bluuebunny/crossref_metadata_2025_split huggingface dataset:
Enzyme kinetics in reversed micelles
Authors: VERHAERT Raymond M. D. , TYRAKOWSKA Bożena , HILHORST Riet , SCHAAFSMA Tjeerd J. , VEEGER Cees 
Published: March 2005

Enoate reductase (EC 1.3.1.31) can stereospecificially reduce a variety of α,β‐unsaturated carboxylates. Its use was extended to apolar media by incorporating the enzyme into a reversed micellar medium. The kinetics of the enzyme in such a medium have been investigated using 2‐methylbutenoic acid as substrate and NADH as a cofactor and compared with the reaction rates in aqueous solution.In aqueous solution the enzyme obeys a ping pong mechanism [Bühler et al. (1982) Hoppe‐Seyler's Z. Physiol. Chem 363, 609–625]. In 50 mM Hepes pH = 7.0 with ionic strength of 0.05 M the Michaelis constants for NADH and 2‐methylbutenoic acid are 20 μM and 6.0 mM respectively. In reversed micelles the kinetics of the reaction (Michaelis constant, maximum velocity as well as inhibitory effects) were markedly different. The rate of the enzymatic reaction of enoate reductase was studied using various concentrations of 2‐methylbutenoic acid and various NADH concentrations. In reversed micelles composed of the anionic detergent sodium di(ethylhexyl)sulphosuccinate, the enzymatic reaction deviates substantially from the values in aqueous solution. Using our model (see preceding paper in this issue of the journal), all kinetics could be explained as evolving from enclosure in reversed micelles without any change in the intrinsic rate parameters of the enzyme. So the enzyme itself is unaffected by incorporation in reversed micelles, but the rate of intermicellar exchange as well as the microheterogeneity of the medium, resulting in very high local concentrations of the substrate, are the most important factors altering the reaction pattern. The effect of the composition of the reversed micellar medium was also investigated using either a nonionic or a cationic surfactant. In these solutions too, exchange and microheterogeneity of the medium proved to be the most important parameters influencing the enzymatic reaction.In all reversed micellar solutions inhibition by the enoate was observed at an overall concentration of 0.5–5 mM, implying that a concentration of substrate equal to the Km value in aqueous solution may already cause inhibition in reversed micelles. At this level no inhibition by NADH was observed. The microheterogeneity of the medium also explains this inhibition of the enzyme at relatively low 2‐methylbutenoic acid concentrations.
Resonance energy transfer between points in a reconstituted skeletal muscle thin filament
Authors: MIKI Masao 
Published: March 2005

The spatial relationships between Lys‐61, Cys‐374 on actin or SH1 on myosin subfragment‐1 (S1) and Cys‐190 on tropomyosin or Cys‐133 on troponin‐I (TnI) in a reconstituted thin filament were studied by fluorescence resonance energy transfer. 5‐(2‐Iodoacetylaminoethyl)aminonaphthalene 1‐sulfonic acid (IAEDANS) attached to Lys‐190 on tropomyosin or to Cys‐133 on TnI was used as a donor. Fluorescein 5‐isothiocyanate (FITC) attached to Lys‐61 or 5‐(iodoacetoamido)fluorescein (IAF) attached to Cys‐374 on actin and 4‐dimethylaminophenyl‐azophenyl 4′‐maleimide (DABMI) attached to SH1 on S1 were used as an acceptor. The transfer efficiency between AEDANS attached to Cys‐190 on tropomyosin and FITC attached to Lys‐61 on actin was 0.42 in the absence of troponin, 0.46 in the presence of troponin and Ca2+ and 0.55 in the presence of troponin and absence of Ca2+. The corresponding distances between the probes were calculated to be 4.7 nm, 4.6 nm and 4.3 nm respectively, assuming a random orientation factor K2= 2/3. A large difference in the transfer efficiency from AEDANS attached to Cys‐133 on TnI to FITC attached to Lys‐61 on actin was observed between in the presence (0.52) and absence (0.70) of Ca2+. The corresponding distances between the probes were calculated to be 4.5 nm in the presence of Ca2+ and 3.9 nm in the absence of Ca2+.The distance between Cys‐190 on tropomyosin and Cys‐374 on actin was measured to be 5.1 nm and the transfer efficiency (0.35) did not change upon addition of troponin whether Ca2+ is present or not, in agreement with the previous report [Tao, T., Lamkin, M. & Lehrer, S. S. (1983) Biochemistry 22, 3059–3064]. The distance between Cys‐133 on TnI and Cys‐374 on actin was measured to be 4.4 nm. No detectable change in transfer efficiency (0.58) was observed between values in the presence and absence of Ca2+. These results suggest that a relative movement of the two domains of actin monomer in a reconstituted thin filament occurs in response to a change in Ca2+ concentration.The transfer efficiencies between DABMI attached to SH1 on S1 and AEDANS attached to Cys‐190 on tropomyosin or Cys‐133 on TnI were too small (less than 2%) for an accurate estimation of the distances, suggesting the distances are longer than 7.3 nm.
Location and function of three sites phosphorylated on rat acetyl‐CoA carboxylase by the AMP‐activated protein kinase
Authors: DAVIES Stephen P. , SIM Alastair T. R. , HARDIE D. Grahame 
Published: March 2005

We have sequenced two tryptic/chymotryptic peptides (TC3 and TC3a) containing a third site phosphorylated on rat acetyl‐CoA carboxylase by the AMP‐activated protein kinase. Comparison with the complete sequence of rat acetyl‐CoA carboxylase predicted from the cDNA sequence [López‐Casillas et al. (1988) Proc. Natl Acad. Sci. USA 85, 5784–5788] shows that this site corresponds to Ser1215.
Comparison of the cDNA sequence with previous amino acid sequence data identifies the other two sites for the AMP‐activated protein kinase as Ser79 and Ser1200. A total of eight serine residues phosphorylated in vitro by six protein kinases can now be identified: six of these (Ser23, Ser25, Ser29, Ser77, Ser79 and Ser95) are clustered in the amino terminal region, while two (Ser1200 and Ser1215) are located in the central region.
Prior phosphorylation of Ser77 and Ser1200 by cyclic‐AMP‐dependent protein kinase prevents subsequent phosphorylation of Ser79 and Ser1200, but not Ser1215, by the AMP‐activated protein kinase. Phosphorylation of Ser1215 under these conditions is not associated with a change in enzyme activity.
Limited trypsin treatment of native acetyl‐CoA carboxylase selectively cleaves off the highly phosphorylated amino‐terminal region containing Ser79.
Phosphorylation at Ser79 and Ser1200 by the AMP‐activated protein kinase dramatically decreases Vmax and increases the A0.5 for citrate. Phosphorylation at Ser77 and Ser1200 by cyclic‐AMP‐dependent protein kinase causes more modest changes in the A0.5 for citrate and the Vmax. Dephosphorylation, or removal of the amino‐terminal region containing Ser77/79 using trypsin, reverses all of these effects.
These results suggest that the effects of the AMP‐activated protein kinase on acetyl‐CoA carboxylase activity are mediated entirely by phosphorylation of Ser79, and not Ser1200 and Ser1215. The smaller effects of cyclic‐AMP‐dependent protein kinase are mediated by phosphorylation of Ser77.
Roles of the AMP‐activated and cyclic‐AMP‐dependent protein kinases in the adrenaline‐induced inactivation of acetyl‐CoA carboxylase in rat adipocytes
Authors: HAYSTEAD Timothy A. J. , MOORE Frances , COHEN Philip , HARDIE D. Grahame 
Published: March 2005

In isolated rat adipocytes, acetyl‐CoA carboxylase is inactivated by treatment of the cells with adrenaline or the β‐agonist isoproterenol, but not by the α‐agonist phenylephine. The inactivation is stable during purification in the presence of protein phosphatase inhibitors, and is associated with a 30–40% increase in the labelling of enzyme isolated from 32P‐labelled cells.
Increased phosphorylation occurs within peptide T1, which was identified by sequencing to be the peptide Ser‐Ser77‐Met‐Ser79‐Gly‐Leu‐His‐Leu‐Val‐Lys, containing Ser‐77 (phosphorylated by cyclic‐AMP‐dependent protein kinase) and Ser‐79 (phosphorylated by the AMP‐activated protein kinase). Analysis of the release of radioactivity as free phosphate during Edman degradation of peptide T1 revealed that all of the phosphate was in Ser‐79 in both basal and hormone‐ or agonist‐stimulated cells. Treatment of adipocytes with various agents which activate cyclic‐AMP‐dependent protein kinase by receptor‐independent mechanisms (forskolin, cyclic AMP analogues, isobutylmethylxanthine) also produced inactivation of acetyl‐CoA carboxylase and increased phosphorylation at Ser‐79.
The (Rp)‐[thio]phosphate analogue of cyclic AMP, which is an antagonist of binding of cyclic AMP to the regulatory subunit of cyclic‐AMP‐dependent protein kinase, opposes the effect of adrenaline on phosphorylation and inactivation of acetyl‐CoA carboxylase. Together with the effects of isobutylmethylxanthine and the stimulatory cyclic AMP analogues, this strongly indicates that cyclic‐AMP‐dependent protein kinase is an essential component of the signal transduction pathway, although clearly it does not directly phosphorylate acetyl‐CoA carboxylase.
As shown by okadaic acid inhibition, > 95% of the acetyl‐CoA carboxylase phosphatase activity in extracts of rat adipocytes or liver is accounted for by protein phosphatase‐2A, with <5% attributable to protein phosphatase‐1. Inhibition of protein phosphatase‐1 via phosphorylation of inhibitor‐1 is therefore unlikely to be the mechanism by which cyclic‐AMP‐dependent protein kinase indirectly increases phosphorylation of acetyl‐CoA carboxylase. Various other potential mechanisms are discussed.
The 2‐oxoglutarate dehydrogenase complex from Azotobacter vinelandii
Authors: WESTPHAL Adrie H. , de KOK Arie 
Published: March 2005

The nucleotide sequence encoding the succinyltransferase component (E2o) of the 2‐oxoglutarate dehydrogenase complex from Azotobacter vinelandii has been determined. Previously the cloning in Escherichia coli of the gene encoding lipoamide dehydrogenase from A. vinelandii was reported [Westphal, A. H. & de Kok, A. (1988) Eur. J. Biochem. 172, 299–305]. The 3.2‐kb fragment used for the sequence determination contained the main part of the gene encoding succinyltransferase. The complete E2o gene, as well as the gene encoding the 2‐oxoglutarate dehydrogenase component, resided on a 14.7‐kb fragment from which the 3.2‐kb fragment was subcloned.The protein‐coding sequence of the gene consists of 1200 bp (400 codons, including the AUG start codon and the UGA stop codon). It is separated from the gene encoding the 2‐oxoglutarate dehydrogenase component by 42 bp. No E. coli‐like promoter sequence was found. A putative ribosome‐binding site is located 9–15 bp upstream from the start codon. No terminator sequences were found downstream of the stop codon. This makes it likely that the three genes of the oxoglutarate dehydrogenase complex are transcribed as a single mRNA transcript analogous to the pyruvate dehydrogenase complex in E. coli. The intact gene was subcloned from the 14.7‐kb fragment and brought to high expression under the influence of the vector‐encoded lacZ promotor.The similarity with the E. coli enzyme is high with 63% identity. Like the enzyme from E. coli, it consists of a single lipoyl‐binding domain, a putative E1‐and E3‐binding domain and a catalytic domain. The main difference is found in a 31‐residue sequence rich in alanine and proline located between the lipoyl domain and the putative E1‐ and E3‐binding domain. This sequence, usually found in acetyltransferases and there identified as a highly mobile region by 1H‐NMR, is replaced by a more polar, charged region in the E. coli enzyme.
Metabolism of adenosine through adenosine kinase inhibits gluconeogenesis in isolated rat hepatocytes
Authors: LAVOINNE Alain , CLAEYSSENS Sophie , CHEDEVILLE Arlette 
Published: March 2005

In isolated hepatocytes from fasted rats, 0.5 mM adenosine inhibited gluconeogenesis from glutamine, lactate and pyruvate. This inhibition was due to adenosine conversion through adenosine kinase. An increase in ketone‐body release was only observed in the presence of lactate or pyruvate, and the two phenomena (i.e. inhibition of gluconeogenesis and increased ketone‐body release) were linked.With alanine, dihydroxyacetone or serine as substrates, adenosine did not change gluconeogenesis; however, its conversion through adenosine kinase also inhibited gluconeogenesis. With asparagine as substrate, 0.5 mM adenosine increased gluconeogenesis; this increase was due to adenosine conversion through adenosine deaminase. However, adenosine conversion through adenosine kinase inhibited gluconeogenesis from asparagine. Thus, whatever the substrate used, adenosine conversion through adenosine kinase inhibited gluconeogenesis.The inhibitory effect of adenosine on gluconeogenesis cannot be related to the decrease in Pi concentration and to the increase in ATP pool.Beside its effect on gluconeogenesis, adenosine inhibited ketogenesis measured without added substrate; adenosine conversion through adenosine kinase was also involved in the inhibition of ketogenesis.
Conformational changes of tarantula (Eurypelma californicum) haemocyanin detected with a fluorescent probe, 7‐chloro‐4‐nitrobenzo‐2‐oxa‐1,3‐diazole
Authors: LEIDESCHER Thomas , DECKER Heinz 
Published: March 2005

Different fluorescent labels were tested in order to monitor conformational transitions of the four‐hexamer haemocyanin from the tarantula Eurypelma californicum during the oxygenation process. When the four‐hexamer was labelled with 7‐chloro‐4‐nitrobenzo‐2‐oxa‐1,3‐diazole, the maximum wavelength λmax of the fluorescence emission spectrum was significantly shifted up to 5 nm, depending on pH and the degree of oxygenation. The values for λmax of the fully oxygenated haemocyanin were 531.5 nm (pH < 7.8) and 530.0 nm (pH > 7.8). For deoxygenated haemocyanin the values were 533.5 nm (pH < 7.2) and 535.2 nm (pH > 7.2). The occurrence of four distinct emission maxima supports the hypothesis of four conformational species for the tarantula haemocyanin, which have been predicted by the nesting model [Robert, C. H., Decker, H., Richey, B., Gill, S. J. & Wyman, J. (1987) Proc. Natl Acad. Sci. USA 84, 1891–1895].Only four amino acids of the four‐hexamer were labelled with 7‐chloro‐4‐nitrobenzo‐2‐oxa‐1,3‐diazole. They were identified as lysine 484 on the purified peptide Leu‐Arg‐Lys‐Phe‐His‐Arg. This amino acid is located on the surface of the four copies of subunit d.The sharp shift of the maxima of the emission wavelengths during oxygenation indicates that the four copies of subunits d synchronously take part in the conformational switch. This points to a concerted mechanism for the conformational transitions of the tarantula haemocyanin.
Regulation by butyrate of the cAMP response to cholera toxin and forskolin in pituitary GH1 cells
Authors: ARANDA Ana , TORO Maria Jose , USERA Fernando , PASCUAL Angel 
Published: March 2005

In pituitary GH1 cells, a rat growth hormone‐producing cell line, butyrate elicited a dose‐dependent increase in cholera toxin receptors as measured by an increased binding of 125I‐labeled cholera toxin to the intact cells. Butyrate did not alter the affinity of cholera toxin binding, the dissociation constant being 0.4 nM for both control and butyrate‐treated cells. Despite the increased binding, the cAMP response to cholera toxin was strongly reduced after exposure to butyrate. This reduction was dose‐dependent and with butyrate 1–5 mM, intracellular and extracellular (medium) cAMP levels were decreased by more than 70% in cells incubated for 24 h with 1 nM cholera toxin. Forskolin (30 μM) elicited a cAMP response similar to that found with the toxin, and a similar inhibition of cAMP was also found after incubation of GH1 cells with butyrate. Butyrate also affected basal cAMP levels which were reduced by 40–60% in cells cultured for 24–48 h with the fatty acid. In order to study whether butyrate influenced cAMP synthesis and/or cAMP degradation, adenylyl cyclase and phosphodiesterase activities were determined in control cells and in cells incubated for 24 h with cholera toxin or forskolin. Butyrate had a dual effect since, besides activating phosphodiesterase by more than twofold, it also inhibited the cyclase by 40–50% in all groups. The in vitro response of adenylyl cyclase to stimulatory (NaF) and inhibitory (carbachol and adenosine) effectors was also examined. The absolute activity of the cyclase was always 40–50% lower in the cells incubated with butyrate, but the percentage change of activity obtained in butyrate‐treated and untreated cells was unaltered. In addition, ADP‐ribosylation of the guanine nucleotide stimulatory component of the cyclase (Gs) was not affected in the cells incubated with butyrate. These results suggest that the catalytic (C) subunit of adenylyl cyclase and/or its interaction with the regulatory components might be altered in butyrate‐treated GH1 cells.The inhibition of the cAMP response in GH1 cells was accompanied by an inhibition of a biological action of the nucleotide, namely growth hormone (somatotropin) production which is primarily controlled by thyroid hormones in these cells. Forskolin alone did not affect the somatotropin levels but potentiated the growth hormone response to triiodothyronine. Butyrate produced a dose‐dependent inhibition of this response, which was totally abolished at concentrations of butyrate higher than 1 mM.
Quantitative estimation of minor mRNAs by cDNA‐polymerase chain reaction
Authors: CHELLY Jamel , MONTARRAS Didier , PINSET Christian , BERWALD‐NETTER Yoheved , KAPLAN Jean‐Claude , KAHN Axel 
Published: March 2005

Amplification of the mRNA polymerase chain reaction is a very sensitive technique to detect low‐abundance transcripts. We describe in this paper conditions necessary to make this technique quantitative. Quantification is performed in the exponential phase of the amplification process and the results are standardized with respect to those obtained with an exogenous mRNA which is co‐reverse‐transcribed and co‐amplified in the same reaction as the analyzed transcripts. The primers are chosen in different exons to distinguish the amplification of mRNA fragments from the amplification of contaminating DNA. Analysis of the kinetics of amplification and parameters influencing this kinetics shows that: (a) in the exponential phase of amplification, the amount of amplified fragments is proportional to the initial amount of transcripts; (b) in a certain range of length fragment, the yield of amplification is inversely proportional to the length of the amplified fragments.Using this method we have demonstrated that the dystrophin gene is already activated at the myoblastic stage. A quantitative estimation of the transcript showed that the expression of this gene increases strongly in the course of in vitro myogenesis. In primary culture of mouse brain cells, the dystrophin gene was found to be more expressed in neuronal than in glial cells.
Chemiosmotic energy conversion and the membrane ATPase of Methanolobus tindarius
Authors: SCHEEL Edgar , SCHÄFER Günter 
Published: March 2005

Electron transport phosphorylation has been demonstrated to drive ATP synthesis for the methanogenic archaebacterium Methanolobus tindarius: Protonophores evoked uncoupler effects and lowered the membrane potential ΔΨ. Under the influence of N,N′‐dicyclohexylcarbodiimide [(cHxN)2C] the membrane potential increased while methanol turnover was inhibited. 2‐Bromoethanesulfonate, an inhibitor of methanogenesis, had no effect on the membrane potential but, like (cHxN)2C and protonophores, decreased the intracellular ATP concentration.Labeling experiments with (cHxN)214C showed membranes to contain a proteolipid, with a molecular mass of 5.5 kDa, that resembles known (cHxN)2C‐binding proteins of F0‐F1 ATPases. The (cHxN)2‐sensitive membrane ATPase hydrolysed Mg · ATP at a pH optimum of 5.0 with a Km (ATP) of 2.5 mM (V= 77 mU/mg). It was inhibited competitively by ADP; Ki (ADP) = 0.65 mM. Azide or vanadate caused no significant loss in ATPase activity, but millimolar concentrations of nitrate showed an inhibitory effect, suggesting a relationship to ATPases from vacuolar membranes. In contrast, no inhibition occurred in the presence of bafilomycin A1.The ATPase was extractable with EDTA at low salt concentrations. The purified enzyme consists of four different subunits, α (67 kDa), β (52 kDa), γ (20 kDa) and δ (< 10 kDa), as determined from SDS gel electrophoresis.
The effect of substituting phosphotyrosine for sulphotyrosine on the activity of hirudin
Authors: HOFSTEENGE Jan , STONE Stuart R. , DONELLA‐DEANA Arianna , PINNA Lorenzo A. 
Published: March 2005

Recombinant hirudin (hirudin), which lacks the sulphate group of Typ‐63, has a tenfold‐reduced affinity for α‐thrombin. Incubation of recombinant hirudin with [γ‐32P]ATP and protein tyrosine kinase III from spleen resulted in incorporation of radioactivity into the protein. Phosphatohirudin was purified to homogeneity (overall yield 5%) and shown to contain 1 mol phosphate/mol protein, as a phosphotyrosyl residue at position 63.The kinetics of the inhibition of human α‐thrombin by phosphatohirudin were determined. It was found that the introduction of the negatively charged phosphate had fully restored the affinity of recombinant hirudin for α‐thrombin to the level of the wild‐type sulphatohirudin. The inhibition constant of phosphatohirudin was 18 fM compared with 20 fM for that of sulphatohirudin. Moreover, the values for the on‐and off‐rate constants of the both forms of hirudin were indistinguishable.
Conformational and thermodynamic effects of naturally occurring base methylations in a ribosomal RNA hairpin of Bacillus stearothermophilus
Authors: HEUS Hans A. , FORMENOY Leo J. , VAN KNIPPENBERG Peter H. 
Published: March 2005

The 3′‐terminal colicin fragments of 16S ribosomal RNA were isolated from Bacillus stearothermophilus and from its kasugamycin‐resistant (ksgA) derivative lacking N6‐dimethylation of the two adjacent adenosines in a hairpin loop. The fragment from the ksgA strain still contains a naturally occurring N2‐methylguanosine in the loop. An RNA molecule resembling the B. stearothermophilus colicin fragment but without modified nucleosides was synthesized in vitro using a DNA template and bacteriophage T7 RNA polymerase.Proton‐NMR spectra of the RNAs were recorded at 500 MHz. The imino‐proton resonances of base‐paired G and U residues could be assigned on the basis of previous NMR studies of the colicin fragment of Escherichia coli and by a combination of methylation‐induced shifts and thermal melting of base pairs. The assignments were partly confirmed by NOE measurements. Adenosine dimethylation in the loop has a distinct conformational effect on the base pairs adjoining the loop.The thermal denaturation melting curve of the enzymatically synthesized RNA fragment was also determined and the transition midpoint (tm) was found to be 73°C at 15 mM Na+. A comparison with previously determined thermodynamic parameters for various colicin fragments demonstrates that base methylations in the loop lead to a relatively strong destabilization of the hairpin helix. In terms of free energy the positive contribution of the methylations are in the order of the deletion of one base pair from the stem. Other data show that recently published free‐energy parameters do not apply for certain RNA hairpins.