bluuebunny/crossref_metadata_2025_split huggingface dataset:
Spontaneous modification of the oxoglutarate translocator in vivo
Authors: DUYCKAERTS Claire , SLUSE‐GOFFART Claudine M. , SLUSE Francis E. , GOSSELIN‐REY Cécile , LIÉBECQ Claude 
Published: March 2005

In studying the oxoglutarate translocator of rat‐heart mitochondria over many years, we have observed an unexpected decrease in its efficiency. It has been divided by 2.48 ± 0.07 (S.E.M.) for the exchange of external oxoglutarate for internal malate at 2 °C when the internal‐malate concentration is 4 mM and is accompanied by an increase in its concentration (multiplied by 1.61 ± 0.02, S.E.M.).The affinity of the external sites of the translocator for the external oxoglutarate is unchanged as well as the binding and kinetic cooperativities of the external oxoglutarate. This shows that the external side of the translocator has not been modified and suggests that its central part has not been modified either. The apparent Michaelis constant of the internal malate is increased (multiplied by 1.74 ± 0.23, S.E.M.) suggesting that the translocator has been modified on its matricial side.Some control experiments show that a change in the diet of the rats, despite its effect on the fatty‐acid content of the mitoplasts, is probably not responsible for the observed modification. As it is nevertheless very likely that changes of the oxoglutarate translocator have occurred in vivo, it is proposed that the observed modification has a genetic origin.The existence of two antagonist changes which are not directly related suggests that one of them is a response of the organism against the other; thus the oxoglutarate translocator may play a regulatory rôle in certain physiological conditions.
Complex structure of human bronchial mucus glycoprotein
Authors: SLAYTER Henry S. , LAMBLIN Geneviève , TREUT André LE , GALABERT Claude , HOUDRET Nicole , DEGAND Pierre , ROUSSEL Philippe 
Published: March 2005

Human bronchial mucus glycoproteins or mucins were isolated from the sputum of two patients by a method avoiding reducing agents and involving water extraction and gel filtration on Sepharose CL‐2B in 6 M guanidinium chloride. The chemical analysis indicated approximately 25–40% lipid. The amino acid and carbohydrate analysis differ quantitatively from that of mucins purified after prior reduction of mucus. These fractions also have a higher proportion of aspartic and glutamic acids than that of the mucins from reduced sputum. These mucins are still contaminated by small amounts of peptides but do not seem to contain disulfide‐attached cross‐linking protein. Human bronchial mucins have a strong tendency to form aggregates except in 6 M guanidinium chloride.Electron microscopy performed with various procedures indicates the presence of both micelles and flexible threads measuring 200–1000nm. Delipidation removes most of the micellar forms. Thereafter mucins appear mainly as polydisperse flexible extended threads and also as aggregates.
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.
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.
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.
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.
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.
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.
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.
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.