bluuebunny/crossref_metadata_2025 huggingface dataset:
Maturation of rabbit reticulocytes: susceptibility of mitochondria to ATP‐dependent proteolysis is determined by the maturational state of reticulocyte
Authors: Rapoport S. , Schmidt J. , Prehn S.
Published: July 2002

(1) A simple procedure is described to separate reticulocytes of different maturity in high yield. (2) It is shown that exhaustion of supply of mitochondria susceptible to degradation by the lipoxygenase‐ATP‐dependent proteolysis system limits the extent of breakdown of mitochondria during in vitro maturation. The susceptibility of mitochondria depends on the maturity of the reticulocytes. (3) Incubation in the presence of calcium ions and calcium ionophore leads to full susceptibility of mitochondria in immature reticulocytes but has no effect on those in mature reticulocytes which are already fully susceptible to degradation. (4) Conditions which lead to rapid degradation of mitochondria do not affect the behaviour of the reticulocyte count. There appears to be no obligatory connection between the breakdown of mitochondria and of ribosomes.
Production of peptide leukotrienes in endotoxin shock
Authors: Hagmann Wolfgang , Denzlinger Claudio , Keppler Dietrich
Published: July 2002

Arachidonate metabolites are potent mediators generated in endotoxin shock. Following endotoxin administration (15 ) into unanesthetized rats, we found a rapid biliary secretion of peptide leukotrienes. Analysis of bile for peptide leukotrienes included organic solvent extractions, reversed phase‐HPLC, radioimmunoassay (RIA), and spectrophotometry. The major immunoreactive endogenous leukotriene (LT) from bile was eluted between LTC4 and LTD4 in three chromatographic systems. It corresponded thereby to a biliary metabolite of injected LTC4 and LTD4 which in turn showed the ultraviolet spectrum of a peptide leukotriene. This demonstration of endotoxin‐induced generation of peptide LTs in vivo was possible by sequential HPLC and RIA analyses in bile into which peptide LTs are eliminated from blood.
One‐domain interaction of histone H4 with nucleosomal core DNA is restricted to a narrow DNA segment
Authors: Ebralidse K.K. , Mirzabekov A.D.
Published: July 2002

The interaction of histone H4 with DNA in the nucleosomal core particle has been studied by crosslinking DNA to proteins through their lysine residues. We have compared the crosslinked peptides of H4 at the detected DNA‐binding sites: H4(55), H4(65), H4(88), located, respectively, at about 55, 65 and 88 nucleotides from the core DNA termini. For all these binding sites, the patterns of crosslinked peptides were shown to be very similar. This suggests the presence of a single DNA‐binding domain in the H4 molecule. The H4‐binding sites are located within a narrow DNA segment close to one another on the complementary strands across the DNA grooves, overlap with sites ± 1 of the DNA sharp bending [(1984) Nature 311, 532‐537] and with the strong binding sites for histone H3: H3(75) and H3(85).
Heterogeneity of adrenocortical ferredoxin
Authors: Hiwatashi Atsuo , Sakihama Naoko , Shin Masateru , Ichikawa Yoshiyuki
Published: July 2002

Bovine adrenocortical ferredoxin (adreno‐ferredoxin) was purified from adrenocortical mitochondria by an improved method that included hydrophobic chromatography on Toyopearl gels. The purified ferredoxin was electrophoretically homogeneous. It was further separated into five fractions by hydrophobic chromatography on a TSK‐gel phenyl‐5PW column with a high‐pressure liquid chromatography system. The properties of the three main fractions were examined. The fractions had identical absorption spectra and almost the same activity in an NADPH‐cytochrome c reducing system. Their amino‐terminal sequences all corresponded to the reported sequence, but the carboxyl‐terminal residues were glycine or serine, not alanine as reported. These results indicate that these adreno‐ferredoxins had additional amino acid residues at the carboxyl end. It seems that adreno‐ferredoxin extracted from mitochondria undergoes proteolytic attack during purification to become heterogeneous.
Transcription control of the 32 kDa‐QB protein of photosystem II in differentiated bundle sheath and mesophyll chloroplasts of maize
Authors: Schuster Gadi , Pecker Iris , Hirschberg Joseph , Kloppstech Klaus , Ohad Itzhak
Published: July 2002

Bundle sheath chloroplasts of maize, a C4 plant, lack a functional herbicide‐binding site and the 32 kDa‐QB thylakoid protein of photosystem II. Measurements of the amounts of QB‐protein mRNA in bundle sheath and mesophyll chloroplasts by hybridization to a cloned psbA probe, and analysis of in vitro translation products of bundle sheath and mesophyll RNA show that during differentiation of maize leaf cells to bundle sheath and mesophyll, the expression of the chloroplast gene, psbA, which encodes the 32 kDa‐QB protein, is controlled at the transcriptional level.
Phylloquinone copurifies with the large subunit of photosystem I
Authors: Schoeder H.-U. , Lockau W.
Published: July 2002

Photosystem I preparations from the cyanobacteriumAnabaenavariabilisand from spinach were analyzed for their quinone content. The dominant quinone was identified as phylloquinone. It was present at a ratio of roughly 2 per P700 (the primary electron donor of photosystem I) both in particles prepared with Triton X‐100 and on the large, P700‐carrying subunit derived from these particles by treatment with SDS. The results suggest a function of phylloquinone in the reaction center.
Studies on inorganic pyrophosphatase using imidodiphosphate as a substrate
Authors: Smirnova Irina N. , Baykov Alexander A. , Avaeva Svetlana M.
Published: July 2002

Baker's yeast inorganic pyrophosphatase has been found to catalyze Mg2+‐dependent hydrolysis of imidodiphosphate yielding phosphate and amidophosphate. The reaction proceeds linearly in the presteady state. The catalytic constant is maximal at pH 9.0 and equals 0.5 min−1. Kinetic titrations of the enzyme with imidodiphosphate and Mg2+ have provided direct evidence for the involvement of three Mg2+ per active site in the transition state of the pyrophosphatase reaction.
Flow‐force relationships in mitochondrial oxidative phosphorylation
Authors: Woelders Henri , Putters Jacques , van Dam Karel
Published: July 2002

The rates of oxidation and phosphorylation in isolated rat‐liver mitochondria have a steep dependence on the protonmotive force (Δ≈sm
h
) across the membrane. These experimentally observed relationships proved to be independent of the way in which δ≈gm
h
was varied. These results were obtained when the membrane potential (ΔΨ) was calculated from the distribution of K+ (in the presence of valinomycin). When triphenyl‐methylphosphonium (TPMP+) was used as a probe for ΔΨ, slightly different flow‐force relationships were obtained. We conclude that unique relationships exist between Δ≈sm
h
+ and the rates of oxidation and phosphorylation, and that under some conditions the behaviour of the probe TPMP+ is anomalous.
The generation of an organic free radical in substrate‐reduced pig kidney diamine oxidase‐cyanide
Authors: Dooley David M. , McGuirl Michele A. , Peisach Jack , McCracken John
Published: July 2002

When the cyanide complex of the copper protein, pig kidney diamine oxidase, is reduced anaerobically by cadaverine (1,5‐diaminopentane), the broad, 480 nm absorption band characteristic of the resting enzyme is bleached and a new absorption spectrum with features at 457, 429, 403 (shoulder), 360 (shoulder) and 332 nm appears. Concomitantly, the EPR spectrum of the enzyme Cu(II)‐CN complex decreases in intensity and a new signal is observed that is attributable to an organic free radical. The g values and hyperfine splittings are similar to those previously assigned to a free radical observed when the cyanide complex of lentil seedling diamine oxidase is reacted with the substrate p‐dimethylaminomethylbenzylamine [(1984) FEBS Lett. 176, 378–380]. The optical absorption and EPR spectra of the organic radical observed in both proteins are consistent with the same semiquinone‐type structure, as expected if pyrroloquinolinequinone (PQQ) is the bound cofactor found in both enzymes.
Identification of the structures of multiple subspecies of protein kinase C expressed in rat brain
Authors: Kikkawa Ushio , Ono Yoshitaka , Ogita Kouji , Fujii Tomoko , Asaoka Yoshinori , Sekiguchi Kazuo , Kosaka Yoshiyuki , lgarashi Koichi , Nishizuka Yasutomi
Published: July 2002

Rat brain protein kinase C purified to apparent homogeneity [(1986) Biochem. Biophys. Res. Commun. 135, 636–643] was resolved into three distinct fractions, type I, II and III, upon chromatography on a hydroxyapatite column connected to high‐performance liquid chromatography. Comparison of each fraction with the four subspecies of protein kinase C, that were separately expressed in COS cells transfected by the respective cDNAs, α, βI,βII an d γ, identified the primary structures of these three fractions of protein kinase C. Type I corresponded to the enzyme encoded by the γ‐sequence; type II was a mixture of the two subspecies determined by the βI‐ and βII‐sequences; and type III had the structure encoded by the α‐sequence. The structures and properties of these subspecies of protein kinase C were similar to each other.
Regulation of thylakoid protein phosphorylation by high‐energy‐state quenching
Authors: Oxborough Kevin , Lee Pam , Horton Peter
Published: July 2002

The possible co‐regulation of light‐induced protein phosphorylation by the redox state of plastoquinone and the transthylakoid ΔpH was investigated in isolated pea chloroplasts. Incorporation of γ‐32P‐labelled ATP into the LHC‐II and 9 kDa phosphoproteins in the coupled state was reduced by 54 and 28%, respectively, when compared to the uncoupled state. Selective inhibition of energy‐state‐dependent quenching of chlorophyll fluorescence by antimycin A in the coupled state greatly reduced this inhibition of protein phosphorylation and subsequent quenching of chlorophyll fluorescence. These results suggest that the protein kinase(s) involved in phosphorylation of the LHC‐II and 9 kDa phosphoproteins is regulated by either the energy state of the thylakoid membrane or the redox state of some component of the electron‐transport chain, rather than by ΔpH per se.
Citrate synthase from the thermophilic archaebacteria Thermoplasma acidophilum and Sulfolobus acidocaldarius
Authors: Smith Leon D. , Stevenson Kenneth J. , Hough David W. , Danson Michael J.
Published: July 2002

Citrate synthase has been purified to homogeneity from the thermophilic archaebacteria Thermoplasma acidophilum and Sulfolobus acidocaldarius. From the relative molecular masses of the native proteins (85 and 83 kDa, respectively) and of their polypeptide chains (43 and 41 kDa, respectively) it is established that they are dimeric enzymes. The N‐terminal sequence of the Thermoplasma citrate synthase was determined to be P‐E‐T‐E‐E‐I‐S‐K‐G‐L‐E‐D‐V‐N‐I‐K. These properties are compared with those of citrate synthases from eubacteria and eukaryotes to extend the pattern of structural and functional diversity previously observed for this enzyme in non‐archaebacterial species.
The dependence of the rate of transhydrogenase on the value of the protonmotive force in chromatophores from photosynthetic bacteria
Authors: Cotton N.P.J. , Myatt J.F. , Jackson J.B.
Published: July 2002

In conditions where the pH gradient is negligible, the rate of the pyridine nucleotide transhydrogenase in chromatophores of Rhodobacter capsulatus has a threshold dependence on membrane potential. The relationship is similar when either antimycin or myxothiazal or carbonyl cyanide p‐trifluoromethoxyphenylhydrazone is used to depress the membrane potential. The relationship is distorted when membrane potential is reduced by lowering the photosynthetic light intensity.
Pi∠ATP exchange in the absence of proton gradient by the H+ ‐ATPase from yeast plasma membranes
Authors: de Meis Leopoldo , Blanpain Jean-Philippe , Goffeau André
Published: July 2002

Purified soluble H+ ‐ATPase from Schizosaccharomyces
pombe catalyzes a Pi∠ATP exchange in the absence of a H+ gradient. When the pH of the assay medium is raised from 5.5 to 8.0 there is a decrease of the ATPase activity and an increase of the rate of Pi∠ATP exchange. At pH 7.0 the addition of the organic solvent dimethyl sulfoxide (20%, ) promotes a decrease of ATPase activity and an increase of the Pi∠ATP exchange reaction. The effect of the organic solvent on the Pi∠ATP exchange is related to a decrease of the apparent K
m for Pi.
Identification of the serotonin‐S2 receptor ligand binding site by photoaffinity labelling with 7‐azido‐8‐[125I]ketanserin ([125I]AZIK)
Authors: Wouters Walter , Van Dun Jacky , Laduron Pierre M.
Published: July 2002

7‐Azido‐8‐[125I]ketanserin ([125I]AZIK) was characterized as a potent photoaffinity probe for serotonin‐S2 receptors. In reversible binding experiments, [125I]AZIK bound with high affinity (Kd = 0.69 nM) to rat frontal cortex membranes. When incubation with [125I]AZIK was followed by UV irradiation, the binding was found to be irreversible. Protection experiments with various drugs demonstrated the serotonin‐S2 nature of the photoaffinity labelling. SDS‐polyacrylamide gel electrophoresis of the photolabelled membranes allowed one to identify the serotonin‐S2 receptor ligand binding site as a single polypeptide with a molecular mass of approx. 67500 Da. [125I]AZIK will be a valuable tool for the elucidation of the serotonin‐S2 receptor structure.
Importance of sulfhydryl group for rabbit gastric lipase activity
Authors: Moreau Hervé , Gargouri Youssef , Pieroni Gérard , Verger Robert
Published: July 2002

We have shown recently that rabbit gastric lipase (RGL) purified from gastric tissue presents catalytic properties comparable with those of human gastric lipase (HGL). We report here that only one sulfhydryl group was modified per molecule of native RGL after incubation at pH 8.0 with 5,5′‐dithiobis(2‐nitrobenzoic acid) (NbS2) for 4 h or 4,4′‐dithiopyridine (4‐PDS) for 60 min. With both reagents, a direct correlation was found between the modification of one sulfhydryl group per enzyme molecule and loss of RGL activity. Incubation of RGL with the new hydrophobic sulfhydryl reagent, dodecyldithio‐5‐(2‐nitrobenzoic acid) (C12‐NbS), at 30‐fold molar excess, at pH 3.0, 5.0 and 8.0, induced immediate and complete inactivation of RGL. Unlike NbS2 and 4‐PDS, C12‐NbS almost instantaneously stopped the course of tributyrin hydrolysis by RGL, in contrast to porcine pancreatic lipase (PPL). RGL can be included with HGL in the group of sulfhydryl enzymes.
High level of expression in the prostate of a human glandular kallikrein mRNA related to prostate‐specific antigen
Authors: Chapdelaine Pierre , Paradis Gilles , Tremblay Roland R. , Dubé Jean Y.
Published: July 2002

Using a synthetic oligonucleotide primer complementary to human prostate‐specific antigen mRNA, we found that an additional sequence possibly similar to human glandular kallikrein‐1 could be read by a primer‐extension sequencing technique. We were able to confirm the identity of that additional sequence with another oligonucleotide primer complementary to a specific region of the human glandular kallikrein‐1 mRNA sequence. Northern blot analysis with 2 oligonucleotide probes respectively specific for prostate‐specific antigen and human glandular kallikrein‐1 mRNAs showed that the length of both mRNAs was similar at 1.5 kb. The level of human glandular kallikrein‐1 mRNA relative to that of prostate‐specific antigen could be estimated as approx. 10–20%. This study constitutes the first evidence that the human glandular kallikrein‐1 gene is expressed at a high level in a human tissue.
Identification of α‐subunit Lys201 and β‐subunit Lys115 at the ATP‐binding sites inEscherichia coli F1‐ATPase
Authors: Tagaya Mitsuo , Noumi Takato , Nakano Kenichi , Futai Masamitsu , Fukui Toshio
Published: July 2002

Binding of about 1 mol of adenosine triphosphopyridoxal to Escherichia coli F1‐ATPase resulted in the nearly complete inactivation of the enzyme [(1987) J. Biol. Chem. 262, 7686–7692]. About two thirds of the label was bound to the α‐subunit, and the rest to the β‐subunit. The present study revealed that Lys201 in the α‐subunit and Lys155 in the glycinerich region of the β‐subunit are the major sites labeled with this reagent. Thus, these two residues might be located close to the γ‐phosphate of the bound ATP.
Characterization of an intronless human calmodulin‐like pseudogene
Authors: Koller Markus , Strehler Emanuel E.
Published: July 2002

We report the isolation and characterization of a human genomic clone encoding a calmodulin‐like pseudogene. It contains an open reading frame of 444 nucleotides, not interrupted by introns. The nucleotide sequence of the open reading frame shows 80%, 71% and 69% identity to the previously reported human calmodulin cDNAs λht6 [17], hCWP [22], and λhCE1 [23], respectively. The derived amino acid sequence has only 85% identity to vertebrate calmodulin, but shows four potentially functional Ca2+‐binding loops. In the human tissues tested, this pseudogene is not expressed, though gene structure including promoter elements and a putative polyadenylation site seems to be intact.
The IFI‐56K and IFI‐54K interferon‐inducible human genes belong to the same gene family
Authors: Wathelet Marc G. , Clauss Isabelle M. , Content Jean , Huez Georges A.
Published: July 2002

The IFI‐56K and IFI‐54K human genes are coordinately regulated by interferon, double‐stranded RNA and viruses in a number of cell lines. These genes encode polypeptides of 56 and 54 kDa, respectively, whose function remains to be determined. We analysed the possible structural relatedness between these syntenic and similarly regulated genes. We found that they are very closely related at the protein, mRNA and promoter levels. This suggests that the IFI‐56K and IFI‐54K genes are members of a gene family, which probably arose from duplication of an ancestor gene.
Applications of a CP‐II model to excitation transfer experiments on light‐harvesting chlorophylla/b‐protein complexes
Authors: Lin Su , Knox Robert S.
Published: July 2002

A theoretical study of energy‐transfer rates between chlorophyllamolecules within light‐harvesting chlorophylla/bcomplexes of photosystem II (LHC‐II) based on transient absorption recovery data [(1985) FEBS Lett. 193, 267–270] has been made using a trimer model of interacting chromophores [(1977) Biochim. Biophys. Acta 462, 642–658]. It provides a kinetic explanation of the data and shows that the experimental results on LHC‐II are consistent with and therefore support the model.
Phosphorylation of the glycogen‐binding subunit of protein phosphatase‐1G in response to adrenalin
Authors: MacKintosh Carol , Campbell David G. , Hiraga Akira , Cohen Philip
Published: July 2002

The glycogen‐binding (G) subunit of protein phosphatase‐1 is phosphorylated in vivo. In rabbits injected with propranolol the serine residue termed site‐1 was phosphorylated in 56% of the molecules isolated, and phosphorylation increased to 82% after administration of adrenalin. It is concluded that the G‐subunit is a physiological substrate for cyclic AMP‐dependent protein kinase. The G‐subunit remained largely bound to glycogen even after injection of adrenalin, whereas half of the protein phosphatase‐1 activity associated with glycogen was released into the cytosol. The results indicate that adrenalin induces dissociation of the catalytic subunit from the G‐subunit in vivo.
Maturation‐dependent changes of the rabbit reticulocyte energy metabolism
Authors: Kostić M.M. , Rapoport S.M.
Published: July 2002

Rabbit reticulocytes were separated into four fractions of different maturity in order to investigate the changes of cellular respiration and glycolysis, adenine nucleotides, 2,3‐biphosphoglycerate (2,3‐BPG) as well as cyclic AMP level during the transition from the youngest to the most mature reticulocytes. A significant reduction of total oxygen consumption, mainly due to depression of coupled respiration was found. The decline of respiration was accompanied by a 2‐fold increase of the rate of aerobic glycolysis indicating a reduced Pasteur effect during maturation. A decline of ATP and an increase of ADP concentration was found. The oxygen‐delivery capacity of the red cells increased by about 26% caused by an increase of the 2,3‐BPG level of about 2 mmol/l cells. Cyclic AMP level in the fraction of youngest reticulocytes was about 60‐fold higher than that in mature rabbit erythrocytes. The biggest decline of cyclic AMP was registered during the transition from youngest to the intermediate stage of maturity.
Phosphorylation of small peptides by spleen TPK‐IIA, a tyrosine protein kinase stimulated by polylysine and by high ionic strength
Authors: Brunati Anna Maria , Marchiori Fernando , Ruzza Paolo , Calderan Andrea , Borin Gianfranco , Pinna Lorenzo A.
Published: July 2002

The specificity of TPK‐IIA, a spleen tyrosine protein kinase whose activity is dramatically enhanced by either polylysine or very high ionic strength, has been inspected with the aid of a variety of very short peptide substrates. Unlike free tyrosine, which is not phosphorylated at all, and the dipeptides YA and AY, whose phosphorylation is hardly detectable, the tripeptides AYA, YYY and YYA, tetrapeptide AYAA and pentapeptide AAYAA are readily phosphorylated, the V
max of AYA being 7‐ and 15‐fold lower vs those of AYAA and AAYAA, respectively. In comparison to AYA, the tripeptides YAA, EYA, GYA and VYV are poorer substrates, while EYH is unaffected by TPK‐IIA. The rate of phosphorylation of the pentapeptide EYAA is negligible as compared to AYAA; the hexapeptide EEYAA, however, is a substrate quite comparable to AAYAA, exhibiting a somewhat lower V
max but also a 4‐fold lower K
m (3.9 vs 18.6 mM). Taken together, the data would indicate that although TPK‐IIA does not exhibit any absolute requirement for a definite consensus sequence it is nevertheless endowed with peptide substrate specificity, mainly resulting from negative determinants that can be variably overcome by additional features of the sequence.
Arctic adaptation in reindeer The energy saving of a hemoglobin
Authors: Giardina Bruno , Brix Ole , Nuutinen Matti , El Sherbini Said , Bardgard Astrid , Lazzarino Giuseppe , Condò Saverio G.
Published: July 2002

Previous results [(1988) Arct. Med. Res. 47, 83–88] have shown that hemoglobin from reindeer is characterized by a low overall heat of oxygenation. This particular aspect has been investigated further in a series of precise oxygen equilibrium experiments. The results obtained show a peculiar dependence of the temperature effect on the fractional saturation of hemoglobin with oxygen, which could be regarded as a very interesting case of molecular adaptation to extreme environmental conditions.
Rat pineal S‐antigen: Sequence analysis reveals presence of α‐transducin homologous sequence
Authors: Abe T. , Yamaki K. , Tsuda M. , Singh V.K. , Suzuki S. , McKinnon R. , Klein D.C. , Donoso L.A. , Shinohara T.
Published: July 2002

S‐antigen (S‐Ag) is a soluble, highly antigenic protein, the administration of which induces autoimmune uveitis. This protein is found in the retina and pineal. Retinal S‐Ag from three species has been sequenced. In this study rat pineal S‐Ag was sequenced. Clones were isolated from a rat pineal λgt11 cDNA library by probing with a 300 bp fragment of mouse retinal S‐Ag cDNA containing the 5′‐coding region. The largest clone isolated (RPS‐118; 1364 bp) contained the entire coding sequence. Comparison of the rat pineal and mouse retinal S‐Ag nucleotide sequences indicated a high homology (95%). The deduced amino acid sequence was found to contain 403 residues (≌44 992 Da). Comparison of the rat pineal and mouse retinal S‐Ag amino acid sequences also revealed high homology (97%). The similarity of both the nucleotide and amino acid sequences of rat pineal and mouse retinal S‐Ag indicates that expression of the S‐Ag gene in both tissues is similar. Further analysis of the rat pineal S‐Ag sequence indicated that it contained essentially the same major uveitopathogenic region of S‐Ag present in bovine retina; minor uveitopathogenic sites were somewhat different. As is true of retinal S‐Ag, rat pineal S‐Ag contains the same consensus phosphoryl‐binding site present in many GTP/ GDP‐binding proteins and a homologous sequence found in the C‐terminus of α‐transducin. These sequences may play a role in the action of pineal S‐Ag in transmembrane signal transduction.
Engineering of microheterogeneity‐resistant p‐hydroxybenzoate hydroxylase from Pseudomonas fluorescens
Authors: Eschrich Klaus , van Berkel Willem J.H. , Westphal Adrie H. , de Kok Arie , Mattevi Andrea , Obmolova Galina , Kalk Kor H. , Hol Wim G.J.
Published: July 2002

By site‐directed mutagenesis, Cys‐116 was converted to Ser‐116 in p‐hydroxybenzoate hydroxylase (EC 1.14.13.2) from Pseudomonas fluorescens. In contrast to wild‐type enzyme, the C116S mutant is no longer susceptible to oxidation by hydrogen peroxide and shows no reactivity towards 5,5'‐dithiobis(2‐nitrobenzoate). Crystals of the C116S mutant are isomorphous with the crystal form of wild‐type enzyme. A difference electron density confirms the mutation made.
Purification, some properties and nucleotide sequence of 5‐carboxymethyl‐2‐hydroxymuconate isomerase of Escherichia coli C
Authors: Roper David I. , Cooper Ronald A.
Published: July 2002

As part of an investigation into the evolution of catabolic pathway enzymes a cloned gene encoding the Escherichia coli C 5‐carboxymethyl‐2‐hydroxymuconate (CHM) isomerase, an enzyme of the homoprotocatechuate catabolic pathway, was used to produce large amounts of the protein. The isomerase was purified to homogeneity and some of its properties determined. The reaction occurred optimally at pH 7.6 and the specificity constant was 5.8 × 105 M−1·s−1 with CHM and 6.0 × 102 M−1·s−1 with 2‐hydroxyhepta‐2,4‐diene‐1,7‐dioate, the substrate of a second isomerase in the pathway. The pure protein showed one type of subunit of M
r 14 000 whilst the molecular mass of the native enzyme was 30 000, suggesting that it was a dimer of identical subunits. The first 19 N‐terminal amino acids were sequenced and the data used to confirm that the open reading frame of 378 bp, identified from the nucleotide sequence, encoded the CHM isomerase.
ATP‐dependent leukotriene export from mastocytoma cells
Authors: Schaub Thomas , Ishikawa Toshihisa , Keppler Dietrich
Published: July 2002

The biosynthesis of leukotrienes (LT) C4 and B4 is followed by an export of these mediators into the extracellular space. This transport was characterized using plasma membrane vesicles prepared from mastocytoma cells and identified as an ATP‐dependent primary active process. The apparent Km
‐values were 110 nM for LTC4 and 48 μM for ATP. The transport rate was highest for LTC4, whereas LTD4, LTE4, and N‐acetyl‐LTE4 were transported with relative rates of 31, 12 and 8%, respectively, at a concentation of 10nM. LTB4 transport was also dependent on ATP, LTC4 transport with inhibited by LTD4 receptor antagonists (IC10 = 1.0 μM for MK‐571 and 1.3 μM for LY245769) and by the inhibitor of leukotriene biosynthesis MK‐886 (IC10 = 1.8 μM). The ATP‐dependent export carrier for leukotrienes in leukotriene‐synthesizing cells represents a novel member of the family of ATP‐dependent exit pumps.
Sequence‐specific binding of the N‐terminal three‐finger fragment of Xenopus transcription factor IIIA to the internal control region of a 5S RNA gene
Authors: Christensen Jorgen Holst , Hansen Peter Kamp , Lillelund Ove , Thøgersen Hans Christian
Published: July 2002

An N‐terminal fragment of Xenopus TFIIIA, containing domains 1–3, (TF 3), was expressed in E. coli. High yields of recombinant zinc finger protein was isolated, and its DNA binding activity for the internal control region (ICR) of the Xenopus 5S RNA gene, was demonstrated by bandshift experiments and DNase I footprinting analysis. TF 3 protects 20 bp of ICR against DNase I digestion. The limits of protection are from +77 to +96 on both coding and noncoding strand. This protection pattern is identical to the protection pattern obtained with TFIIIA in the overlapping region, showing that the 3‐finger fragment accounts fully for the protein‐DNA interactions in TFIIIA‐5S RNA gene over this region.
Status of the cofactor identity in copper oxidative enzymes
Authors: Klinman J.P. , Dooley D.M. , Duine J.A. , Knowles P.F. , Mondovi B. , Villafranca J.J.
Published: July 2002

Much conflicting data have appeared in the literature regarding the nature of the active site structures responsible for catalysis in three classes of copper enzymes: the copper amine oxidases, dopamine β‐monooxygenase and galactose oxidase. Although pyrroloquinoline quinone has been proposed to be the active site cofactor in each instance, new findings indicate this is not the case. Instead, recently available data indicate a spectrum of strategies for substrate activation, which range from direct metal catalysis (dopamine β‐monooxygenase) to the involvement of protein‐derived radicals (galactose oxidase) and protein‐derived quinones (copper amine oxidases).
Basic fibroblast growth factor (bFGF) in rodent testis Presence of bFGF mRNA and of a 30 kDa bFGF protein in pachytene spermatocytes
Authors: Lahr Georgia , Mayerhofer Artur , Seidl Klaus , Bucher Sabine , Grothe Claudia , Knöchel Walter , Gratzl Manfred
Published: July 2002

We have previously described a 30 kDa basic fibroblast growth factor (bFGF)‐like protein in rodent testicular homogenates and have shown that pachytene spermatocytes are the sites of predominant immunoreactivity for this bFGF‐like protein (Mayerhofer, A., Russell, L.D., Grothe, C., Rudolf, M. and Gratzl, M. (1991) Endocrinology 129, 921–924). We have now addressed the question whether this 30 kDa bFGF‐like protein is a large bFGF form and whether it is produced by pachytene spermatocytes. We detected bFGF mRNA in homogenates of isolated mouse spermatocytes (which consisted mainly of pachytene spermatocytes) using S1 nuclease protection assays. As shown by Western blot analyses, the bFGF mRNA in mouse spermatocytes is translated into bFGF of an approximate molecular weight of 30 kDa. Neither bFGF mRNA, nor bFGF itself, was observed in isolated mouse Leydig cells. These results indicate that the immunoreactive bFGF‐like protein observed previously in germ cells of the murine testis is identical to bFGF. Thus, germ cells of the testis produce bFGF, which may exert regulatory function in the process of spermatogenesis.
Tissue specificity of the regulation of ATP hydrolysis by isolated plant mitochondria
Authors: Valeric Marie , Haraux Francis , Gardeström Per , Diolez Philippe
Published: July 2002

Pea leaf mitochondria had a high ATP hydrolase activity following the collapse of the membrane potential by addition of valinomycin in state 4. In mitochondria isolated from potato tubers such ATP hydrolase activity was not observed. Pea leaf mitochondria also had a ΔpH, in contrast to what was previously found for potato tuber mitochondria. This ΔpH could, however, not explain the different results on ATP hydrolysis since this activity was also observed in the presence of nigericin. The results suggest a tissue‐specific regulation of ATP hydrolysis in resting organs (potato tubers) as compared to active organs (leaves).
Cloning and sequencing of rat liver cDNAs encoding the regulatory protein of glucokinase
Authors: Detheux Michel , Vandekerckhove Joël , Van Schaftingen Emile
Published: July 2002

cDNAs encoding the rat liver regulatory protein of glucokinase were cloned and sequenced. The deduced protein contains 568 amino acids for a molecular mass of 62,867 Da. Northern blot analysis showed the presence of a major RNA species of 2.35 kb in rat liver. No signal was observed with muscle, brain, heart, testis, intestine or spleen RNA. Recombinant regulatory protein expressed in Escherichia coli was insoluble and inactive, and was presumably contained in inclusion bodies. Western blot analysis showed that the recombinant protein was recognized by antibodies raised against regulatory protein purified from rat liver.
Myo‐inositol 1,3,4,5‐tetrakisphosphate can independently mobilise intracellular calcium, via the inositol 1,4,5‐trisphosphate receptor: studies with myo‐inositol 1,4,5‐trisphosphate‐3‐phosphorothioate and myo‐inositol hexakisphosphate
Authors: Wilcox Robert A. , Whitham Emma.M. , Liu Changsheng , Potter Barry V.L. , Nahorski Stefan R.
Published: July 2002

Myo‐inositol 1,3,4,5‐tetrakisphosphate [Ins(1,3,4,5)P4] acts as a full agonist for Ca2+ release in saponin‐permeabilised SH‐SY5Y neuroblastoma cells. Studies were conducted in the presence of myo‐inositol hexakisphosphate (InsP6, 10 μM), to inhibit the Ins(1,3,4,5)P4‐3‐phosphatase catalysed back conversion of Ins(1,3,4,5)P4 to Ins(1,4,5)P3. HPLC analysis confirmed that Ins(1,3,4,5)P4 releases the entire content of Ins(1,4,5)P3‐sensitive intracellular Ca2+ stores, independent of 3‐phosphatase activity. Further we utilised racemic myo‐inositol 1,4,5‐trisphosphate‐3‐phosphorothioate [dl‐Ins(1,3,4,5)P4‐3S], a novel intrinsically Ins(1,3,4,5)P4‐3‐phosphatase resistant Ins(1,3,4,5)P4 analogue. DL‐Ins(1,3,4,5)P4‐3S specifically displaced [3H]Ins(1,4,5)P3 from bovine adrenal cortex Ins(1,4,5)P3 binding sites (IC50 = 889 nM, compared to Ins(1,4,5)P3,1C50 = 4.4 nM and Ins(1,3,4,5)P4, IC50 = 152 nM). dl‐Ins(1,3,4,5)P4‐3S was a full agonist for Ca2+ release (EC50 = 4.7 μM), being 90‐ and 2‐fold less potent than Ins(1,4,5)P3 and Ins(1,3,4,5)P4 (with InsP6), respectively. dl‐Ins(1,3,4,5)P4‐3S will be an important tool for identification of potentially exclusive Ins(1,3,4,5)P4 second messenger functions, since its resistance to 3‐phosphatase action precludes the inconvenient artefact of steady state Ins(1,4,5)P3 generation.
Molecular characterization of the red visual pigment gene of the American chameleon (Anolis carolinensis)
Authors: Kawamura Shoji , Yokoyama Shozo
Published: July 2002

The red sensitive visual pigment of the American chameleon, Anolis carolinensis, is unique in having absorption maximum some 50 nm further into the red than any other terrestrial vertebrate examined. We report here the isolation and sequence determination of the genomic DNA clone for the Anolis red visual pigment gene. Phylogenetic analysis shows that this gene is most closely related to the gecko green and chicken red visual pigment genes. We identified nine Anolis‐specific amino acid replacements, seven of which reside in transmembrane domains and might contribute to the red‐shift of the Anolis visual pigments.
Modelling, synthesis and biological activity of a BLV proteinase, made of (only) 116 amino acids
Authors: Précigoux G. , Geoffre S. , Léonard R. , Llido S. , Dautant A. , d'Estaintot B.Langlois , Picard P. , Ménard A. , Guillemain B. , Hospital M.
Published: July 2002

Bovine leukaemia virus (BLV) is the aetiological agent of Leukosis enzootica bovis [Viral Oncology (1980), G. Klein (Ed.) Raven Press, New York, pp. 231–238], a widely spread disease in cattle. BLV is reported as the animal model of human T‐cell leukaemia virus (HLTV) which is the causative agent of adult T‐cell leukaemia and tropical spastic paraparesis. Like the viruses themselves, the two retroviral proteinases (PR) are very closely related [Virology 142 (1985) 357–377]. BLV and HTLV‐I PR are reported as putative proteins made of 126 [J. Virol. 57 (1986) 826 832] and 125 [FEBS Lett. 293 (1991) 106–110] amino acids, respectively (long sequences), belonging to the aspartyl proteinase family [Nature 329 (1987) 351–354] with the aid of molecular modelling, we show that BLV and HLTV‐I proteinases made of only 116 and 115 amino acids, respectively (short sequences), display three‐dimensional structures similar to that observed for other retroviral aspartyl proteinases. The models are based on three‐dimensional structures of the Rous sarcoma virus (RSV PR) and the human immunodeficiency virus (HIV‐I PR). We used solid phase peptide synthesis to produce the putative proteolytic enzyme of BLV (116 amino acids). In this study, we show that the folded synthetic protease accurately hydrolyzes a decapeptide corresponding to the sequence of the Matrice—Capside (MA/CA) cleavage site of the gag polyprotein. In addition, the proteolytic activity is inhibited by a statine ((4S,3S)‐4‐amino‐3‐hydroxyl‐6‐methylheptanoic acid) containing an analogous sequence.
Activation of Na,K‐ATPase by an endogenous peptide, PEC‐60
Authors: Kairane Czeslawa , Zilmer Mihkel , Mutt Viktor , Sillard Rannar
Published: July 2002

Several peptidic and non‐peptidic factors can modulate Na,K‐ATPase activity, among them mainly inhibitors of this enzyme, ouabain being the most effective. In a very few cases only, activation of Na,K‐ATPase by endogenous factors has been recorded. We have investigated the effect on Na,K‐ATPase of a novel regulatory peptide, PEC‐60, recently isolated from porcine intestine. Various biological effects have been described for PEC‐60 in different tissues, including brain. We have found that PEC‐60 caused a dose‐dependent activation of Na,K‐ATPase from rat brain frontal cortex, whereas the carboxymethylated form of PEC‐60 or other hormonal peptides had no effect. The maximal value of activity reaches up to 125% at close to micromolar concentrations of PEC‐60 and the dependence can be described with a bell‐shaped curve, indicating a complex mechanism for the interaction. The activation of the enzyme by PEC‐60 is apparently related to Na+‐dependent steps of the Na,K‐ATPase system. The kinetic parameters for K+‐phosphatase were unaffected. Moreover, the activating effect was enhanced by preincubation at low concentrations of ATP that transform the enzyme into the Na+‐form. Due to the crucial physiological role of Na,K‐ATPase, its activity has to be finely controlled and thus PEC‐60 may be one of the endogenous factors that regulate this enzyme.
Folding and aggregation of DNA chains induced by complexation with lipospermine: formation of a nucleosome‐like structure and network assembly
Authors: Yoshikawa Yuko , Emi Nobuhiko , Kanbe Toshio , Yoshikawa Kenichi , Saito Hidehiko
Published: July 2002

Dioctadecylamidoglycylspermine (DOGS) is a cationic lipid vector capable of efficiently introducing DNA into various eukaryotic cells. We investigated the higher‐order structure of the DNA/DOGS complex using fluorescence and electron microscopy. Our results show that the DNA/DOGS complex exhibits a nucleosome‐like structure in which DNA wraps around an aggregate of DOGS molecules. In addition, DNA/DOGS complexes tend to associate with each other to form network structures. The resulting network assembly may play a role in effective gene transfection.
The international Classification of Impairments, Disabilities, and Handicaps (ICIDH): mental health aspects of its use in rehabilitation
Authors: Üstün TB , van Duuren-Kristen S , Bertolote J , Cooper JE , Sartorius N
Published: July 2002

This paper summarizes several issues related to the mental health aspects of the International Classification of Impairments, Disabilities, and Handicaps (ICIDH) and its use in the field of rehabilitation. Rehabilitation is characterized by multiple interventions, demanding extensive communication and coordination among different care providers. The ICIDH will be useful in the field of rehabilitation by providing a sound framework for consequences of diseases or disorders if clear descriptions and assessment guidelines of the categories are given. The development of dedicated assessment instruments linked to the ICIDH will be useful to many health workers to assess the needs of the patients, to evaluate services and to measure the outcomes of rehabilitation interventions, thus creating a common language for disablements.
The interaction between Alzheimer amyloid β(1–40) peptide and ganglioside GM1‐containing membranes
Authors: Choo-Smith Lin-P'ing , Surewicz Witold K
Published: July 2002

The interaction between Alzheimer amyloid peptide Aβ(1–40) and membrane lipids was studied by circular dichroism spectroscopy under the conditions of physiologically relevant ionic strength and neutral pH. The peptide binds to the membranes containing ganglioside GM1and upon binding undergoes a conformational transition from random coil to an ordered structure rich in β‐sheet. This interaction appears to be ganglioside‐specific as no changes in Aβ(1–40) conformation were found in the presence of various phospholipids or sphingomyelin. The isolated oligosaccharide moiety of the ganglioside was ineffective in inducing alterations in the secondary structure of Aβ(1–40). No interaction was observed between ganglioside GM1and the N‐terminal peptide fragment Aβ(1–28). Binding to the ganglioside is likely to modulate the neurotoxic and/or amyloidogenic properties of Aβ(1–40).
Heterologous coexpression of the blue light receptor psRII and its transducer pHtrII fromNatronobacterium pharaonisin theHalobacterium salinariumstrain Pho81/w restores negative phototaxis
Authors: Lüttenberg B , Wolff E.K , Engelhard M
Published: July 2002

The photophobic receptor (psRII) and its transducer pHtrII fromNatronobacterium pharaoniswere heterologously coexpressed in the phototaxis‐deficientHalobacterium salinariumstrain Pho81/w which lacks all four bacterial rhodopsins, i.e. the two ion pumps bacteriorhodopsin and halorhodopsin as well as the two sensory pigments SRI and SRII. This genetically transformed Pho81/w strain showed a photophobic response upon illumination with blue light. The action spectrum of the psRII/pHtrII mediated phototactic behavior was determined in the range of 420–600 nm. The shape of the action spectrum was similar to the absorption spectrum of psRII, clearly indicating that the psRII‐specific photophobic response in Pho81/w was restored. These results suggest that thepharaonisphotoreceptor‐transducer complex (psRII/pHtrII) is functionally competent to substitute the correspondingsalinariumreceptor system. Although the two archaea are phylogenetically quite distant from each other the two signal transduction chains are homologous systems which can replace each other.
Caloric restriction prevents age‐related decline in skeletal muscle dihydropyridine receptor and ryanodine receptor expression
Authors: Renganathan M. , Delbono O.
Published: July 2002

The dihydropyridine receptor (DHPR), a voltage‐gated L‐type Ca2+channel, and the Ca2+release channel/ryanodine receptor isoform‐1 (RyR1) are key molecules involved in skeletal muscle excitation‐contraction coupling. We have reported age‐related decreases in the level of DHPR expression in fast‐ and slow‐twitch muscles from Fisher 344 cross Brown Norway (F344BNX) rats (Renganathan, Messi and Delbono, J. Membr. Biol. 157 (1997) 247–253). Based on these studies we postulate that excitation‐contraction uncoupling is a basic mechanism for the decline in muscle force with aging (Delbono, Renganathan and Messi, Muscle Nerve Suppl. 5 (1997) S88–92). In the present study, we extended our studies to older ages and we intended to prevent or retard excitation‐contraction uncoupling by restricting the caloric intake of the F344BNX rats from 16 weeks of age. Three age groups, 8‐, 18‐, and 33‐month old caloric restricted rats, were compared with ad libitum fed animals. The number of DHPR and RyR1 and DHPR/RyR1 ratio (an index of the level of receptors uncoupling) in skeletal muscles of 8‐month and 18‐month rats was not significantly different in either ad libitum fed or caloric restricted rats. However, the age‐related decrease in the number of DHPR, RyR1 and DHPR/RyR1 ratio observed in 33‐month old ad libitum fed rats was absent in 33‐month old caloric restricted rats. These results suggest that caloric restriction prevents age‐related decreases in the number of DHPR, RyR1 and DHPR/RyR1 ratio observed in fast‐ and slow‐twitch rat skeletal muscles.
Quantitative structure activity relationships for the electron transfer reactions ofAnabaenaPCC 7119 ferredoxin‐NADP+oxidoreductase with nitrobenzene and nitrobenzimidazolone derivatives: mechanistic implications
Authors: Anusevicius Zilvinas , Soffers Ans E.M.F , Cenas Narimantas , Sarlauskas Jonas , Martinez-Julvez Marta , Rietjens Ivonne M.C.M
Published: July 2002

The steady state single electron reduction of polynitroaromatics by ferredoxin‐NADP+oxidoreductase (EC 1.18.1.2) from cyanobacteriumAnabaena PCC 7119has been studied and quantitative structure activity relationships are described. The solubility of the polynitroaromatics as well as their reactivity towards ferredoxin‐NADP+oxidoreductase are markedly higher than those for previously studied mononitroaromatics and this enabled the independent measurement of the kinetic parameterskcatandKm. Interestingly, the natural logarithm of the bimolecular rate constant,kcat/Km, and also the natural logarithm ofkcatcorrelate with the calculated energy of the lowest unoccupied molecular orbital of the polynitroaromatic substrates. The minimal kinetic model in line with these quantitative structure activity relationships is a ping‐pong mechanism which includes substrate binding equilibria in the second half reaction.
The gonadotropin‐releasing hormone family of neuropeptides in the brain of human, bovine and rat: identification of a third isoform
Authors: Yahalom Dror , Chen Alon , Ben-Aroya Nurit , Rahimipour Shai , Kaganovsky Ella , Okon Eli , Fridkin Mati , Koch Yitzhak
Published: July 2002

The mammalian gonadotropin‐releasing hormone (GnRH‐I), which regulates reproduction, was the first isoform of GnRH that was identified in mammals. Recently, we and others have demonstrated the existence of a second isoform of GnRH in the brain of mammals. The presence of a third isoform of GnRH, GnRH‐III, in the brain of mammals is reported herein. GnRH‐III, extracted from the brain of bovine and human, was purified by high performance liquid chromatography, using two distinct elution programs. In both, GnRH‐III was eluted at the same positions as synthetic salmon GnRH, as demonstrated by radioimmunoassay. The luteinizing hormone‐releasing activity of purified GnRH‐III, using dispersed rat pituitary cells, was found to be similar to that of synthetic salmon GnRH. The total amount of GnRH‐III, determined by radioimmunoassay, in the hypothalamus and midbrain of humans and calves is similar to that of GnRH‐I. Immunohistochemical studies demonstrated GnRH‐III‐containing neurons in the hypothalamus and midbrain of human and GnRH‐III fibers in the median eminence of rats. The distribution of GnRH‐III in the brain suggests that in addition to a putative function as a neurohormone at the hypothalamic–pituitary axis, GnRH‐III may have other functions. Our present results suggest that multiple isoforms of GnRH are present in the brain of mammals, and further studies are required in order to elucidate their biological functions.
NMR structure of the channel‐former zervamicin IIB in isotropic solvents
Authors: Balashova T.A. , Shenkarev Z.O. , Tagaev A.A. , Ovchinnikova T.V. , Raap J. , Arseniev A.S.
Published: July 2002

Spatial structure of the membrane channel‐forming hexadecapeptide, zervamicin IIB, was studied by NMR spectroscopy in mixed solvents of different polarity ranging from CDCl3/CD3OH (9:1, v/v) to CD3OH/H2O (1:1, v/v). The results show that in all solvents used the peptide has a very similar structure that is a bent amphiphilic helix with a mean backbone root mean square deviation (rmsd) value of ca. 0.3 Å. Side chains of Trp1, Ile2, Gln3, Ile5and Thr6are mobile. The results are discussed in relation to the validity of the obtained structure to serve as a building block of zervamicin IIB ion channels.