bluuebunny/crossref_metadata_2025 huggingface dataset:
The effect of N‐methylprotoporphyrin and succinyl‐acetone on the regulation of heme biosynthesis in chicken hepatocytes in culture
Authors: De Matteis Francesco , Marks Gerald S.
Published: July 2002

The essential features of hepatic protoporphyria, namely inhibition of ferrochelatase, accumulation of protoporphyrin and stimulation of 5‐aminolevulinic acid synthase (ALA‐S) were all obtained by treating chicken hepatocytes in culture with small doses of N‐methylprotoporphyrin. Both N‐methylprotoporphyrin and succinyl‐acetone, another inhibitor of heme biosynthesis, stimulated ALA‐S when given on their own and also enhanced the stimulation of ALA‐S caused by phenobarbital.
Epidermal growth factor: exposure and dynamics of the aromatic side chains as investigated by photo‐CIDNP and variable temperature 1H‐NMR
Authors: De Marco Antonio , Menegatti Enea , Guarneri Mario
Published: July 2002

EGF is a 53 residue polypeptide of multiple biological activities. Homonuclear decoupling, spin‐echo multiplet selection and Photo‐CIDNP experiments lead us to fully assign the resonances from the aromatic side chains (5 Tyr, 1 His and 2 Trp). The photochemical experiment gives specific attribution of doublets in tyrosines and multiplets in tryptophans. The resonances from two tyrosines are broadened and shifted at high fields, suggesting the existence of hydrophobic domains in EGF, consistent with the presence of ring current shifted methyl resonances. However, the amide exchange in D2O solution is considerably faster than that observed for globular proteins of the same size, and most of the aromatic residues are accessible to the flavin dye. The combined evidence suggests that EGF possesses a folded structure, but the molecule is rather floppy. From spectra measured at variable temperature a ΔG° at 25°C of about 8 kcal/mol is calculated.
The gramicidin A channel: the energy profile for single and double occupancy in a head‐to‐head β6.33,3‐helical dimer backbone
Authors: Pullman Alberte , Etchebest Catherine
Published: July 2002

The energy profile for Na+ in the channel formed by the gramicidin A β‐helical dimer backbone was computed introducing all the terms in the theory of intermolecular interactions. The effect of allowing the ion to reach its successive optimal positions shows the presence of a series of energy minima associated with different carbonyls. The presence of a second ion lowers the central barrier for the first one and facilitates its progression and exit. The energy profile for double occupancy indicates the presence of two symmetrical minima at about 13 Å from the center.
Neuropeptide Y in neuroblastoma × glioma hybrid cells
Authors: Yeats J.C. , Allen J.M. , Bloom S.R. , Leigh P.J. , MacDermot J.
Published: July 2002

High concentrations of a newly‐identified biologically potent peptide, neuropeptide Y, have been demonstrated in 3 related mouse neuroblastoma‐derived clonal cell lines, N18TG2 0.35 pmol/mg protein, NG108‐15 0.44 pmol/mg protein and NCB‐20 0.39 pmol/mg protein. The NG108‐15 cell line was chosen for further evaluation. Dexamethasone (10 μM) and nerve growth factor (10 ng/ml) resulted in a 2‐fold increase in cellular neuropeptide Y concentrations. The response to dexamethasone was demonstrated to be dose‐dependent. Exposure to both agents in combination resulted in a more than additive effect, indicating synergism.
Electrogeneicity of the lysosomal proton pump
Authors: Dell'Antone Paolo
Published: July 2002

Using acridine orange as a pH gradient probe, the effects of valinomycin and FCCP on the pH gradient across lysosomal membranes in an ATP‐free medium as well as on the rate of the inward ATP‐driven proton translocation were investigated. Both lysosome‐enriched and highly purified lysosomal preparations from rat liver were used with identical results. Ionophore effects were found to be different depending upon whether passive ion fluxes or ATP‐driven H+ translocation were involved and supported the existence of a membrane potential in the latter case. Anions stimulated the rate of ATP‐driven proton translocation and stimulation increased with increasing anion lipophilicity. These results strongly support the electrogenic nature of the lysosomal proton pump.
Activity of nitrogen metabolism enzymes in the process of kernel development in different maize genotypes
Authors: Hadži-Tašković Šukalović Vesna
Published: July 2002

The activity of GDH, GOT, GPT, lysine‐ketoglutarate reductase and GS in the developing endosperm of normal maize, opaque‐2, sugary‐2 and opaque‐2 sugary‐2 mutants was investigated. The results indicate that the o2 gene increases GDH, GOT and GPT activities and decreases lysine catabolism in the developing endosperm. The presence of different mutants in the same genetic background does not affect GS activity.
Regulation of bioactive β‐endorphin processing in rat pars intermedia
Authors: Ham J. , Smyth D.G.
Published: July 2002

Acid extracts of rat pituitary neuro‐intermediate lobes have been shown by ion‐exchange chromatography and radio‐immunoassay to contain predominantly the inactive derivatives of β‐endorphin, αN‐acetyl β‐eadorphin 1–27 and α,N‐acetyl β‐endorphin 1–26; the biologically active form, β‐endorphin 1–31, is a minor component. In contrast, it was found that β‐endorphin generated in neuro‐intermediate lobe cells in monolayer culture was less processed: the principal peptides related to bioactive β‐endorphin 1–31. When the cultured cells were incubated in the presence of 10−5 M dopamine or 10−6 M α‐ergocryptine there was a marked increase in the degree of proteolysis and acetylation: the processing pattern reverted to that characteristic of the neuro‐intermediate lobe in situ, with α‐N‐acetyl β‐endorphin 1–26 and α,N‐acetyl β‐endorphin 1–27 as the prominent peptides. The results demonstrate that dopaminergic agents can influence the processing of β‐endorphin‐related peptides in rat pars intermedia, indicating a new level at which the bioactivity may be regulated.
Binding of a spin‐labeled phenylalanine analog to sickle hemoglobin: EPR and NMR studies
Authors: Lu Hwei-Zu , Currie Bruce L. , Johnson Michael E.
Published: July 2002

We have synthesized a spin‐label analog of phenylalanine as a competitive inhibitor probe of the sickle hemoglobin aggregation process. Sickle hemoglobin gelation measurements indicate that the spin‐label phenylalanine analog is a potent inhibitor of deoxy sickle hemoglobin aggregation. We have also used spin label EPR and high‐resolution proton NMR to study the interaction of the phenylalanine analog with hemoglobin, and find that the kinetic off‐rate is comparable to, or slower than the hemoglobin rotational rate (i.e., ⪸ 108 s−1), and that at least one, and perhaps two significant localized interaction region(s) exist within a few angstroms of the β chain N‐ and C‐termini. Correlation with other known structural information suggests that the observed interaction sites may be relevant to the mechanism for inhibition of sickle hemoglobin aggregation.
Identification of 16‐dehydropregnenolone as an intermediate in 16‐androstene biosynthesis in neonatal porcine testicular microsomes
Authors: Kwan T.K. , Taylor N.F. , Watson D. , Gower D.B.
Published: July 2002

The biosynthesis of 16‐androstenes has been studied in neonatal porcine testicular microsomes using 17‐hydroxypregnenolone and 16‐dehydropregnenolone, separately, as substrates. The metabolites formed after microsomal incubation with these substrates were purified, derivatized as O‐methyloxime‐trimethylsilyl ethers and analysed by capillary gas chromatography‐mass spectrometry. In the incubation of 17‐hydroxy‐pregnenolone with microsomes, 16‐dehydropregnenolone was identified as an intermediate in the biosynthesis of 16‐androstenes. Further microsomal incubation of 16‐dehydropregnenolone has established the intermediary role of this steroid in the production of 16‐androstenes.
Chemically induced antioxidant‐sensitive respiration
Authors: Videla Luis A.
Published: July 2002

The interrelations between the hepatic chemically induced antioxidant‐sensitive respiration and the contents of malondialdehyde (MDA) and of reduced glutathione (GSH), were studied in the isolated hemoglobin‐free perfused rat liver. Antioxidant‐sensitive respiration was induced by the infusion of agents such as ethanol, iron, xanthine or t‐butyl hydroperoxide, or by phenylhydrazine pretreatment in vivo. The development of this respiratory component occurred concomitantly with high levels of MDA in the perfused livers, while those of GSH were diminished.
A C‐terminal fragment of hemoglobin β‐chains in extracts of porcine upper intestine
Authors: Carlquist Mats , Jörnvall Hans , Mutt Viktor
Published: July 2002

A 32‐residue peptide has been isolated from extracts of porcine upper intestine. Amino acid sequence determination showed that the peptide is a fragment of hemoglobin, corresponding to the C‐terminal part of the β‐chain. The region in the β‐chain which precedes the isolated fragment is hydrophobic (8 Leu/Val/Ile) and has no charges at 10 preceding positions. It therefore, to some extent, resembles the structure of ‘signal sequences’ which may suggest a specific cleavage site in hemoglobin β‐chains.
The gramicidin A channel
Authors: Etchebest Catherine , Pullman Alberte
Published: July 2002

The inclusion of the presence and flexibility of the CH2CH2OH end chain in the computation of the energy profile for single occupancy by Na+ of the gramicidin A channel modifies substantially the profile obtained without that chain. The binding site (deepest minimum) in the profile is situated at 10.5 Å from the center of the channel, in satisfactory agreement with the conclusions based on 13C‐NMR studies. The existence of an external minimum at the mouth is confirmed.
Amino acid sequences of allophycocyanin α‐ and β‐subunits isolated from Anabaena cylindrica
Authors: Minami Y. , Yamada F. , Hase T. , Matsubara H. , Murakami A. , Fujita Y. , Takao T. , Shimonishi Y.
Published: July 2002

Allophycocyanin (APC) α‐ and β‐subunits were isolated from Anabaena cylindrica and their amino acid sequences were determined. The α‐ and β‐subunits consisted of 160 and 161 amino acid residues, respectively, and lacked tryptophan. The β‐subunit contained an unknown derivative of aspartic acid. The sequences were compared with those of other allophycocyanins.
Intact human lymphocyte membranes respond to muscarinic receptor stimulation by oxotremorine with marked changes in microviscosity and an increase in cyclic GMP
Authors: Masturzo P. , Salmona M. , Nordstrom O. , Consolo S. , Ladinsky H.
Published: July 2002

The muscarinic agonist oxotremorine produced a linear dose‐dependent increase in membrane fluidity of intact and viable human lymphocytes in vitro. This effect proved to be receptor‐mediated because preincubation with 10−5 M atropine shifted the dose‐response curve one order of magnitude rightward. Pirenzepine preincubation did not affect membrane fluidity variation. A cGMP increase was also found after oxotremorine treatment. The results are discussed in terms of possible modulation ofguanyl cyclase and adenyl cyclase through membrane fluidity variations.
Photoactivation of NH2OH‐treated leaves: reassembly of released extrinsic PS II polypeptides and religation of Mn into the polynuclear Mn catalyst of water oxidation
Authors: Becker David W. , Callahan Franklin E. , Cheniae G.M.
Published: July 2002

Inactivation of the water‐oxidizing enzyme by exposure of leaf segments to hydroxylamine results in a disassembly/perturbation of photosystem (PS) II extrinsic polypeptides (17>23 kDa) and solubilization of the tetra‐Mn complex. Religation of the tetra‐Mn complex, reassembly of these PS II extrinsic polypeptides with thylakoid membranes and reappearance of active water‐oxidizing activity all require light, and all are unaffected by inhibitors of protein synthesis and photophosphorylation. The results indicate, however, that the reassembly of these PS II extrinsic polypeptides into the multimeric water oxidizing enzyme occurs more rapidly than the photoligation of Mn as the tetra‐Mn complex.
Generation and purification of submitochondrial particles of different polarities from plant mitochondria
Authors: Kay Christopher J. , Ericson Ingemar , Gardestrom Per , Palmer John M. , Møller Ian M.
Published: July 2002

Mitochondria were isolated from Jerusalem artichoke (Helianthus tuberosus L.) tubers in a low‐salt medium. Submitochondrial particles (SMP) produced by sonication in a low salt medium + 20 mM MgCl2 were 18% right‐side‐out (RO) as judged by the latency of cytochrome c oxidase assayed ± Triton X‐100. SMP produced by French press treatment in a low‐salt medium + 5 mM EDTA to remove bound divalent cations were 98% RO. Less extreme treatments gave SMP of intermediate polarity. There was a positive correlation between the % RO‐SMP (produced by sonication) and the % NAD+‐malate dehydrogenase enclosed within the SMP indicating that only RO‐SMP contained trapped matrix. When a mixed population of SMP (45% RO) was applied to an aqueous polymer two‐phase system, the top phase contained 76% RO‐SMP and the bottom phase mostly inside‐out SMP (26% RO). By analogy with the models for stacking of photosynthetic membranes, we propose that crista formation in the inner mitochondrial membrane is electrostatically regulated and SMP deriving from the closely stacked crista regions are inside‐out.
Cardiac troponin‐C: a rapid and effective method of purification
Authors: Szynkiewicz Jolanta , Stȩpkowski Dariusz , Brzeska Hanna , Drabikowski Witold
Published: July 2002

A new purification procedure for cardiac troponin‐C is described which has several advantages over previous methods. High purity of the final product was assessed by electrophoretic, enzymatic and spectroscopic methods.
Influence of smooth muscle myosin conformation on myosin light chain kinase binding and on phosphorylation
Authors: Cross Robert A. , Sobieszek Apolinary
Published: July 2002

Conventional smooth muscle myosin preparations contain a tightly bound myosin light chain kinase activity, which is incompletely removed by gel filtration at high ionic strength. We show here that by contrast, this kinase activity is released, together with calmodulin, under conditions in which myosin is in the folded configuration. The conformation‐related release of kinase occurred for dephosphorylated myosin in both the presence and absence of ATP and Ca2+. Binding of kinase to extended phosphorylated myosin was relatively weaker than to dephosphorylated myosin, but was nonetheless detected. The kinetic consequences of this binding behaviour were determined by measuring initial myosin phosphorylation rates as a function of KCl concentration. Rate optima occurred at 60 mM KCl and 300 mM KCl, conditions favouring respectively stable filaments and stable extended monomers. Phosphorylation of the folded monomer was uniformly slow at low KCl concentrations. The folded moysin monomer is thus a relatively poor substrate for the kinase, and is therefore unlikely to represent an analog of the relaxed crossbridge configuration in myosin filaments.
Trimeric structure and other properties of the chloroplast reductase binding protein
Authors: Ceccarelli Eduardo A. , Chan Raquel L. , Vallejos Rubén H.
Published: July 2002

A complex between the ferredoxin‐NADP+ reductase and a 17.5 kDa polypeptide was isolated from lettuce chloroplasts by the same procedure used previously in spinach [(1984) J. Biol. Chem. 259, 8048‐8051]. The stoichiometry of the complex from both sources was determined by quantification of stained protein bands after gel electrophoresis and, in the case of the spinach complex, by rocket inmunolectrophoresis. The results suggest that the binding protein is a trimer of the 17.5 kDa polypeptide. The amino arid composition of the spinach reductase binding protein and partial sequencing of its tryptic peptides clearly show that it is not identical to the 17 kDa polypeptide of the cytochrome b
6‐ƒ complex.
Changes in nuclear proteins induced by heat shock in Drosophila culture cells
Authors: Rabilloud T. , Vincens P. , Hubert M. , Pennetier J.L. , Tarroux P.
Published: July 2002

Nuclear proteins of normal and heat‐shocked Drosophila cells were analysed by two‐dimensional electrophoresis. The computerized processing of the gels allowed us to detect 6 proteins strongly induced by the heat treatment, but which were different from the usually described heat‐shock proteins. The possible role of these proteins in genetic regulation is discussed, as is the value of this type of approach for the study of other genetic regulation phenomena.
Growth‐related variation of α2‐adrenergic receptivity in the HT 29 adenocarcinoma cell‐line from human colon
Authors: Paris H. , Bouscarel B. , Cortinovis C. , Murat J.C.
Published: July 2002

The human colon adenocarcinoma cell‐line HT 29 has been shown to possess functional α2‐adrenergic receptors. Here, [3H] clonidine was used as radioligand to study the evolution of α2‐adrenergic receptivity during the time course of HT 29 cell culture. Scatchard analysis of the saturation curves indicates that the number of [3H]clonidine binding sites increases throughout the 17 day culture period. The maximal number of α2adrenoceptors is found during the stationary phase of growth, when cell density is high and mitotic rate low. Moreover, the use of adrenaline and clonidine as α2‐adrenergic agonists reveals a relationship between the number of receptors and the intensity of the biological effect associated with their stimulation (inhibition of the VIP‐induced cyclic AMP accumulation).
Quantitation of chordin in developing Huso huso embryos and larvae by radioimmunoassay
Authors: Preobrazhensky A.A. , Glinka A.V.
Published: July 2002

Chordin is a protein discovered in the notochord cells of the representatives of Acipenseridae; giant sturgeon, stellate sturgeon and sterlet. Some characteristics of the purified chordin preparation which justify its use in radioimmunoassay are described. A sensitive competitive‐binding double‐antibody radioimmunoassay for chordin is described by which its content in the extracts from giant sturgeon embryos and larvae has been measured. It is shown that chordin biosynthesis started in the embryos from stage 32.
Differential expression of α‐tubulin mRNA in rat cerebellum as revealed by in situ hybridization
Authors: Ginzburg I. , Teichman A. , Griffin W.S.T. , Littauer U.Z.
Published: July 2002

Nucleotide sequence analysis of two rat α‐tubulin cDNA clones showed a marked divergence in their 3'‐untranslated regions. However, each of the α‐tubulin isotypes shows a high interspecies homology in this region, when compared with an isotubulin sequence from human and Chinese hamster. In situ hybridization of rat cerebellum with α‐tubulin cDNA revealed differential expression in various cell layers. The mitotically active cells in the external granular layer show the highest level of α‐tubulin mRNA, while lower levels are observed in the migrating cells in the molecular layer and in the differentiating cells in the internal granular layer. Very low levels of the mRNA are observed in the prenatally differentiated Purkinje cells.
Monoclonal antibody ARC MAC 50.1 binds to a site on the phytochrome molecule which undergoes a photoreversible conformational change
Authors: Thomas Brian , Penn Susan E.
Published: July 2002

Monoclonal antibody from rat hybridoma cell line ARC MAC 50.1 binds more strongly to Pfr than to Pr in a sandwich ELISA of phytochrome in crude Avena sativa L. extracts or highly purified Avena phytochrome. Discrimination is not a consequence of differential modification of Pr and Pfr during the assay. Loss of a 6 kDa peptide from the N‐terminal end of the phytochrome apoprotein during purification does not prevent discrimination. Neither is preferential binding to Pfr a consequence of a steric interaction between ARC MAC 50.1 and the antibody used with it in the sandwich ELISA. It is concluded that the binding site for ARC MAC 50.1 undergoes a reversible conformational change upon photoconversion and may thus represent a functional region of the phytochrome molecule.
Detection of the X gene product of simian T‐cell leukemia virus
Authors: Tsujimoto Atsumi , Tsujimoto Hajime , Yanaihara Noboru , Abe Kaonu , Hayami Masanori , Miwa Masanao , Shimotohno Kunitada
Published: July 2002

The gene product of the X region was examined in simian lymphoid cell lines producing simian T‐cell leukemia virus (STLV), which is closely related to human T‐cell leukemia virus (HTLV). By use of specific antibodies against pX peptides of HTLV‐I, a protein of 41 kDa was identified as a pX product of STLV.
Inhibition by streptozotocin of the activity of succinyl‐Co A synthetase in vitro and in vivo
Authors: Boquist L. , Ericsson I.
Published: July 2002

The activity of succinyl‐CoA synthetase from mouse liver and kidney was inhibited by streptozotocin in vitro. Streptozotocin behaved essentially as a non‐competitive inhibitor, and the following kinetic values were obtained (in the presence of 10 nM streptozotocin): apparent K
m 1.7 mM, apparent K
i 10 nM, and k
cat 440 nkat·kg−1. Compared with non‐diabetic control mice, the succinyl‐CoA synthetase activity was significantly decreased in the islets and kidneys of mice with early (1 h) and manifest (≧ 2 days) streptozotocin diabetes, whereas the activity in the liver was not significantly altered. Inhibited succinyl‐CoA synthetase activity is believed to play a prominent role in the cellular effects of streptozotocin.
Inhibitory diffusible factor IDF45, a G1 phase inhibitor
Authors: Blat Christiane , Chatelain Gilles , Desauty Geneviéve , Harel Louise
Published: July 2002

An inhibitory diffusible factor of 45 kDa (IDF45) was isolated from medium conditioned by dense cultures of 3T3 cells. The procedure involved Bio‐Gel PI 50 chromatography and 2 reverse‐phase FPLC. After the final step of purification, 60 of IDF45 inhibited 50% of α‐globulin‐stimulated DNA synthesis. It was shown that IDF45 acted in the G1 phase of the cell cycle. When added for 8 h in the G1 phase of the cell cycle, it was able to inhibit DNA synthesis in the S phase which followed this G1 phase. Furthermore, IDF45 inhibited the early stimulation of RNA synthesis induced by α‐globulin.
N‐terminal sequence determination and secondary structure analysis of extrinsic membrane proteins in the water‐splitting complex of spinach
Authors: Vater Joachim , Salnikow Johann , Jansson Christer
Published: July 2002

The N‐terminal sequences of the 16 and 23 kDa extrinsic membrane proteins in photosystem II of spinach have been determined by solid‐phase sequencing. The sequence of the 23 kDa protein has been corroborated by a small 3 kDa CNBr fragment confirming partial N‐terminal processing by a protease of unknown nature. A secondary structure analysis of the 23 kDa protein by circular dichroism has been performed indicating a β‐structure content of 61 % with only 14% α‐helix conformation.
Protein‐chemical analysis of pertussis toxin reveals homology between the subunits S2 and S3, between S1 and the A chains of enterotoxins of Vibrio cholerae and Escherichia coli and identifies S2 as the haptoglobin‐binding subunit
Authors: Capiau Carine , Petre Jean , Van Damme Jozef , Puype Magda , Vandekerckhove Joël
Published: July 2002

The purified toxin of Bordetella pertussis was dissociated in 5 M urea in the presence of immobilized haptoglobin. The toxin was dissociated in free S1, free S5 and the free complexes S2‐S4 and S3‐S4, with S2‐S4 as the only haptoglobin‐binding moiety, identifying S2 as the haptoglobin‐binding protein. Partial NH2‐terminal amino acid sequences were obtained from the dissimilar toxin subunits, after separation by SDS‐polyacrylamide gel electrophoresis followed by electroblotting onto polybrene‐coated glass‐fiber sheets. The sequences reveal extensive homology of the N‐terminal portions of the constitutive subunits S2 and S3 and between S1 and the enterotoxin A chains of Vibrio cholerae and Escherichia coli.
Interaction of the high‐spin Fe atom in the photosystem II reaction center with the quinones QA and QB in purified oxygen‐evolving PS II reaction center complex and in PS II particles
Authors: Itoh Shigeru , Tang Xiao-Song , Satoh Kimiyuki
Published: July 2002

EPR signals in the high‐spin region were studied at 10 K in photosystem II (PS II) particles and in a purified oxygen‐evolving PS II reaction center complex under oxidizing conditions. PS II particles showed EPR peaks at g = 8.0 and 5.6, confirming the recent report by Petrouleas and Diner [(1986) Biochim. Biophys. Acta 849, 264‐275]. Addition of 3‐(3',4'‐dichlorophenyl)‐1,1‐dimethylurea (DCMU) or o‐phenanthroline shifted the peaks to be closer to g = 6.0 depending on the medium pH. On the other hand, the PS II reaction center complex showed peaks at g = 6.1 and 7.8, and at g = 6.1 and 6.4, in the absence and presence of o‐phenanthroline, respectively. All these peaks were found to be decreased by the illumination at 10 K. These results suggest that the high‐spin signals are due to Q400, Fe(III) atom interacting with the PS II primary electron acceptor quinone QA as reported and that the Fe atom also interacts with the secondary acceptor quinone QB. This interaction seems to induce the highly asymmetric ligand coordination of the Fe atom and to be affected by DCMU and o‐phenanthroline in a somewhat different manner.
Complete amino acid sequence of the large subunit of the low‐Ca2+‐requiring form of human Ca2+‐activated neutral protease (μCANP) deduced from its cDNA sequence
Authors: Aoki Kazumasa , Imajoh Shinobu , Ohno Shigeo , Emori Yasufumi , Koike Morio , Kosaki Goro , Suzuki Koichi
Published: July 2002

The complete amino acid sequence of the large subunit (catalytic subunit) of human low‐Ca2+ requiring‐calcium‐activated neutral protease (μCANP) was deduced from its cDNA base sequence. It is composed of 714 amino acid residues and its sequence is highly homologous to the chicken CANP sequence determined previously. Human μCANP, like chicken CANP, has a clear 4‐domain structure, and their fundamental structures are essentially the same, although their Ca2+sensitivities are significantly different. The role of each domain in the Ca2+sensitivity and protease activity of CANP is discussed on the basis of sequence comparison.
Activation of the Na+/K+‐ATPase by interleukin‐2
Authors: Redondo Juan Miguel , Rivas Abelardo López , Fresno Manuel
Published: July 2002

Activated B61.SF.1 and CTLL‐2 T lymphocyte clones which are strictly dependent on interleukin‐2 (IL‐2) for growth were used to study the activation of Na+/K+‐ATPase. 50% of [3H]thymidine maximal incorporation was obtained when the extracellular concentration of Na+ or K+ was reduced to 50 or 2 mM, respectively. ‘Quiescent’ CTL clones stimulated with IL‐2 showed an increase of 48–380% in ouabain‐sensitive 86Rb uptake. Furthermore, this stimulation was completely inhibited by a monoclonal antibody PC.61 directed at the IL‐2 receptor. The activation of the pump was dependent on the dose of IL‐2, took place at the same doses of IL‐2 that were required to stimulate cell proliferation and was linear for at least 30 min.
Affinity purification of the opioid receptor in NG 108‐15 cells using an avidin‐biotin system with a novel elution method
Authors: Nakayama Hitoshi , Shikano Hiroyoshi , Aoyama Tomoko , Amano Takehiko , Kanaokat Yuichi
Published: July 2002

Affinity purification of the opioid receptor in NG 108‐15 cells was carried out using an affinity resin based on the avidin‐biotin interactions, but a new elution method was employed with a radioligand of sub‐micromolar concentration. A synthesized biotinyl derivative of leucine‐enkephalin has a high affinity for the δ‐receptor, but the affinity was lowered 10‐fold in the presence of avidin. The new elution method is based on this affinity decrease and resulted in a 100‐fold purification over the initial crude materials in the single step. SDS‐polyacrylamide gel electrophoresis of the purified receptor revealed three polypeptides of 58, 65 and 71 kDa as possible components of the δ‐receptor.
Expression of insulin‐like growth factor‐I and ‐II genes in rat medullary thyroid carcinoma
Authors: Höppener J.W.M. , Steenbergh P.H. , Slebos R.J.C. , de Pagter-Holthuizen P. , Roos B.A. , Jansen M. , Van den Brande J.L. , Sussenbach J.S. , Jansz H.S. , Lips C.J.M.
Published: July 2002

Several types of cancer cells produce polypeptide growth factors and often the same cells have functional receptors for the released growth factor (autocrine secretion). We have studied expression of genes encoding somatomedin‐C/insulin‐like growth factor‐I (Sm‐C/IGF‐I) and IGF‐II, in rat medullary thyroid carcinomas (MTCs) in different stages of tumour differentiation. RNAs hybridizing specifically to an IGF‐I cDNA probe were detected in 6 out of 7 differentiated MTCs and IGF‐II related RNAs were demonstrated in 5 out of these 7 differentiated MTCs. In 5 anaplastic MTCs no IGF RNAs were detected, except for a small amount of IGF‐II related RNA in one tumour.
G‐proteins in Torpedo marmorata electric organ Differential distribution in pre‐ and post‐synaptic membranes and synaptic vesicles
Authors: Toutant Madeleine , Bockaert Joël , Homburger Vincent , Rouot Bruno
Published: July 2002

The nature of the G‐proteins present in the pre‐ and post‐synaptic plasma membranes and in the synaptic vesicles of cholinergic nerve terminals purified from the Torpedo electric organ was investigated. In pre‐ and post‐synaptic plasma membranes, Bordetella pertussis toxin, known to catalyze the ADP‐ribosylation of the α‐subunit of several G‐proteins, labels two substrates at 41 and 39 kDa. The 39 kDa subunit detected by ADP‐ribosylation in the synaptic plasma membrane fractions was immunologically similar to the Go α‐subunit purified from calf brain. In contrast to bovine chromaffin cell granules, no G‐protein could be detected in Torpedo synaptic vesicles either by ADP‐ribosylation or by immunoblotting.
Construction of a hybrid cDNA encoding a major legumin precursor polypeptide and its expression and localization in Saccharomyces cerevisiae
Authors: Yarwood Jennifer N. , Harris Nick , Delauney Ashton , Croy Ron R.D. , Gatehouse John A. , Watson Martin D. , Boulter Donald
Published: July 2002

A hybrid cDNA encoding a major legumin precursor polypeptide was constructed. Using a phosphoglycerate kinase expression vector levels of expression of 1–2% total protein were obtained in Saccharomyces cerevisiae. Immunocytochemical localization demonstrated that the legumin was accumulated within the Golgi vesicles.
Fructose 2,6‐bisphosphate and the control of the energy charge in higher plants
Authors: Mertens Emmanuel , Van Schaftingen Emile , Hers Henri-Géry
Published: July 2002

The mechanism by which cutting Jerusalem artichoke tubers into slices causes a large increase in their content in fructose 2,6‐bisphosphate was analysed by investigating the changes in metabolite concentrations occurring within the first minutes after slicing. The first event was a rapid consumption of ATP, and also of glycerate‐3‐P and phosphoenolpyruvate, two potent inhibitors of phosphofructokinase 2. The synthesis of fructose 2,6‐bisphosphate started after a lag of 5 min, corresponding to the time required for the decrease in concentration of the two inhibitors. This sequence of events is proposed as a new mechanism for the preservation of ATP, which also operates in intact tubers maintained in anoxia. The role of fructose 2,6‐bisphosphate in the Pasteur effect in higher plants is discussed.
Receptor‐phosphoinositidase C coupling Multiple G‐proteins?
Authors: Lo William W.Y. , Hughes John
Published: July 2002

Recent evidence has suggested that receptor‐mediated phosphoinositide turnover, like that of the adenylate cyclase cAMP pathway, is regulated by guanine nucleotides. It is likely that one or more guanine nucleotide‐binding proteins (G‐proteins) couple calcium‐mobilizing receptors to the activation of phosphoinositidase C. Recent studies utilizing various bacterial toxins have strongly suggested the presence of multiple G‐proteins in the regulation of receptor‐phosphoinositidase C coupling in a variety of cell types.
Monoclonal antibodies against chordin Use in structural and immunohistochemical studies
Authors: Preobrazhensky A.A. , Rodionova A.I. , Trakht I.N. , Rukosuev V.S.
Published: July 2002

Eight MAbs have been developed against chordin and designated as At2–At9. It is shown that all antibodies are directed against identical, spatially overlapping or closely positioned epitopes of chordin. The chordin molecule has repetitive sites wherein epitopes for the eight MAbs are located. This site lies within a protein ase‐resistant fragment of chordin, presumably a glycopeptide, of molecular mass between 2 and 10 kDa. Fluorescence staining of cryostat sections from stellate sturgeon with the use of At5 (indirect Coons' method) has revealed a positive reaction with notochord cells and sheath and with the spinal cord. No significant reaction with cartilage, muscle and kidney was detected.
Assessment of salvage pathways utilized for incorporation of exogenous pyrimidine nucleosides into DNA of guinea pig lymphocytes stimulated by Con A
Authors: Ringer David P. , Howell Boyd A. , Etheredge Janet L. , Clouse Joe A. , Kizer Donald E.
Published: July 2002

The organization of specific pyrimidine pathways to channel various nucleoside precursors into DNA is poorly understood. We show that concanavalin A‐stimulated guinea pig lymphocytes incorporate [3H]dThd, [3H]dCyd, [3H]dUrd, [3H]Cyd and [3H]Urd into DNA‐thymines and DNA‐cytosines in a highly conserved distribution pattern. DNA‐thymines were labeled only by dThd and dUrd, while DNA‐cytosines were labeled only by dCyd, Cyd and Urd. The kinetics for the incorporation of the [3H]nucleosides were essentially identical, indicating equivalent abilities to measure DNA synthesis. Pyrazofurin inhibition of the pyrimidine de novo synthetic pathway inhibited cell proliferation and the levels of [3H]nucleoside incorporation by approx. 50%, but did not alter restricted distribution of the [3H]nucleosides among DNA‐thymines and DNA‐cytosines. These findings indicate the absence of Cyd and dCMP deaminase salvage pathways and suggest either subcellular compartmentalization or differential regulation of ribonucleoside diphosphoreductase which permits reduction of CDP but not UDP.
Multiple effects of α‐toxins on the nicotinic acetylcholine receptor
Authors: Bradley R.J. , Pagala M.K. , Edge M.T.
Published: July 2002

Very low concentrations (5 nM) of α‐toxin from the venom of Naja naja atra produced a characteristic fade in muscle compound action potential and tetanus induced by repetitive nerve stimulation which was identical to the effects of curare. High concentrations of α‐toxin and all concentrations of α‐bungarotoxin reduced the response but produced very little fade in comparison to curare. These results suggest that α‐toxins have more than one effect at the neuromuscular junction.
Lipocortin‐like anti‐phospholipase A2 activity of endonexin
Authors: Fauvel Josette , Salles Jean-Pierre , Roques Véronique , Chap Hugues , Rochat Hervé , Douste-Blazy Louis
Published: July 2002

Endonexin (protein II, 32.5 kDa) has been purified to homogeneity from bovine liver in the following steps: selective extraction by EGTA from membranes precipitated with Triton X‐100/calcium; chromatography on DEAE‐TSK 545 at pH 7.0, endonexin being eluted at 0.1 M NaCl; affinity chromatography on polyacrylamide‐immobilized phosphatidylserine; gel filtration on TSK 3000. The amino acid composition was essentially similar to that previously reported. Using [3H]oleic acid‐labelled Escherichia coli membranes as substrate, endonexin inhibited phospholipase A2 from pig pancreas. Maximal inhibition was 55 and 70%, whereas 50% inhibition occurred at 480 and 120 nM endonexin and lipocortin II, respectively. These data could be related to common features shared by both lipocortins/calpactins and endonexin, i.e. the presence of a consensus sequence and the ability to bind to anionic phospholipids in a calcium‐dependent manner.
Isolation of several cDNAs encoding yeast peroxisomal enzymes
Authors: Ueda Mitsuyoshi , Okada Hirofumi , Hishida Tadashi , Teranishi Yutaka , Tanaka Atsuo
Published: July 2002

Several candidate clones carrying partial cDNAs for yeast peroxisomal enzymes, such as catalase, carnitine acetyltransferase, isocitrate lyase, malate synthase and acyl‐CoA oxidase, were efficiently isolated at a single plating from a phage λgt11 recombinant cDNA library prepared with poly(A)‐rich RNA from an n‐alkane‐grown yeast, Candida tropicalis, with a mixture of antibodies against the respective purified enzymes. Among them, one candidate clone carrying partial cDNA for catalase was subcloned and subjected to nucleotide sequence analysis. We succeeded in determining that the amino acid sequence deduced from the nucleotide analysis included the sequences derived from the two peptide fragments obtained from the purified enzyme.
Isolation of two 67 kDa calcium‐binding proteins from pig lung differing in affinity for phospholipids and in anti‐phospholipase A2 activity
Authors: Fauvel Josette , Vicendo Patricia , Roques Véronique , Ragab-Thomas Jeannie , Granier Claude , Vilgrain Isabelle , Chambaz Edmond , Rochat Hervé , Chap Hugues , Douste-Blazy Louis
Published: July 2002

Two 67 kDa proteins adsorbed to membranes in the presence of Ca2+ have been purified to homogeneity from pig lung using conventional procedures, followed by calcium‐dependent affinity chromatography on polyacrylamide‐immobilized phosphatidylserine. The two proteins were, respectively, excluded (67E) and retained (67R) on the column in the presence of Ca2+. On the basis of amino acid composition and isoelectric point, 67R was identified as 67 kDa calelectrin/calcimedin, whereas 67E could be differentiated from albumin, calregulin, 67 kDa fragment of protein kinase C and surfactant‐associated proteins. Only 67R was slightly phosphorylated by protein kinase C, reacted with an antibody raised against 32.5 kDa endonexin and inhibited pig pancreas phospholipase A2 in a way similar to that of lipocortin or endonexin. These data bring further support to the view that inhibition of phospholiphase A2 by lipocortin or other related proteins involves interaction with the lipid/water interface. They also provide evidence for a new kind of Ca2+‐binding protein (67E), whose role still remains to be determined.
Modulation of stimulatory action of follicle stimulating hormone (FSH) and inhibitory action of epidermal growth factor (EGF) on aromatase activity in Sertoli cells by calcium
Authors: Mallea L.E. , Machado A.J. , Navaroli F. , Rommerts F.F.G.
Published: July 2002

Aromatization of testosterone by cultured Sertoli cells isolated from immature rats was stimulated more than 7‐fold by follicle stimulating hormone (FSH) or dcAMP. The effects of FSH and dcAMP could be partly inhibited by epidermal growth factor (EGF) in a dose‐dependent manner (ID50 0.5 nM). The phorbol ester 4β‐phorbol‐12‐myristate‐13‐acetate (PMA) could also inhibit aromatase activity in a fashion similar to EGF. When 3 mM EGTA was present in the culture medium, the inhibitory effect of EGF was abolished but the stimulatory effect of FSH or dcAMP was magnified. These results suggest that EGF exerts a negative control on aromatase via calcium and protein kinase C. The abolishment of the inhibitory effect of EGF and the enhancement of the stimulatory effect of FSH or dcAMP by a calcium deficiency may be an indication that growth factors produced by Sertoli cells negatively control FSH‐induced responses in an autocrine fashion.
Light‐ and nucleotide‐dependent increase in apparent viscosity in a suspension of retinal disks
Authors: Caretta Antonio , Stein Peter J.
Published: July 2002

Light triggers the cyclic nucleotide cascade in photoreceptor disk membranes. We report here that light‐induced changes in the apparent viscosity of disk membrane suspensions can also be observed using either native disk membranes or washed membranes reconstituted with G protein and PDE. The viscosity changes are light‐ and GTP‐dependent and require the presence of G protein and PDE. The magnitude of the viscosity change increases with increasing membrane concentration. Under the same conditions in which light elicits a change in viscosity, we observe a large increase in light scattering by the disk membrane suspension.
Co‐solubilization of asymmetric acetylcholinesterase and dermatan sulfate proteoglycan from the extracellular matrix of rat skeletal muscles
Authors: Brandan Enrique , Inestrosa Nibaldo C.
Published: July 2002

We have previously communicated that heparin released asymmetric acetylcholinesterase (AChE) from cholinergic synapses. Here we report studies showing that heparin, besides releasing asymmetric AChE from the skeletal muscle extracellular matrix (ECM), specifically solubilizes a dermatan sulfate proteoglycan (DSPG) which accounts for more than 95% of the 35S‐released material. The co‐solubilization of AChE and the proteoglycan opens up the possibility that both macromolecules could be involved in the formation of the soluble AChE complex observed after incubation of muscle homogenate with heparin. Our results suggest a possible association between asymmetric AChE and DSPG at the muscle ECM, moreover this work is the first report of the existence of DSPG at the skeletal muscle cell surface.
Guanine nucleotide‐induced Ca2+ release in permeabilized murine thymocytes
Authors: Ueda Teruko , Ichikawa Yoichi , Kusaka Iwao
Published: July 2002

GTP and IP3 induced Ca2+ release from an internal store in permeabilized murine thymocytes loaded with Ca2+ by ATP. Ca2+ release was dependent on the concentration of GTP: half‐maximal release with 0.5 μM and maximal release with 10 μM. The GTP effect was completely abolished by 100 μM GTPγS, GMPPNP and UTP. None of the other nucleotides used except ITP induced Ca2+ release. When GTP was added after the effect of IP3 had virtually subsided, and vice versa, further Ca2+ release occurred, which led to the conclusion that the mechanism of GTP‐mediated Ca2+ release may be different from that of IP3‐mediated release.
Purification and partial characterization of a calmodulin‐like protein from cell suspension cultures of Catharanthus roseus
Authors: Radvanyi Laszlo G. , DiCosmo Frank
Published: July 2002

A calmodulin‐like protein was isolated from suspension cultured cells of Catharanthus roseus and purified by a combination of Ca2+‐facilitated phenyl‐Sepharose affinity chromatography and reverse‐phase HPLC. The HPLC‐purified protein was analysed using SDS‐PAGE and found to be a homogeneous 19.5 kDa band in gels containing 1 mM EGTA unlike a higher plant calmodulin from spinach which migrated as a 17.38 kDa band. Despite this apparent difference in molecular mass, the purified protein showed a similar increase in electrophoretic mobility (4010 Da) to spinach calmodulin (4200 Da) in gels containing 1 mM Ca2+ and had a plant calmodulin‐like UV spectrum and phosphodiesterase‐activation profile.
Gn‐proteins are distinct from ras p21 and other known low molecular mass GTP‐binding proteins in the platelet
Authors: Bhullar Rajinder P. , Haslam Richard J.
Published: July 2002

The 27 kDa platelet membrane protein (Gn27) that binds [α‐32P]GTP on nitrocellulose blots of SDS‐polyacrylamide gels [(1987) Biochem. J. 245, 617–620] was compared with other low molecular mass GTP‐binding proteins. Platelet membranes also contained 21 kDa proteins that bound anti‐ras p21 antibody and 22–23 kDa proteins that could be ADP‐ribosylated by botulinum neurotoxin type D. These groups of proteins were resolved electrophoretically from each other and from Gn27. A low molecular mass GTP‐binding protein from bovine brain [(1987) Biochem. J. 246, 431–439] was also resolved from Gn27. At the levels normally present in cell membranes, only Gn‐proteins bound significant amounts of [32P]GTP after transfer of protein from SDS‐polyacrylamide gels to nitrocellulose.
Binding of monovalent cations induces large changes in the secondary structure of Na+,K+‐ATPase as probed by Raman spectroscopy
Authors: Nabiev I.R. , Dzhandzhugazyan K.N. , Efremov R.G. , Modyanov N.N.
Published: July 2002

Raman spectra of active Na+,K+‐ATPase from pig kidney in media containing Na+ (E1), K+ (E2) or without exogenous ions (E1 conformation) were recorded in order to calculate the changes in the enzyme's secondary structure induced by binding of monovalent cations. It is demonstrated that: (i) K+ binding to the E1 form of the enzyme leads to conversion of 100 peptide groups from the β‐structure to α‐helical conformation; (ii) the transition is reversible and fully reproducible in the E1→E2→E1 and E2→E1→E2 experimental schemes. Predictional calculations revealed polypeptide chain segments involved in the α ↔ β transformations. These segments reside mainly in the two highly conserved regions of the α‐subunit in the cytoplasmic domain of Na+,K+‐ATPase. A possible role for the β‐subunit is discussed.
Production of a truncated human c‐myc protein which binds to DNA
Authors: Naoe Tomoki , Shiku Hiroshi , Yamada Kazumasa , Okazaki Tuneko , Kurosawa Yoshikazu
Published: July 2002

Two kinds of truncated human c‐myc proteins were produced in Escherichia coli. The human c‐myc gene is composed of three exons, exons 2 and 3 having coding capacity for a protein of 439 amino acids. 252 N‐terminal amino acids are encoded by exon 2, the C‐terminal 187 amino acids being encoded by exon 3. One of the proteins (p42) produced in E. coli corresponds to 342 amino acids from the 98th Gln to the C‐terminus, plus 21 amino acids derived from the H‐ras gene at the N‐terminus. The other (p23) corresponds to 155 amino acids from the 98th Gln to the 252nd Ser, plus five amino acids (Gly‐Gly‐Thr‐Arg‐Arg) at the C‐terminus, plus 21 amino acids from the H‐ras gene at the N‐terminus. The p23 protein was produced by using cDNA in which a frame shift occurred at the boundary between exons 2 and 3. We investigated the DNA‐binding activity in p42 and p23 proteins. DNA‐cellulose column chromatography showed that p42 binds to DNA, whereas p23 does not. This DNA‐binding activity of p42 was inhibited by antiserum prepared against p42 but not by antiserum against p23. This indicates that the DNA‐binding activity of c‐myc protein is localized in the portion encoded by exon 3.
A new pathway for phosphatidylserine synthesis in rat liver microsomes
Authors: Baraǹska Jolanta
Published: July 2002

It has been shown that the phosphate moiety of glycerophosphate is incorporated into phosphatidylserine of rat liver microsomes, but not of mitochondria. The reaction is dependent on CMP. This observation suggests that the new pathway of acyl‐specific synthesis of phosphatidylserine proposed by J.P. Infante [(1984) FEBS Lett. 170, 1–14] can proceed in rat liver microsomes.
Single‐channel Ba2+ currents in insulin‐secreting cells are activated by glyceraldehyde stimulation
Authors: Velasco J.M. , Petersen J.U.H. , Petersen O.H.
Published: July 2002

Single‐channel Ba2+ current recordings have been made from the insulin‐secreting cell line RINm5F with the patch‐clamp technique. Depolarization evokes opening of Ca2+ (Ba2+) channels with a relatively high conductance (30 pS) and during the 200 ms depolarizing pulses there is no inactivation. The threshold is high as 50 mV depolarization from the resting membrane potential of −70 mV is required for any channel opening to occur. Glyceraldehyde, a substance evoking insulin secretion from the RINm5F cells, enhances the voltage‐activated Ca2+ channel opening by increasing the mean open time and decreasing the longer of the two mean shut times and also decreases the voltage threshold for channel opening.