bluuebunny/crossref_metadata_2025 huggingface dataset:
Metabolic dependence of glycolytic enzyme binding in rat and sheep heart
Authors: CLARKE Frank M. , STEPHAN Petra , HUXHAM Graham , HAMILTON Dan , MORTON Don J.
Published: March 2005

Perfused rat hearts show a markedly increased binding of phosphofructokinase and fructose‐bisphosphate aldolase as a consequence of ischaemia, but little change in binding of pyruvate kinase, lactate dehydrogenase or glyceraldehyde‐3‐phosphate dehydrogenase. After 10 min ischaemia over one quarter of the phosphofructokinase and three quarters of the aldolase are bound. The effect of anoxia is less well marked in its influence on binding with only aldolase showing a significant increase in binding. These results suggest that one factor involved in the increased binding during ischaemia is the fall in pH of the heart. Binding studies with isolated myofibrils confirm that the affinity and stoichiometry of aldolase binding are considerably increased as the pH is lowered over a range comparable to that which occurs in ischaemic heart. The low level of binding of glyceraldehyde‐3‐phosphate dehydrogenase in perfused rat hearts correlates with the relatively low affinity of this enzyme for binding to rat or rabbit cardiac myofibrils. There are species differences in the enzyme binding response to ischaemia. Sheep hearts show rapid and large increases in the binding of glyceraldehyde‐3‐phosphate dehydrogenase in addition to changes in aldolase and phosphofructokinase binding. The greater binding of glyceraldehyde‐3‐phosphate dehydrogenase reflects the greater affinity of sheep cardiac myofibrils. It is suggested that the altered metabolic demands of ischaemia are satisfied by changes in glycolytic enzyme organisation as the enzymes shift from the soluble to the particulate phase of cardiac muscle.
Interactions in vivo between IIIGlc of the phosphoenolpyruvate: sugar phosphotransferase system and the glycerol and maltose uptake systems of Salmonella typhimurium
Authors: NELSON Stephen O. , POSTMA Pieter W.
Published: March 2005

Our previous studies indicated that the ability of phosphoenolpyruvate: sugar phosphotransferase system (PTS) substrates to inhibit the uptake of glycerol or maltose in Salmonella typhimurium is dependent on the relative cellular content of the PTS‐sensitive uptake system and of the PTS protein IIIGlc. Our present study confirms and extends those observations. The maltose and glycerol uptake systems are rendered (wholly or partially) insensitive to PTS inhibition by the presence of a second PTS‐sensitive uptake system (respectively that for glycerol or maltose) and its substrate. Both the second PTS‐sensitive uptake system and its substrate were needed for this protective effect. Galactose and the galactose permease (a PTS‐insensitive transport system) did not have any effect on PTS‐mediated inhibition of the maltose uptake system. The protective effect of the second PTS‐sensitive uptake system and its substrate is counteracted by increasing the cellular levels of IIIGlc. Overproduction of IIIGlc in crr‐plasmid‐containing strains renders the glycerol and maltose uptake systems hypersensitive to inhibition by PTS substrates. We interpret our results on the basis of a stoichiometric interaction between IIIGlc and a PTS‐sensitive uptake system, in which the IIIGlc– transport‐system complex is inactive. Competition between two PTS‐sensitive transport systems for formation of inactive complex with IIIGlc lowers the free intracellular concentration of IIIGlcresulting in a mutual protective effect against inhibition by IIIGlc.
Alpha secondary structures generate weak but recurrent periodicity in proteins
Authors: WUILMART Christian , URBAIN Jacques
Published: March 2005

Weak but recurrent periodic patterns characterize numerous actual proteins: the rate at which similarity occurs between residues i and i+ 4 or i+ 7 or i+ 11 is very often slightly higher than predicted by chance. That result could indicate that numerous actual proteins derive from ancestral clearcut periodic sequences. Nevertheless, it is shown that this recurrent periodic pattern occurs much more significantly when analyses are restricted to the α‐helical portions of proteins while it never occurs when β‐stranded subsequences are taken into account. This preferential location of periodicity inside protein subregions corresponding to α helices suggests that the recurrent pattern of weak periodicity could result from an ubiquitous physical property of α helices. The regular alternation of hydrophobicity, which is most often displayed by α helices, could then be the origin of weak periodicity.
Accounting for the ATP‐consuming processes in rabbit reticulocytes
Authors: SIEMS Werner , DUBIEL Wolfgang , DUMDEY Renate , MÜLLER Marianne , RAPOPORT Samuel Mitja
Published: March 2005

The report deals with a detailed balance of ATP production and consumption of the rabbit reticulocyte.

1. The sum‐total of ATP produced amounts to 135mmol · l−1· h−1.

2. About 70% of the ATP consumption has been accounted for by specific processes. The main contributing processes are the globin synthesis with about 28%, the Na+, K+‐ATPase with 23% and the proteolysis with more than 15%. 30% of ATP consumption has not been accounted for.

3. Cycloheximide (20 μM) leads to a dissociation between synthesis and degradation of proteins, which argues against any obligatory connection between these processes.

4. More than 90% of the lysine liberated from mitochondria by proteolysis were reutilized for the globin synthesis demonstrating the high nitrogen economy of reticulocytes.

5. Each of the ATP‐consuming processes studied appears to control ATP production in an independent manner without competition with each other.
1H‐NMR studies of calmodulin
Authors: KLEVIT Rachel E. , DALGARNO David C. , LEVINE Barry A. , WILLIAMS Robert J. P.
Published: March 2005

Using assignments of resonances in the 1H NMR spectrum of calmodulin obtained by the use of large tryptic fragments of the molecule [Dalgarno, D. C., Klevit, R. E., Levine, B. A., Williams, R. J. P., Dobrowolski, Z., and Drabikowski, W. (1984) Eur. J. Biochem. 138, 281–289], the spectral changes which occur on Ca2+ binding to calmodulin have been examined in detail. Ca2+ binding occurs in two stages: the first two Ca2+ ions bind at sites III and IV (numbered from the N terminus) and the second two Ca2+ ions bind at sites I and II. The high‐affinity binding causes perturbations of residues in both halves of the molecule, whereas binding at the two N‐terminal sites only affects sidechains in that half of the molecule. The effects of binding Cd2+ to the Ca2+ ‐binding sites of calmodulin have also been studied by 1H NMR. The cation induces spectral changes which are very similar to those seen for Ca2+, but some important differences do exist.
Raman spectroscopy and normal vibrations of peptides
Authors: LAGANT Philippe , VERGOTEN Gérard , FLEURY Guy , LOUCHEUX‐LEFEBVRE Marie‐H.
Published: March 2005

Raman and infrared spectra have been recorded on the peptide Gly‐lPro‐Gly‐Gly in its zwitterionic form and on its protected homolog Z‐Gly‐lPro‐Gly‐Gly‐OMe (Z=N‐benzyloxycarbonyl and OMe = methyl ester). Both peptides adopt a type‐II β‐turn conformation into appropriate solvents, that is in dimethylsulfoxide and chloroform respectively.

A normal coordinate treatment has been performed on Gly‐lPro‐Gly‐Gly using a modified Urey‐Bradley force field and the characteristic normal modes of vibrations. Their corresponding frequencies have been extracted from experimental investigations.
The Shigella flexneri O‐antigenic polysaccharide chain Nature of the biological repeating unit
Authors: CARLIN Nils I. A. , LINDBERG Alf A. , BOCK Klaus , BUNDLE David R.
Published: March 2005

The sequence of monosaccharides in the biological repeating tetrasaccharide unit of Shigella flexneri variant Y O‐antigenic polysaccharide chain was determined by subjecting three oligosaccharides of the polysaccharide, obtained by phage‐Sf6‐mediated enzymatic hydrolysis, to methylation analysis and proton nuclear magnetic resonance spectroscopy. The smallest saccharide was shown to be a tetrasaccharide with the structure α‐l‐Rhap‐(1–3)‐β‐d‐GlcpNAc‐(1–2)‐α‐l‐Rhap‐(1–2)‐l‐Rha. The next saccharide, an octasaccharide, was shown to be a dimer of the tetrasaccharide with the l‐Rha residues linked α1.3. The longest saccharide was shown to be a decasaccharide with the following structure: α‐l‐Rhap‐(1–2)‐α‐l‐Rhap‐(1–3)‐α‐l‐Rhap‐(1–3)‐β‐d‐GlcpNAc‐(1–2)‐α‐l‐Rhap‐(1–2)‐α‐l‐Rhap‐(1–3)‐α‐l‐Rhap‐(1–3)‐β‐l‐GlcpNAc‐(1–2)‐α‐l‐Rhap‐(1–2)‐l‐Rha. Thus the decasaccharide differed from the octasaccharide and tetrasaccharide by having the α‐l‐Rhap‐(1–2)‐α‐Rhap disaccharide added in the terminal non‐reducing end of the saccharide chain. This shows that the α‐l‐Rhap‐(1–2)‐α‐l‐Rhap‐(1–2)‐α‐l‐Rhap‐(1–3)‐d‐GlcpNAc tetrasaccharide is the biological repeating unit of the O chain and that the repeating units are joined through a β‐d‐GlcpNAc‐(1–2)‐l‐Rhap linkage. Inhibition experiments utilizing the enzyme‐linked immunosorbent assay (ELISA) with S. flexneri Y lipopolysaccharide/S. flexneri Y rabbit antiserum showed that the decasaccharide was the best inhibitor (threefold as active as the octasaccharide and sixtyfold as active as the tetrasaccharide); this supports the postulated structure of the biological repeating unit.
Acyltransferase‐catalyzed cleavage of arachidonic acid from phospholipids and transfer to lysophosphatides in macrophages derived from bone marrow Comparison of different donor‐ and acceptor substrate combinations
Authors: FLESCH Inge , ECKER Barbara , FERBER Ernst
Published: March 2005

In a previous paper it was shown that in prelabeled murine thymocytes a direct CoA‐mediated transfer of arachidonic acid from phosphatidylcholine to lysophosphatidylethanolamine occurs which does not involve the intermediate formation of free fatty acid. The transfer is ATP‐independent and is catalyzed by the acyl‐CoA: lysophosphatide acyltransferase operating in reverse. In prelabeled thymocytes phosphatidylcholine was the only arachidonoyl donor and lysophosphatidylethanolamine the only lysoacceptor.

In murine bone‐marrow‐derived macrophages a series of CoA‐mediated transfer reactions were detected leading to a redistribution of arachidonic acid between phospholipids. Using exogenous substrates a bidirectional transfer from 1‐acyl‐2‐arachidonoylglycerophosphocholine to lysophosphatidylethanolamine occurs. An unidirectional transfer from l‐acyl‐2‐arachidonoylglycerophosphoinositol to lysophosphatidylcholine and from 1‐acyl‐2‐arachidonoylglycerophosphoinositol to lysophosphatidylethanolamine was observed. Plasmalogenic lysoacceptors generally have a weaker acceptor capacity than the correspondent acyllysophospholipid. In macrophages the CoA‐mediated transfer of arachidonoyl moieties is independent of ATP and Mg2+ and is totally inhibited by sodium cholate, indicating that it is catalyzed by the acyl‐CoA: lysophosphatide acyltransferase.
Ribosomal RNA‐protein cross‐links, induced by γ‐irradiation of 40S and 60S ribosomal subunits of L cells
Authors: CAZILLIS Michèle , GIOCANTI Nicole , HOUSSAIS Jean‐François , EKERT Bernard
Published: March 2005

The 40S and 60S ribosomal subunits of L cells are γ‐irradiated in the absence of oxygen at low radiation doses to keep the integrity of the ribosomal structure. We show that under these experimental conditions, specific cross‐links are induced in situ between rRNA and ribosomal proteins due to close contact between their reactive groups.

We found that about 15 proteins are cross‐linked to the 28S RNA. Most of them belong to the core proteins of the 60S ribosomal subunits. A few high‐molecular mass proteins which might be factors are also bound to 28S RNA.

Between 8 and 11 proteins are covalently linked to 18S RNA; 8 of these have been previously described as transferable proteins from one subunit to the other. Only 3 are core proteins of the small subunit.

The contribution of these results to the study of the three‐dimensional ribosomal structure is also discussed.
Recognition by restriction endonuclease EcoRI of deoxyoctanucleotides containing modified sugar moieties
Authors: OHTSUKA Eiko , ISHINO Yoshizumi , IBARAKI Kazuhiko , IKEHARA Morio
Published: March 2005

The deoxyribooctanucleotide d(G‐G‐A‐A‐T‐T‐C‐C), containing the recognition sequence for EcoRI, d(G‐A‐A‐T‐T‐C), and analogs containing modified sugar moieties were tested for their activity in cleavage with EcoRI. These analogs, with replacement in the third position from the 5′ end, were synthesized using 9‐β‐d‐arabinosyladenine (aA), 2′‐deoxy‐2′‐fluoroadenosine (Afl) and adenosine (rA). Duplex formation by the three analogs was confirmed by measurements of ultraviolet/temperature profiles. It was found that EcoRI cleaved these duplexes less efficiently than d(G‐G‐A‐A‐T‐T‐C‐C). The adenosine‐containing analog d(G‐G)‐rA‐d(A‐T‐T‐C‐C) was cleaved much more slowly than d(G‐G)‐aA‐d(A‐T‐T‐C‐C) and d(G‐G‐Afl‐A‐T‐T‐C‐C). The corresponding ribooctamer G‐G‐A‐A‐U‐U‐C‐C showed a higher melting temperature than the deoxyoctamers but its duplex was not cleaved by this enzyme. An analog with 2′‐deoxy‐2′‐fluoroguanosine at the second position was cleaved by the endonuclease faster than the natural deoxyoctamer.
Comparison of the tryptic digestion pattern of subfragments 1 from V1 and V3 rat cardiac isomyosins
Authors: LOMPRÉ Anne‐Marie , HAN Kia‐Ki , BOUVERET Pierre , RICHARD Claude , SCHWARTZ Ketty
Published: March 2005

The limited tryptic digestion patterns of the chymotryptic subfragment 1 (S1) of the two rat ventricular isomyosins V1 and V3, were compared under several conditions. Pure S1V1 was obtained from 3‐week‐old rats and pure S1V3 from adult rats 6 weeks after hypophysectomy. To localize the sites of trypsin susceptibility and to determine the distribution of the peptides along the S1 molecule, we used, as a probe, antibodies raised against a pig cardiac 29‐kDa peptide. We demonstrate that this peptide contains the N‐acetyl group located on the N‐terminal part of the cardiac myosin molecule.

In S1V1 we observed two major sites of proteolysis, independently of the digestion conditions: they are located at 27kDa and 80kDa from the N terminus as in skeletal muscle S1. S1V3 appears much more sensitive to the proteolysis conditions: at least two additional sites of cleavage are present in the 50‐kDa peptide when digested at pH 8.0. Decrease in the pH from 8.0 to 7.0 or the presence of Mg‐ATP have no effect on the digestion of S1V1 while these ambient factors protect the 50‐kDa peptide of S1V3 from breakdown. We conclude that the 50‐kDa peptide is a variable portion of the myosin molecule, the conformation of which is sensitive to ambient factors such as the pH or the presence of nucleotides.
Use of a monoclonal antibody to distinguish between precursor and mature forms of human lysosomal α‐glucosidase
Authors: OUDE ELFERINK Ronald P. J. , STRIJLAND Anneke , SURYA Inge , BROUWER‐KELDER Elisabeth M. , KROOS Marian , HILKENS John , HILGERS Jo , REUSER Arnold J. J. , TAGER Joseph M.
Published: March 2005

The maturation of lysosomal α‐glucosidase in cultured human skin fibroblasts was studied using a monoclonal antibody that distinguishes between the precursor and mature forms of the enzyme.

1. Monoclonal antibodies against α‐glucosidase isolated from placenta were produced by the hybridoma technique [Hilkens et al. (1981) Biochim. Biophys. Acta 678, 7–11]. One of these monoclonal antibodies, that synthesized by clone 43G8, reacts with the mature forms, but not with the precursor form of α‐glucosidase isolated from urine.

2. By means of pulse‐labelling studies, it could be shown that monoclonal antibody 43G8 does not react with either the intracellular or the secreted precursor of α‐glucosidase from cultured human skin fibroblasts. However, the antibody does react with the intermediate and mature forms of α‐glucosidase.

3. Endocytosis of the precursor of α‐glucosidase from urine by fibroblasts is followed by its conversion to a form with lower molecular mass.

4. After endocytosis urinary precursor α‐glucosidase is converted to a form that binds to monoclonal antibody 43G8. The t1/2 for this conversion is 2 h. The conversion is inhibited by addition of leupeptin to the culture medium.

5. It is concluded that a thiol proteinase is involved in the maturation of α‐glucosidase in fibroblasts and the appearance of the antigenic determinant for 43G8.
Monoclonal antibodies against placental‐like and intestinal‐like alkaline phosphatases in a malignant human cell line
Authors: WRAY Larry K. , HARRIS Harry
Published: March 2005

Monoclonal antibodies were raised against alkaline phosphatase (ALP) from the malignant human cell line Hep2, a derivative of HeLa, which was established from a cervical carcinoma. Two forms of ALP are found in Hep2 cells resembling, but not identical to, the normal placental and intestinal ALPs. Seven monoclonal antibodies were raised against Hep2 ALP. All of these reacted nearly identically with normal placental ALP and the placental‐like ALP from Hep2 cells. Three of these antibodies exhibited reduced reactivities for either the common type 2 or 3 allelic variants of placental ALP. From these binding studies, the placental‐like ALP from Hep2 cells appears to be a slightly modified form of the normal type 1 placental ALP. One of the seven antibodies bound to the adult and fetal intestinal ALPs, but more strongly to the adult form, suggesting that the Hep2 intestinal‐like ALP is a form of the normal adult intestinal ALP.
Comparison of effects of inhibitors on adenosine triphosphatase and adenosine diphosphatase activities in rat‐liver mitochondria
Authors: MONTAGUE David J. , PETERS Timothy J. , BAUM Harold
Published: March 2005

Adenosine diphosphatase (ADPase) activity and ATPase activity were assayed in rat liver mitochondria and outer mitochondrial membrane preparations with [β‐32P]ADP and [γ‐32P]ATP as substrates. Inhibition studies were performed with the mitochondrial ATPase inhibitor oligomycin and the adenine nucleotide transport inhibitor, carboxyatractyloside. Kinetic studies were also performed with the nucleotide thiophosphate analogs adenosine 5′‐O‐thiophosphate, adenosine 5′‐O‐(2‐thiodiphosphate) and adenosine 5′‐O‐(3‐thiotriphosphate) which can act as inhibitors of phosphohydrolases. It is concluded that part of the apparent ADPase activity of intact mitochondria is mediated via ATPase, presumably in conjunction with adenylate kinase. In addition the outer mitochondrial membrane appears to show a distinct ADPase not attributable to contamination by inner membrane ATPase.
Metabolism of tRNA in near‐ultraviolet‐illuminated Escherichia coli The tRNA repair hypothesis
Authors: BLANCHETOT Alain , HAJNSDORF Eliane , FAVRE Alain
Published: March 2005

The relA+‐dependent stringent response is an important component of the mechanism of the near‐ultraviolet‐induced growth delay. However, the behaviour of the intracellular level of ppGpp is unexpected [Thomas et al. (1981) Eur. J. Biochem. 118, 381–387] and this led us to examine the metabolism of tRNAs during the illumination period and the growth lag that follows. Analysis of the gel electrophoresis migration profiles of tRNA molecules, synthesized prior to the illumination period, provides no evidence for tRNA degradation. Rather, it is suggestive of the rearrangement of some cross‐linked tRNA species during the growth lag. By the same technique the neosynthesis of one or several tRNA species escaping the stringent response could be ruled out at the beginning of the growth lag. The behaviour of the cross‐linked tRNAs was followed by a chromatographic procedure allowing the quantitative evaluation of the 8–13 link present in vivo. Upon illumination of growing cells, one observes an initial linear increase of the 8–13 link content. Unexpectedly this is followed during the illumination period by an abrupt decrease. The 8–13 link content then remains stable. The data above suggest that part of the 8–13 link (25–40 %) is eliminated from tRNA without degradation of the molecules involved.

A tRNA repair hypothesis is proposed: elimination of the 8–13 link would occur by scission of the N1–C1′ glycosidic bonds at positions 8 and 13 of tRNA. It would be followed by reinsertion of uracil and cytosine in their respective positions.
Methionine sulfoxide formation: the cause of self‐inactivation of reticulocyte lipoxygenase
Authors: RAPOPORT Samuel , HÄRTEL Brigitte , HAUSDORF Gert
Published: March 2005

Under conditions leading to inactivation of reticulocyte lipoxygenase by 13ls‐hydroperoxylinoleic acid a single methionine, presumably in the active center of the enzyme, is oxidized to methionine sulfoxide.
Steady‐state kinetics of horse‐liver alcohol dehydrogenase with a covalently bound coenzyme analogue
Authors: KOVÅŘ Jan , ŠIMEK Karel , KUČERA Igor , MATYSKA Luděk
Published: March 2005

The steady‐state kinetics of the enzyme modified by affinity labelling with an NAD analogue, nicotinamide‐N6‐[N‐(6‐aminohexyl)carbamoylmethyl]‐adenine dinucleotide, has been investigated using a recycling reaction with p‐nitrosodimethylaniline and n‐butanol as substrates and compared to the kinetics of native alcohol dehydrogenase. The modified enzyme obeys a ping‐pong mechanism involving two inactive enzyme forms (enzyme‐NAD and enzyme‐NADH complexes in the ‘open’ conformations, the nicotinamide moieties of the coenzymes being out of the active center). The rate of p‐nitrosodimethylaniline reduction in the reaction catalyzed by the modified enzyme is comparable to that observed in the presence of the native enzyme. On the other hand, the oxidation of butanol by the modified enzyme is essentially slower under our experimental conditions (pH 8.5). The measurements in the presence of specific alcohol dehydrogenase inhibitors competing with substrates and coenzymes (isobutyramide, pyrazole and AMP) revealed that the relative portion of the inactive ‘open’ form of the enzyme‐NADH complex is negligible, whereas the ‘open’ form of the enzyme‐NAD complex seems to represent a more significant portion (about 30%) under the conditions used.
Inhibition of cell proliferation by interferons
Authors: MOORE Graham , GEWERT Dirk R. , CLEMENS Michael J.
Published: March 2005

The inhibition of proliferation of Daudi cells in culture by human interferons is characterized by a change in the kinetics of labelling of different size classes of newly synthesized DNA. Initially, labelled precursors are incorporated exclusively into small DNA (Okazaki fragments) in both control and interferon‐treated cells, as revealed by alkaline sucrose gradient centrifugation. In the interferon‐treated cells, there is enhanced labelling of this small DNA after short periods of incorporation and slower conversion to larger DNA size classes, in comparison with the DNA of control cells. This effect is apparent after 12 h of interferon treatment, coincident with the onset of the inhibition of cell proliferation. It becomes progressively more marked up to 4 days, by which time cell growth has ceased completely.

Experiments using bromodeoxyuridine as a density label and analysis of radioactive DNA on caesium chloride/caesium sulphate gradients also reveal that some newly replicated DNA may be unstable and may turn over within a few hours of its synthesis. The label derived from DNA breakdown is efficiently reincorporated into newly synthesized molecules. It is suggested that interferon treatment inhibits DNA replication by activating DNA turnover rather than by directly inhibiting synthesis. This effect, together with the progressive retardation of conversion of Okazaki fragments to larger DNA, may lead to the eventual failure of cell proliferation.
The importance of monopole‐monopole and monopole‐dipole interactions on the binding of NADPH and NADPH analogues to p‐hydroxybenzoate hydroxylase from Pseudomonas fluorescens
Authors: WIJNANDS Robert A. , VAN DER ZEE Jolanda , VAN LEEUWEN Johan W. , VAN BERKEL Willem J. H. , MÜLLER Franz
Published: March 2005

NADPH binding to p‐hydroxybenzoate hydroxylase from Pseudomonas fluorescens is found to be strongly dependent on pH and ionic strength. In the ionic strength range of 0.02–0.15 M, optimal NADPH binding is observed at a pH value of 6.4. Extrapolation of the dissociation constants to infinite ionic strength shows that under these conditions optimal binding occurs at pH values < 8. Similar results were obtained for complexes between the enzyme and two NADPH analogues in the presence or absence of the substrate.

The experimental data can be explained by a theoretical model in which monopole‐monopole or monopole‐dipole interactions between the enzyme and the ligand are dominant. Changes in the former interaction prevail at low ionic strength and low pH values while the changes in the latter prevail at high ionic strength and high pH values. The dipole moment of the enzyme in the direction of the NADPH binding site was calculated from the ionic strength and pH dependence of the complex formation. The calculated dipole moment of the enzyme is about 2000 Debye at pH 6 and decreases to about 1100 Debye at pH 8.5. The results are discussed with respect to published results, including data obtained from the enzyme from a different source.
Topological location of diamine oxidase in the transverse plane of rabbit liver microsomal membranes
Authors: ARGENTO‐CERÙ Maria Paola , BARGAGLI Anna Maria , SARTORI Claudia
Published: March 2005

The transverse topology of diamine oxidase within rabbit liver microsomal membranes was studied by examining the proteolytic digestion of sealed or detergent‐permeabilized microsomal vesicles. Trypsin and pronase had no effect on diamine oxidase activity in any incubation conditions tested, while nagarse treatment reduced by 60–70% the enzymic activity of intact microsomes; no further loss of activity was observed in the presence of detergent.

These results demonstrate that the active site of diamine oxidase faces the cytoplasmic membrane surface, and suggest that it does not possess or expose on either membrane surfaces bonds susceptible to the proteolytic attack by trypsin or pronase. The possible significance and the biological implications of the results are discussed.
Pyridine‐nucleotide oxidation, Ca2+cycling and membrane damage duringtert‐butyl hydroperoxide metabolism by rat‐liver mitochondria
Authors: BELLOMO Giorgio , MARTINO Anna , RICHELMI Plinio , MOORE Gregory A. , JEWELL Sarah A. , ORRENIUS Sten
Published: March 2005

Astert‐butyl hydroperoxide is metabolized by the glutathione peroxidase–glutathione reductase enzyme system present in liver mitochondria, rapid and extensive oxidation of NADH and slow NADH oxidation are observed. This NAD(P)H oxidation can be prevented, or reversed, more effectively by 2‐hydroxybutyrate than by isocitrate, indicating an important role of mitochondrial NAD(P)+transhydrogenase activity in maintaining a high NADPH/NADP+ratio for glutathione reductase.

In Ca2+‐loaded mitochondriatert‐butyl hydroperoxide‐induced NAD(P)H oxidation is followed by Ca2+release from the mitochondria. If either 2‐hydroxybutyrate or isocitrate is present, no Ca2+release can be induced by the hydroperoxide. Following Ca2+efflux the NAD(P)H oxidation process becomes irreversible and membrane damage occurs. These late effects do not take place if ruthenium red is added to prevent re‐uptake of released Ca2+by the mitochondria. Thus, we conclude that the metabolism oftert‐butyl hydroperoxide leads to a release of mitochondrial Ca2+via oxidation of pyridine nucleotides, and that subsequent membrane damage is not directly associated with this Ca2+efflux but results from continued cycling of released Ca2+.
Nucleotide sequence organization in the very small genome of a tetraodontid fish, Arothron diadematus
Authors: PIZON Véronique , Cuny Gérard , BERNARDI Giorgio
Published: March 2005

We have investigated the sequence organization of the very small genome (DNA content/haploid cell c= 0.4–0.5 pg) of a tetraodontid fish, Arothron diadematus, by using two main experimental approaches. The first one, renaturation kinetics, showed that slowly reassociating, intermediate, fast and foldback sequences represented 87%, 7%, 5% and 1%, respectively, of A. diadematus DNA, which is, so far, the vertebrate DNA lowest in repeated sequences. The second approach, centrifugation in Cs2SO4/BAMD density gradients [BAMD = bis(acetate‐mercurimethyl)dioxane], showed that A. diadematus DNA can be resolved into several components, characterized by buoyant densities of 1.700, 1.7045, 1.708, 1.712 and 1.723 g/cm3, and representing 15%, 73%, 4%, 4% and 2.5%, respectively, of total DNA. The last component comprised a satellite DNA and ribosomal DNA. A family of interspersed repeats, possibly related to the AluI family of warm‐blooded vertebrates, showed an extremely specific genomic distribution, being present in only the 1.708 g/cm3 component, which it matched in base composition.
Catalytical mechanism of the phenylalanyl‐tRNA synthetase from yeast
Authors: THIEBE Rainer
Published: March 2005

Phenylalanyl‐tRNA synthetase catalyses an AMP‐ATP exchange under conditions where no aminoacylation of tRNA occurs. A plausible explanation for this reaction had not been given so far. The results of the present investigation provide evidence for the following interpretation.

tRNAphe induces a polarisation of the ATP in complex with the enzyme; this stimulates (a) the formation of phenylalanyl‐adenylate in the presence of phenylalanine, (b) the hydrolysis of ATP in the absence of phenylalanine and AMP and (c) the transfer of diphosphoryl onto AMP in the presence of AMP, especially when phenylalanine is absent.
Nuclear‐magnetic‐resonance studies on the conformations of tridecapeptide α‐mating factor from yeast Saccharomyces cerevisiae and analog peptides in aqueous solution
Authors: HIGASHIJIMA Tsutomu , MASUI Yoshihiko , CHINO Naoyoshi , SAKAKIBARA Shumpei , KITA Hiroshi , MIYAZAWA Tatsuo
Published: March 2005

The conformation of tridecapeptide α‐mating factor from yeast Saccharomyces cerevisiae in aqueous solution was analyzed, in comparison with those of active analog and inactive analog peptides. 270‐MHz 1H‐NMR spectra of these peptides were observed and the spectral patterns of main‐chain N‐H proton resonances were classified into three groups, α‐mating factor and Trp1‐bearing active peptides belong to the group A1, active des‐Trp1‐peptides belong to the group A2 while the peptides of group B are inactive. The main‐chain N‐H proton resonances of the groups A1 and A2 and side‐chain N‐H proton resonances were all assigned to individual residues. The 13C‐NMR analysis of α‐mating factor indicates that the Lys7‐Pro8 and Gln10‐Pro11 peptide bonds exclusively take the trans form. From the temperature and pH dependences of chemical shifts and Gd(III)‐induced relaxation enhancements of amide proton resonances, α‐mating factor is found to take partly a folded conformation in aqueous solution, with an α‐helical form in the N‐terminal domain and two β‐turn forms in the central and C‐terminal domain. The pH dependence of fluorescence intensity indicates that, in this folded conformation, the C‐terminal carboxylate group lies close to the N‐terminal domain. The presence of the folded form in the N‐terminal domain and the β‐turn form in the central domain correlates with the biological activity of α‐mating factor and analog peptides. However, the folded conformation of α‐mating factor is in equilibrium with predominantly unordered form, as found from the circular dichroism and NMR analyses. The N‐H proton and C‐α proton resonances of free α‐mating factor as assigned in the present study allow the transferred nuclear Overhauser enhancement (NOE) analysis of the membrane‐bound conformation that is more directly related with the activity.
Mechanism of pertussis toxin action on the adenylate cyclase system
Authors: JAKOBS Karl H. , AKTORIES Klaus , SCHULTZ Günter
Published: March 2005

The activation reaction of the inhibitory guanine nucleotide‐binding regulatory site of the adenylate cyclase system was studied in membranes of rat adipocytes, S49 lymphoma wild‐type cells and their cyc− variants, pretreated without and with the Bordetella pertussis toxin, islet‐activating protein (IAP), by measuring the kinetics of adenylate cyclase inhibition by the stable GTP analogue, guanosine 5′‐[γ‐thio]triphosphate (GTP[S]). The IAP treatment, which caused a loss of GTP and hormone‐induced adenylate cyclase inhibition, did not prevent enzyme inhibition by the stable GTP analogue. However, in either cell type studied, pretreated with IAP, the lag phase of GTP[S] inhibitory action was largely increased by about fivefold compared to control membranes. Similar to the controls, the lag phase of GTP[S] inhibition of adenylate cyclase in membranes of IAP‐pretreated cells was shortened in the presence of an inhibitory hormone. Furthermore, the lag phase of inhibition by GTP[S] was decreased with increasing concentrations of Mg2+. The data indicate that the pertussis toxin does not principally prevent an interaction of the inhibitory guanine nucleotide regulatory site of the adenylate cyclase system with either the catalytic moiety or an inhibitory hormone receptor. The data, furthermore, suggest that the toxin inhibits the activation reaction (turn‐on reaction) of the inhibitory coupling component. This inhibition, which may take place at a Mg2+ ‐binding site, can account for the observed functional loss of GTP and hormone‐induced adenylate cyclase inhibition after IAP treatment.
A differential effect of trypsin on pituitary gonadotropin‐releasing hormone receptors from intact and ovariectomized rats
Authors: LISCOVITCH Mordechai , BEN‐AROYA Nurit , MEIDAN Rina , KOCH Yitzhak
Published: March 2005

The present report demonstrates that pituitary receptors for gonadotropin‐releasing hormone generated following ovariectomy are susceptible to the action of trypsin while those present in intact metestrous females are not. The action of trypsin of gonadotropin‐releasing hormone receptors in pituitary cells derived from ovariectomized rats was dependent on time and dose. Trypsin exerted a maximal effect of 40–50% reduction without causing full abolition of the binding activity, indicating that two populations of gonadotropin‐releasing hormone receptors can be differentiated in pituitaries derived from ovariectomized rats: one which is sensitive to limited proteolysis by trypsin and another which is resistant to it. Scatchard analysis of gonadotropin‐releasing hormone antagonist binding to homogenate of trypsin‐treated hemipituitaries derived from ovariectomized rats, revealed that trypsin reduced receptor density while slightly increasing binding affinity. The in vitro responsiveness of hemipituitaries from ovariectomized rats to gonadotropin‐releasing hormone and to K+‐induced depolarization was reduced following trypsin treatment, while that of hemipituitaries derived from metestrous females was not affected or increased. Trypsin reduced the maximal response level by 30–50 % but did not modify the median effective dose of gonadotropin‐releasing hormone (1 nM). The reduced gonadotropin‐releasing hormone receptor density and the lower responsiveness caused by trypsin in pituitary tissue from ovariectomized rats are not due to a selective loss of gonadotrophs, as trypsin treatment did not affect the lutropin content of the glands. These findings indicate that two populations of gonadotropin‐releasing hormone receptors can be distinguished on the basis of their susceptibility or resistance to limited proteolysis by trypsin and suggest that these two gonadotropin‐releasing hormone receptor populations are reciprocally affected by ovariectomy.
Affinity chromatography, on fructose‐bisphosphatase–Sepharose, of two chloroplastic thioredoxins F
Authors: BUC Jean , RIVIÈRE Mireille , GONTERO Brigitte , SAUVE Paul , MEUNIER Jean‐Claude , RICARD Jacques
Published: March 2005

A new method of purification of chloroplastic thioredoxins has been presented. This method is based on affinity chromatography on fructose‐bisphosphatase—Sepharose columns.

Two thioredoxin, fA and fB, may be extracted and purified to homogeneity from the same leaf extract. Whereas fA is monomeric and has an Mr of 11400 ± 500, fB is dimeric with an Mr of 18000 ± 600. The dimer dissociates in two halves in the ultracentrifuge under the effect of high pressures. Raising the ionic strength results in the same effect.

Thioredoxins fA and fB activate to similar extents chloroplastic fructose bisphosphatase and NADP–malate dehydrogenase. Chloroplastic sedoheptulose bisphosphatase is activated by thioredoxin fB but not by thioredoxin fA.
Chemical behaviour of cytidine 5′‐monophospho‐N‐acetyl‐β‐d‐neuraminic acid under neutral and alkaline conditions
Authors: BEAU Jean‐Marie , SCHAUER Roland , HAVERKAMP Johan , KAMERLING Johannis P. , BORLAND Lambertus , VLIEGENTHART Johannes F. G.
Published: March 2005

The chemical behaviour of CMP‐N‐acetylneuraminic acid under neutral and different alkaline conditions has been investigated. The products formed were isolated by ion‐exchange chromatography and gel filtration and analysed by colorimetric methods, thin‐layer chromatography, combined gas‐liquid chromatography/mass spectrometry and/or 360‐MHz 1H‐NMR spectroscopy. A maximum stability of CMP‐N‐acetylneuraminic acid was observed at pH 8–11. In the tested pH range of 6–13, CMP and N‐acetylneuraminic acid were formed in variable amounts as decomposition products. 2‐Deoxy‐2,3‐dehydro‐N‐acetylneuraminic acid was produced at pH > 7; the amount of this substance increased with increasing pH. In anhydrous triethylamine its yield was 50%. A new neuraminic acid derivative, N‐acetyl‐β‐d‐neuraminic acid 2‐phosphate, could be isolated from the mixture of alkaline decomposition products of CMP‐N‐acetylneuraminic acid. The yield of this compound was maximum 22% in anhydrous triethylamine.

Because 2‐deoxy‐2,3‐dehydro‐N‐acetylneuraminic acid was formed under simulated physiological conditions, it is assumed that this compound, which occurs in tissues and fluids of man and animals, is derived from CMP‐N‐acetylneuraminic acid non‐enzymically also under conditions in vivo.
Interaction of the HB protein of Bacillus globigii with nucleic acids
Authors: WATANABE Fumiyuki , STANKOWSKI Stefan , SCHWARZ Gerhard
Published: March 2005

Fluorescence of the phenylalanine residues of the HB protein of Bacillus globigii was found to be quenched upon binding to nucleic acids. Using this phenomenon, the binding properties were investigated on the basis of an approach published recently.

The stoichiometric numbers were about 10 basepairs per bound protein molecule for double‐stranded DNA and about 10 bases for single‐stranded polynucleotides, independent of salt concentration. Cooperativity parameters were in the range of 50–250. Binding constants were about 5 × 106M−1 (at 0.1 M NaCl) and decreased with increasing salt concentration. From the salt dependence it is inferred that about one Na+ ion is displaced upon binding of a protein molecule to DNA. Therefore the binding site should contain one positively charged amino acid residue.

The protein was found to bind with comparable strength to double‐stranded and single‐stranded DNA as well as to poly(rA). Hence it is concluded that the HB protein does not belong to the category of ‘melting proteins’.
The cytoplasmic isoenzyme of horse liver aldehyde dehydrogenase
Authors: von BAHR‐LINDSTRÖM Hedvig , John HEMPEL  , JÖRNVALL Hans
Published: March 2005

The structural divergence between the cytoplasmic isoenzymes of aldehyde dehydrogenase from different species was investigated by analysis of peptides from the horse protein, and correlation of the results with the complete primary structure of the human isoenzyme. The amino acid sequences of these two proteins show a high degree of homology (91% of residues compared are identical). The differences observed are spread over the entire polypeptide chains, with only one cluster, which is close to a reactive cysteine residue and also adjacent to the most conserved region (covering 68 residues) in the primary structures of the whole enzymes. The secondary structure predicted for the human isoenzyme is mainly unaffected by the residue differences in the horse isoenzyme, although limited conformational changes might be compatible with an unexpected overrepresentation of differences involving isoleucine (12 of 43 exchanges represent a loss of He in the horse protein). Two cysteine residues that correlate with catalytic activity are identically positioned in the enzyme from the two species.
Membrane crystals of Ca2+‐ATPase in sarcoplasmic reticulum of fast and slow skeletal and cardiac muscles
Authors: DUX Laszlo , MARTONOSI Anthony
Published: March 2005

Crystalline arrays of Ca2+ transport ATPase develop in sarcoplasmic reticulum membranes after treatment with Na3VO4 in a calcium‐free medium [Dux, L. and Martonosi, A. (1983) J. Biol. Chem. 258, 2599–2603). The proportion of vesicles containing Ca2+‐ATPase crystals in microsome preparations isolated from rat muscle of different fiber types (semimembranosus, levator ani, extensor digitorum longus, diaphragm, soleus, and heart) correlates well with the Ca2+‐ATPase content and Ca2+‐modulated ATPase activity. This implies that the concentration of Ca2+‐ATPase in sarcoplasmic reticulum membranes of fast and slow skeletal or cardiac muscles. differs only slightly, and the low Ca2+ transport activity of ‘sarcoplasmic reticulum’ preparations isolated from slow‐twitch skeletal and cardiac muscles is due to the presence of large amount of non‐sarcoplasmic‐reticulum membrane elements. This is in accord with the relatively small differences in the density of 8.5‐nm intramembranous particles seen by freeze‐etch electron microscopy in sarcoplasmic reticulum of red and white muscles. The dimensions of the Ca2+‐ATPase crystal lattice are similar in sarcoplasmic reticulum membranes of different fiber types; therefore if structural differences exist between ‘isoenzymes’ of Ca2+‐ATPase, these are not reflected in the crystal‐lattice.
Rat‐liver lysosomal sialidase
Authors: MIYAGI Taeko , TSUIKI Shigeru
Published: March 2005

Purified liver lysosomes, prepared from rats previously injected with Triton WR‐1339, exhibited sialidase activity towards sialyllactose, fetuin, submaxillary mucin (bovine) and gangliosides, and could be disrupted hypotonically with little loss in these activities. After centrifugation, the activities with sialyllactose and fetuin were largely recovered in the supernatant, demonstrating that they were originally in the intralysosomal space. The activities towards submaxillary mucin and gangliosides, on the other hand, remained in the pellet. In the supernatant, activity with fetuin or orosomucoid was markedly reduced by protease inhibitors, suggesting that proteolysis of these glycoproteins may be prerequisite to sialidase activity.

The intralysosomal sialidase was solubilized from the mitochondrial‐lysosomal fraction of rat liver and partially purified by Sephadex G‐200, or Sephadex G‐200 followed by CM‐cellulose. The enzyme was maximally active at pH 4.7 with sialyllactose as substrate and had a minimum relative molecular mass of 60000 ± 5000 by gel filtration; if hydrolyzed a variety of sialooligosaccharides, those containing (α2 → 3)sialyl linkages being better substrates than those with (α2 → 6)sialyl linkages. The enzyme failed to attack submaxillary mucin and gangliosides. It was also inactive towards fetuin, orosomucoid and fransferrin but capable of hydrolyzing glycopeptides from pronase digest of fetuin.

In contrast to the intralysosomal silaidase, the sialidase partially purified from rat liver cytosol by (NH4)2SO4 fractionation followed by chromatography on DEAE‐cellulose and CM‐cellulose hydrolyzed fetuin and orosomucoid to the extent about half that for sialyllactose. The enzyme was maxitoally active at pH 5.8 and had a relative molecular mass of approximately 60000. It also hydrolyzed gangliosides but not submaxillary mucin.
Processing of the 3′ sequence extensions upon the 5S rRNA of a mutant yeast in Xenopus laevis germinal vesicle extract
Authors: PIPER Peter W. , PATEL Nita , LOCKHEART Alan
Published: March 2005

A processing endonuclease acts to remove a short sequence from the 3′ end of transcripts of the Saccharomyces cerevisiae 5S ribosomal RNA gene in generating the mature sequence of 5S RNA. Cells bearing the nuclear mutation rna82.1 lack this activity and accumulate 5S forms with additional nucleotides at their 3′ termini. 5S RNAs labelled during short pulse‐labellings of the mutant are essentially primary transcripts that mostly have the sequence U‐U‐A‐U‐U‐U‐C[U‐U‐U‐U(U‐U)] added to the 3′ end of normal yeast 5S RNA. They are subjected in vivo to a series of slow processing events whereby this sequence is ultimately replaced by: U‐U(A)1–9 in a substantial proportion of the 5S RNA molecules of the mutant [Piper, P. W., Bellatin, J. A. and Lockheart, A. (1983) EMBO J. 2, 353–359].

In higher eukaryotes no endonuclease cleavage occurs during 5S RNA maturation, yet processing at the 3′ ends of certain transcripts made by RNA polymerase III, most notably transfer RNA precursors, is still important. Since the enzymes involved in this processing have not been well characterised, we investigated how the additional sequences upon rna82.1 yeast 5S RNA are processed in vitro in a system from a higher eukaryote that is often used for studying transcription by RNA polymerase III, the Xenopus laevis germinal vesicle extract. Our results are consistent with slow digestion of these 5S molecules by a 3′→ 5′ exonuclease until they become 122–123 nucleotides in length, whereupon digestion ceases. This activity probably participates in the processing of certain Xenopus RNA polymerase III transcripts.
Primary structures of the α‐crystallin A chains of twenty‐eight mammalian species, chicken and frog
Authors: de JONG Wilfried W. , ZWEERS Anneke , VERSTEEG Marlies , NUY‐TERWINDT Eugénie C.
Published: March 2005

The amino acid sequences of the α‐crystallin A chains of 28 mammalian species, representing 14 different orders, have been analyzed, mainly on the basis of amino acid compositions of the composing peptides. The αA sequences of chicken and a frog have been completely determined by Edman degradation. A method is described to transport eye lenses, to be used for protein sequence studies, at ambient temperature in a solution of guanidine · HCl. The number of cysteine residues in different αA chains could be determined by alkaline urea gel electrophoresis after aminoethylation. In some cases the αA chains have been isolated from total lens extracts in a single ion‐exchange chromatographic step. The average rate of substitutions in the evolution of the αA chains is moderately slow, approximately 3 amino acid substitutions per 100 residues in 100 million years, but varies considerably in different lineages. Substitutions involving changes in charge are strongly underrepresented; the αA chains tend to keep their net charge constant throughout evolution. Analysis of the types of substitutions suggests a directional trend leading to an increase in functional density of αA in the course of evolution.