bluuebunny/crossref_metadata_2025 huggingface dataset:
EPR of a novel high‐spin component in activated hydrogenase from Desulfovibrio vulgaris (Hildenborough)
Authors: Hagen W.R. , van Berkel-Arts A. , Krüse-Wolters K.M. , Dunham W.R. , Veeger C.
Published: July 2002

The EPR of reoxidized hydrogenase from Desulfovibrio vulgaris (H.) has been reinvestigated. In contrast to other workers [(1984) Proc. Natl. Acad. Sci. USA 81, 3728‐3732] we find the axial signal with g = 2.06; 2.01 to be only a minor component of concentration 0.03 . In the spectrum of fully active reoxidized enzyme this signal is overshadowed by a rhombic signal (0.1 ) with g = 2.11; 2.05; 2.00 reminiscent of the only signal found for other oxidized bidirectional hydrogenases. In addition, a novel signal has been detected with g
eff = 5.0 which, under the assumptions that S = 2 and ¦Δm
s¦= 2, quantitates to roughly one . Ethylene glycol affects the relative intensity of the different signals. It is suggested that O2 sensitization parallels a spin‐state transition of an iron‐sulfur cluster.
Abolishment of inhibitory effects of 3'‐deazaadenosine on Superoxide generation of guinea pig phagocytes by pre‐exposure to phorbol myristate acetate
Authors: Yagawa Katsuro , Nakanishi Masayuki , Hayashi Shinichiro , Kaku Mariko , Ichinose Yukito , Itoh Tokuo , Tomoda Akio , Yoneyama Yoshimasa , Shigematsu Nobuaki
Published: July 2002

Superoxide generation
3'‐Deazaadenosine
Phorbol myristate acetate
Wheat germ agglutinin
Immune complex
Phagocyte
Microelectrode studies of D‐glucose‐ and K+‐induced changes in membrane potential of electrofused insulin‐producing cells
Authors: Berggren Per-Olof , Sohtell Morgan
Published: July 2002

In the present study electrical field‐induced fusion has been applied to both normal pancreatic islet cells isolated from obese hyperglycemie mice and clonal insulin‐producing cells (RINmSF) derived from a transplantable rat insulinoma. The fused cells were then punctured with microelectrodes to measure changes in membrane potential after exposure to stimulatory concentrations of D‐glucose or K+. Fused cells of normal islet cellular origin revealed a resting membrane potential of −60 mV and were depolarized by 24 or 27 mV after exposure to 11 mM D‐glucose or 30 mM K+. Although D‐glucose induced depolarization, it was not possible to establish the existence of an oscillating burst pattern superimposed by action potentials. The resting membrane potential of the fused RINm5F cells was also −60 mV and decreased to −30 mV after exposure to 30 mM K+. As judged from the membrane potential measurements, the reconstitution of the plasma membrane subsequent to electrical breakdown is essentially the same whether the giant cells originated from normal islet cells or RINm5F cells.
Stimulation of cyclic GMP synthesis in human cultured glomerular cells by atrial natriuretic peptide
Authors: Ardaillou Nicole , Nivez Marie-Paule , Ardaillou Raymond
Published: July 2002

Recently a stimulatory effect of atrial natriuretic peptide (ANP) on the particulate guanylate cyclase system has been reported in the glomeruli from different species. Using cultures of homogeneous human glomerular cell lines, we found that rat and human ANP stimulated markedly cGMP formation in epithelial cells with a threshold dose of 1 nM. A 20‐fold increase was obtained at 5 μM. Stimulation was also present but less substantial (2‐fold at 5 μM) in mesangial cells. cGMP was formed rapidly and released in the medium. ANP and sodium nitroprusside, an activator of soluble guanylate cyclase, had additive effects on cGMP formation. ANP did not inhibit cAMP formation in both cell lines. These results demonstrate that, at least in the human species, epithelial cells represent the main target of ANP in the glomerulus. Synthesis of cGMP in the glomerular epithelial cells in response to ANP also suggests that the excess of urinary cGMP produced by the kidney which is observed after ANP administration is of glomerular rather than of tubular origin.
The inhibitory effect of nitrite on the oxidase activity of cells of Paracoccus denitrificans
Authors: Kucera I. , Kozák L. , Dadák V.
Published: July 2002

It was observed with anaerobically grown cells of Paracoccus denitrificans which possess dissimilatory nitrite reductase that on increasing the flux of electrons into the terminal part of the respiratory chain by means of N,N,N',N‐tetramethyl‐p‐phenylenediamine (TMPD) plus ascorbate the sensitivity of the oxidase activity towards the inhibitory action of nitrite markedly increased. It was shown that under these conditions the reduction of nitrite gave rise to the formation of an agent strongly inhibiting the terminal oxidases, the presence of which was detected using aerobically grown cells or membrane vesicles derived from them. The strong inhibition of TMPD‐oxidase activity of membranes was transient in time and could also be brought about by addition of nitric oxide (NO) but not by nitrous oxide (N2O).
Turnover of rat liver ornithine transcarbamylase
Authors: Wallace Ruth , Knecht Erwin , Grisolía Santiago
Published: July 2002

The relative half‐life of ornithine transcarbamylase from rat liver has been determined using the double isotope technique and affinity chromatography. The calculated half‐life (6–9 days) is similar to that of mitochondria and of the other mitochondrial enzyme of the urea cycle, carbamoyl‐phosphate synthase. Therefore, both mitochondrial urea cycle enzymes are most probably degraded mainly via the lysosomal (autophagic) pathway of mitochondrial protein degradation.
Scorpion venom inhibits selectively Ca2+‐activated K+ channels in situ
Authors: Leneveu Estelle , Simonneau Michel
Published: July 2002

Using patch‐clamp techniques, a study was made of the component of Leiurus quinquestriatus scorpion venom which caused a blockade of one class of membrane potassium channels, the calcium activated potassium (BK) channels. This blockade was obtained on channels in their native lipidic environment and was specific for this class of channels as other types of potassium channels were not affected by this venom.
A new sensitive Edman‐type reagent: 4‐(N‐1‐dimethylaminonaphthalene‐5‐sulfonylamino)phenyl isothiocyanate
Authors: Jin Shan-Wei , Chen Gui-Xiang , Palacz Zbigniew , Wittmann-Liebold Brigitte
Published: July 2002

A new fluorescent PITC homologue Edman‐type reagent, 4‐(N‐1‐dimethylaminonaphthalene‐5‐sulfonylamino) phenyl isothiocyanate (DNSAPITC), was synthesized following a simple three‐step synthetic route. The reagent was crystallized and characterized by thin‐layer chromatography, IR and electron impact mass spectrometry. Reference DNSAPTH‐amino acid derivatives were prepared and a two‐dimensional chromatography system on micro‐polyamide sheets was developed for separating this mixture. On these sheets the sensitivity was 1–5 pmol, by exposure at 366 nm. Model peptides and proteins were subjected to Edman degradations with this new reagent. A similar coupling efficiency and repetitive degradation yield to those of PITC were found with this reagent. The advantages and limitations of this reagent for sensitive microsequencing are discussed.
Photoaffinity labelling of melanoma cell MSH receptors
Authors: Scimonelli Teresa , Eberle Alex N.
Published: July 2002

UV‐irradiation at 365 nm of cultured Cloudman S91 mouse melanoma cells in the presence of photoreactive α‐MSH analogues induced longlasting receptor stimulation as revealed by the ensuing activation of tyrosinase. Receptor labelling was more efficient with 4‐diazirinophenyl and 2‐nitro‐4‐azidophenyl photolabels than with 4‐azidophenyl, and was further increased when superpotent [Nle4,D‐Phe7]‐α‐MSH was used as ligand. Incubation of B16 melanoma cell membranes with mono‐iodinated [Nle4,D‐Phe7 Trp‐(Naps)9]‐α‐MSH followed by UV‐irradiation at 310–550 nm labelled a single band on SD‐PAGE with a molecular mass ⋍ 45 kDa. The displacement curve obtained in a competitive photolabelling experiment paralleled that of the binding assay, demonstrating that the labelling was specific.
The binding of glucosylceramidase to glucosylceramide is promoted by its activator protein
Authors: Vaccaro A.M. , Muscillo M. , Salvioli R. , Tatti M. , Gallozzi E. , Suzuki K.
Published: July 2002

A protein activator of glucosylceramidase (EC 3.2.1.45) has been previously identified by us in human placenta [(1985) Biochim. Biophys. Acta 836, 157–166]. In the present paper we report that its function in vitro is to stimulate the binding of the enzyme to its substrate, glucosylceramide. After the purification step which frees the enzyme of most of its activator protein (octyl‐Sepharose 4B chromatography), the capacity of glucosylceramidase to bind to the glucosylceramide micelles is dramatically decreased. The addition of the activator protein to the purified enzyme restores this binding.
Regional distribution of leukotriene and mono‐hydroxyeicosatetraenoic acid production in the rat brain Highest leukotriene C4 formation in the hypothalamus
Authors: Miyamoto Tsumoru , Lindgren Jan Åke , Hökfelt Tomas , Samuelsson Bengt
Published: July 2002

The regional distribution of ionophore A23187‐induced synthesis of leukotrienes and mono‐hydroxyeicosatetraenoic acids in the rat brain in vitro was investigated. Pronounced differences in leukotriene C4 formation were observed, with the highest synthetic capacity in the hypothalamus. The formation of leukotriene C4 was about 12‐times higher in the hypothalamus as compared to the cerebellum. This finding is in agreement with a possible neuroendocrine role for leukotriene C4. In contrast, the activity of leukotriene B4 synthesis was widely distributed without pronounced regional differences in the rat brain. Formation of 5‐, 9‐, 11‐, 12‐ and 15‐monohydroxyeicosatetraenoic acid was detected in all regions. The major lipoxygenase product in the hypothalamus and thalamus was 5‐hydroxyeicosatetraenoic acid, while other monohydroxyeicosatetraenoic acids predominated in the remaining regions tested.
Measurement of glucose and 2‐deoxy‐2‐[18F]fluoro‐D‐glucose transport and phosphorylation rates in myocardium using dual‐tracer kinetic experiments
Authors: Huang S.C. , Williams B.A. , Barrio J.R. , Krivokapich J. , Nissenson C. , Hoffman E.J. , Phelps M.E.
Published: July 2002

To examine the use of 2‐deoxy‐2‐[18F]fluoro‐D‐glucose (2‐FDG) as a glucose analog for measuring glucose utilization rate in myocardium, dual‐tracer kinetic experiments with 2‐FDG and 2‐[3H]glucose were performed in the perfused, isolated rabbit interventricular septum to measure simultaneously the transport and phosphorylation rates of glucose and 2‐FDG. Results of the present study indicated that, in the septum, (i) the transport rate constants of 2‐FDG and glucose were similar in magnitude, (ii) the phosphorylation rate constant for 2‐FDG was about 60% of that of glucose, (iii) hypoxia caused an increase in phosphorylation rates of glucose and 2‐FDG without affecting transport.
Nitrofuran inhibition of microsomal lipid peroxidation
Authors: Dubin M. , Grinblat L. , Villamil S.H.Fernandez , Stoppani A.O.M.
Published: July 2002

Two nitrofuran compounds, nifurtimox and nitrofurantoin, inhibited in a concentration‐dependent manner the NADPH‐, iron‐induced lipid peroxidation in rat liver microsomes, as shown by the decreased rate of MDA accumulation. Other nitro compounds (benznidazole and chloramphenicol) were relatively inactive. Nifurtimox inhibition affected polyenoic fatty acids and cytochrome P‐450 degradation that follows lipid peroxidation. The ascorbate‐ or tert‐butyl hydroperoxide‐dependent lipid peroxidations were much less inhibited than the NADPH‐dependent one. Nifurtimox and nitrofurantoin, but not benznidazole and chloramphenicol, strongly stimulated the microsomal NADPH‐oxidase activity, thus supporting electron diversion, as the main cause of the inhibition of peroxidation initiation.
A stopped‐flow study of the reaction of cytochrome c peroxidase with hydroperoxides
Authors: Balny Claude , Anni Helen , Yonetani Takashi
Published: July 2002

Transient kinetic measurements show that cytochrome c peroxidase reacts with excess of hydroperoxides to produce compound ES in two phases. The activation energies for the fast and slow phases are calculated to be 6.3 and 20.5 kcal·mol−1, respectively. The fast phase is assigned to the reaction of native active (pulsed) cytochrome c peroxidase with peroxides, whereas the slow phase is due to the presence of an inactive (aged, resting) enzyme. As the active species is exhausted, the equilibrium between the active and inactive enzymes is shifted by a slow conformational change to replenish the active enzyme. Since the rate‐limiting step of the reaction of the inactive enzyme with peroxides is the conformation change, the overall reaction rate is independent of the nature and concentration of peroxides.
Fibronectin‐binding 36 kDa protein in human fibroblasts
Authors: Salonen Eeva-Marjatta , Vaheri Antti
Published: July 2002

A 36 kDa fibronectin‐binding protein was identified from electrophoretically separated proteins of the deoxycholate‐soluble fraction of cultured fibroblasts by blotting with fibronectin and using poly‐ or monoclonal antibodies and immunoperoxidase staining to detect the bound fibronectin. The 36 kDa protein was purified by preparative electrophoresis and used to raise specific antibodies. Solid‐phase 36 kDa protein bound plasma and fibroblast fibronectins equally well. The 36 kDa protein is an amphipathic protein with pI 5.9. It is monomeric with a tendency to dimerize and appears to be distinct from the cell surface fibronectin receptors which interact with the Arg‐Gly‐Asp recognition site in the fibronectin molecule.
Deletion analysis of the c‐Ha‐ras oncogene promoter
Authors: Trimble William S. , Hozumi Nobumichi
Published: July 2002

Using transient assays of chloramphenicol acetyltransferase expression in CV‐1 cells, we have localized the human c‐Ha‐ras oncogene promoter to a 550 bp fragment located about 1 kb upstream from the ras coding sequence. Deletion analysis has revealed that a 100 nucleotide region 200 base pairs upstream of the putative initiation sites for transcription is essential for high levels of expression. Within this sequence lie two Spl binding sites and a consensus CCAAT box.
Functional expression of cloned cDNA encoding the α‐subunit of adenylate cyclase‐stimulating G‐protein
Authors: Nukada Toshihide , Mishina Masayoshi , Numa Shosaku
Published: July 2002

Cloned cDNA encoding the α‐subunit of the adenylate cyclase‐stimulating G‐protein (Gs), carried by a simian virus 40 vector, has been introduced into the cyc− variant of S49 lymphoma cells by electroporation. In contrast to untransfected cys− cells, clones transformed with the cDNA exhibit an increase in intracellular cyclic AMP concentration in response to a β‐adrenergic agonist.
Characterization of lactogen receptors in lactogenic hormone‐dependent and independent NB2 lymphoma cell lines
Authors: Ashkenazi A. , Cohen R. , Gertler A.
Published: July 2002

A comparative study of lactogen receptors in lactogenic hormone‐dependent (Nb2‐11C) and independent (Nb2‐SP) rat lymphoma cell lines revealed no significant differences in their binding and structural characteristics. The affinity for human growth hormone (hGH) and ovine prolactin is identical (K
d) = 1.3 × 10−10M). Exposure to hGH results in a rapid receptor down‐regulation. This process is faster in Nb2‐SP cells, however, similar receptor levels are reached within 3 h. Affinity labeling revealed 2 major binding species (Mr
75 000 and 29000) in both cell lines. These observations suggest that the spontaneous proliferation of Nb2‐SP cells probably results from an aberration in some post‐receptoral event of the mitogenic pathway.
The tissue form of chicken type VI collagen
Authors: Schreier Thomas , Winterhalter Kaspar H. , Trüeb Beat
Published: July 2002

We have purified intact type VI collagen from chicken gizzard. The protein was found to consist of a 130 kDa, a 140 kDa and a 180–200 kDa subunit. The 130 kDa and 140 kDa subunits were obtained in equimolar amounts and identified as the α2 (VI) and the α1 (VI) chains, respectively. The third subunit was usually obtained in the form of 3–4 closely related polypeptides, which may represent different processing or modification products of the α3 (VI) chain.
Porcine brain natriuretic peptide, another modulator of bovine adrenocortical steroidogenesis
Authors: Hashiguchi Takashi , Higuchi Kazumi , Ohashi Masao , Minamino Naoto , Kangawa Kenji , Matsuo Hisayuki , Nawata Hajime
Published: July 2002

Porcine brain natriuretic peptide (pBNP) significantly inhibited aldosterone production stimulated by an angiotensin II analog and ACTH‐stimulated cortisol secretion, together with simultaneously increasing the formation of cGMP in dispersed bovine adrenocortical cells. Receptors for pBNP were identified in bovine adrenal gland using an in vitro receptor autoradiographic technique and studies of 125I‐pBNP binding. In vitro receptor autoradiography demonstrated specific binding sites for 125I‐pBNP in bovine adrenal cortex. Complete displacement of 125I‐pBNP by unlabeled pBNP or human atrial natriuretic peptide (hANP) can take place at these sites. Analysis of 125I‐pBNP binding to bovine adrenocortical membrane fractions showed that the adrenal cortex had high‐affinity, low‐capacity pBNP‐binding sites, with a dissociation constant (K
d) of 2.32 ± 0.33 × 10−10 M (mean ± SE) and a maximal binding capacity (B
max) of 36.7 ± 1.6 fmol/mg protein. Moreover, the specific binding sites for 125I‐pBNP were completely displaced not only by unlabeled pBNP but also by unlabeled hANp. The hANP dose required for 50% inhibition of specific 125I‐pBNP binding was almost identical to that for pBNP (IC50 values for hANP and pBNP: 8.5 × 10−10 and 6.5 × 10−10 M, respectively). These results suggest that pBNP exerts a suppressive effect on bovine adrenocortical steroidogenesis via a receptor which may be shared with ANP.
Fc receptor‐mediated transcytosis of IgG‐coated liposomes across epithelial barriers
Authors: Patel Harish M. , Wild Arthur E.
Published: July 2002

Drug carriers such as liposomes are not readily transported across cellular barriers that constitute epithelia. However, certain epithelia (rabbit yolk sac endoderm and enterocytes of suckling rat gut proximal small intestine) are well known to transcytose maternal IgG by Fc receptor‐mediated endocytic events. We have shown that coating liposomes with appropriate IgG enhances their transport across these epithelia, as measured both by radioactivity indicative of liposomal membrane or entrapped 125I‐PVP and [3H]inulin, and by the hypoglycemic effect of entrapped insulin. It is suggested that these transported liposomes follow a pathway of transcytosis in clathrin‐coated vesicles, thus escaping lysosomal degradation.
Thyroxine and testosterone transcriptionally regulate renin gene expression in the submaxillary gland of normal and transgenic mice carrying extra copies of the Ren2 gene
Authors: Tronik Diana , Rougeon François
Published: July 2002

Expression of the mouse renin genes (Ren1 and Ren2) in the submaxillary gland of female mice has been analyzed following administration of thyroxine (T4) or dihydrotestosterone (DHT). Both hormones appear to act independently on mRNA accumulation which increases about 5 fold over basal level. In vitro transcription assays in isolated nuclei demonstrate that both hormones act at the transcriptional level. The effects of DHT and T4 were also analyzed in transgenic mice obtained by microinjection of the Ren2 gene. We show that T4 is as efficient as DHT in promoting renin mRNA accumulation in these transgenic animals, in spite of their low basal level of Ren2 mRNA. Structural comparison of the Ren1 and Ren2 promoters with those of other genes regulated by T4 shows the conservation of two discrete regions.
Pertussis toxin stimulates delayed‐onset, Ca2+‐dependent catecholamine release and the ADP‐ribosylation of a 40 kDa protein in bovine adrenal chromaffin cells
Authors: Brocklehurst Keith W. , Pollard Harvey B.
Published: July 2002

Pertussis toxin was found to stimulate catecholamine release from bovine adrenal chromaffin cells in a Ca2+ ‐dependent manner and in the absence of any stimulatory or inhibitory agonists for this cell. The release of catecholamine was associated with the ADP‐ribosylation of an approx. 40 kDa protein present in the total membrane fraction. These results are consistent with the existence of an exocytosis‐linked G‐protein.
A stretch‐activated anion channel in tobacco protoplasts
Authors: Falke Lee C. , Edwards Kathryn L. , Pickard Barbara G. , Misler Stanley
Published: July 2002

Stretch‐activated ion channels have been described in animal cells, where they might serve as mechanoreceptors, baroreceptors or osmoreceptors, as well as in yeast and bacteria, where osmoregulatory functions have been suggested. Here we report a large conductance, stretch‐activated, anion‐selective channel in protoplasts of a higher plant, tobacco, and discuss its possible role in osmoregulation.
Identification of a Glu > Lys substitution in the activation segment of human pepsinogen A‐3 and ‐5 isozymogens by peptide mapping using endoproteinase Lys‐C
Authors: Bank Ruud A. , Crusius Bart C. , Zwiers Toon , Meuwissen Stephan G.M. , Arwert Fre , Pronk Jan C.
Published: July 2002

The isozymogens PGA‐3 and PGA‐5 of human pepsinogen A were digested with endoproteinase Lys‐C. The peptides were separated by reverse‐phase HPLC. PGA‐5 showed a peak strongly absorbing at 254 nm absent in PGA‐3. Analysis of amino acid composition using the Pico‐Tag methodology combined with DABITC‐sequencing reveals the sequence Tyr‐Phe‐Pro‐Gln‐Trp‐Lys (peptide 37–43 of the activation segment). This confirms a study at the DNA level by our group [16] suggesting a Glu > Lys mutation at position 43 in the activation segment of PGA‐5. Furthermore, it is proposed that the number of genetic variants of PGA is higher than is actually seen by electrophoresis.
Inhibition of N‐acetylneuraminate lyase by N‐acetyl‐4‐oxo‐D‐neuraminic acid
Authors: Groß H.J. , Brossmer R.
Published: July 2002

We show that the 4‐oxo analogue of N‐acetyl‐D‐neuraminic acid strongly inhibits N‐acetylneuraminate lyase (NeuAc aldolase, EC 4.1.3.3) from Clostridum perfringens (K
i = 0.025 mM) and Escherichia coli (K
i = 0.15 mM). In each case the inhibition was competitive.
Localization of donor and acceptor sites of NADH dehydrogenase activities using inside‐out and right‐side‐out plasma membrane vesicles from plants
Authors: Askerlund Per , Larsson Christer , Widell Susanne
Published: July 2002

Inside‐out and right‐side‐out plasma membrane vesicles from sugar beet (Beta vulgaris L.) leaves, prepared by aqueous two‐phase partitioning, were used to localize donor and acceptor sites and to determine substrate affinities for plasma membrane‐bound NADH dehydrogenase activities. NADH‐ferricyanide and NADH‐cytochrome c reductase activities were approx. 30% latent with inside‐out vesicles and about 80% latent with right‐side‐out vesicles, indicating that both donor and acceptor sites for these activities are located on the cytoplasmic surface of the plasma membrane, and that a possible transplasma membrane electron transport would constitute only a minor proportion of the total activity.
Complete amino acid sequence of human placental 17β‐hydroxysteroid dehydrogenase deduced from cDNA
Authors: Peltoketo H. , Isomaa V. , Mäentausta O. , Vihko R.
Published: July 2002

CDNA clones for 17β‐hydroxysteroid dehydrogenase (17‐HSD; EC 1.1.1.62) were isolated from a placental λgt11 expression library using polyclonal antibodies against placental 17‐HSD. The largest cDNA contained 1325 nucleotides, consisting of a short 5′‐noncoding segment, a coding segment of 987 nucleotides terminated by a TAA codon, and a 329 nucleotide long 3′‐noncoding segment. The open reading frame encoded a polypeptide of 327 amino acid residues with a predicted M
r of 34853. The amino acid sequence of 23 N‐terminal amino acids determined from purified 17‐HSD agreed with the sequence deduced from cDNA. The deduced amino acid sequence also contained two peptides previously characterized from the proposed catalytic area of placental 17‐HSD.
Glycogen synthase from human and bovine polymorphonuclear leukocyte Immunochemical characterization and comparison to glycogen synthase from rat and rabbit muscle and liver cells
Authors: Rasmussen Lars Hvilsted , Juhl Henning , Esmann Viggo , Petersen Hans Uffe
Published: July 2002

Glycogen synthase from human and bovine polymorphonuclear leukocytes was purified to homogeneity. Rabbit antisera were raised against the two glycogen synthases and used for immunochemical analysis. Western blotting analysis showed that the subunit of glycogen synthase in crude homogenates of human and bovine leukocytes in both cases has an M
r of 85 000. The existence of a cross‐reactivity between the two enzymes and the corresponding antisera demonstrates immunological similarities between bovine and human leukocyte glycogen synthase. In addition, both antisera recognize glycogen synthase in crude cellular extracts from rabbit and rat liver and from skeletal muscle. Leukocyte glycogen synthase, therefore, cannot be classified as either muscle (M‐type) or liver (L‐type) glycogen synthase and our results do not support the proposed immunochemical distinction between M‐ and L‐type glycogen synthase.
Number of tRNA binding sites on 80 S ribosomes and their subunits
Authors: Rodnina M.V. , El'skaya A.V. , Semenkov Yu.P. , Kirillov S.V.
Published: July 2002

The ability of rabbit liver ribosomes and their subunits to form complexes with different forms of tRNAPhe (aminoacyl‐, peptidyl‐ and deacylated) was studied using the nitrocellulose membrane filtration technique. The 80 S ribosomes were shown to have two binding sites for aminoacyl‐ or peptidyl‐tRNA and three binding sites for deacylated tRNA. The number of tRNA binding sites on 80 S ribosomes or 40 S subunits is constant at different Mg2+ concentrations (5–20 mM). Double reciprocal or Scatchard plot analysis indicates that the binding of Ac‐Phe‐tRNAPhe to the ribosomal sites is a cooperative process. The third site on the 80 S ribosome is formed by its 60 S subunit, which was shown to have one codon‐independent binding site specific for deacylated tRNA.
Cloning and sequencing of spinach cDNA clones encoding the 20 kDa PS I polypeptide
Authors: Lagoutte Bernard
Published: July 2002

Apart from the 8 kDa subunit, which is of chloroplast origin, most of the small polypeptides of the PS I reaction center from higher plants are encoded in nuclear genes. We describe here the first nucleotide sequence of a nuclear component of this photosystem, the precursor of the 20 kDa protein. The deduced sequence of the large transit peptide (55–60 amino acids) is rich in serine/threonine residues and has a net positive charge, which are classical features of these precursors. The sequence itself is mainly hydrophilic, with no possibility of classical membrane‐spanning α‐helices; it exhibits an interesting stretch of five basic amino acids in close vicinity: Thr‐Arg‐Leu‐Arg‐Ser‐Lys‐Tyr‐Lys‐Ile‐Lys‐Tyr.
Lateral motion of fluorescent molecules embedded into cell membranes of clonal myogenic cells, L6, changes upon cell maturation
Authors: Kawasaki Yukishige , Wakayama Nobuyuki , Seto-Ohshima Akiko
Published: July 2002

The lateral motion of fluorescent molecules embedded into cell membranes of myogenic cell line, L6, was measured. The motion of S‐F‐ConA became faster at cell fusion stage, and became slower after fusion. On the other hand, the motion of lipid analog, F18, was not changed at cell fusion stage. However, after fusion when myotubes were formed, the motion of F18 became slower. At cell fusion stage, there was a large variation in the motion of S‐F‐ConA. This means that at this stage the properties of myoblasts change drastically and rapidly.
Polylysine activates and alters the divalent cation requirements of the insulin receptor protein tyrosine kinase
Authors: Rosen Ora M. , Lebwohl David E.
Published: July 2002

Protamine and poly(Lys) activate the protein tyrosine kinase of both the human placental insulin receptor and its purified recombinant cytoplasmic domain. Spermidine, poly(Arg) (average molecular mass 15 kDa), poly(Glu), Arg or Lys are not effective. Activation is stable, reversible, and optimal when the enzyme is preincubated with activator, divalent cation and ATP prior to the addition of exogenous protein substrates. The most striking feature of the activation is that it results in 20–30‐fold stimulation of the kinase in the presence of 0.2–0.4 mM Mn2+ and induces equivalent activity in the presence of Mg2+ alone (0.4–4.0 mM). The activated protein tyrosine kinase has a specific activity (0.25–0.5 μmol/mg protein) that approaches that of well characterized protein serine kinases.
Expression of K channels in Xenopus laevis oocytes injected with poly(A+) mRNA from the insulin‐secreting β‐cell line, HIT T15
Authors: Ashcroft Frances M. , Ashcroft S.J.H. , Berggren P.O. , Betzholz C. , Rorsman P. , Trube G. , Welsh M.
Published: July 2002

Two types of exogenous K channel were identified in Xenopus laevis oocytes injected with poly(A+) mRNA from the insulin‐secreting cell line HIT T15. One of these was the ATP‐regulated K channel (G channel) as evidenced by its conductance and inhibition by tolbutamide. The other resembled the Ca‐activated K channel from β‐cells.
Identification of lysine residue 199 of human serum albumin as a binding site for benzylpenicilloyl groups
Authors: Yvon Mireille , Wal Jean-Michel
Published: July 2002

Cyanogen bromide and tryptic fragments of penicilloylated serum albumin from penicillin‐treated patients were separated by HPLC. Determinations of benzylpenicilloyl (BPO) were performed on the different fractions. A BPO containing peptide was identified by its amino acid sequence and the bound BPO group was located on the lysine residue 199.
N‐terminal amino acid sequence of the chromosomal dihydrofolate reductase purified from trimethoprim‐resistant Staphylococcus aureus
Authors: Hartman Peter G. , Stähli Marija , Kocher Hans P. , Then Rudolf L.
Published: July 2002

The existence of two distinct dihydrofolate reductases (DHFR) in highly trimethoprim‐resistant clinical isolates has been unequivocally demonstrated. The enzymes have been characterized with regard to the affinity for substrates and sensitivity to inhibitors. The chromosomal, trimethoprim‐sensitive DHFR was purified to homogeneity by a new simple two‐step procedure. Its N‐terminal amino acid sequence, determined up to the first 35 amino acids, showed 69% homology with the Escherichia coli DHFR.
Spectroscopic studies on the mode of binding of ATP, UTP and α‐amanitin with yeast RNA polymerase II
Authors: Bhargava Purnima , Chatterji Dipankar
Published: July 2002

The binding affinity between the substrates ATP and UTP with the purified yeast RNA polymerase II have been studied here in the presence and absence of Mn2+. In the absence of template DNA, both ATP and UTP showed tight binding with the enzyme without preference for any specific nucleotide, unlike Escherichia coli RNA polymerase. Fluorescence titration of the tryptophan emission of the enzyme by nucleoside triphosphate substrates gave an estimated K
d value around 65 μM in the absence of Mn2+ whereas in the presence of Mn2+, the K
d was 20 μM. The effect of substrates on the longitudinal relaxation of the HDO proton in enzyme‐substrate complex also yielded a similar K
d value.
Characterization of an isolated chloroplast membrane Fe◀S protein and its identification as the photosystem I Fe◀SA/Fe◀SB binding protein
Authors: Wynn R.Max , Malkin Richard
Published: July 2002

An iron‐sulfur protein of approx. 9 kDa has been isolated from spinach chloroplast membranes. Based on iron and sulfide content (∼900 nmol Fe and S2− per mg protein), optical absorbance and low‐temperature EPR spectra, it appears that this protein contains 8Fe and 8S2− in two [4Fe◀4S] clusters. The protein cross‐reacts with an antibody raised against a 9 kDa PS I subunit previously identified as the psaC (frxA) gene product. This identity was confirmed by the N‐terminal amino acid sequence of the isolated Fe◀S protein. It is concluded that the isolated Fe◀S protein binds PS I centers Fe◀SA and Fe◀SB and that each of these centers contains 4Fe and 4S2−.
Ectoenzymes of the kidney microvillar membrane Aminopeptidase P is anchored by a glycosyl‐phosphatidylinositol moiety
Authors: Hooper Nigel M. , Turner Anthony J.
Published: July 2002

The mode of membrane anchorage of three kidney microvillar membrane ectoenzymes has been examined. The release of aminopeptidase P (EC 3.4.11.9) from kidney membranes by bacterial phosphatidylinositol‐specific phospholipase C (PI‐PLC) and the pattern of detergent solubilization of this ectoenzyme implies that it is anchored to the membrane via a covalently attached glycosyl‐phosphatidylinositol moiety. As deduced by phase separation in Triton X‐114, octyl‐glucoside solubilized the amphipathic form of aminopeptidase P, whereas the PI‐PLC‐released form displayed hydrophilic properties. In contrast, the pattern of detergent solubilization of two microvillar carboxypeptidases and their resistance to release from the membrane by bacterial PI‐PLC suggest that these two ectoenzymes are not anchored via phosphatidylinositol.
Purification, composition and Ca2+‐binding properties of the monomeric protein of the S‐layer of Thermus thermophilus
Authors: Faraldo Maria Luisa M. , de Pedro Miguel A. , Berenguer Jose
Published: July 2002

The S‐layer of Thermus thermophilus is apparently formed by a single protein of M
r 100 000 called P100. In the cell envelope of the organism, P100 forms oligomeric complexes of exceptional thermostability in the presence of Ca2+. A simple and rapid method for the purification of P100 is reported. The amino acid composition of the protein was found to be comparable to that of other S‐layer proteins. Binding of Ca2+ to P100 occurs with a dissociation constant of 50 μM apparently at 12 high‐affinity sites per P100 molecule.
Direct electrochemistry in the characterisation of redox proteins: Novel properties of Azotobacter 7Fe ferredoxin
Authors: Armstrong Fraser A. , George Simon J. , Thomson Andrew J. , Yates M.Geoffrey
Published: July 2002

Fast diffusion‐dominated electron transfer between Azotobacter chroococcum 7Fe ferredoxin, FdI, and pyrolytic graphite ‘edge’ electrodes, promoted by aminoglycosides, permits detailed voltammetric studies and preparation of oxidation states inaccessible by chemical titration. The [3Fe‐4S] cluster exhibits pH dependent E

values (30°C); E

(alkaline) = 460 ± 10 mV vs NHE, −dE

/d(pH) = 55 mV, pK = 7.8. The [4Fe‐4S] cluster is characterised by an unusually low reduction potential, −645 ± 10 mV vs NHE, at pH 8.3, with a slight pH dependence, −dE

/d(pH) ∼25 mV over the pH range 8.5‐7.0. No redox couple is observed at potentials between −300 and +600 mV vs NHE. This shows that the [4Fe‐4S] cluster is not an HIPIP‐type centre. The electron paramagnetic resonance spectrum, centred at g = 1.93, of the product resulting from bulk electrolysis at −835 mV is assigned to a [4Fe‐4S]+ cluster interacting magnetically with a reduced [3Fe‐4S] cluster.
Isolation and characterization of restriction endonuclease BstYI from Bacillus stearothermophilus Y406
Authors: Chen Zhongfu , Kong Huimin
Published: July 2002

BstYI, an isoschizomer of XhoII and MflI, has been purified from Bacillus stearothermophilus Y406. This enzyme recognized 5′…Pu/GATCPy…3′ in DNA and cleaved between Pu and G in this sequence. BstYI can be easily isolated and purified by heparin‐agarose column chromatography in a high yield (8000 units BstYI can be obtained per g wet wt of cells).
Isolation of two blood type A and N specific isolectins from Moluccella laevis seeds
Authors: Lis Halina , Latter Hedva , Adar Rivka , Sharon Nathan
Published: July 2002

Extraction of Moluccella laevis seed flour with phosphate‐buffered saline, followed by ammonium sulfate (40%) precipitation and affinity chromatography of the dialysed precipitate on a column of galactose‐bound Sepharose, afforded a single protein peak with high agglutinating activity for human AMM and ONN erythrocytes and poor activity for BMM and OMM cells. Gel filtration of the affinity‐purified lectin on a column of Superose 12 gave two peaks that differed markedly in their activities for AMM and ONN erythrocytes. Whereas the ratio of A/N activities in the slow‐moving peak (isolectin I) was 0.25‐0.5, that of the fast‐moving peak (isolectin II) was 2–3. Molecular mass estimation by gel filtration on Sephadex G‐150 gave a value of 130 kDa for isolectin I and 240 kDa for isolectin II. SDS‐polyacrylamide gel electrophoresis of isolectin I without or with mercaptoethanol revealed that it contains a subunit of 67 kDa, consisting of two chains (46 and 28 kDa, after reduction) held together by SS bonds, and two noncovalently linked subunits of 42 and 26 kDa. The same subunits are also present in isolectin II, where the 67 kDa seems to be predominant. The M. laevis isolectins are unique in that they are specific for determinants of two different blood group systems, and also because they contain both covalently and noncovalently linked subunits. The pattern of inhibition by different sugars of the two blood type activities of the isolectins, assayed with either AMM or ONN erythrocytes, was similar. Of the monosaccharides tested, N‐acetylgalactosamine was the best inhibitor, being 300–600 times more active than galactose. Methyl α‐galactoside was a better inhibitor than the corresponding β‐galactoside. The sugar specificity fits well with the specifity of the isolectins for blood type A erythrocytes, but does not account for their type N specificity.
Selective activation of the γ‐subspecies of protein kinase C from bovine cerebellum by arachidonic acid and its lipoxygenase metabolites
Authors: Shearman Mark S. , Naor Zvi , Sekiguchi Kazuo , Kishimoto Akira , Nishizuka Yasutomi
Published: July 2002

The γ‐subspecies of protein kinase C (PKC) apparently is expressed only in central nervous tissues, and at a high level in the cerebellum and hippocampus. γ‐PKC from bovine cerebellum, but not the α‐or βI/βII‐subspecies, is activated by micromolar concentrations of arachidonic acid (AA), in the absence of both phospholipid and diacylglycerol. A significant component of this activation is also calcium independent. Other unsaturated fatty acids are much less active in this respect. Among the AA metabolites tested, lipoxin A (5(S),6(R),15(S)‐11‐cis‐isomer) was a potent, selective activator of the γ‐subspecies, and also, to a lesser extent, 12(S)‐hydroxy‐5,8,10,14‐eicosatetraenoic acid could support activation. These results raise the possibility that AA and some of its lipoxygenase metabolites may function as messenger molecules in neurones to activate the γ‐subspecies of PKC.
Mechanism of inhibition of papain by chicken egg white cystatin
Authors: Machleidt Werner , Thiele Ulrich , Laber Bernd , Assfalg-Machleidt Irmgard , Esterl Anna , Wiegand George , Kos Janko , Turk Vito , Bode Wolfram
Published: July 2002

N‐terminally truncated forms of chicken egg white cystatin and its cyanogen bromide fragments were isolated and assayed for inhibition of papain. Truncated forms beginning with Gly‐9 and Ala‐10 had a 5000‐fold lower affinity for papain than the two isoelectric forms (pI=6.5 and 5.6) of the full‐length inhibitor (K
i=6 pM and 7 pM) or a truncated form beginning with Leu‐7 (K
i=6 pM), indicating the outstanding importance of one or two residues preceding conserved Gly‐9 for binding. A weak inhibition of papain (K
i=900 nM) was exhibited by the intermediate cyanogen bromide fragment (residues 30–89) containing the chicken cystatin QLVSG variation of the QVVAG segment which is conserved in almost all members of the cystatin superfamily. The obtained affiffity data provide independent evidence for the validity of the proposed docking model of a chicken cystatin‐papain complex [(1988) EMBO J. 7, 2593–2599].
Inhibition of stimulus‐dependent epidermal growth factor receptor and transforming growth factor‐α mRNA accumulation by the protein kinase C inhibitor staurosporine
Authors: Bjorge Jeffrey D. , Kudlow Jeffrey E. , Mills Gordon B. , Paterson Andrew J.
Published: July 2002

The ability of staurosporine, a potent inhibitor of protein kinase C, to block certain cellular events initiated by 12‐O‐tetradecanoylphorbol‐13‐acetate (TPA) and epidermal growth factor (EGF) was examined. Treatment of MDA468 breast cancer cells with TPA decreases EGF binding to the cell surface and this effect is blocked by pretreatment with staurosporine with an IC50 of 30 nM. Either 10−9 M EGF or 100 ng/ml TPA stimulated the accumulation of both EGF receptor and TGF‐α mRNA and staurosporine (50 nM) completely abolished these mRNA accumulations. Staurosporine did not block EGF‐stimulated tyrosine phosphorylation of its receptor as measured by immunoblotting with anti‐phos‐photyrosine antibodies. The ability of staurosporine to block the mRNA responses of either EGF or TPA suggests that these two agents have common signaling pathways and it implies a role for protein kinase C in the control of EGF receptor and TGF‐α expression.
Cellular distribution of cholesterogenesis‐linked, phosphoisoprenylated proteins in proliferating cells
Authors: Sepp-Lorenzino Laura , Azrolan Neal , Coleman Peter S.
Published: July 2002

A set of isoprenylated proteins has been detected in rapidly proliferating, suspension‐grown murine lymphoma cells. Our evidence indicates that all of these isoprenylated proteins are phosphorylated. Subsequent to a 24 h incubation with mevinolin to deplete the intracellular mevalonate (MVA) level, cells were incubated with [3H]MVA and/or 32Pi and both total cell and subcellular fraction proteins were resolved via 1‐ and 2‐D gel electrophoresis, then assessed via subsequent autoradiography. The phospho‐isoprenylated proteins comprise a set spanning a molecular mass range of 21–69 kDa and all dispay acidic pI. MVA‐derivatized proteins of 21–24 kDa, which consist of multiple isoforms, are present in both cytosolic and nuclear fractions. Larger phospho‐isoprenylated protein species (44–69 kDa) are specifically localized within the nucleus, where applicable extraction protocols indicate that they are part of or closely affiliated with the nuclear matrix‐intermediate filament (NM‐IF) components. The localization of the 69 kDa prenylated species within the NM‐IF fraction, together with evidence of its phosphorylation, supports recent indications that this protein is the nuclear matrix component lamin B.
Inhibition by cyclic AMP of guanine nucleotide‐induced activation of phosphoinositide‐specific phospholipase C in human platelets
Authors: Yada Yukihiro , Nagao Seiji , Okano Yukio , Nozawa Yoshinori
Published: July 2002

Phosphoinositide‐specific phospholipase C (PLC) activity of human platelet membranes was activated by the nonhydrolyzable guanine nucleotide GTPγS. This activation did not occur in either membranes prepared from dibutyryl cyclic AMP‐pretreated platelets (A‐membranes) or those prepared from untreated cells and subsequently incubated with cyclic AMP (cAMP) (B‐membranes). This cAMP‐mediated inhibition was abolished in the presence of inhibitors of cAMP‐dependent protein kinase (A‐kinase), suggesting that the inhibition was due to phosphorylation of (a) protein component(s). No significant differences were observed in the basal PLC activity and the extent of pertussis toxin‐catalyzed ADP‐ribosylation among control membranes and the two types of phosphorylated membranes (A‐ and B‐membranes). GTP‐binding activities of Gs, Gi and GTP‐binding proteins of lower molecular masses were not altered by the phosphorylation of the membranes. These findings suggest that a GTP‐binding protein is involved in the GTPγS‐mediated activation of PLC and that cAMP (plus A‐kinase) inhibits this activation by phosphorylating a membrane protein (probably a 240‐kDa protein), rather than the GTP‐binding protein or PLC itself. It is likely that this phosphorylation uncouples the GTP‐binding protein from PLC.
Comprehensive, human cellular protein databases and their implication for the study of genome organization and function
Authors: Celis Julio E. , Ratz Gitte P. , Madsen Peder , Gesser Borbala , Lauridsen Jette B. , Kwee Sianette , Rasmussen Hanne Holm , Nielsen Henrik V. , Crüger Dorthe , Basse Bodil , Leffers Henrik , Honoré Bent , Møller Olaf , Celis Ariana , Vandekerckhove Joel , Bauw Guy , van Damme Jozef , Puype Magda , Van den Bulcke Marc
Published: July 2002

Comprehensive, computerized databases of cellular protein information derived from the analysis of two‐dimensional gels, together with recently developed techniques to microsequence proteins offer a new dimension to the study of genome organization and function. In particular, human protein databases provide an ideal framework in which to focus the human genome sequencing effort.
Forskolin stimulates pinealocyte cGMP accumulation Dramatic potentiation by an α1‐adrenergic → [Ca2+]i mechanism involving protein kinase C
Authors: Ho Anthony K. , Chik Constance L. , Klein David C.
Published: July 2002

The effect of forskolin on cGMP regulation was investigated using dispersed rat pinealocytes. Forskolin stimulated cGMP accumulation in a concentration‐dependent manner; this response was strongly potentiated by an α1‐adrenergic → [Ca2+]i mechanism involving protein kinase C. These findings provide further evidence that activation of two receptor‐regulated signal transduction mechanisms may be commonly required for maximal stimulation of cGMP accumulation, and establish a new experimental approach to the study of cGMP regulation.
Myosin isoforms in mackerel (Scomber scombrus) red and white muscles
Authors: Martinez I. , Olsen R.L. , Ofstad R. , Janmot C. , d'Albis A.
Published: July 2002

Myosin extracts of red and white muscles from mackerel (Scomber scombrus) were analyzed by native electrophoresis to investigate the existence of myosin isoforms in both kind of muscles, and by two‐dimensional and SDS gel electrophoresis to study their subunit composition. Two isoforms were found in red muscle comprising one type of heavy chain and two light chains. Four isoforms were found in white muscle made up of one type of heavy chain and three types of light chains. The heavy chains from white muscle showed a higher electrophoretic mobility than that of red muscle in SDS‐PAGE. Both heavy chains had an intermediate mobility between those of slow and fast myosins from rat diaphragm.
Light chain of tetanus toxin intracellularly inhibits acetylcholine release at neuro‐neuronal synapses, and its internalization is mediated by heavy chain
Authors: Mochida Sumiko , Poulain Bernard , Weller Ulrich , Habermann Ernst , Tauc Ladislav
Published: July 2002

The ability of the two‐chain form of tetanus toxin (TeTx), its constituent light (LC) or heavy (HC) chains, and papain fragment to block evoked acetylcholine (ACh) release in the buccal ganglia of Aplysia californica was studied electrophysiologically. Extracellularly applied, TeTx or its B fragment (consisting of LC and β2, the amino‐terminal portion of HC) blocked ACh release, whereas LC, HC, or the β2 fragment did not affect it. Toxicity was restored when LC was bath applied together with HC or the β2 fragment. When injected into the presynaptic neuron, TeTx, the B fragment or LC, but not HC, induced inhibition of ACh release. These results indicate that the blockade of ACh release by TeTx is mimicked by intracellular action of LC, the internalization of which is mediated by the HC via its amino‐terminal moiety.