bluuebunny/crossref_metadata_2025_split huggingface dataset:
Uptake of Bromosulfophthalein byIsolated Liver Cells
Authors: SCHWENK Michael , BURR Reinhold , SCHWARZ Leslie , PFAFF Erich 
Published: March 2005

Uptake of the hepatodiagnostic dye bromosulfophthalein into isolated hepatocytes was studied with special regard to the kinetics of transport. The following results were obtained.

The uptake of bromosulfophthalein follows Michaelis‐Menten kinetics only at low substrate concentrations with an apparent Km= 7 ± 2 μM and V= 2.6 ± 1.7 nmol × mg protein−1× min−1′. At higher bromosulfophthalein concentrations a second mechanism of uptake is observed as indicated by the deviation from linearity in the Lineweaver‐Burk plot.
The activation energy of uptake was found to be 11 kcal/mol at 10 μM bromosulfophthalein.
Uptake is independent of metabolic energy and of the Na+ gradient across the membrane.
Taurocholate does not inhibit uptake while indocyanine green inhibits competitively at low bromosulfophthalein concentrations and activates uptake at high bromosulfophthalein concentrations (> 201 μM).
Amino acid reagents, such as dinitrofluorobenzene, mersalyl, N‐ethylmaleimide, and dithionitrobenzene, which modify specific functional groups, did not affect uptake at a concentration of 100 μM.
No pH optimum for bromosulfophthalein uptake was observed in the physiological pH range.
Adsorption of bromosulfophthalein to the liver cell membrane has two distinguishable site, with affinities K1= 5.7 × 10−6 M and K2=7 × 10−5 M and binding capacities n1= 1.2 nmol/mg protein and n2= 7 nmol/mg protein. Adsorption is inhibited by indocyanine green.

The results do not indicate the mediation of bromosulfophthalein uptake by a carrier protein and are consistent with the hypothesis that bromosulfophthalein is bound in an energy‐consuming process to a translocating site, possibly in the undissociated form or as ion pair. The consecutive transfer across the membrane appears to require little additional energy.
Studies on the Control of Development
Authors: RHAESE Hans‐Jürgen. , GRADE Reinhardt , DICHTELMÜLLER Herbert 
Published: March 2005

Unusual highly phosphorylated nucleotides are found in sporulating cells of Bacillus subtilis. Adenosine 3′(2′)‐diphosphate 5′‐diphosphate, ppApp (highly phosphorylated nucleotide I), and adenosine 3′(2′)‐diphosphate 5′‐triphosphate, pppApp (highly phosphorylated nucleotide II), are detectable in vivo when cells are starved for carbon and nitrogen sources. These nucleotides are correlated with sporulation because only ribosomes from sporulating but not vegetative cells are able to synthesize ppApp and pppApp in vitro. Two other nucleotides, adenosine 3′(2′)‐triphosphate 5′‐triphosphate; pppAppp (highly phosphorylated nucleotide IV), and a nucleotide with a tentative structure of, ppZpUp (highly phosphorylated nucleotide III), where Z is an undetermined sugar, also seem to be involved in regulation of sporulation, especially initiation of sporulation. Sporulation can be initiated even in the presence of amino acids, salts, vitamins etc. in logarithmically growing or stationary‐phase cells when carbon sources, i.g. glucose, are used up or artifically removed from the medium. A drastic increase in spore titer is observed 4–5 h later. Also, carbon starvation causes accumulation of the highly phosphorylated nucleotides pppAppp and ppZpUp. On the other hand, sporulation is prevented under the same conditions when excess glucose is maintained in the medium. Correlated with this inhibition of sporulation is the inhibition of formation of highly phosphorylated nucleotides, pppAppp and ppZpUp. Since synthesis of these nucleotides is closely related to sporulation, we anticipate that these substances can cause initiation of development in B. subtilis.Further evidence for our hypothesis on initiation of sporulation by highly phosphorylated nucleotides is that phosphate starvation also causes sporulation with prior accumulation of pppAppp and ppZpUp. Apparently, as long as phosphate is present to synthesize phosphorylated metabolites of glucose, formation of highly phosphorylated nucleotides is repressed. Derepression occurs when either lack of glucose or phosphate or both prevents synthesis of phosphorylated metabolites of glucose allowing synthesis of highly phosphorylated nucleotides. These nucleotides, representing the signal lack of glucose or phosphate′, then somehow cause changes in gene activity, initiating the complex process of sporulation. Whether or not pppAppp alone or together with ppZpUp or even further substances (nucleotides, proteins etc.) is necessary for the above described processes will be answered with the help of suitable mutants lacking the ability to synthesize either one or both regulatory nucleotides.Guanosine 3′(2′)‐diphosphate 5′‐diphosphate, ppGpp, and guanosine 3′(2′)‐diphosphate 5′triphosphate, pppGpp, are not involved in regulation of development as is shown by using a normally sporulating mutant of B. subtilis, unable to synthesize these nucleotides.
The Influence of Insulin on Various Enzyme Activities in Human and Rat Hepatoma Cells
Authors: GRIMM Johanna 
Published: March 2005

Incubation of human and rat hepatoma cells with insulin (1 mU/106 cells) decreases their content of adenosine 3′:5′‐monophosphate by more than half after 1 h and by about a quarter after 4 h.
The activities of the ATP‐metabolising enzymes, adenylate kinase and Mg2+‐adenosine triphosphatase are significantly increased by insulin within I h and after 4 h. Activity of succinate dehydrogenase. and lactic dehydrogenase showed no change at either time interval.
Insulin markedly stimulated glucose 6‐phosphate dehydrogenase activity within I h but by 4 h the increase was less apparent. Glutamate dehydrogenase activity by contrast was not increased by 1,h but was elevated at 4 h.
Acetic Anhydride: an Intermediate. Analogue in the Acyl‐Exchange Reaction of Citramalate Lyase
Authors: BUCKEL Wolfgang 
Published: March 2005

Reactivation of deacetyl citramalate lyase by acetic anhydride proceeds through an enzymeanhydride complex prior to actual acetylation. The reaction is inhibited by citramalate which is competitive with acetic anhydride.
A corresponding complex is an intermediate in the carboxymethylation of deacetyl enzyme by iodoacetate. However, the inhibition of this reaction by S‐citramalate appears to be non‐competitive with iodoacetate.
The results lead to the conclusion that acetic anhydride can be regarded as a structural analogue of citramalic acetic anhydride, the proposed intermediate in the acyl exchange reaction on citramalate lease.
The formation of 6‐citryl thiolester from the 1‐thiolester via the cyclic citric anhydride provides a chemical model for enzymic acyl exchange.
The data suggest that anhydrides are of general importance in acyl exchange reactions of thiolesters.
Small‐Angle X‐Ray and Light‐Scattering Study of Native and Trypsin‐Modified Methionyl‐tRNA Synthetase from Escherichia coli
Authors: GULIK Annette , MONTEILHET Claude , DESSEN Philippe , FAYAT Guy 
Published: March 2005

Small‐angle X‐ray scattering experiments were performed on an absolute scale on solutions of methionyl‐tRNA synthetase from Escherichia coli in its native and trypsin‐modified forms. A light‐scattering study was performed on the same solutions to verify monodispersity.The structural parameters for the trypsin‐modified enzyme, radius of gyration (2.48 nm), volume (90 nm3), surface/volume (1.5 nm−1) and the distribution of chords can account for an equivalent prolate ellipsoid of revolution having an axial ratio 2.3 and a maximum length of 9 nm, with a creviced surface.The results obtained for the native enzyme [i.e. radius of gyration (4.3 nm), volume (244 nm3), distribution of the scattering intensity and distribution of chords] exclude the possibility of a very compact quaternary structure and suggest that the enzyme consists of at least two globular parts, probably the two protomers, linked together by interactions involving a limited region of the structure.
Studies of Glutamate Dehydrogenase. Regulation of Glutamate Dehydrogenase from Candida utilis by a pH and Temperature‐Dependent Conformational Transition
Authors: NEUMANN Peter , MARKAU Klaus , SUND Horst 
Published: March 2005

Glutamate dehydrogenase from Candida utilis undergoes a reversible conformational transition between an active and an inactive state at low pH and low temperature. This conformational transition can also be followed by fluorescence measurements.The temperature‐dependent equilibrium between the active and the inactive state is characterized by a transition temperature of 10.7 °C and a ΔH value of 148 kcal/mol (620 kJ/mol). The temperature dependence of the enzymic activity above 15 °C yields an activation energy of 15 kcal/mol (63 kJ/mol), a larger value than that for the beef liver enzyme (9 kcal/mol; 38 kJ/mol). In contrast to the yeast enzyme the Arrhenius plot is linear and, therefore, the beef liver enzyme is not transformed into an inactive conformation at low temperatures. Sedimentation analysis shows that the inactivation of the Candida utilis enzyme is not caused by change in the quaternary structure.The pH dependence of the conformational transition at low pH measured by fluorescence change is characterized by a pK value of 7.01 for the enzyme in the absence and of 6.89 for the enzyme in the presence of 2‐oxoglutarate with a Hill coefficient of 3.4 in both cases. Similar results are found when the pH dependence of the enzymic activity is analyzed. With the beef liver enzyme the same pK value is obtained but with a Hill coefficient of 1 indicating cooperativity only in the case of the Candida utilis enzyme.The best fit of the pH dependence of the rate constants of the fluorescence changes was obtained with pK values of 7.45 and 6.45 for the active and the inactive state respectively. In this model the lowest time constant which is obtained at the pH of the equilibrium was found to be 0.05 s−1.Preincubation experiments with the substrate 2‐oxoglutarate but not with the coenzyme shift the equilibrium to the active conformation. The coenzyme obviously reduces the rate constant of the conformational transition.The sedimentation coefficient (s°20, W) and the molecular weight were found to be 11.0 S and 276000, respectively. The enzyme molecule is built up by six polypeptide chains each having a molecular weight of 47000.
Baker's Yeast Flavocytochrome b2 [l‐(+)‐Lactate Dehydrogenase]
Authors: GUIARD Bernard , LEDERER Florence 
Published: March 2005

Baker's yeast flavocytochrome b2 has been suggested before to be a polyglobular protein formed after a gene fusion process. It is known that one of the potential globules, the heme‐binding core, can exist in the absence of the rest of the protein. Using antibodies elicited against the whole enzyme acid against the core, we show in this paper that part of the core surface, and in particular the mouth of the heme‐binding crevice, must be exposed in the complete enzyme molecule.Antibodies inhibit the activity of intact and proteolytically‐cleaved enzymes, which normally show a number of differences in some kinetic parameters. Interestingly, antibodies against the core induce a modification of some kinetic constants for the cleaved enzyme (in particular the Km for the substrate and Ki for d‐(−)‐lactate), bringing them back to values similar to those for the intact enzyme. These results can be interpreted as a tightening of the cleaved enzyme by anti‐core antibodies. The conformational effect is transmitted from the heme‐binding region to other parts of the molecule. This implies some intimate contacts between the core and the rest of the protein.
AMP Analogs: Their Function in the Activation of Glycogen Phosphorylase b
Authors: MORANGF Michel , GARCÍA BLANCO Francisco , VANDENBUNDER Bernard , BUC Henri 
Published: March 2005

A series of AMP analogs has been selected in order to better understand the structural requirements (a) for the efficient binding of the activator molecule at the correct site on phosphorylase b from rabbit skeletal muscle and (b) for the activation which is observed. Two types of activation are known, according to Black and Wang [J. Biol. Chem. 243, 5892—5898 (1968)]: either a cooperative response with respect to the activator concentration (like the one which is obtained for AMP itself) or a non‐cooperative response observed in the case of IMP.It is shown that the 5′‐phosphate moiety is absolutely required for the analog to bind at the correct site (adenine or adenosine bind at another enzymic site), and that the free enthalpy, ΔG, corresponding to the association process varies in a complex manner with respect to the substitution of the different positions of the AMP molecule. Moreover, the differences ΔG (analog) ‐ ΔG (AMP) =δG obtained for two types of substitution separately do not add up to the same energy difference as the one obtained when the two substitutions are made simultaneously on the AMP molecule.It appears that all the mononucleotides which have been tested up to now may be divided into two classes. Class I (AMP class) is characterized, apart from a strong activation, by the following features: (a) one molecule of analog expels two molecules of bound glucose 6‐phosphate as it binds on the enzyme; (b) bound analog protects slowly one crucial cysteinyl residue against attack by 5.5′‐dithio‐bis(2‐nitrobenzoic acid) at 4 °C; (c) association of two molecules of dimer is strengthened at 4 °C in the presence of the analog. Class II (IMP class) is associated with a weak activation and with the following set of properties: (a) a single molecule of bound glucose 6‐phosphate is released as the first molecule of analog binds on the dimer; (b) two slowly reacting cysteinyl residues per subunit are immediately protected against 5,5′‐dithio‐bis(2‐nitrobenzoic acid) by the binding of the analog at 4 °C; (c) the analog dissociates the low amount of tetramer which is present at 4 °C in the absence of AMP into two molecules of dimer.These results are discussed according to a plausible scheme of transconformations taking place in glycogen phosphorylase b, a model which has been derived earlier by relaxation studies.
The State of ADP or ATP Fixed to the Mitochondria by Bongkrekate
Authors: KLINGENBERG Martin 
Published: March 2005

The reported studies are intended to clarify the binding state of ADP fixed to mitochondria under the influence of bongkrekate, and thus to discern between the affinity increase and reorientation mechanism proposed for the bongkrekate effect.(a) The composition of the intramitochondrial adenine nucleotide pool is not changed under the influence of bongkrekate with and without added nucleotides. (b) The added ADP and ATP fixed by bongkrekate can be identified as AMP, ADP and ATP in the same proportions as in the endogenous pool. (c) The bound nucleotides respond to oxidative phosphorylation or uncoupler stimulated dephospitorylation similar as endogenous nucleotides.It can be concluded that the ADP or ATP fixed under the influence of bongkrekate to the mitochondria are equilibrated with the intramitochondrial adenine nucleotide pool and are active in intramitochondrial phosphate transfer reactions.The results disagree with the affinity increase mechanism but support the reorientation mechanism which postulates that ADP and ATP are trapped in the mitochondria under the influence of bongkrekate in the same amount as there are carrier sites available outside before bongkrekate addition.
Purification and Properties of an N‐Formylmethionyl‐tRNA Hydrolase
Authors: GANOZA M. Clelia , BARRACLOUGH Nada , WONG J. Tze‐Fei 
Published: March 2005

The isolation and properties of a novel N‐formylmethionyl‐tRNA hydrolase (hydrolase II) from Escherichia coli are described. This enzyme is difficult to detect in crude extracts; purification, however, unmasks the activity. Sedimentation and gel filtration parameters of this enzyme differ from those of the previously described peptidyl‐tRNA hydrolase (hydrolase I), and preparations can be obtained where the two activities are free of each other. A mutant of hydrolase I has wild‐type levels of hydrolase II. These data indicate that hydrolase II is a different enzyme, or an altogether different form of hydrolase 1.The bulk of the enzymic activity occurs in the ribosome‐free cytoplasm; the remainder is found on intact or dissociated 70‐S ribosomes.Purified preparations of hydrolase II analyzed by two‐dimensional gel electrophoresis contain 2 protein bands. These 2 proteins do not coincide in electrophoretic mobility with any known ribosomal proteins. Analysis after mixing experiments verifies this conclusion.The purified enzyme (hydrolase II) is inhibited by ribosomes bearing bound N‐formylmethionyl‐tRNA. The inhibition is potentiated by sparsomycin and other antibiotics that block specifically peptide‐bond synthesis. The relationship of this enzyme to other hydrolytic activities, including a newly described ribosome‐dependent hydrolase, are discussed.
On the Transport of Tripeptide Antibiotics in Bacteria
Authors: DIDDENS Heyke , ZÄHNER Hans , KRAAS Ekkehard , GÖHRING Walter , JUNG Günther 
Published: March 2005

The two tripeptide antibiotics l‐2‐amino‐4‐met hylphosphinobutyryl‐alanyl‐alanine (L‐phosphinothricyl‐alanyl‐alanine) and l ‐(N5‐phosphono)methionine‐S‐sulfoximinyl‐alanyl‐alanine, both inhibitors of the glutamine svnthetase, are transported into the cell of Esclierichia coli K 12 via the oligopeptide transport system. The uptake by this system is proved first of all by cross resistance with tri‐ l ‐ornithine using oligopeptide‐transport‐deficient mutants, and secondly by antagonism tests demonstrating competitive reversal of the action of the antibiotic by several peptides which have been shown to be transported via the oligopeptide transport system, e.g. tri‐l‐alanine, tri‐l‐alanine, tri‐l‐lysine, tri‐ l ‐serine, tri‐glycine, glycyl‐glycyl‐ l‐alanine and the synthetic tripeptide l ‐azaadenyl‐aminohexanoyl‐alanyl‐alanine. On the other hand, there is no effect on the action of the antibiotic in antagonism tests with compounds which use different transport systems, such as l.‐alanyl‐alanine, l‐lysyl‐lysine, glutathione and the synthetic amino acid azaadenylaminohexanoic acid, i.e. 2‐amino‐6‐(7‐amino‐3H‐v‐triazolo‐[4,5‐d]‐pyrimidin‐3‐yl)hexanoic acid.Another inhibitor of the glutamine synthetase, l‐methionine‐S‐dioxide (methioninesulfone) could be converted into a tripeptide form by linkage to l‐alanyl‐alanine analogously to the tripeptide antibiotics described above. Whereas the free l‐methionine‐S‐dioxide seems to be transported via the methionine transport system, the tripeptide form is transported via the oligopeptide transport system. Thus, this glutamine synthetase inhibitor can be taken up by the cell via two different transport mechanisms. Our results indicate that this could provide a synergistic effect.The syntheses of the new tripeptides l‐azaadenylaminohexanoyl‐alanyl‐alanine and l ‐methionine‐S‐dioxidyl‐alanyl‐alanine were performed by dicyclohexylcarbodiimide couplings of the unusual N‐protected l‐α‐amino acids azaadenylaminohexanoic acid and l‐methionine‐S‐dioxide to l‐alanylalanine‐tert‐butyl ester followed by common deprotection steps. Tri‐ l‐ornithine was synthesized without carboxyl protection via two successive couplings of hydroxybenzotriazol esters of Nδ‐butoxycarbotlyl‐Nδ‐benzyloxycarbonyl‐l: ornithine to Nδ‐benzyloxycarbonyl‐l ‐ornithine
The Tentative Identification in Escherichia coli of a Multienzyme Complex with Glycolytic Activity
Authors: MOWBRAY John , MOSES Vivian 
Published: March 2005

Penicillin spheroplasts of Escherichia coli were ruptured osmotically, by freezing and thawing, or mechanically. Differential centrifugation sedimented 20–30 % of the glycolytic enzymes without increasing their specific activities. There was, however, evidence of distinct groups of sedimenting enzymes; growth on different carbon sources could influence the distribution. Sucrose gradient studies gave no evidence of enzyme association but provided estimations of the molecular weight of each enzyme which were close to those subsequently observed on gel filtration. Using the determined molecular weight and a literature value for specific activity, the measured activity ratio of the enzymes was compared with that expected from an equimolar mixture. All values agreed within a factor of five, except for hexokinase. The relative roles of hexokinase and phosphotransferase in E. coli are briefly considered.An equimolar multienzyme aggregate of all the enzymes of glycolysis would have a molecular weight of about 1.6 × 106. Chromatography on a Biogel column yielded one fraction, corresponding to a molecular weight of 1.6 × 106, which contained a proportion of all the glycolytic enzyme studied, the remaining portion of each enzyme activity was eluted from the column at the position expected from its individual molecular weight. The fraction of mol. wt 1600 000 was tested for complete glycolysis pathway activity and found not to be different from a reconcentrated mixture of the separated enzymes. Both‐the eluted and the reconstructed systems showed unexpected activity changes at different protein concentrations. The specific radioactivity of pyruvate formed by these systems from [14C]glucose 6‐phosphate was reduced by the presence of unlabelled 3‐phosphoglycerate, but by less than would have been expected had the latter been able to participate fully in glycolytic activity. This result indicates that these preparations were capable of selectivity compartmenting glycolytic intermediates.Electron microscope investigation of both systems showed large numbers of regular 30 nm, diameter particles which, on disruption, appeared to be composed of smaller units: it is possible that these particles may have been aggregates containing glycolytic enzymes. The possible advantages of a glycolytic multienzyme complex are briefly discussed.
Yeast Hexokinase: Substrate‐Induced Association‐ Dissociation Reactions in the Binding of Glucose to Hexokinase P‐II
Authors: HOGGETT James G. , KELLETT George L. 
Published: March 2005

A method is described for the purification of native hexokinases P‐I and P‐II from yeast using preparative isoelectric focussing to separate the isozymes.The binding of glucose to hexokinase P‐II, and the effect of this on the monomer‐dimer association‐dissociation reaction have been investigated quantitatively by a combination of titrations of intrinsic protein fluorescence and equilibrium ultracentrifugation.Association constants for the monomer‐dimer reaction decreased with increasing pH, ionic strength and concentration of glucose. Saturating concentrations of glucose did not bring about complete dissociation of the enzyme showing that both sites were occupied in the dimer.At pH 8.0 and high ionic strength, where the enzyme existed as monomer, the dissociation constant of the enzyme glucose complex was 3 × 10−4 mol1 −2 and was independent of the concentration of enzyme. Binding to the dimeric form at low pH and ionic strength (I = 0.02 mol1−1, pH < 7.5) was also independent of enzyme concentration (in the range 10–1000.μg ml−1) but was much weaker. The process could be described by a single dissociation constant, showing that the two available sites on the dimer were equivalent and non‐cooperative; values of the intrinsic dissociation constant varied from 2.5 × 10−3 mol 1−1 at pH 7.0 to 6 × 10−3 at pH 6.5.Under intermediate conditions (pH 7.0, ionic strength = 0.15 mol l−1), where monomer and dimer coexisted, the binding of glucose showed weak positive cooperativity (Hill coefficient 1.2); in addition, the binding was dependent upon the concentration of enzyme in the direction of stronger binding at lower concentrations.The results show that the phenomenon of half‐sites reactivity observed in the binding of glucose to crystalline hexokinase P‐II does not occur in solution; the simplest explanation of our finding the two sites to be equivalent is that the dimer results from the homologous association of two identical subunits.