bluuebunny/crossref_metadata_2025_split huggingface dataset:
Effect of Funiculosin and Antimycin A on the Redox‐Driven H+‐Pumps in Mitochondria: on the Nature of ‘Leaks’
Authors: PIETROBON Daniela , AZZONE Giovanni F. , WALZ Dieter 
Published: July 2010

The effect of antimycin A and funiculosin, two inhibitors which block electron transfer in the b‐c1 complex, on electron flow and electrochemical potential difference of H+ ions in mitochondria at static head (state 4) is investigated. In addition, the respiratory control ratio is determined as the ratio between uncoupler stimulated and static‐head electron flow. Malonate, a competitive inhibitor of succinic dehydrogenase, is used for comparison.All three inhibitors cause an extensive depression of static‐head electron flow but only a limited decrease in the electrochemical potential difference of H+ ions. With the antimycin‐type of inhibitors, the respiratory control ratio slightly increases up to about 50% inhibition of electron flow and then steeply declines. With malonate, a strong decrease of the respiratory control ratio is observed in a concentration range where the electron flow is inhibited less than 10%.It is shown that the data do not compiy with the generally accepted hypothesis of a leak conductance being regulated by the electrochemical potential difference of H+ ions. They can be interpreted in terms of not tightly coupled redox‐driven H+‐pumps. A non‐vanishing electron flow at static head then arises predominantly from molecular slipping in the pumps, and the (constant) leak conductance yields only a minor contribution.
Regulatory Properties of Phosphofructokinase 2 from Escherichia coli
Authors: KOTLARZ Denise , BUC Henri 
Published: July 2010

Escherichia coli K12 contains two phosphofructokinases: phosphofructokinase 1, the most studied one, appears to behave as an allosteric enzyme, while phosphofructokinase 2 presents the features of a Michaelian enzyme.We show in the present paper that, in fact, phosphofructokinase 2 also presents some regulatory properties in vitro: at high concentrations, ATP is an inhibitor of phosphofructokinase 2 and it provokes the tetramerization of the dimeric native enzyme.The binding of the two substrates to phosphofructokinase 2 is sequential and ordered as for phosphofructokinase 1, but in the former case fructose 6‐phosphate is the first substrate to be bound and ADP the first product to be released. Each dimer of phosphofructokinase 2 binds two molecules of fructose 6‐phosphate but only one molecule of the product fructose 1,6‐bisphosphate.Although both phosphofructokinases of E. coli K12 present regulatory properties in vitro, the mechanism of regulation of the activity of the two enzymes is strikingly different. It can be asked whether or not these mechanisms operate in vivo.
Reconstitution of a Functional Acetylcholine Receptor
Authors: POPOT Jean‐Luc , CARTAUD Jean , CHANGEUX Jean‐Pierre 
Published: July 2010

Highly purified plasma membrane fragments rich in acetylcholine (nicotinic) receptor were prepared fromTorpedo marmorataelectric organ, depleted at alkaline pH of the 43000‐Mr, protein and solubilized with sodium holate in the presence or absence of exogeneous lipids. The solubilized receptor (essentially in its 9‐S light form) was reinserted into artificial lipid vesicles in the course of detergent removal by two methods yielding reconstituted vesicles with mean diameters of about 40 nm and 100 nn respectively. The influence of the composition of the lipid and aqueous phases on the physical reinsertion of the receptor protein was studied by gel filtration. gradient centrifugation and electron microscopy. At low ionic strength, the α‐toxin binding sites were quantitatively reintegrated into vesicles from soy‐bean lipids, but not into egg phosphatidylcholine vesicles. Raising the ionic strength during detergent removal decreased receptor incorporation.The permeability properties of the reconstituted vesicles were investigated by a Millipore filtration method using radioactive tracer ions. Passive equilibration of22Na+or134Cs+ions between internal and external compartments was slow and remained incomplete after several days. Addition of the agonists acetylcholine or carbamylcholine stimulated the exchange of cations which reached a plateau value within less than 15 s. These agomsts did not affect the rate of36Cl−exchange. The permeability response was totally blocked by snake α‐toxins. trimethisoquin, histrionicotoxin, and by preincubation with agonists (desensitization). It is concluded that these properties do not depend on the dimerization of the receptor protein into the 13‐S form nor on its association with the 43000‐Mrprotein and therefore appear to be intrinsic to the 9‐S form of the receptor.
Synthetic Monoacylphospholipids as Reactivators of the Calcium‐Dependent ATPase of Enzymatically Delipidated Sarcoplasmic Membranes
Authors: THE Rudy , HUSSEINI Hussein Shakib , HASSELBACH Wilhelm 
Published: July 2010

The reactivating effect of synthetic analogues of natural lysophosphatidylcholines, acyldeoxyglycerophosphocholines, with acyl chain lengths between C10–C18 on enzymatically delipidated sarcoplasmic membranes has been analyzed.
The calcium‐dependent ATPase is fully restored by myristoyldeoxyglycerophosphocholine. The restoring effect of deoxyglycerophosphocholines declines when the length of the saturated acyl chain becomes shorter or longer.
In contrast to the weakly effective palmitoyldeoxyglycerophosphocholine, its cis‐9‐mono unsaturated analogue oleyldeoxyglycerophosphocholine proved to be a highly effective reactivating compound.
The transfer of the terminal phosphate residue of ATP to the transport protein and the phosphate exchange between ADP and ATP displays the same dependence on the acyl chain length of the deoxyglycerophosphocholines. In contrast to the ATPase activity the ATP‐supported phosphoryltransfer reactions can only partially be restored.
A significant restoration of the phosphorylation of the protein by inorganic phosphate could be achieved with none of the deoxyglycerophosphocholines.
Below 23°C the apparent activation energy of the calcium‐dependent ATPase increases with increasing chain length of the deoxyglycerophosphocholines while above 23°C the activation energies were identical for all restituted preparations.
Enzymic Synthesis of Juvenile Hormone in Locust Corpora Allata: Evidence for a Microsomal Cytochrome P‐450 Linked Methyl Farnesoate Epoxidase
Authors: FEYEREISEN Rene , PRATT Grahame E. , HAMNETT Anthony F. 
Published: July 2010

Homogenates of corpora allata from adult Locusta migratoria in phosphate‐buffered EDTA have been analysed by sucrose‐density‐gradient centrifugation. Succinate‐cytochrome c reductase activity (mitochondrial) bands between d204 1.13–1.15, whereas NADPH‐cytochrome c reductase and NADPH–dependent methyl farnesoate 10, 11‐epoxidase activities band identically between d204 1.06–1.12. We conclude that the methyl farnesoate epoxidase is exclusively microsomal. Farnesoic acid O‐methyltransferase is an exclusively soluble enzyme which stoichiometrically transfers the S‐methyl group from S‐adenosylmethionine to farnesoic acid. No carboxyl esterase activity was found.Isolated microsomes were used to obtain an apparent Km= 7.7 × 10−6 M for the epoxidase, although substrate solubility limits the rate to 0.5 V As expected, the product (juvenile hormone III) is chiral (10 R). The epoxidase is inhibited by excess NADP+ and oxidised cytochrome c, but neither inhibited nor synergised by NADH. NADH supports less than 10% of the NADPH rate of epoxidation. The epoxidase is inhibited by a carbon monoxide/oxygen atmosphere, half‐maximal inhibition occurring at a CO/O2 ratio of 4.0. This inhibition is reversed by white‐light irradiation.
Allotropism in Aspartyl‐tRNA Synthetase from Porcine Thyroid
Authors: VELLEKAMP Gary J. , KULL Fredrick J. 
Published: July 2010

Three forms of aspartyl‐tRNA synthetase, PC‐1, PC‐2 and PC‐3 (based on their order of elution from phosphocellulose columns), were purified from porcine thyroid utilizing fractional pH precipitations and column chromatography. Two forms, PC‐1 and PC‐2, eluted identically from gel filtration columns and migrated the same electrophoretically and in glycerol density gradients. They were estimated to have sedimentation coefficients of 7 S and molecular weights of about 150000. Both PC‐1 and PC‐2 were equally inhibited by KCl. Their elution positions from phosphocellulose were unchanged upon rechromatography, indicating that they were nonequilibrium forms and they were present in post‐mitochondrial supernatants in roughly equal amounts. All three forms were present in the same ratios whether or not phenylmethylsulphonyl fluoride was present in homogenizing buffers.PC‐3 had the characteristics of an aminoacyl‐tRNA synthetase complex. It contained aminoacyl‐tRNA synthetase activity for 16 of 18 amino acids tested and was about 4% low‐molecular‐weight RNA. The complex did not significantly penetrate 5% polyacrylamide gels, was excluded by Sephacryl S‐200 and had a sedimentation coefficient greater than 20 S indicating a particles mass in excess of 500000 daltons. In addition, the PC‐3 fraction contained a ribonuclease capable of degrading both aminoacyl‐tRNA and tRNA.After storage, rechromatography of PC‐3 on phosphocellulose showed that some dissociation of the enzymes had taken place. Aspartyl‐tRNA synthetase activity was found in positions analogous to PC‐1 as well as PC‐3, and ribonuclease activity was found in two positions on the columns, one coincident with PC‐3 and one preceding the position of elution of PC‐1. Dissociation of the complex was also observed using glycerol density gradient centrifugation and by chromatography on Sepharose4B.When present in the PC‐3 complex, the aspartyl‐tRNA synthetase activity and the ribonuclease activity were both stimulated by KC1. However, when dissociated to their free forms, the aspartyl‐tRNA synthetase activity was inhibited by KC1, whereas the ribonuclease remained stimulated. The effects on aspartyl‐tRNA synthetase, appeared to be potassium‐ion‐specific but were more pronounced in the presence of phosphate dianion than in chloride ion.The differential effects of potassium ion on the free and complexed forms of aspartyl‐tRNA synthetase and the prescence of a ribonuclease in the aminoacyl‐tRNA complex that is active toward tRNA could have functional significance in the intact cell.
Latent Phosphorylase Phosphatases from Rat Liver: Relationship with the Heat‐Stable Inhibitory Protein
Authors: JETT Mary‐Frances , HERS Henri‐Géry 
Published: July 2010

A high‐speed supernatant from rat liver contains at least two latent phosphorylase phosphatases the activities of which are revealed by treatment with ethanol, urea, mercaptoethanol or trypsin. This fraction also contains at least one protein which, after heating, inhibits to various degrees the activated form(s) of the two phosphatases. The two latent enzymes can be separated by cellulose‐phosphate chromatography and can be differentiated by their preferential activation by ethanol or trypsin and by their different sensitivity to the inhibitory protein after ethanol activation.Activation of the latent phosphorylase phosphatases by ethanol, urea or mercaptoethanol is not accompanied by the destruction of the precursor of the inhibitory protein whereas activation by trypsin is. However, trypsin treatment of fractions previously activated by ethanol decreases their activity and also increases their sensitivity to the inhibitory protein in a way which is unrelated to the destruction of this inhibitor. Furthermore, some protein fractions, almost free of the precursor of the inhibitory protein can be readily activated by trypsin. It is concluded that the activation of the latent phosphorylase phosphatases is unrelated to the destruction of the inhibitory protein.
Glutaconate CoA‐Transferase from Acidaminococcus fermentans
Authors: BUCKEL Wolfgang , DORN Ulrike , SEMMLER Rosuitha 
Published: July 2010

Glutaconate CoA‐transferase catalyses the transfer of CoAS− from acetyl‐CoA preferentially to (E)‐glutaconate, but glutarate, (R)‐2‐hydroxyglutarate, acrylate and propionate are also good acceptors. No reaction was observed with (Z)‐glutaconate and C4‐dicarboxdic acids.
The product of the reaction of acetyl‐CoA with (E)‐glutaconate is the 1‐isomer of glutaconyl‐CoA, i.e. the thiol ester is conjugated with the double bond. Other results indicate. however, that with (R)‐2‐hydroxyglutarate as substrate both possible isomers are generated.
Glutaconate CoA‐transferase was purified from cell‐free extracts of Acidaminococcus fermentans to apparent homogeneity and crystallized. The relative molecular mass of the enzyme is approximately 275000. It consists of two different polypeptide chains (Mr 32000 and 34000). On the catalytic pathway a thiolester is formed between CoASH and a carboxylate of the smaller polypeptide chain.
The structural and functional relationships between glutaconate CoA‐transferase and other CoA‐transferases are discussed.
Glutaconate CoA‐transferase is also present in other bacteria fermenting glutamate via hydroxyglutarate. Experiments with an antiserum against the enzyme indicate that the transferase is necessary for the decarboxylation of glutaconate but not for the dehydration of (R)‐2‐hydroxyglutarate.
Ribosomal Protein S6 from Xenopus laevis Ovaries
Authors: KALTHOFF Holger , DARMER Dorothea , TOWBIN Harry , GORDON Julian , AMONS Reinout , MÖLEER Wim , RICHTER Dictmar 
Published: July 2010

Ribosomal protein S6 from Xenopus laevis ovaries was prepared by ion‐exchange chromatography on phosphocellulose and gel filtration on Sephadex G‐75. The protein was identified as S6 from its position on two‐dimensional polyacrylamide gels and from its immunological cross‐reaction with monoclonal antibody raised against chicken liver S6, and from the fact that it is the major phosphorylated protein of the small subunit.When oocytes were incubated with [32P]orthophosphate in the presence of progesterone. 32P incorporation of 40‐S ribosoniul proteins was stimulated about 10‐fold over controls without hormone. The bulk of the 32P radioactivity was incorporated into protein S6.
Molecular Cloning of the 8000‐Base Thyroglobulin Structural Gene
Authors: CHRISTOPHE Daniel , MERCKEN Luc , BROCAS Huguette , POHL Viviane , VASSART Gilbert 
Published: July 2010

Bovine tliyroglobulin mRNA was reverse‐transcribed into full‐length double‐stranded cDNA. The existence of three HindIII restriction endonuclease sites in the 8000‐base tliyroglobulin structural gene had allowed the easy cloning of the two internal HindIII fragments [Cliristophe et al. (1980) Eur. J. Biochem. 111, 419‐423], In the present study, the central portion of the structural gene was cloned in Escherickia coii as two individual recombinant plasmids containing 2000‐base‐pair and 4700‐base‐pair segments located respectively 5′ and 3′ relative to the unique BamHI site of the cDNA. BumHI linkers were added to the double‐stranded cDNA and, following restrictions with HindIII. selective cloning of the 5′ (2600‐base‐pair) and 3′ (1000‐base‐pair) terminal HindIII fragments was achieved by inserting them between the HindIII and BamHI sites of the plasmid pBR322. Partial sequencing of the 1000‐base‐pair 3′‐terminal fragment demonstrated the presence of an A‐A‐U‐A‐A‐A sequence in the inRNA 14 bases upstream from a poIy(A) tract corresponding to the 3′ end of the mRNA.Together, the four clones represent about 99%of the thyroglobulin structural gene and provide the starting material for the determination of thyroglobulin primary structure.
Transport of the Precursor for Rat‐Liver Ornithine Carbamoyltransferase into Mitochondria in vitro
Authors: MORITA Tetsuo , MIURA Satoshi , MORI Masataka , TATIBANA Masamiti 
Published: July 2010

Transport of the precursor form of rat liver ornithine carbanioyltransferase was studied in a cell‐free reconstitution system. The precursor. synthesized in vitro. was converted to an apparently mature form of the enzyme by incubation with a mitochondria1 preparation of rat liver. On further purification of the preparation by isopycnic sucrose gradient centrifugation, the processing activity cosedimented with mitochondria. The processed pro‐duct was no longer susceptible to externally added proteases.
The activity responsible for the conversion of the precursor to the mature form disappeared when the mitochondria were disrupted by digitonin treatment. sonication or freeze‐thawing. The inactivation apparently pardleled the leakage of the marker enzyme of the intermembrane space from the mitochondria.
Processing of the precursor by intact mitocliondria was strongly inhibited by both 0.1 mM dinitroplienol and 1 μM carbonylcyanide p‐trifluoromethoxyphenylhydrazone. However. neither KCN, NaN3. antimycin A nor oligomycin had any marked effect on the processing.
Mitochondria from rat spleen and ascites hepatonin cells, which lack ornithine carbamoyltransferase. were able to take up the enzyme precursor and to process it to the mature form of the enzyme. Mouse liver kind kidney mitochondria also took up and processed the rat precursor.
In pulse and pulse‐chase experiments with isolated rat hepatocytes. the labeled precursor first appeared in the cytosol and then disappeared with a half‐life of less than 3 min. Dinitrophenol and carbonylcyanide p‐trifluoromethoxyphenylhydrazone failed to inhibit the disappearance of the precursor from the cytosol.

The results indicate that the ornithine carbamoyltransferase precursor is transported into isolated mitochondria in association with the proteolytic processing to the mature enzyme. The transport‐processing system requires mitochondria of high integrity. It was sensitive to uncouplers but not much to respiratory inhibitors. and may be common to more than one mitochondria1 enzyme precursor.
Kinetic Equivalence of the Subunits of Liver Alcohol Dehydrogenase
Authors: ANDERSON Pia , PETTERSSON Gösta 
Published: July 2010

The kinetics of two‐substrate enzyme reactions proceeding by an ordered ternary‐complex mechanism have been analyzed theoretically with respect to the precise magnitude of amplitudes of the two slowest transients governing such reactions under single‐turnover conditions. Relationships derived have been applied to the liver alcohol dehydrogenase system for detailed interpretation of amplitudes of the biphasic absorbance changes associated with the enzymic reduction of aromatic aldehydes in catalytic‐suicide experiments.
Amplitudes of the slow phase 328–330‐nm absorbance changes occurring during the enzymic reduction of various para‐substituted benzaldehydes by NADH or NAD2H correspond to 6–26% of the total absorbance change under conditions of substrate and coenzyme saturation. Saturation values of amplitudes determined in the present and previous investigations agree within 2 % of the total absorbance change with those calculated theoretically for an ordered mechanism of enzyme action at equivalent sites. In particular, amplitudes observed exhibit the expected sensitivity to electronic substituent and deuterium isotope effects.
Enzyme recycling, aldehyde binding, and alcohol desorption provide negligible contributions to the 328–330‐nm absorbance changes observed during aldehyde reduction in the presence of 20 mM pyrazole. The maximum slow phase amplitude contribution provided by pyrazole binding corresponds to 5–6% of the total absorbance change. Additional slow phase absorbance changes persisting at saturating concentrations of coenzyme and substrate reflect incomplete accumulation of the enzymic intermediate formed through hydride transfer in the rapid reaction phase. This intermediate contains no detectable amounts (less than 2 %,) of NADH, but exhibits spectral properties indistinguishable from those of enzyme. NAD+. alcohol complexes examined.
It has been concluded in recent publications that an ordered ternary‐complex mechanism involving equivalent sites is insufficient to account for the transient‐state kinetics of liver alcohol dehydrogenase catalysis. These conclusions are shown to be based on an inadequate interpretation of the observations made. Results reported up to date provide no evidence whatsoever for the kinetic significance of subunit interactions leading to a non‐equivalence of the enzymic sites. Subunit interactions resulting in a “half‐site7rdquo; reactivity of the enzyme are definitely not at hand.
Differences in the Binding of Thyroid Hormones and Indoles by Rat α1‐Fetoprotein and Serum Albumin
Authors: HERVÉ Françoise , GRIGOROVA Anne‐Marie , RAJKOWSKI Krzysztof , CITTANOVA Nicole 
Published: July 2010

The transport of small molecules in the blood, normally assured by serum albumin in the adult. is not well known in the fetus since the albumin concentration is low in fetal serum and inversely related to the α1‐fetoprotein concentration.In order to investigate whether rat α1‐fetoprotein might be a fetal counterpart to albumin. the binding properties of these two proteins have been compared with respect to a series of molecules of biological importance, especially during fetal development: thyroid hormones and indole analogues.Though high‐affinity binding of thyroxine was found with both rat α1‐fetoprotein and albumin, a significant difference in the number of binding sites for this hormone was found with the two protcins. Further, while rat serum albumin strongly bound L‐tryptophan and indolyl‐3‐acetic acid (Ka∼ 105 M−1), rat α1‐fetoprotein did not bind any of the indoles tested.These results are discussed with respect to the physiological and pharmacological significance of the transport role of these proteins.
Occurrence of a Catabolic l‐Serine (l‐Threonine) Deaminase in Saccharomyces cerevisiae
Authors: RAMOS Fernando , WIAME Jean‐Marie 
Published: July 2010

Saccharomyces cerevisiae mutants lacking the anabolic l‐threonine deaminase, the ilvl−mutants, have been found to exhibit a normal ability to grow, without auxotrophy towards isoleucine, on l‐threonine or l‐serine as only nitrogen nutrient. Starting from a strain carrying a ilvl−mutation, which also impairs the ability to utilize l‐serine, has been denominated cha−, for ‘catabolism of hydroxyamino acids’ and was found to result in the lack of a catabolic l‐serine (l‐threonine) deaminase.This enzyme which, unlike the anabolic threonine deaminase, is more active towards serine that towards threonine, differs from the latter enzyme by a number of biochemical and regulatory properties. Whereas the anabolic enzyme is an allosteric enzyme sensitive to feedback inhibition by isoleucine, the catabolic enzyme exhibits Michaelian kinetics; no control of its activity has been detected. Its synthesis is induced by l‐serine and l‐threonine.These two enzymes, which thus can be easily differentiated by means of their regulations, display a limited ability to compensate for one another's absence and appear to play clearly distinct roles under normal physiological conditions.