bluuebunny/crossref_metadata_2025 huggingface dataset:
Synthesis of prostanoids and cyclic nucleotides by phagocytosing rat Kupffer cells
Authors: BIRMELIN Manfred , DECKER Karl
Published: March 2005

Rat Kupffer cells in monolayer culture were allowed to phagocytose unopsonized zymosan granules. They responded with a strongly stimulated synthesis and release of prostanoids, mainly the immunologically determined prostaglandins PGE2 and PGF2α. The same response could be obtained by treatment with the calcium ionophore A23187. The effects of the ionophore and the zymosan particles were of the same magnitude but not additive. The rapid uptake of Ca2+ after contact with phagocytosable material recently described by us [(1983) Eur. J. Biochem. 131, 539–543] appears to mediate the enhanced prostaglandin synthesis. That response was suppressed not only by indomethacin but also by trifluoperazine which does not inhibit Ca2+ entry in the Kupffer cells. Similar effects by R24571 and 4‐bromophenacyl bromide support the participation of calcium‐calmodulin and of phospholipase A2.

The calcium channel blocker Verapamil® did not influence the zymosan‐provoked production of prostaglandin PGE2 nor were any indications obtained for a feedback inhibition by PGE1 or PGE2.

Contact with zymosan resulted in a rapid but transient rise of the intracellular levels of cAMP and cGMP: 10 nM indomethacin completely blocked the increase of both cyclic nucleotides while trifluoperazine elicited different responses in the cAMP and cGMP levels.

The stimulated release of prostaglandin E2 was inhibited in a dose‐dependent manner by nordihydroguaiaretic acid, an inhibitor of 5‐lipoxygenase and by FPL 55712, known as a receptor antagonist for some leukotrienes. This suggests a regulatory role for its metabolites on prostaglandin synthesis.
Guanine‐nucleotide‐dependent inhibition of adenylate cyclase of rabbit heart by glucagon
Authors: KISS Zoltan , TKACHUK Vsevolod A.
Published: March 2005

The present study demonstrates an inhibitory effect of glucagon on the adenylate cyclase system of rabbit heart. Inhibition was maximal (22–40%) at 0.1 –0.01 μM glucagon and required the presence of 0.01 –0.1 mM GTP or guanosine 5′‐[β,γ‐imido]triphosphate (GuoPP[NH]P). Reduced or no inhibitor effect of glucagon was observed: (a) after limited proteolysis of plasma membrane proteins by trypsin, (b) in the presence of 1 mM Mn2+, (c) in the absence of Na +, and (d) during the first 10 min of incubation if GuoPP[NH]P was the activating ligand. With GTP as the activating ligand, inhibition of cyclase by glucagon occurred without delay. These data are consistent with a mediation of glucagon inhibition by a guanine‐nucleotide‐binding protein.

In the presence of ethanol (0.2 M) or benzyl alcohol (0.05 M), agents which are known to increase the fluidity of biological membranes, glucagon increased the enzyme activity in a guanine‐nucleotide‐dependent manner. Activation of cyclase in the presence of alcohols was maximal (30–60%) at 0.1–1.0 μM glucagon and 0.01 mM guanine nucleotides.

Data suggest that glucagon receptors can interact with both the activatory and inhibitory guanine‐nucleotide‐binding proteins and the physical state of membranes may play a role in determining which interaction will be preferential.
The nature of the reaction of an essential tyrosine residue of bovine heart mitochondrial ATPase with 4‐chloro‐7‐nitrobenzofurazan and related compounds
Authors: SUTTON Raul , FERGUSON Stuart J.
Published: March 2005

Bovine heart mitochondrial ATPase is inhibited after covalent modification with 4‐chloro‐7‐nitro‐benzofuroxan. The kinetics of the reaction are indistinguishable from those for the reaction of an essential tyrosine residue of the ATPase with 4‐chloro‐7‐nitrobenzofurazan that have been described previously [Ferguson et al. (1975) Eur. J. Biochem. 54, 117–126]. 4‐Fluoro‐7‐nitrobenzofurazan inhibits the ATPase with a pseudo‐first‐order rate constant that is tenfold greater than that for 4‐chloro‐7‐nitrobenzofurazan. These data indicate that the rate‐limiting step for reaction of the enzyme with these reagents is formation of a Meisenheimer complex at the C‐4 position and that the modified tyrosine is probably on the surface of the protein. Noevidence was found for more complex patterns of reactivity of 4‐chloro‐7‐nitrobenzofurazan and its analogues.

Both ammonium 4‐chloro‐7‐sulphobenzofurazan and ammonium 4‐fluoro‐7‐sulphobenzofurazan fail to react with the ATPase. The utility of these reagents as alternatives to the nitro derivatives may be limited owing to their slow reaction rates.

After modification on tyrosine by 4‐chloro‐7‐nitfobenzofurazan, the nitrobenzofurazan group can be transferred by an intramolecular process to lysine [Ferguson et al. (1975) Eur. J. Biochem. 54, 127–133]. ATPase with the lysine thus modified is shown to be reactive towards 4‐chloro‐7‐nitrobenzofurazan in a manner indistinguishable from the native enzyme. This indicates that the intramolecular transfer occurs at sufficient distance to avoid steric hindrance to the second reaction, and that the lysine does riot participate in a neighbouring group effect to enhance the reactivity of the tyrosine.
Developmental regulation of the cyclic‐nucleotide‐phosphodiesterase mRNA of Dictyostelium discoideum
Authors: MULLENS Isabel A. , FRANKE Jakob , KAPPES Dietmar J. , KESSIN Richard H.
Published: March 2005

Extracellular cyclic‐nucleotide phosphodiesterase of Dictyostelium discoideum has previously been purified and characterized [Orlow et al. (1981) J. Biol. Chem. 256, 7620–7627]. Antisera have been raised against the purified enzyme. Following cell‐free translation of RNA extracted from cells at various stages of development and immunoprecipitation with anti‐phosphodiesterase serum, cAMP phosphodiesterase synthesized in vitro and labeled with l‐[35S]methionine can be detected by sodium dodecyl sulfate/polyacrylamide gel electrophoresis and fluorography. The cell‐free translation product is an Mr‐48000 polypeptide and can be immunoprecipitated with antiserum raised against active Mr‐50000 cAMP phosphodiesterase or antiserum raised against heat‐denatured cAMP phosphodiesterase. Purified native cAMP phosphodiesterase blocks immunoprecipitation of the cAMP‐phosphodiesterase polypeptide synthesized in vitro.

A detectable level of cAMP‐phosphodiesterase mRNA is present in axenically grown cells. After starvation of the cells in phosphate buffer for 1 h an increase of translatable cAMP‐phosphodiesterase mRNA occurs, followed by a decrease and another increase. When cells are starved in the presence of the slowly hydrolyzed cAMP analogue, adenosine 3′,5′‐thiophosphate, the level of translatable cAMP‐phosphodiesterase mRNA increases about tenfold and does not show a temporary decline. A maximum of 0.015% of the total acid‐insoluble radioactivity is incorporated into the Mr‐48000 cAMP‐phosphodiesterase polypeptide.
How do enzyme activities control metabolite concentrations?
Authors: WESTERHOFF Hans V. , CHEN Yi‐Der
Published: March 2005

A simple theorem is derived relating the extent to which enzymes in a metabolic pathway control the steady‐state concentration of metabolites to the kinetic properties of those enzymes. The theorem gives insight into the mechanism by which the concentration of a second messenger is controlled by the enzymes that form and degrade it, and provides an alternative to the ‘cross‐over theorem’
Monoclonal antibodies that block cell adhesion in Polysphondylium pallidum: reaction with L‐fucose, a terminal sugar in cell‐surface glycoproteins
Authors: TODA Katsumi , THARANATHAN Rudrapatham N. , BOZZARO Salvatore , GERISCH Günther
Published: March 2005

By permethylation analysis the linkages of L‐fucose and D‐mannose in the oligosaccharide residues of cell surface glycoproteins of Polysphondylium pallidum were determined. Mannose was found in terminal positions, in 1,2‐, 1,3‐ and 1,6‐intra‐chain linkages, and at branch points. Fucose was exclusively located at nonreducing ends. Fab of a monoclonal antibody, mAb 293, has been previously shown to inhibit cell adhesion in P. pallidum completely. Binding of this antibody to glycoprotein was blocked by L‐fucose, and at very high concentrations also by D‐mannose. The dissociation constant for the antibody‐fucose complex was Kd= 70μM, which was two orders of magnitude higher than estimated for the natural oligosaccharide. Antibody‐glycoprotein complexes dissociated in the presence of 100 mM L‐fucose with a half‐time of about 56s. The blockage by L‐fucose is taken as evidence that the adhesion‐blocking antibody binds to oligosaccharide end groups containing L‐fucose as the terminal sugar.
Immunochemical identification of a two‐subunit NADH‐ubiquinone oxidoreductase from Paracoccus denitrificans
Authors: GEORGE Christina L. , FERGUSON Stuart J.
Published: March 2005

Analysis by crossed‐immunoelectrophoresis of Paracoccus denitrificans membrane vesicles has shown that only one antigen stains for NADH dehydrogenase activity. This activity could be partially purified by a combination of gel filtration and ion‐exchange chromatography of membrane vesicles that had been solubilised in the non‐ionic detergent Nonidet P‐40. From the limited number of precipitates observed after crossed immunoelectrophoresis of this partially purified preparation of NADH dehydrogenase it was possible to excise specifically part of the precipitate that stained for NADH dehydrogenase. Excised precipitates containing NADH dehydrogenase that had been radiolabelled by growth of cells in the presence of [35S]SO42‐ allowed the polypeptide composition of the enzyme to be determined by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulphate followed by fluorography. Two subunits were identified with estimated relative molecular masses of 48000 and 25000. Subunits of similar molecular weight are found in the flavoprotein fragment of the NADH dehydrogenase of the mammalian mitochondrial respiratory chain. The latter has general similarities with the respiratory chain in the plasma membrane of P. denitrificans.
Similarities between human and rat leukocyte elastase and cathepsin G
Authors: VIRCA G. Duke , METZ Gabrielle , SCHNEBLE Hans Peter
Published: March 2005

Rat is a likely test animal for determining the efficacy of proteinase inhibitor drugs directed toward human leukocyte elastase and cathepsin G. We therefore sought to assess and compare relevant properties of both human and rat leukocyte elastase and cathepsin G. Some differences between the pairs of proteinases from the two species were found, however both pairs of enzymes displayed comparable specificity toward various natural (plant and animal) proteinase inhibitors and also toward specific peptide substrates and a serine proteinase‐specific reagent. Such overlapping specificity implies similarity of reactive center topography and sequence homology around the extended substrate/inhibitor binding regions of these proteinases. This apparent homology leads us to conclude that a pharmacologically effective inhibitor of leukocyte proteinases in the rat would probably also be effective in man.
Primary structure of β subunit precursor of calf muscle acetylcholine receptor deduced from cDNA sequence
Authors: TANABE Tsutomu , NODA Masaharu , FURUTANI Yasuji , TAKAI Toshiyuki , TAKAHASHI Hideo , TANAKA Ken‐ichi , HIROSE Tadaaki , INAYAMA Seiichi , NUMA Shosaku
Published: March 2005

Clones harbouring cDNA sequences for the β subunit precursor of the acetylcholine receptor from calf skeletal muscle have been isolated. Nucleotide sequence analysis of the cloned cDNA has disclosed the primary structure of this polypeptide, which consists of 505 amino acids including a hydrophobic prepeptide of 24 amino acids. The β subunit of the calf muscle acetylcholine receptor, like the α and γ subunits of the same receptor and the a subunit of its human counterpart, exhibits structural features common to all four subunits of the Torpedo electroplax receptor, apparently being oriented across the membrane in the same manner as proposed for the fish receptor subunits. The degree of sequence homology between the calf and Torpedoβ subunits (59%) is comparable to that between the γ subunits (56%), but is lower than that between the α subunits of the two species (81%). Some regions of the β subunit molecule, including the region corresponding to the putative acetylcholine binding area on the α subunit and the region encompassing the clustered putative transmembrane segments M1, M2 and M3, are relatively well conserved between the two species.
Escherichia coli 5S RNA A and B conformers
Authors: GÖRINGER Hans Ulrich , SZYMKOWIAK Christof , WAGNER Rolf
Published: March 2005

The structures of the two stable conformers of Escherichia coli 5 S RNA, the A and B form, were compared. Information about the structures were obtained using the methods of limited enzymatic hydrolysis and chemical modification of accessible nucleotides. Base‐specific modifications were performed for adenosines and cytidines using diethylpyrocarbonate and dimethylsulfate in combination with a strand‐scission reaction at the modified site. Base‐specific (RNase T1) as well as conformation‐specific (nuclease S1, cobra venom nuclease) enzymes were employed for the limited enzymatic hydrolysis.

Clear differences in the accessibility of the two 5S RNA conformers to the enzymes and the chemical reagents were established and the regions with altered reactivities were localized in the 5S RNA structure.

The results are consistent with the disruption of the secondary structural interactions in helix II and partly in helices III and IV during the transition from the A to the B form. (The numbering of the helices is according to the generally accepted Fox and Woese model.) In addition some regions presumably involved in the tertiary structure are distorted. There is evidence, however, for the new formation of structural regions between two distant sites in the 5S RNA B form. The results enable us to refine the existing 5S RNA A‐form model and provide insight into the structural dynamics that lead to the formation of the 5S RNA B form.
Cell‐free synthesis of ornithine decarboxylase
Authors: KAMEJI Takaaki , FUJITA Kazunobu , NOGUCHI Tamio , TAKIGUCHI Masaki , MORI Masataka , TATIBANA Masamiti , HAYASHI Shin‐ichi
Published: March 2005

Ornithine decarboxylase (ODC) mRNA associated with free polysomes of rat liver was translated in a reticulocyte lysate cell‐free system. Newly synthesized ODC protein was identified by specific immunoprecipitation, molecular size as determined by polyacrylamide gel electrophoresis with sodium dodecyl sulfate, and competition by excess unlabeled ODC in the immunoprecipitation. A single injection of thioacetamide was found to cause several fold increases in both immunotitratable ODC protein and polysomal ODC‐mRNA activity, while it provoked a much larger increase in ODC activity in rat liver. The results indicate that the induction of hepatic ODC activity by thioacetamide treatment is due not only to an increase in the activity of polysomal ODC‐mRNA but also to a translational and/or posttranslational control.
Spectral intermediates of prostaglandin hydroperoxidase
Authors: NASTAINCZYK Wolfgang , SCHUHN Dietmar , ULLRICH Volker
Published: March 2005

Microsomes from ram seminal vesicles or purified prostaglandin H synthase supplemented with either arachidonic acid or prostaglandin G2 formed an unstable spectral intermediate with maxima at 430 nm, 525 nm and 555 nm and minima at 410 nm, 490 nm and 630 nm. At −15°C the band at 430 nm disappeared within 4 min whereas the trough at 410 nm increased three fold. At higher temperatures (10–37°C) spectral complex formation and decay were observed in less than 1 s.

An apparent KS value of about 3 μM was determined for the titration of purified prostaglandin synthase with prostaglandin G2 at −20°C.

Substrates for cooxidation reactions of prostaglandin synthase such as phenol, hydroquinone and reduced glutathione as well as the peroxidase inhibitors cyanide and azide inhibited the prostaglandin G2‐induced spectral complex formation.

The oxene donor iodosobenzene and hydrogen peroxide formed a spectral intermediate analogous to the complex observed with prostaglandin G2 or arachidonic acid in ram seminal vesicle microsomes as well as with the purified prostaglandin synthase.

These results are interpreted as the formation of a ferryl‐oxo complex (FeO)3+ of the heme of prostaglandin synthase with prostaglandin G2 analogous to the formation of compound I of horseradish peroxidase.
The action of anoxia and cyanide on glycogen breakdown in the liver of the gsd/gsd rat
Authors: CONAGLEN John V. , MALTHUS Rex S. , REDSHAW‐LOTEN Jane C. , SNEYD John G. T.
Published: March 2005

Perfusion of normal rat livers under anoxic conditions or the addition of KCN to aerobic perfusions activated phosphorylase and stimulated glycogen breakdown and glucose output. Livers from rats with a deficiency of liver phosphorylase kinase (gsd/gsd) showed a much smaller activation of phosphorylase with anoxia or KCN and produced glucose at about half the rate of normal livers. The increase in phosphorylase a in gsd/gsd livers was insufficient to account for the increase in glucose output. The addition of KCN to normal hepatocytes, activated phosphorylase and stimulated glucose output almost as effectively as glucagon. Hepatocytes from gsd/gsd rats showed only a very small increase in phosphorylase a on the addition of KCN, and glucose output did not increase. We conclude that in the perfused liver, anoxia and KCN stimulate glycogen breakdown and glucose output, at least in part, by a mechanism that does not involve conversion of phosphorylase b to phosphorylase a. In isolated hepatocytes KCN stimulates glucose output only by increasing the content of phosphorylase a.
Two‐dimensional 1H NMR of two chemically modified analogs of the basic pancreatic trypsin inhibitor
Authors: STASSINOPOULOU Chariclia I. , WAGNER Gerhard , WÜTHRICH Kurt
Published: March 2005

Two‐dimensional nuclear magnetic resonance was used to obtain sequence specific assignments for the 1H NMR spectra of two chemically modified analogs of the basic pancreatic trypsin inhibitor. In one analog the disulfide bond 14–38 was cleaved, in the second derivative the N‐terminus was transaminated. From measurements of the chemical shifts and dermination of the sequence locations of slowly exchanging backbone amide protons it was found that conformational differences between the native inhibitor and the chemical modifications occur exclusively near the modification sites and that the internal hydrogen bonds are nearly fully preserved. Intriguing conformation differences with respect to the native protein are that for five residues in the transaminated inhibitor and for one residue in the reduced inhibitor multiple local conformers are indicated, and that the four internal water molecules observed in the crystal structure of the native inhibitor appear not to be preserved after reduction of the disulfide bond 14–38.
Sequential ADP‐ribosylation pattern of nucleosomal histones
Authors: HULETSKY Ann , NIEDERGANG Claude , FRÉCHETTE André , AUBIN Rémy , GAUDREAU Alain , POIRIER Guy G.
Published: March 2005

The pattern of nucleosomal histones poly(ADP‐ribosyl)ation is changed under conditions which affect the poly(ADP‐ribosyl)ation state of the enzyme. At low NAD concentrations the enzyme can poly(ADP‐ribosyl)ate histones H1 and H1°, H2A, A2A, and H2B. However at NAD concentrations above 10 μM the enzyme preferentially poly(ADP‐ribosyl)ates histone H1 to a hyper ADP‐ribosylated form. Furthermore we have observed hyper ADP‐ribosylation of histone H2B at NAD concentrations of 10 μM suggesting that histone H2B can undergo the same type of ADP‐ribosylation pattern as histone H1. Also at higher NAD concentrations an elongation of the polymer attached to the enzyme and other nuclear proteins takes place.
A calcium‐dependent protease associated with the neural cytoskeleton
Authors: ISHIZAKI Yasuki , KUROKAWA Masanori , TAKAHASHI Kenji
Published: March 2005

Calcium‐dependent protease activity was found associated with a neurofilament‐enriched cytoskeleton isolated from the bovine spinal cord. The protease was extracted from the cytoskeleton by 0.6 M KCl, and purified to apparent homogeneity (3300‐fold) by chromatography on organomercurial‐Sepharose 4B, casein‐Sepharose 4B, and Sepharose CL‐6B. A cytosolic calcium‐dependent protease was similarly purified from the bovine spinal cord, after the cytosol was fractionated on DEAE‐cellulose. Both cytoskeleton‐bound and cytosolic enzymes had an apparent molecular mass of 100 kDa as judged by gel filtration, and consisted of two subunits (79 kDa and 20 kDa) upon sodium dodecyl sulfate/polycrylamide gel electrophoresis. Both enzymes exhibited caseinolytic activity with 0.5 mM Ca2+, and above, and the activity was strongly inhibited by various thiol protease inhibitors. In the presence of 0.1–0.2 mM Ca2+, the 68‐kDa and 160‐kDa components, and to a lesser extent the 200‐kDa component, of the neurofilament triplet polypeptides were degraded by the cytosolic protease, whereas the cytoskeleton‐bound protease needed two‐fold higher concentration of Ca2+ to degrade the neurofilaments. Nevertheless, the cytoskeleton‐bound protease in situ, i.e. before its extraction from the cytoskeleton by 0.6 M KCl, preferentially degraded the 160‐kDa component in the presence of 0.1–0.2 mM Ca2+, suggesting that a proper locational relation of this enzyme to the neurofilament structure is a prerequisite to its preference for the 160‐kDa component. It appears that a factor or factors involved in such an interaction between the protease and the neurofilament were eliminated during the course of enzyme purification. The glial fibrillary acidic protein was almost insensitive to the proteases purified in the present study.
On the regulation of cold‐labile cytosolic and of mitochondrial acetyl‐CoA hydrolase in rat liver
Authors: SÖLING Hans‐Dieter , RESCHER Christa
Published: March 2005

The discovery of a cold‐labile cytosolic acetyl‐CoA hydrolase of high activity in rat liver by Prass et al. [(1980) J. Biol. Chem. 255, 5215–5223] has questioned the importance of mitochondrial acetyl‐CoA hydrolase for the formation of free acetate [Grigat et al. (1979) Biochem. J. 177, 71–79] under physiological conditions. Therefore this problem has been reevaluated by comparing various properties of the two enzymes.

Cold‐labile cytosolic acetyl‐CoA hydrolase bands with an apparent Mr of 68000 during SDS/polyacrylamide gel electrophoresis, while the native enzyme elutes in two peaks with apparent MR of 136000 and 245000 during gel chromatography in the presence of 2 mM ATP. The mitochondrial enzyme elutes under the same conditions with an apparent Mr of 157000. Under conditions where the cold‐labile enzyme binds strongly to DEAE‐Bio‐Gel and ATP‐agarose, the mitochondrial enzyme remains unbound.

The cold‐labile enzyme can be activated 14‐fold by ATP, half‐maximal activation occurring already at 40 μM ATP. AdoPP[NH]P, AdoPP[CH2]P and GTP have a similar though weaker effect. ADP as well as GDP can completely inhibit the cold‐labile enzyme with 50% inhibition occurring for both nucleotides at about 1.45 μM. The binding of ATP and ADP is competitive. Acetyl phosphate and pyrophosphate have no effect on the activity of the cold‐labile enzyme. The mitochondrial acetyl‐CoA hydrolase is not affected by these nucleotides. CoASH is a strong product inhibitor (∼ 80% inhibition at 40 μM CoASH) of the cold‐labile enzyme, but only a weak inhibitor of the mitochondrial enzyme.

Under in vivo conditions the activity of the cold‐labile cytosolic acetyl‐CoA hydrolase can be no more than 7% of the activity calculated for mitochondrial acetyl‐CoA hydrolase under the same conditions. Accordingly the mitochondrial enzyme seems to be mainly responsible for the formation of free acetate by the intact liver, especially in view of the fact that the substrate specificity of the mitochondrial enzyme is much higher (activity ratios acetyl‐CoA/butyryl‐CoA 4.99 and 1.16 for the mitochondrial and the cold‐labile enzyme respectively). Alloxan diabetes neither increased the activity of the cold‐labile enzyme nor that of the mitochondrial enzyme. No experimental support has been found yet for the hypothesis that the acetyl‐CoA hydrolase activity of the cold‐labile enzyme represents the side‐activity of an acetyl‐transferase.
Phytochrome control of in vitro transcription of specific genes in isolated nuclei from barley (Hordeum vulgare)
Authors: MÖSINGER Egon , BATSCHAUER Alfred , SCHÄFER Eberhard , APEL Klaus
Published: March 2005

The transcriptional rates of four different genes in shoots of barley grown under different light regimes were quantified by monitoring nuclear RNA transcripts using gene‐specific hybridization probes. Isolated nuclei were pulse‐labelled with [α‐32P]UTP and the relative rates of light‐harvesting chlorophyll a/b protein (LHCP) mRNA, NADPH: protochlorophyllide oxidoreductase mRNA, B1 hordein mRNA, and 26‐S rRNA synthesis were measured. Irradiation of dark‐grown plants with a red light pulse increased the rate of LHCP mRNA synthesis tenfold within 3 h, and the rate of rRNA synthesis more than twofold within 9 h. The relative rate of synthesis of the oxidoreductase mRNA decreased following a red light pulse reaching a minimum after 3–6 h. As a direct proof of phytochrome involvement in the light‐induced stimulation of LHCP and the repression of the oxidoreductase transcripts for both responses, red/far‐red reversibility could be demonstrated. We conclude that phytochrome is able both to increase the transcription of certain nuclear genes and decrease the transcription of others.
Fructose‐1,6‐bisphosphatase from Synechococcus leopoliensis
Authors: GERBLING Klaus‐Peter , STEUP Martin , LATZKO Erwin
Published: March 2005

Extracts of Synechococcus leopoliensis(Anacystis nidulans) contain two forms of D‐fructose‐1,6‐bisphosphatase(EC 3.1.3.11) previously designatedas forms A and B [Gerbling, K.‐P., Steup, M., and Latsko, E. (1984) Arch. Microbiol. 137, 109–114]. Form B, which probably represents the mahor part of the total extractable fructose‐1,6‐bisphosphatase activity, has been purified to apparent homogeneity. Gel filtration, non‐denaturing polyacrylamide gel electrophoresis, and cross‐linking with bis(sulfosuccinimidyl) suberate revealed that the fructose‐1,6‐bisphosphatase B exists in either a dimeric or in a tetrameric subform, depending upon the absence or presence of fructose‐1,6‐bisphosphate and Mg2+. The dimer–tetramer interconversion was readily reversible. The results provide evidence for a two‐step activation of fructose‐1,6‐bisphosphatase B involving the reduction of the dimeric subform and the subsequent substrate‐dependent conversion of the reduced dimer to a reduced tetramer, which is the only catalytically active state. In contrast to form B, no substrate‐dependent interconversion was detected with form A from S. leopoliensis.
Chiral discrimination of 5,6‐epoxy‐3‐dehydroretinal by aporetinochrome and cattle opsin
Authors: SEKI Takaharu , SHINGU Keiji , KITO Yuji
Published: March 2005

The combination of a racemic all‐trans 5,6‐epoxy‐3‐dehydroretinal (EDR) with aporetinochrome formed a mixture of two diastereomeric pigments. One of the diastereomeric pigments which contained the all‐trans EDR with a negative circular dichroic (CD) band, hereafter called EDR(−)‐chrome, has its visible absorption maximum around 438 nm, while the other pigment, called EDR(+)‐chrome, has its maximum at 464 nm. These were substantiated by measuring the optical activities of the two EDR isomers which were extracted from a mixture of racemic all‐trans EDR and a smaller amount of aporetinochrome following exposure to orange light (> 530 nm) that irradiates EDR(+)‐chrome selectively. The extracted all‐trans EDR had a negative CD band around 240 nm and the extracted 11‐cis EDR had a positive band in that region with two negative bands on either side of the main band.

In the case of both pigments, the effect of alkalinization on the increase of absorbance in the near‐ultraviolet and the decrease of absorbance in the visible region was proportionate, qualitatively, to that on the positive CD intensities in both regions. These results suggest that the chromophore EDR in each pigment binds to the same binding site via a Schiff base.

The EDR(+)‐chrome exhibited properties similar to those of retinochrome, but EDR(−)‐chrome showed some different properties, i.e. its formation rate was slower than that of the former one and its absorption band in the near‐ultraviolet appeared even at neutral pH. Moreover, by exposing EDR(–)‐chrome to yellow light (>480 nm), only a part of its prosthetic all‐trans EDR was isomerized and resulted in the formation of 11‐cis and 13‐cis isomers. This variation in photoisomerizing activity was supposed to be due to the difference in conformation of the side chain between EDR(+) and EDR(–) in aporetinochrome.

Combination of 11‐cis EDR with cattle opsin was also shown to result in the formation of two diastereomeric pigments. The absorption maxima of the diastereomers containing 11‐cis EDR(+) and EDR(−) were at about 446 and 474 nm, respectively.
The nucleotide sequences of the ponA and ponB genes encoding penicillin‐binding proteins 1A and 1B of Escherichia coli K12
Authors: BROOME‐SMITH Jenny K. , EDELMAN Alex , YOUSIF Samira , SPRATT Brian G.
Published: March 2005

Penicillin‐binding proteins 1A and 1B of Escherichia coli are the major peptidoglycan transglycosylase‐transpeptidases that catalyse the polymerisation and insertion of peptidoglycan precursors into the bacterial cell wall during cell elongation. The nucleotide sequence of a 2764‐base‐pair fragment of DNA that contained the ponA gene, encoding penicillin‐binding protein 1A, was determined. The sequence predicted that penicillin‐binding protein 1A had a relative molecular mass of 93 500 (850 amino acids). The amino‐terminus of the protein had the features of a signal peptide but it is not known if this peptide is removed during insertion of the protein into the cytoplasmic membrane. The nucleotide sequence of a 2758‐base‐pair fragment of DNA that contained the ponB gene, encoding penicillin‐binding protein 1B, was also determined. Penicillin‐binding protein 1B consists of two major components which were shown to result from the use of alternative sites for the initiation of translation. The large and small forms of penicillin‐binding protein 1B were predicted to have relative molecular masses of 94100 and 88 800 (844 and 799 amino acids). The amino acid sequences of penicillin‐binding proteins 1A and 1B could be aligned if two large gaps were introduced into the latter sequence and the two proteins then showed about 30% identity. The amino acid sequences of the proteins showed no extensive similarity to the sequences of penicillin‐binding proteins 3 or 5, or to the class A or class C β‐lactamases. Two short regions of amino acid similarity were, however, found between penicillin‐binding proteins 1A and 1B and the other penicillin‐binding proteins and β‐lactamases. One of these included the predicted active‐site serine residue which was located towards the middle of the sequences of penicillin‐binding proteins 1A, 1B and 3, within the conserved sequence Gly‐Ser‐Xaa‐Xaa‐Lys‐Pro. The other region was 19–40 residues to the amino‐terminal side of the active‐site serine and may be part of a conserved penicillin‐binding site in these proteins.
Detergent‐soluble form of acetylcholinesterase in the electric organ of electric rays
Authors: SAKAI Michizo , SAISU Hideo , KOSHIGOE Naoya , ABE Teruo
Published: March 2005

The detergent‐soluble form of acetylcholinesterase was purified from the electric organ of the electric rays Narke japonica and Torpedo californica, and its properties were examined.

The electric organ of N. japonica and T. californica contains three types of acetylcholinesterase: low‐salt‐soluble, asymmetric or tailed, and detergent‐soluble forms. Results showed that in N. japonica, asymmetric forms were predominant, whereas in T. californica the detergent‐soluble form was predominant. Low‐salt‐soluble acetylcholinesterase constituted 10% of the total acetylcholinesterase in both species.

Detergent‐soluble acetylcholinesterase was purified by immunoaffinity chromatography with a monoclonal antibody (Nj‐601) to acetylcholinesterase. Triton X‐100 extracts of these electric organs were applied to a column of Nj‐601 – Sepharose, and the bound acetylcholinesterase was eluted quantitatively by lowering the pH to 2.8. This simple procedure gave good yields.

The purified enzymes gave single peaks at 6 S on sucrose gradients in the presence of detergent and polydisperse aggregates in the absence of detergent. Reduction of disulfide bonds gave peaks at 4.4 S.

On polyacrylamide gel electrophoresis in sodium dodecyl sulfate, the purified acetylcholinesterases gave bands with Mr of about 130 000 in the unreduced state and with Mr of 66 000 in addition to a very faint band of Mr 130000 in the reduced state. The Mr‐66000 polypeptides were labeled with diisopropylfluorophosphate. Thus, the detergent‐soluble acetylcholinesterases exist as dimers of the Mr‐66000 components.

Two‐dimensional electrophoresis of the purified enzymes indicated their homogeneity. The isoelectric points of both enzymes were 5.1 under the conditions employed.

The two enzymes had very similar amino acid compositions, and contained more than 14% of neutral sugars and glucosamine.

Monoclonal antibodies were raised to detergent‐soluble acetylcholinesterase by the hybridoma technique; eight were obtained. All of them recognized the catalytic subunits of detergent‐soluble and asymmetric acetylcholinesterases. One antibody showed considerable (about 100‐fold) preference for the detergent‐soluble acetylcholinesterase, and reacted only with detergent‐soluble acetylcholinesterase in immunoblots.

Four of the monoclonal antibodies inhibited the activities of both the detergent‐soluble and asymmetric forms of acetylcholinesterase.
Cloning, nucleotide sequencing and expression of cDNAs encoding mouse urokinase‐type plasminogen activator
Authors: BELIN Dominique , VASSALLI Jean‐Dominique , COMBÉPINE Chantal , GODEAU François , NAGAMINE Yoshikuni , REICH Edward , KOCHER Hans P. , DUVOISIN Robert M.
Published: March 2005

Controlled extracellular proteolysis is catalyzed in part by the secretion of plasminogen activators. As a step in the study of the expression of these enzymes in mouse tissues, we have isolated five cDNAs encoding the mouse urokinase‐type plasminogen activator from a cDNA library prepared with size‐selected mRNA from MSV‐transformed 3T3 cells. The longest cDNA insert contains the entire coding region of mouse urokinase, 58 base pairs of the 5′ non‐coding region, and the entire 3′ non‐coding region, which is 942 base pairs long. The deduced protein sequence, which starts with a signal peptide of 20 amino acids, shows extensive homology to that of human and porcine urokinase. However, in contrast to these enzymes, mouse urokinase contains no N‐glycosylation site. Bacteria harbouring one of the recombinant plasmids synthesize and secrete into their periplasm a protease indistinguishable from mouse urokinase.
Glycerol formation after the breaking of dormancy of Phycomyces blakesleeanus spores
Authors: VAN SCHAFTINGEN Emile , VAN LAERE André J.
Published: March 2005

The breaking of dormancy of Phycomyces blakesleeanus spores by a heat shock was followed by a transient production of glycerol, which culminated within 5–10 min and was terminated at 20 min. Extracts of spores contained a magnesium‐dependent glycerol‐3‐phosphatase active on both l‐glycerol 3‐phosphate and dihydroxyacetone phosphate but having more affinity for the first substrate than for the second. In extracts from dormant spores, the phosphatase was profoundly inhibited by physiological concentrations of inorganic phosphate, which induced cooperativity for the substrate, whereas the enzyme from heat‐activated spores was much less inhibited and this difference in kinetic properties persisted after gel filtration of the enzymic preparation. When measured at 1 mM phosphate and 0.1 mM glycerol 3‐phosphate, the phosphatase activity was undetectable in dormant spores, increased sharply during the heat treatment and the following 5 min at 25°C, then fell again to a low value by 20 min. A similar transient activation of the enzyme was observed following the breaking of dormancy by incubation of the spores in the presence of 0.1 M ammonium acetate. Incubation of a cell‐free extract or of the partially purified glycerol‐3‐phosphatase in the presence of ATP‐Mg and the catalytic subunit of cyclic‐AMP‐dependent protein kinase released the enzyme from inhibition by phosphate and endowed it with the same kinetic properties as did the heat treatment of the spores. It appears therefore most likely that phosphorylation of glycerol‐3‐phosphatase by cyclic‐AMP‐dependent protein kinase causes its activation and that this transient process explains the equally transient formation of glycerol by the spores after the heat shock.
Complete amino acid sequence of bovine plasminogen
Authors: SCHALLER Johann , MOSER Peter W. , DANNEGGER‐MÜLLER Gabrielle A. K. , RÖSSELET Susanne J. , KÄMPFER Urs , RICKLI Egon E.
Published: March 2005

The amino acid sequence of the single polypeptide chain of bovine plasminogen (786 residues, Mr 88092) was determined. Cleavage with CNBr yielded 13 fragments of which six originated from cleavage sites different from human plasminogen. Digestion with elastase gave three major fragments: kringles (1+2+3) and kringle 4. both with intact lysine binding sites, and mini‐plasminogen. Subfragmentation was achieved mainly with 2‐(2‐nitrophenylsulfenyl)‐3‐methyl‐3′‐bromoindolenine (BNPS‐skatole), Staphylococcus aureus V8 protease and trypsin. The sequences of fragments which were determined by automated Edman degradation, were aligned with overlapping sequences, or, in a few instances, by homology with the known sequence of human plasminogen. Sequence comparison with the human protein showed varying degrees of homology in the different functional and structural domains. The overall identity (78%) is practically the same as that found in those regions corresponding to the heavy (79%) and the light chain (80%) of plasmin. The average degree of identity among the kringles is 83%. Outside the kringle structures the extent of identity decreases, to 65% in the N‐terminal region and to about 50% in the connecting strands between the kringles except for the strand between kringles 2 and 3, where only one out of 12 residues is exchanged. The results reported show that bovine plasminogen apparently contains the same structural and functional domains as human plasminogen.

Bovine plasminogen also contains two carbohydrate moieties. The only partially substituted N‐glycosidic site, Asn289, corresponds to partially glycosylated Asn288 in human plasminogen, whereas the O‐glycosidic site of the human sequence, Thr345, is shifted to Ser339 in bovine plasminogen.
Differential expression of thyroglobulin gene in normal and transformed thyroid cells
Authors: AVVEDIMENTO Vittorio Enrico , MONTICELLI Antonella , TRAMONTANO Donatella , POLISTINA Claudio , NITSCH Lucio , DI LAURO Roberto
Published: March 2005

We have mesured the synthesis of thyroglobulin in two differentiated cell lines, FRTL‐5 and FRTL‐424, and two transformed thyroid cell lines, FRA and 1‐5G. The untransformed cells actively synthesized and exported thyroglobulin in the medium: however, the FRTL‐5 cell line synthesized seven times less thyroglobulin than the FRTL‐424 cell line, even though both cell types contained equal amounts of functional thyroglobulin mRNA. In contrast the transformed cells expressed extremely low levels of thyroglobulin mRNA, even though there was no detectable change in gene structure or copy number as determined by Southern blot analysis. On the basis of these data we conclude that (a) the different levels of thyroglobulin synthesis in the two untransformed cell lines are due to stable post‐transcriptional alterations in the biosynthesis of thyroglobulin and (b) the transformation of thyroid cells results in a substantial reduction in thyroglobulin gene expression.
Component X
Authors: DE MARCUCCI Olga , LINDSAY John Gordon
Published: March 2005

The mammalian pyruvate dehydrogenase multi‐enzyme complex contains a tightly‐associated 50 000‐M, polypeptide of unknown function (component X) in addition to its three constituent enzymes, pyruvate dehydroge‐ nase (El), lipoate acetyltransferase (E2) and lipoamide dehydrogenase (E3) which are jointly responsible for production of CoASAc and NADH. The presence of component X is apparent on sodium dodecyl sulphate/polyacrylamide gel analysis of the complex, performed in Tris‐glycine buffers although it co‐migrates with the E3 subunit on standard phosphate gels run under denaturing conditions. Refined immunological techniques, employing subunit‐specific antisera to individual components of the pyruvate dehydrogenase complex, have demonstrated that protein X is not a proteolytic fragment of E2 (or E3) as suggested previously. In addition, anti‐X serum elicits no cross‐reaction with either subunit of the intrinsic kinase of the pyruvate dehydrogenase complex.

Immune‐blotting analysis of SDS extracts of bovine, rat and pig cell lines and derived subcellular fractions have indicated that protein X is a normal cellular component with a specific mitochondrial location. It remains tightly‐associated with the ‘core’ enzyme, E2, on dissociation of the complex at pH 9.5 or by treatment with 0.25 M MgC12. This polypeptide is not released to any significant extent from E2 by p‐hydroxymercuriphenyl sulphonate, a reagent which promotes dissociation of the specific kinase of the complex from the ‘core’ enzyme.

Incubation of the complex with [2‐14C]pyruvate in the absence of CoASH promotes the incorporation of radio‐label, probably in the form of acetyl groups, into both E2 and component X.
Prokaryotic triterpenoids
Authors: ZUNDEL Magali , ROHMER Michel
Published: March 2005

3β‐Methylbacteriohopanepolyol derivatives were isolated from three bacteria, Acetobacter pasteurianus ssp. pasteurianus, Methylococcus capsulatus and Nostoc muscorum, and identified by spectroscopic methods and direct comparison with 3β‐methyldiplopterol and 3β‐methylhopan‐29‐ol synthesized from 22‐hydroxyhopan‐3‐one. The 3β‐methylhopanoid content of A. pasteurianus ssp. pasteurianus could be dramatically increased (up to 60% of the total hopanoid content) by addition of L‐methionine, the actual methyl donor, to the culture medium.
Secretion of high‐mannose‐type α1‐proteinase inhibitor and α1‐acid glycoprotein by primary cultures of rat hepatocytes in the presence of the mannosidase I inhibitor 1‐deoxymannojirimycin
Authors: GROSS Volker , STEUBE Klaus , TRAN‐THI Thuy‐Anh , MCDOWELL William , SCHWARZ Ralph T. , DECKER Karl , GEROK Wolfgang , HEINRICH Peter C.
Published: March 2005

Two different forms of α1‐proteinase inhibitor and α1‐acid glycoprotein were found in primary cultures of rat hepatocytes. After a 2.5‐h labeling period with [35S]methionine the high‐mannose‐type precursor of α1‐proteinase inhibitor (Mr 49000) and α1‐acid glycoprotein (Mr 39000) and the mature‐complex‐type α1‐proteinase inhibitor (Mr 54000) and α1‐acid glycoprotein (Mr 43000–60000) could be immunoprecipitated from the cells, but only the complex‐type forms of the two glycoproteins were secreted into the hepatocyte media. When hepatocytes were incubated with the mannosidase I inhibitor 1‐deoxymannojirimycin at a concentration of 4 mM, the 49000‐Mr form of α1‐proteinase inhibitor and the 39000‐Mr form of α1‐acid glycoprotein could be detected in the cells as well as in their media. Neither the secretion of α1‐proteinase inhibitor nor that of α1‐acid glycoprotein was impaired by 1‐deoxymannojirimycin. While α1‐proteinase inhibitor and α1‐acid glycoprotein, secreted by control cells, were resistant to endoglucosaminidase H, α1‐proteinase inhibitor and α1‐acid glycoprotein, secreted by hepatocytes treated with 4mM 1‐deoxymannojirimycin, could be deglycosylated by endoglucosaminidase H. When the [3H]mannose‐labeled oligosaccharides of α1‐proteinase inhibitor, secreted by 1‐deoxymannojirimycin‐treated hepatocytes, were cleaved off by endoglucosaminidase H and analyzed by Bio‐Gel P‐4 chromatography, they eluted at the position of Man9GlcNAc, indicating that mannosidase I had been efficiently inhibited. 1‐Deoxymannojirimycin did not inhibit the synthesis or the cotranslational N‐glycosylation of α1‐proteinase inhibitor or α1‐acid glycoprotein.
Nucleotide sequence of the Escherichia coli gap gene
Authors: BRANLANT Guy , BRANLANT Christiane
Published: March 2005

A 1523‐base‐pair DNA fragment, spanning the gap gene from Escherichia coli, has been sequenced. It contains an open‐reading frame whose length (330 amino acids) is in agreement with D‐glyceraldehyde‐3‐phosphate dehydrogenase (GAPDH) molecular mass. This coding sequence is preceded by a Shine‐Dalgarno complementary sequence and by two overlapping promoter‐like structures. The codon usage within gap is consistent with that expected for a gene which is strongly expressed. The amino acid sequence of the E. coli GAPDH, deduced from the DNA sequence, contains all the amino acids postulated to play a functional role in GADPH. Comparison of the E. coli enzyme with enzymes from other species reveals different evolutionary behaviour of the NAD+‐binding domain and of the catalytic domain of GAPDH. The E. coli enzyme is found to be more similar to eucaryotic enzymes than to enzymes from thermophilic bacteria. This observation is discussed in terms of adaptation to growth at high temperature.
Biosynthesis and in vitro translation of type IV procollagens
Authors: SAWHNEY Rajinder S. , DIXIT Saryu N.
Published: March 2005

The present paper describes how epitheilial cells, cultured from bovine anterior lens capsule explants, synthesize and secrete procollagen type IV polypeptide chains α1(IV). and α2(IV). Metabolic labeling of these cells with [14C]proline for different time intervals and subsequent analysis by SDS/polyacrylamide gel electrophoresis revealed the presence of two polypeptide chains with apparent molecular masses of 180 kDa and 170 kDa. The procollagens were bacterial‐collagenase‐sensitive and were specifically immunoprecipitated by antibodies raised against the 7S domain of type IV collagen. Type IV procollagen poly(A)‐rich RNA was was isolated from cultured lens capsule cells and translated in a reticulocyte lysate cell‐free system. Two polypetptides with apparent molecular lens capsule cells and translated in a reticulocyte lysate cell‐free system. Two polypeptides with apparent molecular masses of 152 kDa and 145 kDa were identified as procollagen type IV unmodified chains by gel electrophoresis, collagenase digestion and specific immunoprecipitation. During experiments in which cells were labeled in the presence of α,α′‐bipyridyl, type IV procollagen appeared as one major band comigrating with a 145 kDa polypeptide on SDS‐gel electrophoresis.
Inhibition, by lanthanides, of neutral proteinases secreted by human, rheumatoid synovium
Authors: EVANS Christopher H. , RIDELLA John D.
Published: March 2005

Fragments of human, rheumatoid synovium were maintained on organ culture for three days under serumless conditions. Their conditioned media contained collagenolytic, gelatinolytic and caseinolytic activities, which were susceptible to inhibition by lanthanide ions. Of the four lanthanides tested, Sm3+ proved the best inhibitor of gelatinase and caseinase, while La3+ inhibited collagenase the most strongly. Inhibition of collagenase by La3+ was uncompetitive. A direct binding assay confirmed the greater association between collagen fibrils and collagenase in the presence of La3+. Ca2+ was not required for binding of the uninhibited enzyme to collagen, but acted to stabilize collagenase against thermoinactivation.