polypeptides of 9–15, 29, 32–34 and 40–45 kDa. This kinase(s) activity was light‐dependent and could be elicited by addition of duroquinol in the dark. Thylakoids from both WT and mutant phosphorylated histone III‐S at comparable rates. However, the redox‐controlled phosphorylation of the LHCII polypeptide which could be demonstrated in vitro and in vivo in the WT thylakoids could not be detected under any experimental condition in the cyt b
6/f‐less thylakoids. Halogenated quinone analogues known to inhibit reduction of the cyt b
6/f complex inhibited both the electron flow and duroquinol‐activated LHCII phosphorylation, but had no effect on the duroquinol‐dependent phosphorylation of the other thylakoid polypeptides. These results indicate that the Lemna thylakoids contain at least two redox‐activated protein kinase(s). A quinone‐binding site is involved in the activation of the LHCII kinase system which is rendered inactive in the absence of the cyt b
6/f complex.
Perivenous localisation of Na‐dependent glutamate transport in perfused rat liver
Authors: Taylor P.M. , Rennie M.J.
Published: July 2002

Periportal and perivenous hepatocytes differ in their metabolism of blood glutamate (Glu). Uncertainty about the mechanisms of Glu blood‐liver exchange led us to characterise, by paired‐tracer dilution, a sodium‐dependent dicarboxylate transporter (resembling system X−ag) in sinusoidal membranes of perfused rat liver (V
max = 0.18 μmol Glu/g per min, K
m = 0.29 mM Glu). Tracer Glu transport was depressed 65% after necrosis of perivenous hepatocytes by acute CCl4 treatment, indicating that X−ag transporter activity is located mainly in these cells, the sites of glutamine (Gln) synthesis from glutamate and ammonia. Modulation of Glu transport may influence the extent of hepatic Gln release.