bluuebunny/crossref_metadata_2025_split huggingface dataset:
The Mr‐50000 polypeptide of mammalian pyruvate dehydrogenase complex participates in the acetylation reactions
Authors: DE MARCUCCI Olga G. L. , HODGSON Jeffery A. , LINDSAY John G. 
Published: March 2005

The mammalian pyruvate dehydrogenase complex, Mr 8.5 × 106, contains an additional tightly bound 50000‐Mr polypeptide, component X, which copurifies with the intact assembly.Small amounts of the individual E2 and X polypeptides were obtained by elution of the protein bands from SDS/polyacrylamide gels. One‐dimensional peptide mapping studies with 125I‐labelled lipoyl acetyltransferase (E2) and component X subunits indicate that these two proteins are structurally distinct entities. Similar analysis of purified subunits, initially radiolabelled in the intact complex in the presence of [2‐14C]pyruvate and N‐ethyl‐[2,3‐14C]maleimide confirm that distinct 14C‐labelled peptides are generated from these two species. These protein‐chemical data supplement recent immunological findings, which demonstrate that component X is not a proteolytic fragment of the larger lipoyl acetyltransferase (Mr 70000) subunit.Incubation of the native PDC in the presence of [2‐14C]pyruvate leads to rapid uptake of radiolabel, presumably as acetyl groups, into both E2 and protein X. Specific incorporation of acetyl groups declines to a similar extent on both polypeptides after inhibiting pyruvate dehydrogenase (E1) activity by phosphorylation or omitting thiamine diphosphate (TPP) from the assay mixture. Addition of CoASH promotes the parallel deacetylation of both lipoyl acetyltransferase and protein X in a reaction which displays sensitivity to N‐ethylmaleimide.
Cytosolic adenylates and adenosine release in perfused working heart
Authors: BÜNGER Rolf , SOBOLL Sibylle 
Published: March 2005

Free cytosolic adenylates were examined in relation to adenosine plus inosine released from perfused working guinea‐pig hearts. Whole‐tissue adenylate data from freeze‐clamped hearts were quantitatively compared with corresponding values obtained by subcellular fractionation of homogenized myocardium in non‐aqueous media. Adenosine and inosine in venous cardiac effuents were measured by high‐performance liquid chromatography. Hearts, perfused at their natural flows, were subjected to various workloads, substrates and catecholamines to alter myocardial energy metabolism and respiration over a wide physiological range. Non‐aqueous cytosolic ATP and creatine phosphate (CrP) accounted for more than 80% of the respective total myocardium content. The cytosolic CrP/Pi ratio was in near‐quantitative agreement with the overall tissue CrP/Pi ratio when the latter parameter was corrected for extracellular Pi. This was conclusive evidence that ATP, CrP and Pi were pre‐dominantly located in the cytosol of the well‐oxygenated cardiomyocyte. Measured myocardial oxygen uptake (MVO2) was reciprocally related to the phosphorylation state of CrP ([CrP]/[Cr] × [Pi]) and hence that of ATP ([ATP]/[ADP] × [Pi]) assuming the creatine kinase at near‐equilibrium at a near‐constant pH of 7.2. On the other hand, calculated mean free cytosolic ADP concentrations increased essentially linearily up to threefold with increasing MVO2 in the presence of virtually unchanged or only slightly decreased ATP levels; this was found both according to the whole tissue and the special subcellular fractionation data. Employing the myokinase mass‐action ratio and substituting total cardiac ADP by the mean free cytosolic ADP concentrations, the mean free cytosolic AMP concentrations proved to be in the nanomolar range, i.e. up to three orders of magnitude lower than the overall tissue AMP content. We propose, therefore, that in the normoxic heart, AMP is located predominantly in the mitochondrial compartment. Nevertheless, both free cytosolic AMP concentration and release of adenosine plus inosine were apparently square or even higher‐power functions of the rate of cardiac respiration. On the other hand, the mean purine nucleoside release seemed linearily correlated (r= 0.920) with the calculated free cytosolic AMP concentration.Our observations seem to suggest that the concentrations of free ADP and AMP in the cytosol are major determinants of the production of inosine and coronary vasodilator adenosine. Such kinetic control of AMP degradation can conceivably render the free adenylate/adenosine system highly adaptive to mechanical perfor‐mance, total energy output, and/or the energy state of the myocardium. The energy‐linked changes in the free ATP/ADP system also seemed to produce an obligatory association between stimulated respiration and decreased cytosolic ATP potential concomitant with an increase in the concentration of free ADP.
Multispecific DNA methyltransferases from Bacillus subtilis phages
Authors: GÜNTHERT Ursula , LAUSTER Roland , REINERS Luzia 
Published: March 2005

Temperate Bacillus subtilis phages SPR, φ3T, ϱ11 and SPβ code for DNA methyltransferases, each having multiple sequence specificities. The SPR wild‐type and various mutant methyltransferases were overproduced 1000‐fold in Escherichia coli and were purified by three consecutive chromatographic steps.The stable form of these multispecific enzymes in solution are monomers with a relative molecular mass (Mr) of about 50000. The methyl‐transfer kinetics of the SPR wild‐type and mutant enzymes were determined with DNA substrates carrying either none or one of the three recognition sequences (GGCC, CCGG, CCATGG).Evaluation of the catalytic properties for DNA and S‐adenosylmethionine binding suggested that the NH2‐terminal part of the protein is important for both non‐sequence‐specific DNA binding and S‐adenosylmethionine binding as well as transfer of methyl groups. On the other hand, mutations in the COOH‐terminal part lead to weaker site‐specific interactions of the enzyme.Antibodies raised against the purified SPR enzyme specifically immunoprecipitated the φ3T, ϱ11 and SPβ methyltransferases, but failed to precipitate the chromosomally coded enzymes from B. subtilis (BsuRI) and B. sphaericus (BspRI). Immunoaffinity chromatography is an efficient purification step for the related phage methyltransferases.
Sulfite oxidase from chicken liver
Authors: RITZMANN Martin , BOSSHARD Hans Rudolf 
Published: March 2005

Oxidation of sulfite to sulfate by sulfite oxidase is inhibited when the enzyme is treated with reagents known to modify imidazole and carboxyl groups. Modification inhibits the oxidation of sulfite by the physiological electron acceptor cytochrome c, but not by the artificial acceptor ferricyanide. This indicates interference with reaction steps that follow the oxidation of sulfite by the enzyme's molybdenum cofactor.Reaction with diethylpyrocarbonate modifies ten histidines per enzyme monomer. Loss of activity is concomittant to the modification of only a single histidine residue. Inactivation takes place at the same rate in free sulfite oxidase and in the sulfite‐oxidase – cytochrome‐c complex.Blocking of carboxyl groups with water‐soluble carbodiimides inactivates the enzyme. But none of the enzyme's carboxyl groups seems to be essential in the sense that its modification fully abolishes activity.The pattern of inactivation by chemical modification of sulfite oxidase is quite similar to that observed previously for cytochrome c peroxidase from yeast [Bosshard, H. R., Bänziger, J., Hasler, T. and Poulos, T. L. (1984) J. Biol. Chem. 259, 5683–5690; Bechtold, R. and Bosshard, H. R. (1985) J. Biol. Chem. 260, 5191–5200]. The two enzymes have very different structures yet share cytochrome c as a common substrate of which they recognize the same electron‐transfer domain around the exposed heme edge.
Carbohydrate‐dependent induction of fatty acid synthase in primary cultures of rat hepatocytes
Authors: GIFFHORN‐KATZ Susanne , KATZ Norbert R. 
Published: March 2005

The glucose and insulin‐dependent long‐term regulation of fatty acid synthase was studied in primary cultures of hepatocytes from adult female rats.
Under basic culture conditions, i.e. 5.5 mM glucose and 0.5 nM insulin, the enzyme activity was continuously decreased over 6 days. In the presence of 100 nM insulin this decrease was reduced but it was not prevented. Enhancement of glucose to 20 mM was followed by an increase of the enzyme activity; after 5 days of treatment the activity was three times higher than under basic culture conditions. The simultaneous presence of 20 mM glucose and of 100 nM insulin resulted in a much more pronounced increase of the activity; after 5 days of treatment the activity was eight times higher than under basic culture conditions.
The enhancement was prevented by inhibition of glycolysis. This may indicate that the increase of fatty acid synthase was mediated by a metabolite of glucose rather than by glucose itself.
The coordinate regulation of fatty acid synthase and of other lipogenic enzymes was specific as demonstrated by comparison with the activity of lactate dehydrogenase and with synthesis and degradation of cytosolic proteins.
The enhancement of the enzyme protein, demonstrated by rocket immunoelectrophoresis, was due to an increase in the rate of enzyme synthesis by 600% as well as to a prolongation of the apparent half‐life of the enzyme by 50% (45 h).
The effect of citrate/cis‐aconitate on oxidative metabolism during transformation of Trypanosoma brucei
Authors: OVERATH Peter , CZICHOS Joachim , HAAS Claudia 
Published: March 2005

Monomorphic bloodstream forms of Trypanosoma brucei, grown in the mammal, are deficient in aconitase and 2‐oxoglutarate dehydrogenase and they do not respire in the presence of the substrates citrate, cis‐aconitate, succinate, proline or 2‐oxoglutarate. When grown in vitro low levels of aconitase, succinate oxidase and proline oxidase are detected.Addition of citrate/cis‐aconitate at 37°C to bloodstream forms leads to the formation of aconitase and proline oxidase. Most cells undergo an ‘abortive’ transformation to non‐dividing procyclic‐like cells while some cells adapt to the presence of the citric acid cycle intermediates and continue to multiply as bloodstream forms.At 27°C and in the presence of citrate/cis‐aconitate bloodstream forms transform synchronously to dividing procyclic cells. Within 72 h the rate of respiration with proline, succinate and 2‐oxoglutarate becomes similar to that in established procyclic cells while the rate of glucose oxidation decreases.The possible role of citric acid cycle intermediates in determining whether a trypanosome will retain the properties of a bloodstream trypomastigote or differentiate to a procyclic trypomastigote is discussed.
Effects of guanine nucleotides on the properties of 5‐hydroxytryptamine secretion from electropermeabilised human platelets
Authors: KNIGHT Derek E. , SCRUTTON Michael C. 
Published: March 2005

1. Guanosine 5′‐[γ‐thio]triphosphate and guanosine 5′‐[β,γ‐imido]triphosphate enhance Ca2+‐dependent 5‐hydroxytryptamine secretion from electropermeabilised human platelets. GTP has little such effect except when the platelets are permeabilised, and incubated with this nucleotide, at 2°C and pH 7.4.2. The lag phase observed in the time course of 5‐hydroxytryptamine secretion induced by addition of guanosine 5′‐[γ‐thio]triphosphate is markedly longer than that characterising secretion induced by Ca2+ alone, by thrombin ± GTP or by guanosine 5′‐[γ‐thio]triphosphate in the presence of thrombin.3. GTP causes competitive inhibition of the enhancement of the Ca2+‐dependent secretory response induced by guanosine 5′‐[γ‐thio]triphosphate when both nucleotides are added simultaneously. The extent of inhibition is decreased if guanosine 5′‐[γ‐thio]triphosphate is added prior to GTP.4. GTP markedly enhances the effect of thrombin on Ca2+‐dependent 5‐hydroxytryptamine secretion by increasing the maximal extent of the response and decreasing the thrombin concentration required to give half‐maximal response. A similar effect is observed on addition of guanosine 5′‐[γ‐thio]triphosphate in the presence of thrombin at short incubation times. On more prolonged incubation the effects of thrombin and guanosine 5′‐[γ‐thio]triphosphate are additive.5. Guanosine 5′‐[β‐thio]diphosphate completely inhibits the response induced by guanosine 5′‐[γ‐thio]triphosphate or guanosine 5′‐[β,γ‐imido]triphosphate but has little effect on the response induced by Ca2+ when added alone or in the presence of thrombin. Partial inhibition is observed for the response induced by thrombin + GTP.6. Cyclic‐AMP effectively inhibits the response induced by thrombin + GTP but has little effect on that induced by guanosine 5′‐[γ‐thio]triphosphate or guanosine 5′‐[β,γ]imidotriphosphate.7. The results provide further support for the proposal [Haslam, R. J. & Davidson, M. M. L. (1984) FEBS Lett. 170, 90–95], that receptor–phospholipase‐C coupling in platelets is mediated in part by a guanine‐nucleotide‐binding (Np) protein but that a coupling mechanism may also exist which is independent of such a protein. The properties of guanine‐nucleotide‐dependent coupling resemble those previously described for receptor–adenylate‐cyclase coupling.
Rat liver nuclear protein kinases NI and NII
Authors: DELPECH Marc , LEVY‐FAVATIER Florence , MOISAND Françoise , KRUH Jacques 
Published: March 2005

Rat liver nuclear protein kinases NI and NII have been purified to homogeneity by an improved method. This method includes a casein‐phosvitin‐Sepharose column step, which separates the enzymes from the other chromosomal non‐histone proteins, and a gel filtration at high ionic strength in the presence of a high concentration of protease inhibitors to separate the two enzymes from each other. NI has an apparent molecular mass of approximately 50 kDa and is composed of a single subunit. NII has an apparent molecular mass of 133 kDa and is composed of two subunits of identical molecular mass. The V and the Km of the two enzymes were determined for several substrates. Both enzymes phosphorylate chromosomal non‐histone proteins with partly different specificities as shown by two‐dimensional electrophoreses. When incubated in the absence of protease inhibitors, the enzymes were degraded into discrete polypeptides.Autophosphorylation of a polypeptide derived from NII was observed after incubation of the enzyme with ATP. This phosphorylation stimulated the enzyme activity.Several chromosomal proteins coeluted with NII from the casein‐phosvitin‐Sepharose column. They remained associated with the enzyme in sucrose gradients, during gel filtration performed at physiological ionic strength, and are dissociated at high ionic strength. These proteins were highly phosphorylated when the protein‐NII complex was incubated with ATP.
A Raman spectroscopic study on the interaction of an ion‐channel protein with a phospholipid in a model membrane system (gramicidin A/l‐α‐lysophosphatidylcholine)
Authors: ASLANIAN Dimitrina , NÉGRERIE Michel , CHAMBERT Régis 
Published: March 2005

The interaction of the ion channel polypeptide gramicidin A with the L‐α‐lysophosphatidylcholine micelles in a membrane state association (approximative molar ratio 1:9) was investigated by Raman spectroscopy. Studies were carried out over the spectral ranges of 700–1700 cm−1 and 2800–3100 cm−1 at 10°C.The Raman spectrum of L‐α‐lysophosphatidylcholine micelles indicated a disordered structure of the lipid acyl chains by the high intensities of the gauche conformation vibrations. Changing from the micellar phase to the membrane state of association with gramicidin A, the intensities of all‐trans stretching modes increased whereas the intensities of gauche conformation vibrations decreased, reflecting the emergence of ordered lipid chains.Hydrophobic interactions between the acyl chains and the polypeptide side chain residues were demonstrated. The absence of modifications in intensities of the very strong tryptophan vibrations in the complex spectrum indicated that, if the tryptophan‐stacking interactions suggested by some authors exsist, they are very weak ones.
Specificity of human glucosylceramide β‐glucosidase towards synthetic glucosylsphingolipids inserted into liposomes
Authors: SARMIENTOS Francesco , SCHWARZMANN Günter , SANDHOFF Konrad 
Published: March 2005

The behaviour of highly purified glucosylceramide β‐glucosidase (glucosylceramidase, EC 3.2.1.45) from human placenta [Furbish, F. S., Blair, H. E., Shiloach, J., Pentchev, P. G. & Brady, R. B. (1977) Proc. Natl Acad. Sci. USA 74, 3560–3563] was investigated in the absence of detergents with structurally modified glucosyl‐ceramides inserted into unilamellar liposomes. The reaction between the water‐soluble enzyme and the liposomal substrates was significantly dependent on the structure of the lipophilic aglycon moiety of glycolipids: glucosyl‐N‐acetyl‐sphingosines (d‐erythro and l‐threo) were better substrates than the corresponding glucosylceramides. The l‐threo derivatives were poorer substrates with higher apparent Km values than the corresponding d‐erythro derivatives. For glucosyl‐3‐keto‐ceramide and glucosyl‐dihydro‐ceramide (d‐erythro), higher Km values were found than for glucosylceramide. Sphingosine, glucosylsphingosine and glucosyl‐N‐acetyl‐sphingosine were the most effective inhibitors of the hydrolysis of glucosylceramide. d‐erythro‐Ceramide and d‐galactosyl‐N‐acetyl‐d‐erythro‐sphingosine inhibited the hydrolyis of amphiphilic glucosylceramide but not that of water‐soluble 4‐methyl‐umbelliferyl‐β‐glucoside, suggesting a hydrophobic binding site of the enzyme for the aglycon moiety of its membrane‐bound substrate.Dilution experiments suggested that at least a fraction of the enzyme associates with the liposomes and degrades the lipid substrate even in the absence of activator proteins.Acidic phospholipids incorporated into liposomes caused a powerful stimulation (30–40‐fold) of the glucosylceramide β‐glucosidase, whereas acidic sphingolipids (sulphatide, gangliosides GM1 and GD1a) incorporated into liposomes stimulated this enzyme only moderately (3–10‐fold).
Characterization of two carnosine‐degrading enzymes from rat brain
Authors: KUNZE Norbert , KLEINKAUF Horst , BAUER Karl 
Published: March 2005

From rat brain extracts, two carnosine‐degrading enzymes have been identified and partially purified by ion‐exchange chromatography, hydrophobic interaction chromatography on phenyl‐Sepharose CL‐4B and gel filtration. These enzymes, exhibit distinct differences in their chemical characteristics and substrate specificities.One enzyme, designated carnosinase, preferentially hydrolyzes carnosine and exhibits a low Km value (0.02mM) towards this substrate. Carnosinase also degrades anserine but not homocarnosine or homoanserine. The other carnosine‐degrading enzyme hydrolyzes βAla‐Arg considerably faster than carnosine and, therefore, has been tentatively designated βAla‐Arg hydrolase. This enzyme exhibits high Km values with carnosine (Km= 25 mM) and βAla‐Arg (Km= 2 mM). Homocarnosine and γ‐aminobutyryl‐arginine are not degraded by βAla‐Arg hydrolase.Neither enzyme is inhibited by agents reactive on activated hydroxyl groups, such as diisopropyl fluorophosphate, and also not by a variety of peptidase inhibitors of microbial origin or from other sources. Carno‐sinase is also not inhibited by bestatin but βAla‐Arg hydrolase, although not an aminopeptidase, is strongly inhibited by this aminopeptidase inhibitor (IC50= 50 nM). While carnosinase is strongly inhibited by thiol‐reducing agents such as dithioerythritol and 2‐mercaptoethanol, βAla‐Arg hydrolase is stabilized and activated by these substances. Both enzymes are strongly inhibited by metal‐chelating agents. Carnosinase, however, is not dependent on exogeneously added metal ions and is strongly inhibited by Mn2+ as well as by heavy metal ions. In contrast, βAla‐Arg hydrolase requires Mn2+ ions for full enzymatic activity.Based on these differences, selective incubation conditions could be evaluated in order to determine specifically both enzyme activities in crude tissue extracts. In rat, both enzymes are present in all tissues tested, except skeletal muscles, but considerable differences in their relative distribution among different tissues are also observed.
Accessibility of F0 subunits from Escherichia coli ATP synthase
Authors: DECKERS‐HEBESTREIT Gabriele , ALTENDORF Karlheinz 
Published: March 2005

1. Antisera have been raised against denatured and non‐denatured subunits a, b and c of the F0 complex of the ATP synthase from Escherichia coli. The subunit specificity of the antibodies has been established with immunoblot analysis or enzyme‐linked immunosorbent assay (ELISA).2. In inside‐out oriented membrane vesicles the binding avidities of both sets of antisera, against denatured and non‐denatured subunits of F0, were similar in the presence as well as in the absence of the F1 part. F1‐depleted everted membrane vesicles always produced an efficient binding of the different antisera. In the presence of F1 no antibody recognition could be observed with the anti‐a antisera, while anti‐b and anti‐c antisera showed strong binding. However, a higher membrane protein concentration was necessary for the same antibody binding as in F1‐stripped vesicles.3. In membrane vesicles with right‐side‐out orientation the recognition of the three F0 subunits was dependent on the antisera set used. Antisera raised against denatured subunits showed no binding to the membrane vesicles, only in case of anti‐(dedecylsulfate‐denatured b) antiserum could a slight affinity be detected. An antigen‐antibody recognition with all three F0 subunits occurred when the antisera against non‐denatured subunits were incubated with membrane vesicles of right‐side‐out orientation. The membrane protein concentration which was necessary to produce a significant binding was 10–100‐fold higher compared to that of F1‐depleted everted membrane vesicles.
Transcription of the phosphoglycerate kinase gene of Saccharomyces cerevisiae increases when fermentative cultures are stressed by heat‐shock
Authors: PIPER Peter W. , CURRAN Brendan , DAVIES Mark W. , LOCKHEART Alan , REID Georg 
Published: March 2005

The single gene for phosphoglycerate kinase (PGK) in the haploid genome of Saccharomyces cerevisiae is expressed to a very high level in cultures fermenting glucose. Despite this it responds to heat‐shock. When S. cerevisiae growing exponentially on glucose media was shifted from 25°C to 38°C transient increases of 6–7‐fold in cellular PGK mRNA were observed. This elevation in PGK mRNA still occurred in the presence of the protein‐synthesis inhibitor cycloheximide, but was not observed in cells bearing the rnal.1 mutation. From the kinetics of continuous labelling of PGK mRNA, relative to the labelling of other RNAs in the same cultures whose levels do not alter with heat‐shock, it was shown that the elevation in PGK mRNA in response to temperature upshift reflects primarily an increased synthesis of this mRNA and not an alteration of its half‐life. PGK mRNA synthesis is therefore one target of a response mechanism to thermal stress.Synthesis of PGK enzyme in glucose‐grown cultures is efficient after mild (25°C to 38°C) or severe (25°C to 42°C) heat‐shocks. Following the severe shock, the synthesis of most proteins is abruptly terminated, but synthesis of PGK and a few other glycolytic enzymes continues at levels comparable to the levels of synthesis of most of those proteins dramatically induced by heat (heat‐shock proteins). Cells that overproduce PGK due to the presence of multiple copies of the PGK gene on a high‐copy‐number plasmid continue their overproduction of this enzyme during severe thermal stress. Therefore PGK mRNA is both elevated in level in response to heat‐shock and translated efficiently at supra‐optimal temperatures.