bluuebunny/crossref_metadata_2025_split huggingface dataset:
Enzymes Involved in Ketone Utilization in Different Types of Muscle: Adaptation to Exercise
Authors: WINDER William W. , HOLLOSZY John O. , BALDWIN Kenneth M. 
Published: March 2005

Activity levels of the enzymes involved in ketone utilization were compared in heart, fast‐twitch red, slow‐twitch red, and fast‐twitch white types of muscles in rats. 3‐Hydroxybutyrate dehydrogenase, 3‐ketoacid CoA‐transferase, and acetoacetyl‐CoA thiolase activities were lowest in white muscle, higher in slow red than in fast red muscle, and highest in the heart. The large differences between the four muscle types in the levels of these enzymes reflects differences in both mitochondrial content and composition. Differences in composition were evidenced by large dissimilarities between the different muscle types in the ratios of the activities of the enzymes of ketone oxidation to each other and to citrate synthase and cytochrome c. Of particular interest is the finding that, despite the fact that in the rat slow red muscle has a lower content of mitochondria than fast red muscle, 3‐hydroxybutyrate dehydrogenase activity is 140% higher and 3‐ketoacid CoA‐transferase is 70% higher in slow red than in fast red muscle.A 14‐week‐long program of treadmill running induced increases in the levels of ketone utilization enzymes in all three types of skeletal muscle but not in heart; 3‐hydroxybutyrate dehydrogenase became measurable, though at very low levels, in white muscle; increased 2.6‐fold in slow red muscle, and 6‐fold in fast red muscle. 3‐Ketoacid CoA‐transferase increased 2‐fold in both fast red and white types of muscle, but only 26% in slow red muscle. Acetoacetyl‐CoA thiolase activity increased 40–45% in all three types of skeletal muscle. In contrast, citrate synthase and cytochrome c increased approximately 2‐fold in all three types of skeletal muscle. These adaptive changes tend to make skeletal muscles more like heart muscle in their enzyme patterns and may help to explain why physically trained, as compared to untrained, individuals do not develop post‐exercise ketosis.
Acetate, a Major End Product of Fatty‐Acid Oxidation in Hamster Brown‐Adipose‐Tissue Mitochondria
Authors: BERNSON Vibeke S. M. , NICHOLLS David G. 
Published: March 2005

1. Brown‐adipose‐tissue mitochondria prepared from the cold‐adapted rat and guinea‐pig require malate for significant palmitoyl‐l‐carnitine oxidation. In contrast the mitochondria prepared from the cold‐adapted hamster (a hibernator) rapidly oxidize palmitoyl‐l‐carnitine in the absence of added malate.2. The major end product of malate‐independent fatty acid oxidation in the hamster is identified as acetate. In the presence of malate or l‐carnitine, citric acid cycle intermediates or acetyl‐l‐carnitine are also produced.3. Factors controlling the formation of these products are investigated. Acetate production is highly dependent on the rate of fatty acid oxidation, and on the acetyl‐carnitine/carnitine ratio.4. The rate of acetate production in the presence of malate is independent of the incubation temperature over the range from 37°C to 10°C, while the entry into the citric acid cycle is decreased 10‐fold.5. Acetate production is most pronounced under conditions where a high acetyl‐CoA/CoA ratio would be predicted to occur.6. It is suggested that the acetate pathway is of particular significance in brown adipose tissue from hibernators as a means of allowing fatty acid oxidation, and hence heat production, to occur at temperatures where the citric acid cycle is apparently unable to function.
Utilization by Yeast of d‐Lactate and l‐Lactate as Sources of Energy in the Presence of Antimycin A
Authors: PAJOT Patrick , CLAISSE Maurice L. 
Published: March 2005

Saccharomyces cerevisiae cells contain d and l‐lactate: cytochrome c oxidoreductases. The physiological role of these enzymes is investigated by inhibiting the respiratory chain with antimycin A and observing how the vital functions (respiration, protein biosynthesis, growth) are altered. When d or l‐lactate are provided as the source of energy, stoichiometric amounts of the antibiotic decrease the growth rate about five‐fold, the oxygen uptake two‐fold and the energetic efficiency eleven‐fold.In the presence of antimycin A, it was often proposed that only a very short chain consisting of one of the lactate dehydrogenases, cytochrome c and cytochrome oxidase could be involved in the oxidation of lactate. Consistent with this scheme are the measurements of lactate and oxygen uptake and the observation that keto acids are released into the external medium, which also allow one to rationalize the growth yield and the amino acid incorporation results.
Flavocytochrome b2: Simulation Studies of the Electron‐Transfer Reactions among the Prosthetic Groups
Authors: CAPEILLÈRE‐BLANDIN Chantal 
Published: March 2005

Simulation studies by digital computer were undertaken in order to test and clarify the interpretations deduced from experimental data concerning the electron transfer mechanism from l‐lactate to flavocytochrome b2, which were presented in a preceding paper in this journal.The reaction scheme proposed as the “best” one is composed of 7 steps. It allows the best fitting of the time courses established for the oxidized flavin (Flox), the flavin semiquinone (Flsq), the fully reduced flavin (Flred), and the reduced haem (Hred); it can be extended to 1 s. This scheme also allows a good simulation of the general shape of preequilibrium titration curves obtained at a 200‐ms reaction time for Hred and Flsq, and a valuable simulation of the reduction electron paramagnetic resonance time course established for Hred and Flsq at low lactate concentration. The agreement between experimental and simulated curves led to an estimation of some rate constants experimentally unknown, relative (in particular) to the electron exchange between flavin and haem and between couples of flavins. Another interest of these stimulation studies was to point out the obligatory involvement of a slow final step to perform the flavocytochrome b2 full reduction; this step could be controlled by some conformational change of the protein.
Stimulation of Proteinase K Action by Denaturing Agents: Application to the Isolation of Nucleic Acids and the Degradation of ‘Masked’ Proteins
Authors: HILZ Helmuth , WIEGERS Ulrich , ADAMIETZ Peter 
Published: March 2005

Hydrolysis of serum albumin by proteinase K was strongly (> 7‐fold) stimulated by urea and dodecylsulfate in a dose‐dependent manner. With an oligopeptide as substrate, however, proteinase K was inactivated by dodecylsulfate. This indicates that the apparent activation of proteinase K by urea and dodecylsulfate is caused primarily by denaturation of the protein substrates.Although dodecylsulfate inhibited ribonuclease activity in the test‐tube completely, it could not prevent RNA degradation during isolation of polysomal RNA, to which ribonuclease had been added, because of the reversible nature of the dodecylsulfate inhibition. Complete protection of RNA, however, was achieved by a combination of dodecylsulfate and proteinase K.The combined action of the detergent and proteinase K was also effective in degrading “masked” proteins in a poly(adenosine diphosphoribose) preparation which could not be attacked by the proteinase alone.
Two‐Dimensional Polyacrylamide‐Gel Electrophoresis of the Proteins and Glycoproteins of the Human Erythrocyte Membrane
Authors: ANSELSTETTER Volker , HORSTMANN Hans‐Joachim 
Published: March 2005

A two‐dimensional polyacrylamide gel electrophoresis technique has been developed, improving the analytical separation of some proteins and glycoproteins of the human erythrocyte membrane.Freshly prepared membranes are totally solubilized, subjected to dodecylsulfate – polyacrylamide gel electrophoresis in the first dimension, followed by electrophoresis in the second dimension, using a detergent‐free polyacrylamide gradient gel.By this method the proteins of the human erythrocyte membrane could be resolved into a two‐dimensional pattern, which has been shown to be highly reproducible with respect to various blood‐groups and within one blood‐group from specimen to specimen.The method enables especially the investigation of the hydrophobic and very likely integrated membrane proteins and glycoproteins. Thus, band III [Fairbanks, G., Steck, Th. & Wallach, D. F. H., Biochemistry, 10, 2606–2617 (1971)] could be shown to consist of five proteins, one of them being the major glycoprotein of the human erythrocyte membrane. The two spectrin bands differed considerably in their two‐dimensional patterns.The value of the given method for the investigation of membrane defects, which may be linked with various diseases of human erythrocytes, could be demonstrated in the case of two patients suffering from congenital dyserythropoetic anaemia.
Initiation of Protein Synthesis in Ehrlich Ascites Tumour Cells
Authors: PAIN Virginia M. , HENSHAW Edgar C. 
Published: March 2005

Binding of methionyl‐tRNAf to native 40‐S ribosomal subunits is thought to be an early stage in the process of polypeptide chain initiation, and [35S]Met‐tRNAf· 40‐S‐subunit complexes can be isolated from Ehrlich ascites tumour cells following a brief incubation with [35S]methionine. To determine whether this step is subject to modulation by physiological conditions, we have estimated the extent of binding of Met‐tRNAf to native 40‐S ribosomal subunits in Ehrlich cells under nutritional conditions known to affect the rate of protein synthesis in these cells. Deprivation of either an essential amino acid, lysine, or of glucose, results in a substantial reduction in the proportion of native 40‐S subunits which have Met‐tRNAf associated with them, and refeeding of lysine to cells deprived of this amino acid partially reverses this effect within 10 min. These effects on the concentration of Met‐tRNA · 40‐S‐subunit complexes are paralleled by changes of similar magnitude in the rate of protein synthesis and in polyribosome profiles.Native 40‐S subunits can be separated by equilibrium density gradient analysis on caesium chloride into two species, with buoyant densities approximately 1.40 and 1.49 g × cm−3. In cells deprived of either lysine or glucose, the radioactivity from [35S]methionine is bound exclusively to the particle of buoyant density 1.40 g × cm−3. In well‐fed cells, or in starved cells shortly after refeeding, a significant proportion of the label is associated with a region of the CsCl gradient corresponding to a particle of higher density.The results suggest that the binding of Met‐tRNAf to native 40‐S ribosomal subunits can be greatly affected by physiological conditions which alter the rate of protein synthesis. This is consistent with a regulatory role for this step in the sequence of reactions involved in initiation of translation.
Amino‐Acid Sequence of Toxin III of Naja haje
Authors: KOPEYAN Charles , MIRANDA François , ROCHAT Hervé 
Published: March 2005

The complete amino acid sequence of toxin III of Naja haje (72 residues) has been established mainly by use of a protein sequenator (identification of 70 residues). The two C‐terminal residues have been determined by digestion with carboxypeptidases A and B.Addition of succinylated protein or peptide greatly improved the performance of the sequenator for the Edman degradation of peptides: on one peptide (39 residues) degradation went to step 34 with a protein program and on two peptides (10 and 13 residues) degradation reached the last amino acid with a peptide program (use of dimethylbenzylamine).Amino acid analysis of tryptic peptides obtained by digestion of the C‐terminal cyanogen bromide peptide are in full agreement with the sequence established by automatic degradation. The sequence of toxin III of Naja haje is unique and is very similar to that of Naja niveaα (although there are 9 differences), of Naja melanoleuca b (11 differences) and also to that of Naja naja A (18 differences).
Heart Hexokinase: Quaternary Structure Changes Accompanying the Binding of Regulatory Molecules
Authors: EASTERBY John S. 
Published: March 2005

Heart hexokinase monomer has a molecular weight of 97000 and s020,w 5.2 S. It exists in equilibrium with dimer of 194000 molecular weight and s020,w 8.1 S. The proportions of monomer and dimer present in the absence of added ligands are 91% and 9% respectively. The existence of these forms may be demonstrated by separation on electrophoresis or chromatography.In the presence of the regulatory molecule glucose 6‐phosphate, the dimer form of the enzyme is favoured. The glucose 6‐phosphate mediated dimerisation is abolished in the presence of phosphate or ATP · Mg and less effectively by free ATP. Glucose has no effect on the monomer‐dimer equilibrium.On prolonged storage of hexokinase in glucose 6‐phosphate polymers are also formed and polymerisation is further enhanced by removal of the ligand.
The Photochemical Inactivation of Peptidyl Transferase Activity
Authors: WAN Kwong K. , ZAHID Nasir D. , BAXTER Ross M. 
Published: March 2005

The photochemical oxidation of the 50‐S ribosomal subunit results in a rapid irreversible loss of peptidyl transferase activity. The first‐order rate of inactivation occurring during the first forty minutes suggests that a single reactive group is being inactivated. The pH profile of inactivation exhibits a maximum at pH 7.5.Erythromycin at a low concentration (0.04 μmol) affords significant protection. Puromycin also exerts a protective effect but at higher concentrations. Chloramphenicol, sparsomycin and lincomycin did not exert a protective effect.The loss in catalytic activity was not accompanied by a loss in substrate binding affinity of the donor and acceptor substrates.
Enzymic Properties of Nitrated α‐Chymotrypsin and δ‐Chymotrypsin
Authors: SVENSSON K. Birte , IMHOFF Jean‐Marie , KEIL Borivoj 
Published: March 2005

Chymotrypsinogen A and α‐chymotrypsin are both nitrated at tyrosines 146 and 171 by reaction with tetranitromethane. This substitution was essentially without influence on the overall rate constant for hydrolyses of N‐acetyl‐l‐tryptophan methyl ester and N‐acetyl‐l‐tyrosine ethyl ester catalyzed by α‐chymotrypsin and δ‐chymotrypsin, prepared by fast tryptic activation of nitrated chymotrypsinogen. With both ester substrates Km was doubled for nitrated α‐chymotrypsin.Nitrated α‐chymotrypsin, nitrated δ‐chymotrypsin and δ‐chymotrypsin could all bind Nacetyl‐L‐tryptophan methyl ester at alkaline pH, in contrast to α‐chymotrypsin. The dissociation constant, Kd, of the complex of α‐chymotrypsin and basic pancreatic trypsin inhibitor was lowered ten‐fold relative to the constant obtained with unmodified α‐chymotrypsin. The nitrated δ‐chymotrypsin and δ‐chymotrypsin showed identical Kd values.The nitrated α‐chymotrypsin is inactivated faster at pH 8.0 and 8.5 than α‐chymotrypsin and apparently by a different mechanism.
The Pyruvate‐Dehydrogenase Complex from Azotobacter vinelandii
Authors: GRANDE Hans J. , BRESTERS Tjarda W. , DE ABREU Ronney A. , DE KOK Arie , VEEGER Cees 
Published: March 2005

Labelling studies with N‐ethylmaleimide show that either in the presence of Mg2+, thiamine pyrophosphate (TPP) and pyruvate or in the presence of NADH the overall activity of the pyruvate dehydrogenase complex from Azotobacter vinelandii is inhibited without much inhibition of the partial reactions. The complex undergoes a conformational change upon incubation with NADH. The inhibition by bromopyruvate is less specific.Specific incorporation of a fluorescent maleimide derivative was observed on the two transacetylase isoenzymes. Binding studies with a similar spin label analogue show that 3 molecules/FAD are incorporated by incubation of pyruvate, Mg2+ and TPP, whereas 2 molecules/FAD are incorporated via incubation with NADH. The spin label spectra support the idea that in the complex the active centres of the component enzymes are connected by rapid rotation of the lipoyl moiety.Three acetyl groups are incorporated in the complex by incubation with [2‐14C]pyruvate. Time‐dependent incorporation supports the view that the two transacetylase isoenzymes react in non‐identical ways with the pyruvate dehydrogenase components of the complex. The results show that the complex contains 2 low‐molecular‐weight transacetylase molecules and 4 molecules of the high‐molecular‐weight isoenzyme.Mn2+‐binding studies show that the complex binds 10 ions, with different affinities. 2 Mn2+ ions are bound with a 20‐fold higher affinity than the remaining 8 Mn2+ ions. The latter 8 ions bind with equal affinities and are thought to reflect binding to the pyruvate dehydrogenase components of the complex.It is concluded that the complex contains 8 pyruvate dehydrogenase molecules, 4 high‐molecular‐weight transacetylase molecules, 2 low‐molecular‐weight transacetylase molecules and 1 dimeric (2‐FAD‐containing) symmetric molecule of lipoamide dehydrogenase. Evidence comes from pyruvate‐dependent inactivation and labelling studies that the pyruvate dehydrogenase components contain either an –SH group or an S‐S bridge which participates in the hydroxyethyl transfer to the transacetylase components.
The Renaturation of Reduced Polyalanyl‐chymotrypsinogen and Chymotrypsinogen
Authors: ORSINI Gilbert , GOLDBERG Michel E. , SKRZYNIA Cécile 
Published: March 2005

Chymotrypsinogen has been successfully renatured in solution, after reduction of its 5 disulfide bonds in 6 M guanidine‐HCl. This has been made possible by the study of the renaturation of a model derivative, polyalanyl‐chymotrypsinogen. The reduced derivative is shown to refold and reoxidize spontaneously, with a 30–40% yield, into molecules which are monomeric and fully susceptible to activation by trypsin.Chymotrypsinogen can also be renatured but only in the presence of reagents allowing disulfide interchange and of moderate concentrations of guanidine‐HCl or urea. These results illustrate how the kinetic trapping of incorrectly folded molecules by wrong S‐S bonds and aggregation can be overcome, thus allowing the correct refolding of the protein.
Amino‐Acid Sequence of lac Repressor from Escherichia coli
Authors: BEYREUTHER Konrad , ADLER Klaus , FANNING Ellen , MURRAY Carolyn , GEISLER Norbert , KLEMM Alex 
Published: March 2005

The lac repressor from Escherichia coli, composed of four identical subunits with a molecular weight of 37 160, was carboxymethylated and fragmented by tryptic digestion and cyanogen bromide treatment. Using ion‐exchange chromatography, gel filtration and preparative thin‐layer electrophoresis and chromatography 29 of the 30 tryptic peptides were isolated in pure form. Direct Edman degradation and the dansyl‐Edman technique were used to determine the sequence of the small tryptic peptides. Special emphasis was put on the sequence determination of the six large tryptic fragments which together account for 177 residues, corresponding to 51% of the repressor subunit with its 347 residues. The large tryptic fragments were analyzed after fragmentation with chymotrypsin, thermolysin and dipeptidyl aminopeptidase I. Thus the sequence of all 30 tryptic peptides could be deduced. The complete sequences of all cyanogen bromide fragments were deduced from peptides obtained by tryptic, chymotryptic and thermolytic digestion of the individual fragments and by automated stepwise Edman degradation of lac repressor and of the large cyanogen bromide fragments. The order of the cyanogen bromide fragments was given by overlapping tryptic peptides. The resulting amino acid composition of the monomer is Asp15, Asn11, Thr18, Ser30, Glu14, Gln27, Pro13, Gly22, Ala44, Cys3, Val33, Met9, Ile17, Leu40, Tyr8, Phe4, Trp2, Lys11, His7, Arg19.The sequence of lac repressor shows no similarities with that of other proteins known to bind to DNA or RNA. The N‐terminal 55 residues contain two homologous regions. This part of the sequence which is involved in lac operator binding might have been formed by gene duplication.
The Hormonal Control of Activity of Skeletal Muscle Phosphorylase Kinase
Authors: YEAMAN Stephen J. , COHEN Philip 
Published: March 2005

Phosphorylase kinase was activated 5–10‐fold in vivo by an intravenous injection of adrenalin. Sodium fluoride, an inhibitor of phosphorylase kinase phosphatase, was required to prevent the reversal of this process; the activated and non‐activated forms of the enzyme were indistinguishable by dodecylsulphate gel electrophoresis. This suggested that the activation had resulted from a phosphorylation of the enzyme, and that it was not a consequence of the well known activation by proteolytic cleavage that can be demonstrated in vitro.Phosphorylase kinase activated in vivo was purified and digested with trypsin, and the two tryptic peptides which contain the serine residues which are phosphorylated in vitro by the action of cyclic‐AMP (adenosine 3′: 5′‐monophosphate) dependent protein kinase, were isolated. It was found that the same nine‐amino‐acid segment of the β chain and the same seven‐amino‐acid segment of the α chain had become phosphorylated in vivo in response to adrenalin, as were phosphorylated in vitro. The degree of phosphorylation of each of the two sites was at least 50%. The data provide direct proof that the activation of phosphorylase kinase which occurs in vivo in response to adrenalin results from a phosphorylation of the enzyme. They also indicate that the novel form of regulation associated with the phosphorylation of the α subunit, the stimulation of protein dephosphorylation by “second site phosphorylation”, can now be regarded as a new form of enzyme control mechanism which operates in vivo.The regulation of phosphorylase kinase activity was studied in the protein · glycogen complex from skeletal muscle. The enzyme could be rapidly converted to a phosphorylated form in a cyclic‐AMP‐stimulated reaction upon addition of magnesium ions and ATP, but the conversion of phosphorylase b to phosphorylase a in the complex still showed an absolute requirement for calcium ions.The implications of these findings and major problems in the hormonal control of skeletal muscle glycogenolysis which are not yet resolved, are discussed.