bluuebunny/crossref_metadata_2025_split huggingface dataset:
Mechanism of Reactivation and Refolding of Glyceraldehyde‐3‐Phosphate Dehydrogenase from Yeast after Denaturation and Dissociation
Authors: RUDOLPH Rainer , HEIDER Ingrid , JAENICKE Rainer 
Published: March 2005

Glyceraldehyde‐3‐phosphate dehydrogenase from yeast has been reversibly dissociated utilizing various denaturants. Applying comparable experimental conditions, dissociation, denaturation, and deactivation run parallel. Dissociation and reassociation occur at different limiting concentrations of the denaturant causing a range of metastability where irreversible aggregation competes with the process of reconstitution. In order to realize a high yield in kinetic reactivation experiments, solvent conditions far from the tetramer ⇌ monomer transition were chosen. Maximum recovery of enzymatic activity was obtained after denaturation in strong denaturants (citrate pH 2.3 + 6 M guanidine · HCl), and reactivation in the presence of 10 mM NAD++ 0.1–10 mM dithiothreitol at pH 7.6, c < 0.2 μM.Removal of the denaturant by dilution, and separation of high aggregates, e.g. by gelchromatography, lead to 100% regain of specific enzymatic activity and complete recovery of the hydrodynamic and spectral properties characterizing the native tetrameric enzyme.The analysis of the time course of reactivation and renaturation suggests that transconformation of inactive monomers and their subsequent association are responsible for the sigmoidal kinetic traces. A sequential uni‐bimolecular mechanism with first and second order rate constants k1= (11 ± 2) × 10−3 s−1 and k2= 45 ± 10 mM−1· s−1 (15°C) makes it possible to fit the reactivation kinetics in a quantitative manner. Within the limits of error k1 and k2 hold for the reactivation in the absence and in the presence of NAD+. NADH leads to complex kinetic profiles which cannot be fitted by a simple mechanism due to instability of the enzyme in the presence of the reduced coenzyme.
Regulation in vitro and in vivo of Adenosine 3′:5′‐monophosphate‐Dependent Inactivation of Rat‐Liver Pyruvate Kinase Type L
Authors: FELÌU Juan E. , HUE Louis , HERS Henri‐Géry 
Published: March 2005

The cyclic‐AMP‐dependent inactivation of pyruvate kinase L has been studied in a crude Sephadex filtrate of isolated hepatocytes. This inactivation requires the presence of Mg‐ATP (apparent Km= 0.1 mM) and a half‐maximal rate of inactivation was obtained in the presence of 0.15 μM cyclic AMP. It was inhibited by physiological concentrations of phosphoenolpyruvate and by micromolar concentrations of fructose bisphosphate and these inhibitory effects were counteracted by Mg‐ATP and by several l‐form amino acids such as cysteine, alanine, serine, phenylalanine, which are also ligands of pyruvate kinase. As a rule, it appears that effectors that increase the activity of the enzyme are also those which prevent its cyclic‐AMP‐dependent inactivation and vice‐versa.Considering the potentially important role of phosphoenolpyruvate concentration in the control of gluconeogenesis, experiments were performed to check if the inhibitory action of this metabolite on the inactivation of pyruvate kinase was of importance in vivo. We found that the concentration of phosphoenolpyruvate was higher than normal in the livers of anesthetized rats in conditions like fasting and diabetes in which the rate of gluconeogenesis is known to be higher and the activity of pyruvate kinase lower than normally. The reverse was true upon sucrose feeding. Inactivation of pyruvate kinase was induced in vivo by the administration of glucagon. This inactivation was less important in the livers with high concentrations of phosphoenolpyruvate and vice‐versa. It is proposed that phosphoenolpyruvate exerts an important buffering effect in the variations of the rate of gluconeogenesis.
Heterogeneity in the Kinetics of Oxygen Binding to Partially Reduced Human Methemoglobin
Authors: RAAP Adriaan , van LEEUWEN Johan W. , van ECK‐SCHOUTEN Tony , ROLLEMA Harry S. , de BRUIN Simon H. 
Published: March 2005

The pulse‐radiolysis technique has been introduced because it permits a rapid reduction (in a few microseconds) of one heme group of the methemoglobin tetramer by hydrated electrons. The kinetics of the binding of oxygen to this particular valence intermediate (Hb3+) with one reduced α or β subunit has been studied. It appears that the hydrated electrons preferentially reduce one type of subunit of methemoglobin at acid and neutral pH‐values as is shown by the biphasic behaviour of Hb3+ on oxygenation. The second‐order on‐rate constants measured for the binding of oxygen to Hb3+ are 14 ± 3 mM−1 ms−1 and 56 ± 9 mM−1 ms−1, respectively. The relative contribution of the faster fraction is about 0.63 ± 0.08 of the total oxygenation process.A comparison of the kinetic absorbance difference spectrum for the reduction of methemoglobin with the static difference spectrum of deoxyhemoglobin and methemoglobin in the Soret‐region revealed a decreased absorbance of the unliganded subunit of Hb3+ at 430 nm. This fact suggests that Hb3+ is in the relaxed quaternary conformation, which is in agreement with the observed on‐rate constants.
Glyceraldehyde‐3‐Phosphate Dehydrogenase(NADP) from Sinapis alba L.
Authors: CERFF Rüdiger 
Published: March 2005

Partially purified glyceraldehyde‐3‐phosphate dehydrogenase (NADP) (EC 1.2.1.13) from Sinapis alba seedlings was filtered on Sephadex G‐200 in the presence of NADP(H) and NAD(H). With NADP(H) the enzyme elutes as a dissociated species (Mr∼ 170 000), whereas with NAD(H) it travels as an aggregate in the void volume (Mr≥ 800 000). The NAD‐specific enzyme (EC 1.2.1.12) elutes with an Mr of about 160 000, regardless of whether NADP(H) or NAD(H) is present.
If the NADP‐dependent enzyme is first filtered with NADP+ on Sephadex G‐200 and then with NAD+ on Sepharose 6B, only part of the enzyme aggregates during the second filtration. The other part (fraction S) travels as a narrow band slightly ahead of the marker cytochrome c. Refiltration of fraction S with NADP+ and with increasing [NADP+]/[NAD+]ratios on Sepharose 6B demonstrates that this transport retardation is NAD‐dependent and counteracted by NADP. The effect is also eliminated by inorganic cations and is not observed on Sephadex G‐200.
Dodecylsulfate/polyacrylamide gel electrophoresis of fraction S before and after affinity chromatography on NADP‐agarose shows that the enzyme is pure after transport retardation on Sepharose 6B. It is active with both NADP and NAD and contains at least three different polypeptide chains with apparent Mr of 39 000, 42 000, and 43 000. The NAD‐specific enzyme is shown to be a tetramer of four 38 000‐Mr subunits.
Kinetic investigations show that the binding of NADP(H) and NAD(H) with glyceraldehyde‐3‐phosphate dehydrogenase (NADP) is mutually exclusive.
The present data indicate that the NADP‐dependent enzyme, a species structurally distinct from the NAD‐specific enzyme, occurs in two different conformations in the presence of NADP(H) and NAD(H) respectively. The existence of a ‘sticky’ NAD conformer is suggested, which interacts with Sepharose 6B and/or aggregates with other molecules in crude or partially purified extracts.
Evidence for a Dimeric Form of Artemia salina Extracellular Hemoglobins with High‐Molecular‐Weight Subunits
Authors: MOENS Luc , KONDO Masatoshi 
Published: March 2005

The brine shrimp, Artemia salina, produces at least three chemically and ontogenetically distinct extracellular hemoglobins (Hb‐I, Hb‐II and Hb‐III). The estimated molecular weights of these hemoglobins are 240 000–260 000, containing 14 heme groups based on the iron and heme contents of a major species, Hb‐II, which corresponds to a minimal molecular weight of about 18 000 per heme [Moens, L. and Kondo, M. (1977) Biochem. J. 165, 111–119]. Denaturation of the reduced and alkylated hemoglobins with 8 M guanidine hydrochloride revealed apparently one polypeptide chain having a molecular weight of 126 000. Thus a single native hemoglobin molecule should be composed of two of these high‐molecular‐weight subunits each of which is bound with seven hemes. Upon sodium dodecyl sulfate/polyacryamide gel electrophoresis of either native hemoglobins or isolated subunits it was found that the 126 000‐Mr polypeptide was cleaved specifically into two unequally‐sized fragments of Mr 50 000 and 80 000. Further denaturation of native hemoglobins with urea at pH 2.5 or 11 followed by sodium dodecyl sulfate gel electrophoresis confirmed these results.The amino acid compositions determined for native Hb‐II and its subunit and fragments are found to be very similar, implying that no specifically localized amino acid sequences are present and that the subunit globin chain could be composed of seven similar repeat units (Mr∼ 18 000) being linked covalently to one another. The amino acid compositions of Hb‐I and Hb‐III showed only minor differences to that of Hb‐II.
A Spectrophotometric Rapid Kinetic Study of Reactions Catalysed by Coenzyme‐B12‐Dependent Ethanolamine Ammonia‐Lyase
Authors: HOLLAWAY Michael R. , WHITE Hugh A. , JOBLIN Keith N. , JOHNSON Alan W. , LAPPERT Michael F. , WALLIS O. Caryl 
Published: March 2005

Spectral changes during the transient states and the steady states of the reactions catalysed by the adenosylcobalamin‐dependent ethanolamine ammonia‐lyase with l‐2‐aminopropanol and 2‐aminoethanol as substrates were followed by using a rapid‐wavelength‐scanning, stopped‐flow spectrophotometer (800 spectra per second and each spectrum 340–560 nm).
During the steady state of the reaction with the ‘poor’ substrate, l‐2‐aminopropanol (kcat∼ 1 s−1), under conditions of V, the spectrum of the coenzyme closely resembled that of cob(II)alamin. With the ‘good’ substrate, 2‐aminoethanol (kcat∼ 140 s−1), under the same conditions, the spectrum corresponded to a mixture of 58% cob(II)alamin and 42% cob(III)alamin. The latter may be either unchanged adenosylcobalamin or a coenzyme derivative with a covalent bond between a substrate carbon and the cobalt atom. The magnitude of a reported kinetic deuterium isotope effect favours the second possibility. There was no evidence for the presence of a cob(I)alamin intermediate.
The transient phases of the reactions were dependent on the order of mixing of reactants. When enzyme was placed in one syringe of the stopped‐flow apparatus and coenzyme and substrate in the other, the cob(II)alamin intermediate formed slowly (first‐order rate constants of about 5 s−1 with l‐2‐aminopropanol as substrate and about 2 s−1 with 2‐aminoethanol). When the enzyme and coenzyme were placed in one syringe and the substrate in the other, the formation of the cob(II)alamin occurred much more rapidly (90 s−1 with l‐2‐aminopropanol and greater than 300 s−1 with 2‐aminoethanol). It is concluded that binding of adenosylcobalamin to the enzyme is followed by a slow change in the conformation of the enzyme molecule to give a catalytically active species (there is no observable change in the spectrum of the coenzyme concommitant with this process). The formation of active holoenzyme is slow on the time scale of subsequent catalytic steps when 2‐aminoethanol is the substrate.
The spectra of the intermediates formed in the reactions with both substrates, the rates of their formation and their lifetimes allowed a detailed kinetic analysis of the reactions in terms of a three‐step mechanism involving binding of substrate, cob(II)alamin formation (k+2 step) and cob(II)alamin breakdown (k+3 step). In catalysis the difference between the substrates is expressed mainly in the k+3 step (240 s−1 and about 1 s−1 respectively for 2‐aminoethanol and l‐2‐aminopropanol, the respective k+2 values being 336 s−1 and 90 s−1).
Transport of d‐Glucose and 3‐O‐Methyl‐d‐Glucose in the Cyanobacteria Aphanocapsa 6714 and Nostoc strain Mac
Authors: BEAUCLERK Alan A. D. , SMITH Arnold J. 
Published: March 2005

The cyanobacterium Aphanocapsa 6714 which grows in the dark on D‐glucose, will take up D‐glucose and the analogue 3‐O‐methyl‐D‐glucose; uptake of each of these compounds was inhibited competitively by the other and by 6‐deoxy‐D‐glucose.
This cyanobacterium accumulated 3‐O‐methyl‐D‐glucose up to 100‐fold relative to the medium but did not modify or metabolize it to a significant degree.
Intracellular 3‐O‐methyl‐D‐glucose was rapidly displaced from Aphanocapsa 6714 by exogenous D‐glucose and 3‐O‐methyl‐D‐glucose.
Although not characterized to the same extent, D‐glucose and 3‐O‐methyl‐D‐glucose uptake by Nostoc strain Mac, another cyanobacterium capable of growth in the dark on D‐glucose, was similar.
Other cyanobacteria that do not grow on D‐glucose take up this compound at much lower rates which were unaffected by analogues of D‐glucose that greatly reduced carbohydrate uptake by Aphanocapsa 6714 and Nostoc strain Mac.
It is therefore proposed that Aphanocapsa 6714 and Nostoc strain Mac possess a mechanism for the active transport of D‐glucose. The absence of this transport mechanism is suggested as the reason why other strains fail to grow in the dark on this substrate. These latter organisms are therefore naturally cryptic with respect to D‐glucose as a growth substrate.
Major Proteins of the Escherichia coli Outer Cell Envelope Membrane. Interaction of Protein II* with Lipopolysaccharide
Authors: SCHWEIZER Margarete , HINDENNACH Ingrid , GARTEN Wolfgang , HENNING Ulf 
Published: March 2005

One of the major proteins of the Escherichia coli outer cell envelope membrane, protein II*, is shown to interact specifically with the same membrane's lipolysaccharide. In cell envelopes the protein (Mr∼ 33 000) is partially degraded by trypsin or pronase, and fragments with molecular weights of 24 000 or 19 000, respectively, are left with the outer membrane. The protein and these fragments in the cell envelope can act as receptors for a phage, and, most likely, as receptors in F‐mediated conjugation. Lipopolysaccharide protects isolated protein II* (in solution) in the same way against proteolytic attack, and the tryptic or pronase fragments are identical with those fragments obtained from protease‐digested cell envelopes. All fragments represent the N‐terminal moiety of the protein, and are, in the presence of lipopolysaccharide, active as phage receptors. Protein II* is heat‐modifiable in that its apparent molecular weight upon boiling in dodecylsulfate is 33 000 and without boiling is 28 000. Addition of lipopolysaccharide to the boiled species causes a renaturation to the species of molecular weight 28 000. Total E. coli phospholipid, synthetic dimyristoyl phosphatidylcholine, or the oligosaccharide moiety of lipopolysaccharide do not show any of these effects. However, the lipid part of lipopolysaccharide (lipid A) exhibits essentially the same activity as complete lipopolysaccharide.All results taken together strongly suggest that the interaction observed in vitro reflects such an interaction in vivo, and the possibility exists that this interaction is at least partially responsible for the specific cellular localization of the protein. Very likely a number of other major outer membrane proteins also interact specifically with lipopolysaccharide.
The Binding of Calcium and Magnesium to Sarcoplasmic Reticulum Vesicles as Studied by Manganese Electron Paramagnetic Resonance
Authors: KALBITZER Hans Robert , STEHLIK Dietmar , HASSELBACH Wilhelm 
Published: March 2005

The binding pattern of the biologically relevant ions calcium and magnesium has been investigated via the binding of the ion‐analogue manganese. The binding parametes of manganese are obtained conveniently by standard electron paramagnetic resonance techniques, the binding of calcium and magnesium is inferred from competition experiments.The quantitative analysis was carried out with a computer program which was able to treat the competition of three kinds of ions for three classes of independent and one class of cooperative binding sites.It was possible to correlate specific binding classes with biological functions of the sarcoplasmic reticulum membrane. The magnesium and manganese specific binding class of medium affinity is related to the catalytic function of these ions in the ATP‐splitting. For the first time a manganese and calcium specific class of cooperative binding sites has been observed and it can be related to the inhibition of the calcium transport at high concentrations of manganese or calcium.From the binding results a model can be developed which allows a full description of the role of the divalent ions and their specific binding sites during calcium transport.
Properties of 3‐(3,4‐Dichlorophenyl)‐1,1‐dimethylurea and Other Inhibitors of the Cytochromebc1Segment of the Mitochondrial Respiratory Chain inSaccharomyces cerevisiae
Authors: CONVENT Bernard , BRIQUET Michel 
Published: March 2005

The properties of 3‐(3,4‐dichlorophenyl)1,1‐dimethylurea (diuron) are compared to those of antimycin,n‐heptyl‐hydroxyquinoline‐N‐oxide (HpHOQnO), mucidin and funiculosin in submitochondrial particles ofSaccharomyces cerevisiae.Diuron inhibits the transport of electrons between cytochromesbandc1of the respiratory chain ofS. cerevisiaeas do the other inhibitors.Kinetics of inhibition of NADH oxidase are non‐sigmoidal for the inhibitors of low affinities: diuron and HpHOQnO.Mucidin and funiculosin exhibit high apparent affinities similar to that of antimycin. However the sigmoidicity of the kinetics of inhibition is not apparent for these two inhibitors.Diuron and funiculosin do not induce the ‘red‐shift’ of cytochromeb562.In contrast to beef heart, HpHOQnO does induce the ‘red‐shift’ of cytochromeb562inS. cerevisiae.Diuron induces the ‘extra‐reduction’ of cytochromeb.Mucidin induces a ‘red‐shift’ but yields a very limited ‘extra‐reduction’ of cytochromeb.Our results demonstrate that the inhibition of respiration of thebc1segment of the respiratory chain are not necessarily related to the ‘red‐shift’ and the ‘extra‐reduction’ of the cytochrome(s)b. They raise the possibility of the existence in yeast mitochondria of two distinct inhibitor‐binding sites, each of which would be able to control the transfer of electrons between cytochromesbandc1; in addition, one site would be responsible for the ‘red‐shift’ and the other would control the ‘extra‐reduction’ of cytochrome(s)b.
Energy Metabolism of Various Substrates and the 2,3‐Bisphosphoglycerate Bypass in Human Erythrocytes
Authors: QUADFLIEG Karl‐Hugo , BRAND Karl 
Published: March 2005

Using different experimental conditions and applying various substrates, a wide range of metabolic rates could be observed in incubations with human erythrocytes.
Lower rates of substrate utilization resulted in a decrease of the ATP and, more pronounced, of the 2,3‐bisphosphoglycerate concentration while carbon utilization rates beyond 14 μmol C ml packed cells in 120 min yielded constant levels of ATP and 2,3‐bisphosphoglycerate indicating that steady state had been achieved.
It can be concluded, that a carbon utilization rate of 14 μmol C/ml cells in 120 min is able to cover the energy requirement of the red cells under steady state conditions which could be calculated to amount to 3.6 μmol ATP/ml cells in 120 min at 37°C.
The contribution of the 2,3‐bisphosphoglycerate bypass to carbon metabolism could be calculated to range between 15–25% at metabolic rates below 14 μmol C/ml cells in 120 min (presteady state). When steady state has been achieved, the share of this bypass increases up to 61% with increasing metabolic rates.
Some Properties of Sialyltransferase in Plasma and Lymphocytes of Patients with Chronic Lymphocytic Leukemia
Authors: KESSEL David , CHOU Ta‐Hsu , ALLEN Janet 
Published: March 2005

Some properties of sialyltransferase activity in plasma and lymphocytes from patients with chronic lymphocytic leukemia were compared. Three distinct enzyme fractions were identified in plasma: (1) cation independent, irreversibly bound to agarose; (2) cation dependent, weakly bound to agarose; (3) strongly bound to agarose, lost upon dialysis. Lowering of the peripheral lymphocyte count by leukapheresis markedly decreased the level of serum sialyltransferase, suggesting the circulating lymphocyte is a source of the serum enzyme.The enzyme solubilized by detergent from lymphocytes showed a substantially lower Km for CMP‐sialic acid than did the serum enzyme, was less sensitive to several inhibitors, was not irreversible bound to Agarose, and had a substantial cation requirement. The enzyme solubilized from the lymphocyte therefore generally resembles fraction 2 of serum.
H+ and Cation Movements Associated with ADP, ATP Transport in Mitochondria
Authors: WULF Rainer , KALTSTEIN Andrej , KLINGENBERG Martin 
Published: March 2005

H+ and K+ movement associated with the mitochondrial ADP‐ATP exchange have been measured by glass electrode recordings in order to investigate the energy control of the ADP‐ATP exchange. All measurements are taken in the presence of oligomycin and inhibitors of electron transport in order to exclude other types of H+ transfer.

ATP exchange against endogenous ADP as induced by addition of ATP gives only a small H+ uptake which is partially suppressed in a KCl medium. H+ uptake is increased strongly on addition of uncoupler. On addition of valinomycin, K+ is taken up instead of H+. With carbonylcyanide p‐trifluoromethoxyphenylhydrazone (FCCP) and valinomycin respectively, equivalent amounts of H+ and K+ are taken up over a wide range of protein and ATP concentration. The ATP dependence gives a ratio H+ per ATP added = 0.38 and K+ per ATP added = 0.27. Saturation with ATP is reached at about 14 μmol ATP/g protein for H+ uptake and 20 μmol ATP/g protein for K+ uptake.The FCCP‐facilitated, ATP‐induced H+ uptake increases with endogenous content of ADP, giving a ratio H+/ADP = 0.67. Kinetics of the H+ uptake and ATP‐induced nucleotide exchange are closely correlated to about 0.62 H+/nucleotide released. The temperature dependence of the H+ uptake rate measured between 4 and 11 °C gives an activation energy = 33 kcal (138 kJ), similar to that found for the exchange rate.
In the exchange of ADP against endogenous ATP as started by addition of ADP, H+ are released in the absence of FCCP. In the presence of FCCP, H+ release is only marked when endogenous nucleotides are preloaded with external ATP. This H+ release is decreased by Pi and enhanced by N‐ethylmaleimide, blocking Pi transfer. It varies according to endogenous ATP content reaching the ratio H+ released/endogenous ATP = 0.3 to 0.5.
These data have been interpreted to indicate that the ATPe‐ADPi exchange is largely electrical and the opposite ADPe‐ATPi exchange is only between 50 to 70% electrical. The implications for energy transfer are discussed.