bluuebunny/crossref_metadata_2025 huggingface dataset:
Liver Glycogen Synthase in Rats with a Glycogen‐Storage Disorder
Authors: WATTS Colin , MALTHUS Rex S.
Published: March 2005

1. A strain of rats with a genetically‐determined liver glycogen‐storage disorder (gsd/gsd) caused by a deficiency of liver phosphorylase kinase has a very high concentration of glycogen in the liver with a total glycogen synthase activity higher than in liver of fed normal animals, but only a small amount of the enzyme in the active form. In the livers of both starved and fed normal rats and gsd/gsd rats, there is a positive correlation between glycogen content and total glycogen synthase (r= 0.79) and an inverse correlation between glycogen content and active glycogen synthase (r= 0.86).

2. Homogenates of livers from the gsd/gsd rats have lower glycogen‐synthase phosphatase activities than homogenates from fed normal rats which in turn have lower activities than those from starved rats. The addition of glycogen to homogenates of liver from starved rats reduced the glycogen‐synthase phosphatase activity.

3. Dilution of homogenates of gsd/gsd rat liver produced an increase in glycogen‐synthase phosphatase activity which could be reversed by adding glycogen.

4. The gsd/gsd rats were shown to have liver glycogen‐synthase phosphatase activity comparable to normal rats when the enzyme was measured in dilute homogenates from both groups with the same glycogen concentration.

5. It is suggested that inhibition of glycogen‐synthase phosphatase by high glycogen concentrations is responsible for the low levels of activation of glycogen synthase in the gsd/gsd rats and that glycogen itself is probably the major factor controlling glycogen synthesis in gsd/gsd and fed normal rats.
Isomeric Incorporation of the Haem into Monomeric Haemoglobins of Chironomus thummi thummi
Authors: RIBBING Wilfried , RÜTERJANS Heinrich
Published: March 2005

The Bohr effect in the monomeric haemoglobin component III of Chironomus thummi thummi is described on a molecular basis using various pH‐dependent proton resonances. One of the three titratable histidines changes its pK from 7.9 in the unligated state to 7.7 upon ligation with CO and up to 7.0 upon ligation with O2. It can be concluded from an analysis of the C‐2 proton resonance intensities that these shifts in pK occur in only one of the two conformational isomers of the component III, whereas in the second isomer these pK shifts are not observed. The differences between these functional properties are also derived from pH‐dependent changes of the mesoproton resonances as well as of the methyl resonances of Ile‐E11. The conformational change of the component III, which is connected with the Bohr effect, influences a variety of other resonances which could not be assigned. The pK values derived from the pH dependence of these changes agree exactly with the pK value of the Bohr proton donating group, which is supposed to be His‐G2. Also the corresponding signal intensities indicate the presence of two conformational isomers in the haemoglobin component III.

Although the change in chemical shift of the His‐G2 C‐2 proton resonance is only small upon deprotonation of the imidazole ring, it has been shown with potentiometric and calorimetric methods that this histidine is deprotonated in this pH range.

Considering the tertiary structure obtained from an X‐ray study of Steigemann and Weber we can readily explain the conformational changes in the Bohr active isomer of the component III. With binding of ligands dislocations of side chains at the proximal site produce a change in the interactions of His‐G2 with the carboxyl group of the C‐terminal methionine‐H22.
Identification of Binding Sites of Turnip Yellow Mosaic Virus Protein and RNA by Crosslinks Induced in situ
Authors: EHRESMANN Bernard , BRIAND Jean‐Paul , REINBOLT Joseph , WITZ Jean
Published: March 2005

Turnip yellow mosaic virus RNA and protein could be crosslinked in situ by ultraviolet irradiation at pH 4.8 but not at pH 7.3, and by bisulphite treatment at pH 7.3. Crosslinked peptides could be located in the primary structure of the viral coat protein. Three regions were bound covalently by ultraviolet irradiation, and two of these three regions were bound also by bisulphite treatment. The yield of the crosslinking reaction could be high, indicating that almost all protein subunits of each virion reacted with the viral RNA. The crosslinked peptides contain most of the acidic and basic amino acids of the protein, often associated into pairs of opposite charge. Implications for the folding of the RNA into the virion and for models of RNA–protein interactions are discussed.
Synthesis of a Dicyclohexylcarbodiimide‐Binding Proteolipid by Isolated Pea Chloroplasts
Authors: DOHERTY Andrew , GRAY John C.
Published: March 2005

1. A proteolipid (Mr 8000) has been purified to homogeneity from pea chloroplast membranes by extraction into butan‐1‐ol.

2. The proteolipid from chloroplasts that had been incubated in the light with [35S]methionine or [3H]leucine gave a single radioactive band, coincident with the staining protein band, when analysed by polyacrylamide gel electrophoresis in the presence of sodium dodecylsulphate.

3. In washed chloroplast membranes, or in the purified state, the proteolipid covalently bound [14C]dicyclohexylcarbodiimide. The reaction produced two new forms of the proteolipid, with decreased mobility in polyacrylamide gels containing dodecylsulphate.

4. On analysis by dodecylsulphate/polyacrylamide gel electrophoresis, proteolipid purified from [35S]methionine‐labelled chloroplasts and treated with non‐radioactive dicyclohexylcarbodiimide gave a pattern of protein stain and radioactivity similar to that obtained when purified proteolipid was labelled with [14C]dicyclohexylcarbodiimide.

5. Incorporation of radioactivity into the proteolipid, during incubation of chloroplasts with [35S]methionine, occurred in the light but not in the dark and was inhibited by D(–)threo‐chloramphenicol but not by cycloheximide or ribonuclease. We conclude that intact isolated chloroplasts from pea synthesise a dicyclohexylcarbodiimide‐binding proteolipid.
Small Differences in Tryptophan Fluorescence Spectra of ‘Sodium’ and ‘Potassium’ Forms of (Na+, K+)‐Dependent Adenosinetriphosphatase
Authors: CHETVERIN Alexander B. , AGALAROV Sultan Ch. , EMELYANENKO Viktor I. , BURSTEIN Edward A.
Published: March 2005

A detailed comparative analysis of tryptophan fluorescence spectra of ‘sodium’ and ‘potassium’ forms of (Na+, K+)‐activated ATPase was carried out. The ‘potassium’ form spectrum is shifted relatice to that of the ‘sodium’ form by approximately 0.5–1 nm towards shorter wavelengths. The maximal amplitude of the difference spectrum for these forms makes up about 2% of maximal fluorescence intensity of any of the forms. The shape of the difference spectrum does not depend on the solution temperature or ionic strength. The spectral differences between the forms are reversible upon addition of a functionally opposite cation (K+ for ‘sodium’ form and vice versa) into the medium. The results suggest that if the differences in fluorescence spectra of the ‘sodium’ and ‘potassium’ forms of (Na+, K+)‐ATPase resulted from the differences in the protein structure, they may be caused by an alteration in local environment of no more than one or two tryptophan residues.
Labelling in vivo and in vitro of Molecular Species of Lipids from Chloroplast Envelopes and Thylakoids
Authors: SIEBERTZ Heinz Peter , HEINZ Ernst , JOYARD Jacques , DOUCE Roland
Published: March 2005

Lipid mixtures from chloroplast envelope and thylakoid membranes were isolated after different labelling times in vivo and in vitro and separated into major components. The isolated compounds were subjected to analyses such as separation of molecular species, determination of radioactivity in fatty acids and water‐soluble hydrolysis products and radio gas‐liquid chromatography of fatty acid mixtures. In the case of monogalactosyl and digalactosyl diacylglycerol these procedures were also applied to several individual molecular species. To investigate the extent of de novo synthesis these species were also used for methylation studies and their fatty acids subjected to α‐oxidation. In envelope membranes diacylglycerols and monogalactosyl diacylglycerols may each be separated into several distinct and non‐mixing pools. Molecules made de novo with oligoene fatty acids are very efficient substrates for galactosylation in vivo. The time‐dependent changes in patterns of galactolipid molecular species may indicate a desaturation of acyl chains operating in close contact to intact lipids. After isolation, envelopes incorporated UDP‐[14C]galactose into completely different patterns of galactolipids and molecular species pointing to changed properties of this membrane system or to a loss of regulatory factors
Comparison of the Nuclear Ribonucleoproteins Containing the Transcripts of Adenovirus‐2 and HeLa Cell DNA
Authors: GATTONI Renata , STEVENIN James , JACOB Monique
Published: March 2005

A method was devised allowing the preparation of hnRNA‐containing ribonucleproteins (hnRNP) from HeLa cells infected with adenovirus type 2 under conditions where the extraction of viral replication complexes was minimal. Approximately 60% of the RNA from such hnRNP hybridized with adenovirus DNA. The hnRNP from infected cells had the same general characteristics as those from uninfected cells. Their size was heterogeneous (30–260 S) and depended upon that of their RNA. Their CsCl densities were identical (1.39–1.40 g/ml), indicating the same protein: RNA ratio. Their proteins were found in the same molecular weight range, between 25000 and 200000.

The major proteins of hnRNP from HeLa cells were present in hnRNP from adenovirus‐infected cells. As 60% of the cellular RNA was replaced by adenovirus RNA in hnRNP, this indicated that there was not stringent specificity in the RNA‐protein interactions. The relative proportions of the proteins were identical in both cases, suggesting that the hnRNP assembly was independent of nucleotide sequences at least for the major proteins. The hnRNP from infected and uninfected cells differed by the size of their RNA, which was larger after infection, and by the presence of six additional minor polypeptides after infection. However, it cannot be excluded that the presence of these polypeptides in hnRNP resulted from non‐specific adsorption.
The Complex Formation between Escherichia coli Aminoacyl‐tRNA, Elongation Factor Tu and GTP
Authors: WAGNER Thomas , SPRINZL Mathias
Published: March 2005

The interaction between Escherichia coli aminoacyl‐tRNAs and elongation factor Tu (EF‐Tu) · GTP was examined. Ternary complex formation with Phe‐tRNAPhe and Lys‐tRNALys was compared to that with the respective misaminoacylated Tyr‐tRNAPhe and Phe‐tRNALys. There was no pronounced difference in the efficiency of aminoacyl‐tRNA · EF‐Tu · GTP complex formation between Phe‐tRNAPhe and Tyr‐tRNAPhe. However, Phe‐tRNALys was bound preferentially to EF‐Tu · GTP as compared to Lys‐tRNALys. This was shown by the ability of EF‐Tu · GTP to prevent the hydrolysis of the aminoacyl ester linkage of the aminoacyl‐tRNA species. Furthermore, gel filtration of ternary complexes revealed that the complex formed with the misaminoacylated tRNALyS was also more stable than the one formed with the correctly aminoacylated tRNALys.

Both misaminoacylated aminoacyl‐tRNA species could participate in the ribosomal peptide elongation reaction. Poly(U)‐directed synthesis of poly(Tyr) using Tyr‐tRNAPhe occurred to a comparable extent as the synthesis of poly(Phe) with Phe‐tRNAPhe. In the translation of poly(A) using native Lys‐tRNALys, poly(Lys) reached a lower level than poly(Phe) when Phe‐tRNALyS was used.

It is concluded that the side‐chain of the amino acid linked to a tRNA affects the efficiency of the aminoacyl‐tRNA · EF‐Tu · GTP ternary complex formation.
Phosphorylation of Human Erythrocyte Pyruvate Kinase by Soluble Cyclic‐AMP‐Dependend Protein Kinases
Authors: MARIE Joelle , BUC Hélène , SIMON Marie‐Pierre , KAHN Axel
Published: March 2005

Human red cell contain soluble adenosine‐3′,5′‐phosphate‐dependent protein kinases, which are able to phosphorylate the L′ subunits of erythrocyte pyruvate kinase. Efficiency and maximum level of phosphorylation are very comparable in human liver and red cells. Phosphorylation of red cell pyruvate kinase results in the same kinetic modifications as for liver enzyme, namely a shift towards a T′ allosteric state characterized by a decreased affinity for phosphoenolpyruvate and increased inhibition by the allosteric inhibitors ATP and alanine. In the course of red cell aging a small amount of partially proteolysed pyruvate kinase, devoid of the phosphorylatable site, appears; it resembles the subtilisin‐treated L′4 enzyme and accounts for less than 20% of total pyruvate kinase subunits.

Endogenous phosphorylation of pyruvate kinase from erythrocytes incubated in the presence of cyclic nucleotides produces the same kinetic modifications as phosphorylation in partially purified extract; this, however, does not change glucose consumption, lactate production and glycolytic intermediate concentrations of the incubated cells.
Single‐Strand‐Specific Nuclease from the Nucleoplasm of Rye Germ Nuclei
Authors: PRZYKORSKA Anna , SZARKOWSKI Jan W.
Published: March 2005

The localization of DNAase activity in rye germ nuclei revealed 48% of the total nuclear activity in the nucleoplasm and 4% in the chromatin.

2. The endonuclease present in the nucleoplasm was purified more than 300‐fold as compared with the specific activity in homogenized nuclei by means of ammonium sulphate fractionation and CM‐cellulose and CM‐Sephadex column chromatography. The preparation gave a single band on polyacrylamide gels. Its molecular weight is 45000, pH optimum 5.4. The preparation does not exhibit activity of non‐specific phosphomonoesterase and phosphodiesterase.

3. The nuclease catalyses the hydrolysis of denatured DNA and RNA to acid‐soluble 5′‐phoshate‐terminated products and cleaves the phosphomonoester linkage of 3′AMP.

4. Synthetic polyribonucleotides are hydrolysed at a rate decreasing in the order: poly(U) > Poly(A) > Poly(C) > Poly(G).

5. The endonuclease shows a high preference for single‐stranded nucleic acids. It does not depolymerise double‐stranded poly(U)· poly(A).

6. The nuclease attacks covalently closed circular bacteriophage PM2 DNA, plasmid ColE1 and plasmid pBR322 DNA, converting it initially into the open‐circular (relaxed) form and subsequently into the linear form. This reaction is strongly dependent on ionic strength.

7. The enzyme nicks the supercoiled DNA molecule of phage PM2 in only one place. At high enzyme concentrations several nicks in such a molecule are observed.
Energy‐Linked Reactions Catalyzed by the Purified ATPase Complex (F0F1) from Rhodospirillum rubrum Chromatophores
Authors: SCHNEIDER Erwin , SCHWULÉRA Udo , DOSE Klaus , FRIEDL Peter
Published: March 2005

1. The isolation of the F0F1‐ATPase complex from Rhodospirillum rubrum chromatophores by the use of taurodeoxycholate is described.

2. The enzyme preparation contains about 12 polypeptides; five are subunits of the F1 moiety.

3. The ATPase activity of the purified enzyme is dependent on the addition of phospholipids.

4. Km‐vales for Mg2+‐ATP and Ca2+‐ATP are similar to the values obtained for the membrane‐bound enzyme.

5. The F0F1‐ATPase complex is more than 70% inhibited by oligomycin and N,N′‐dicyclohexyl‐carbodiimide.

6. The F0F1‐ATPase complex was integrated into liposomes. The reconstituted proteoliposomes catalyzed energy transduction as shown by ATP‐dependent quenching of acridine dye fluorescence and ATP‐32Pi exchange.
Reconstitution of a Functional Acetylcholine Receptor
Authors: SOBEL André , HEIDMANN Thierry , CARTAUD Jean , CHANGEUX Jean‐Pierre
Published: March 2005

The ‘reconstitution cycle’ is composed of the following sequence of operations. Highly purified receptor‐rich membranes prepared from Torpedo marmorata electric organ are exposed to pH 11 to remove the 43000‐Mr protein and dispersed into solution by sodium cholate under conditions where more than 85% of the receptor protein is in its 9‐S form. Elimination of the detergent by filtration on a Sephadex column (or dialysis) yields a ‘reconstituted receptor’ fraction, under conditions which conserve part of the endogenous lipids, or ‘reconstituted vesicles’ in the presence of an excess of exogenous lipids.

The polypeptide composition of these fractions was analysed by sodium dodecylsulfate gel electrophoresis. Conditions are defined for quantitative measurements of the various polypeptide chains. The 40000‐Mr chain, which is labelled by the affinity reagent 4‐(N‐maleimido)phenyl [3H]trimethyl‐ammonium and therefore carries the acetylcholine receptor site, is the dominant polypeptide in the alkaline‐treated membranes and the reconstituted acetylcholine receptor.

Electron microscopy discloses that many of the alkaline‐treated membranes no longer form closed vesicles and do not show the transverse asymmetry of the native membranes observed after tannic acid fixation. In the reconstituted receptor fractions, the receptor molecules reaggregate into discs and may be exposed on both faces of the discs. In the reconstituted vesicles, receptor rosettes are integrated to the lipid vesicles.

With native membranes, the radioactive local anesthetic [3H]trimethisoquin binds to three classes of sites: non‐specific, low‐affinity and high‐affinity. Carbamylcholine causes an increase in the number of high‐affinity sites up to approximately 0.7 times the number of α‐125‐bungarotoxin sites. This ratio, the three classes of binding sites, and their regulation by carbamylcholine are conserved through the reconstitution cycle.
Reconstitution of a Functional Acetylcholine Receptor
Authors: HEIDMANN Thierry , SOBEL André , POPOT Jean‐Luc , CHANGEUX Jean‐Pierre
Published: March 2005

The ‘functional’ state of the acetylcholine receptor protein has been followed during reconstitution with the fluorescent agonist [1‐(5‐dimethylaminonaplithalene)‐sulfonamido]‐n‐hexanoic acid‐β‐N‐trimethylammonium bromide ethyl ester (Dns‐C6‐Cho) and rapid‐mixing techniques. Under appropriate conditions, a majority of the acetylcholine receptor sites can be recovered in a low‐affinity state(s) for Dns‐C6‐Cho, similar to that found with the native membrane‐bound receptor. This state can be slowly interconverted to a high‐affinity state after rapid mixing with the agonist, and the non‐competitive channel blockers, like the local anesthetics, still regulate this transition in an allosteric manner.

Several experimental conditions commonly used for the solubilization of the receptor and for its purification in the presence of sodium cholate result in the failure of reconstitution: the soluble receptor protein is stabilized in a low‐affinity state which can no longer be interconverted to a high‐affinity state in the presence of agonists or local anesthetics. On the other hand, it is demonstrated that if the concentration of lipids remains elevated in the presence of sodium cholate, a soluble (9 S) low‐affinity form of the receptor protein can be obtained which shows most of the characteristic properties of the membrane‐bound receptor and in particular the slow interconversion to the high‐affinity state and the effect of local anesthetics on this transition; furthermore, in these conditions the soluble protein can be manipulated ad libitum and submitted, for instance, to column filtrations and sucrose gradient centrifugations in the presence of detergent, without losing its characteristic conformational and allosteric transitions. After elimination of the detergent this form yields a reconstituted receptor which presents binding properties identical to those of the native membrane‐bound receptor and leads to the formation of vesicles which exhibit carbamylcholine‐sensitive ion fluxes.

A necessary and sufficient condition for functional reconstitution is therefore the conservation, in the presence of lipids, of the allosteric properties of the receptor protein in its detergent‐soluble form.
The Histones of Yeast
Authors: BRANDT Wolf F. , PATTERSON Keith , von HOLT Claus
Published: March 2005

The four core histones of yeast chromatin have been isolated. Amino acid composition, electrophoretic mobility and partial sequences identify one variant each of the histones H3 and H4, whereas the histones H2A and H2B are represented by two variants each. In the yeast histones H3 and H4 7% of the residues positioned in the partial sequences vary if compared with the corresponding histones from higher plants and animals, for the histones H2A and H2B from yeast this figure is 20%.
Effect of Membrane Cholesterol on Action of Phospholipase A2 in Mycoplasma mycoides var. Capri
Authors: RIGAUD Jean‐Louis , LEBLANC Gérard
Published: March 2005

Cells of Mycoplasma mycoides var. Capri grown in a medium containing 10 μg/ml cholesterol (native organisms) or in cholesterol‐free medium (adapted organisms) were treated with phospholipase A2. Hydrolysis of polar lipids (phosphatidylglycerol and diphosphatidylglycerol) only occurred in the adapted cells. Cholesterol replenishment of the membranes of these adapted cells in vitro which involves an increase from 7 μg to 66 μg cholesterol/mg membrane protein, completely abolished hydrolysis of polar lipid pools by phospholipase A2. This suggests that cholesterol incorporated either during growth or under conditions in vitro has an identical disposition and function in the membrane. This observation further indicates that cholesterol incorporation in M. mycoides var. Capri can be explained in terms of a simple physical adsorption process.

Polar‐lipid breakdown products resulting from phospholipase A2 action on intact cells, isolated membranes and lipids extracted from adapted organisms were analyzed. In experiments with intact cells, [14C]oleic lysoderivatives but not [3H]palmitic lysoderivatives are accumulated within the membranes. In membrane preparations, again only [14C]oleic lysoderivatives are accumulated but transiently. Finally, both [14C]oleic and [3H]palmitic lysoderivatives were produced in phosphatidylglycerol‐diphosphatidylglycerol liposomal preparations. From these results it can be concluded that: (a) 80% of the phosphatidylglycerol and diphosphatidylglycerol have an unusual positional distribution of their fatty acid (unsaturated oleic acid in position 1) and (b) membranes of M. mycoides var. Capri contain an active lysophospholipase which more efficiently hydrolyzes palmitic‐acid‐containing lysoderivatives.
Role of Lipids in Functions of Photosynthetic Membranes Revealed by Treatment with Lipolytic Acyl Hydrolase
Authors: RAWYLER André , SIEGENTHALER Paul‐André
Published: March 2005

1. Thylakoid membranes and subchloroplast particles I and II enriched in photosystem I and photosystem II, respectively, were treated with a potato lipolytic acyl hydrolase.

2. In the thylakoid membrane fraction, this treatment inhibited electron flows involving both photosystems and the associated photophosphorylations. However, electron flows involving either photosystem I of photosystem II were still preserved. The treatment of thylakoid membranes by lipolytic acyl hydrolase brought about a temporal convergence of different events such as maximal activity of reduced dichloroindophenol‐supported electron flows, complete inhibition of photophosphorylations and electron transport activities through photosystem II + I, onset of the decay N,N,N′,N′‐tetramethyl‐p‐phenylenediamine‐supported activity of photosystem activity and of the stimulation of photosystem activity (from reduced dichloroindophenol to NADP+ by exogenous plastocyanin.

3. Lipolytic acyl hydrolase catalyzed a limited hydrolysis of each lipid but in a stepwise manner, the galactolipids being attacked before the ionic lipids. The extent of the hydrolysis was not more than 50% for each lipid. Most of the hydrolytic process occurred before any significant change in photochemical activities could be observed.

4. In subchloroplast particles I, a treatment by lipolytic acyl hydrolase did not greatly affect the electron transport whilst lipid hydrolysis was almost complete.

5. In subchloroplast particles II, neither the electron flow activities nor lipid content were significantly altered by lipolytic acyl hydrolase.

6. The sites of lipolytic acyl hydrolase action appeared to be localized between plastoquinones and P700. It is suggested that it is not possible to establish a quantitative and/or temporal correlation between the extent of lipid hydrolysis and the inhibition of photochemical activities.

7. The profile of the hydrolysis of lipids in thylakoid membranes suggests that ionic lipids are less accessible to lipolytic acyl hydrolase than galactolipids.
Origin of the Ammonia Found in Protein‐Free Extracts of Rat‐Liver Mitochondria and Rat Hepatocytes
Authors: WANDERS Ron J. A. , HOEK Jan B. , TAGER Joseph M.
Published: March 2005

1. Protein‐free extracts of isolated rat‐liver mitochondria contain 5.17 ± 0.19 nmol ammonia/mg protein [cf. Harris, E. J. and Bassett, D. J. (1971) FEBS Lett. 19, 214–2171.

2. The ammonia found in the protein‐free extracts does not originate from lysosomes contaminating the mitochondrial preparation.

3. When isolated mitochondria are incubated with ornithine, 14CO2 and a source of ATP a small amount of citrulline is formed. This amount is stoichiometrically equivalent to the ammonia that disappears from the extramitochondrial space, whereas the amount of ammonia found in the protein‐free extracts of the mitochondria remains unchanged. Similar results were obtained when the reductive amination of 2‐oxoglutarate was used as an ammonia‐consuming reaction.

4. When isolated mitochondria are incubated under conditions such that the glutamate dehydrogenase and 3‐hydroxybutyrate dehydrogenase reactions reach equilibrium, the thermodynamically active concentration of ammonia is not equal to the concentration measured in the protein‐free extracts.

5. About 80% of the ammonia found in protein‐free extracts of rat‐liver mitochondria is derived from a component or components with a molecular weight of ≧ 50000.

6. Protein‐free extracts of isolated rat‐liver cells contain considerable amounts of ammonia. After digitonin fractionation this ammonia is found in the protein‐free extract of the particulate fraction.

7. It is concluded that the ammonia found in protein‐free extracts of rat‐liver tissue is derived from a component or components in the mitochondria and is released during deproteinization.
Early Kinetics of Glucagon Action in Isolated Hepatocytes at the Mitochondrial Level
Authors: SIESS Elmar A. , WIELAND Otto H.
Published: March 2005

The temporal relationship between the effect of glucagon on respiratory functions and the changes in metabolites related to gluconeogenesis has been studied. Mitochondria prepared from hepatocytes after incubation with glucagon for 1 min already displayed a maximal stimulation of state‐3 respiration. The increase in succinate dehydrogenase activity was almost fully expressed 3 min after glucagon. With respect to the utilization of pyruvate, 2‐oxoglutarate and glutamate, glucagon produced a significant effect within 1 min. The rate of this decrease was linear for about 3 min slowing down thereafter. The stimulation of glucose production from lactate became significant within 1 min and remained constant up to 15 min. The influence of glucagon on the mitochondrial redox state also was an early event. It was maximally shifted to the more reduced state within 2 min and declined within 15 min. Under the conditions employed no effect of glucagon on urea synthesis or branched‐chain amino acid release up to 15 min incubation time was discernible. Glucagon influenced the respiratory parameters virtually independent of Ca2+, in contrast to its action on intermediary metabolism. As to the hormone specificity, no enhancement of state‐3 respiration and succinate dehydrogenase activity was caused by phenylephrine or isoproterenol. From the time course studies presented, it appears that the mitochondrial effects of glucagon might be causally interrelated with the regulation of gluconeogenesis. Moreover, our results indicate that the stimulation of state‐3 respiration represents the earliest, specific action of glucagon at the mitochondrial level.
The Energy‐State of Mitochondria during the Transport of Ca2+
Authors: LÖTSCHER Hans‐Ruedi , WINTERHALTER Kaspar H. , CARAFOLI Ernesto , RICHTER Christoph
Published: March 2005

The fluctuations of the membrane potential during mitochondrial Ca2+ transport have been monitored with an electrode sensitive to tetraphenylphosphonium. The following conclusions have been reached.

1. The membrane becomes depolarized during the influx of Ca2+. When the bulk of the Ca2+ pulse has been taken up, it repolarizes, but not completely.

2. If all of the accumulated Ca2+ is released from mitochondria and cycling is inhibited, the membrane repolarizes completely.

3. The accumulation of Ca2+ alone does not induce mitochondrial damage. In the presence of inorganic phosphate, the uptake of Ca2+ may lead to complete and irreversible depolarization, depending on the amount of Ca2+ and phosphate accumulated. The irreversible damage observed in the presence of phosphate is prevented by Mg2+.
Proton Nuclear‐Magnetic‐Resonance and Resonance Raman Studies of Thermophilic Cytochrome c‐552 from Thermus thermophilus HB8
Authors: HON‐NAMI Koyu , KIHARA Hiroshi , KITAGAWA Teizo , MIYAZAWA Tatsuo , OSHIMA Tairo
Published: March 2005

The pH and temperature dependences of the 270‐MHz proton nuclear magnetic resonance and resonance Raman spectra of Thermus thermophilus cytochrome c‐552 were studied. Observation of the NMR methyl signal of the iron‐bound methionine indicates that a methionine residue is the sixth ligand of heme iron in both ferric and ferrous states, although the environment of this methionine is not similar to that in mitochondrial cytochrome c. The NMR methyl signal of the coordinated methionine in the ferrous state was observed even at 87°C, indicating the retention of the methionine ligand at the sixth coordination position. None of resonance Raman lines in ferrous cytochrome c‐552 at higher temperatures showed a prominant temperature‐dependent frequency shift, which implies that the heme iron was still bound with strong ligands and retained the low‐spin state. In either redox state overall thermal denaturation did not occur even at 87°C, although the ferric from existed in thermal spin mixture of the low‐spin and high‐spin species at higher temperatures. The hyperfine‐shifted NMR resonances of the ferric form indicated rapid exchange of the sixth ligand at alkaline pH in the process of a single‐step alkaline isomerization.
Reconstitution of Mitochondrial Oligomycin and Dicyclohexylcarbodiimide‐Sensitive ATPase
Authors: GLASER Elzbieta , NORLING Brigitta , ERNSTER Lars
Published: March 2005

1. Oligomycin and dicyclohexylcarbodiimide‐sensitive ATPase was isolated from beef‐heart mitochondria and treated with 3.5 M NaBr in order to remove F1. The residue, called F0, was found to consist of seven components. Five of these are stained by Coomassie blue after dodecyl‐sulfate‐polyacrylamide‐gel electrophoresis. Two of them correspond to the oligomycin‐sensitivity‐conferring protein and coupling factor F6, with apparent molecular weights of 21000 and 9400, respectively. Three additional polypeptides of molecular weights 23000, 10500 and 8600 were not identified with known proteins. Two components not stained with Coomassie blue were detected by autoradiography of the gels of F0 preincubated with [14C]dicyclohexylcarbodiimide. These two components probably represent monomeric and oligomeric forms of the dicyclohexylcarbodiimide‐binding protein.

2. F0 induced an oligomycin and dicyclohexylcarbodiimide‐sensitive enhancement of K++ valinomycin‐driven proton translocation across the membrane of artificial phospholipid vesicles.

3. The interaction of F0 with purified, soluble beef heart F1 was investigated. F0 was capable of binding F1 and conferring oligomycin and dicyclohexylcarbodiimide sensitivity and cold stability on its ATPase activity. Furthermore F0 was found to diminish the specific activity of F1‐ATPase. A comparison of these effects at varying F0/F1 ratios shows that F0 binds F1 in both an oligomycin‐sensitive and an oligomycin‐insensitive manner, and that both types of binding involve a conferral of cold stability and a decrease in specific activity. High F0/F1 ratios favoured the oligomycin‐sensitive type of binding, indicating that F1 binds preferentially to oligomycin‐sensitivity‐conferring sites. Treatment of ATPase complex with trypsin resulted in an F0 with a decreased proportion of oligomycin‐sensitivity‐conferring binding sites and a diminished ability to lower the specific activity and cold lability of F1.

4. Reconstitution of F0 treated with trypsin and F1, oligomycin‐sensitivity‐conferring protein and F6 showed that at a constant amount of F1 bound, both oligomycin‐sensitivity‐conferring protein and F6 increased the oligomycin sensitivity of ATPase activity. It was therefore concluded that both of these coupling factors are involved in the conferral of oligomycin sensitivity.

5. The effect of the order of addition of F1, oligomycin‐sensitivity‐conferring protein and F6 to F0 on the reconstitution of oligomycin‐sensitive ATPase activity, and of F1 and oligomycin‐sensitivity‐conferring protein to submitochondrial particles on the reconstitution of respiratory control, was investigated. The highest values of oligomycin sensitivity and respiratory control were obtained when F1 was added as the first component, indicating that F1 plays a directing role in the organisation of the components.
Pyruvate Carboxylase and Propionyl‐CoA Carboxylase as Anaplerotic Enzymes in Skeletal Muscle Mitochondria
Authors: DAVIS E. J. , SPYDEVOLD Ø. , BREMER Jon
Published: March 2005

Oxygen uptake in skeletal muscle mitochondria respiring on pyruvate or on acetylcarnitine plus propionylcarnitine is stimulated 3–4‐fold by bicarbonate. The stimulation is highly dependent on ATP. The respiration rate obtained amounts to 1/4–1/3 of the rate obtained with pyruvate‐malate in the presence of ADP. With decreasing ATP/ADP ratios in the medium, a decreasing stimulation by bicarbonate is obtained. Similar results were obtained with heart mitochondria.

With ATP added, a pyruvate‐dependent build up of citric acid cycle intermediates takes place in incubations with skeletal muscle mitochondria amounting to about 0.5 nmol × min−1× mg protein−1.

In 14CO2‐fixation experiments, the activity of pyruvate carboxylase (EC 6.4.1.l) amounts to about 3 nmol × min−1× mg protein−1 under similar conditions. With propionylcarnitine plus acetylcarnitine a similar stimulation of respiration and fixation of bicarbonate is observed. In this case the respiration and the propionyl‐CoA carboxylase (EC 6.4.1.3) is less inhibited by ADP.

The results are discussed in relation to the regulation of the level of citric acid cycle intermediates in muscle tissues.

It is concluded that pyruvate carboxylase is an important anaplerotic enzyme in skeletal muscle mitochondria.
Affinity Chromatography on Immobilised Nucleotides
Authors: CLONIS Yannis D. , LOWE Christopher R.
Published: March 2005

The synthesis and characterisation of two IMP analogues, 8‐(6‐aminohexyl)‐ionosine 5′‐monophosphate, Ahx8IMP, and inosine 2′,3′‐O‐[1‐(6‐aminohexyl)‐levulinic acid amide]‐acetal 5′‐monophosphate, (AhxLvn)2′,3′ IMP, is described. These analogues were attached to CNBr‐activated agarose through the terminal amino group of the spacer molecule. The immobilised‐IMP analogues displayed specificity for the inosine‐nucleotide‐dependent enzyme, IMP dehydrogenase (IMP: NAD+ oxidoreductase, EC 1.2.1.14) but not for the NAD+‐dependent enzymes, L‐alanine and L‐acetate dehydrogenases. Escherichia coli IMP dehydrogenase could be eluted biospecifically from immobilised 8‐substituted and ribose‐substituted IMP adsorbents with IMP, XMP and GMP. Multiple peaks of enzyme activity in the elution profiles were interpreted in terms of aggregation of the enzyme. A protocol for the large‐scale purification of E. coli IMP dehydrogenase is proposed. Homogeneous enzyme of specific activity 9.1 units/mg was obtained in 50% overall yield, representing 14 mg pure protein from a 20‐l culture of E. coli. The two IMP analogues were inactive as substrates in the IMP dehydrogenase reaction.
Activities of Enzymes Involved in Amino‐Acid Metabolism in Developing Rat Placenta
Authors: REMESAR Xavier , AROLA Lluís , PALOU Andreu , ALEMANY Marià
Published: March 2005

Aspartate transaminase, alanine transaminase, glutamate dehydrogenase, arginase, serine dehydratase, tyrosine transaminase, glutamine synthetase, glutaminase and adenylate deaminase activities were measured in crude homogenates of 12, 19 and 21‐day rat placentae. There is a considerable quantitative importance in enzymes able to produce free ammonia, such as adenylate deaminase and glutamate dehydrogenase, activity that progressively decrease with the age of placenta. The glutamine synthetase and tyrosine transaminase activities increase with age, while serine dehydratase decreases considerably and aspartate and alanine transaminase do not change practically. Arginase shows a maximum at 19, with lower 12 and 21‐day activities. No measurable glutaminase activity has been found.

The possible implications of the enzymes studied upon the ammonia‐producing activity of rat placenta are discussed together with the relative decreasing role of placenta for the overall metabolic activity of the foetus, especially during the last phases of its development.
Hydroxyl‐Ion‐Induced Subunit Dissociation of Yeast Cytoplasmic Pyruvate Decarboxylase
Authors: HOPMANN Rudolf F. W.
Published: March 2005

Cytoplasmic pyruvate decarboxylase (EC 4.1.1.1, from Saccharomyces carlsbergensis) exhibits in its circular dichroic spectrum in the 250–320‐nm range a multiple two‐signal band. This couplet disappears on increasing the pH up to pH 8.5. Two classes of two protons each can be quantified by these spectral changes. The first class dissociates rapidly and the apparent pK is 7.84. The thermodynamic data are ΔG= 87.7 kJ mol−1, ΔH=+ 56.0 kJ mol−1, ΔS=–108 J mol−1 K−1, very characteristic for the deprotonation of an amino‐acid side chain. The second class of the protons has the following thermodynamic data: ΔG= 88.3 kJ mol−1, ΔH=–64.3 kJ mol−1, ΔS=– 520 J mol−1 K−1 which, in conjunction with kinetic reasoning and in view of enzyme stoichiometry and symmetry, suggests a conformational equilibrium exposing the second two protons. The enzyme dissociates into two dimeric subunits. This dissociation step is considered to be rate‐determining for the overall process. The data are: kp= 1.4 · 10−3, ΔH≠=+ 128.3 kJ mol−1, ΔS≠=+ 136 J mol−1 K−1. If there is a conformational equilibrium, the rate constant of product formation kp will be modified by a factor β=Kc/(1 +Kc) (0 < β 1) where Kc is the conformational equilibrium constant. The subunit dissociation appears to be controlled by the enthalpy of activation indicating that a number of interactions, i.e. ionic, hydrogen and hydrophobic bridges, are to be broken. Optimal conditions for the preparation of the apo‐enzyme are derived from the data.
The Role of Inorganic Phosphate in the Release of Ca2+ from Rat‐Liver Mitochondria
Authors: ROOS Isabelle , CROMPTON Martin , CARAFOLI Ernesto
Published: March 2005

The effect of inorganic phosphate on Ca2+ retention has been investigated using phosphate‐depleted liver mitochondria. Phosphate induces the release of Ca2+ through an efflux route insensitive to ruthenium red. This effect is not due to functional or structural damage, since mitochondria maintain their membrane potential during phosphate‐induced Ca2+ efflux. Direct enzymatic measurement of mitochondrial pyridine nucleotides has established that changes in their redox state (i.e. increased oxidation) do not play a role in the phosphate‐effect. The phosphate‐induced Ca2+ efflux requires transport of phosphate out of mitochondria. However, the fluxes of Ca2+ and phosphate do not coincide: the release of phosphate preceeds that of Ca2+.
Dissociation of the DNAse‐I · Actin Complex by Formamide
Authors: ZECHEL Kasper
Published: March 2005

Rabbit skeletal muscle actin labeled with 125 iodine by an enzymic method is shown to be capable of polymerization and to bind to matrix‐bound pancreatic DNAse I like unlabeled G‐actin. It was used to demonstrate that actin can be released from DNAse‐I–agarose by 35–40% formamide. Actin which was only shortly exposed to this solvent was able to bind again to DNAse I and to form filaments indicating that it has been recovered functionally intact from the affinity matrix.
The Purine‐Nucleotide Cycle
Authors: ARAGÓN Juan J. , LOWENSTEIN John M.
Published: March 2005

The contents of citric acid cycle compounds were measured in rat hind limb muscle in situ during rest, exercise, and recovery from exercise. The following changes in citric acid cycle intermediates were observed during exercise for 15 min. The contents of fumarate and malate increased fourfold over resting values. The contents of citrate, isocitrate, and succinate rose 68%, 48% and 87% respectively, but the increase in these intermediates was delayed relative to the increase in fumarate and malate. The content of 2‐oxoglutarate did not change significantly. The content of oxaloacetate increased from less than 1 nmol/g dry weight in resting muscle to 8 nmol/g dry weight during exercise. The orthophosphate content doubled. The sum of the contents of citric acid cycle intermediates doubled. The ratios of the contents of malate/fumarate and citrate/isocitrate remained constant. The constancy of the citrate/isocitrate ratio indicates that the concentration of intra‐cellular free Mg2+ is not affected by exercise. All metabolite levels returned to resting values during recovery. Comparison of these data with the rate of operation of the purine nucleotide cycle leads us to conclude that the increase in citric acid cycle intermediates can be accounted for by the operation of the purine nucleotide cycle.
Processing of Two Forms of the Common Precursor to α‐Melanotropin and β‐Endorphin in the Rat Pars Intermedia
Authors: CRINE Philippe , SEIDAH Nabil G. , ROUTHIER Richard , GOSSARD Francis , CHRÉTIEN Michel
Published: March 2005

Whole neurointermediate lobes were used to study the processing of the common precursor to β‐endorphin and α‐melanotropin (melanotropin) during pulse and pulse‐chase incubations with various radioactive amino acids. After a 30‐min pulse with radioactive amino acids, two major radioactive protein bands with apparent molecular weights of 34000 and 36000 were observed on a 10–30% acrylamide/sodium dodecylsulfate slab gel electrophoretogram. After a 2‐h chase, most of the radioactivity associated with these two protein bands had disappeared and was recovered in smaller peptides. Analysis of the tryptic fragments from the 34000‐Mr and 36000‐Mr proteins showed that both contained amino acid sequences characteristic of adrenocorticotropin (corticotropin) and β ‐lipotropin. Processing of the two precursor forms involves several proteolytic steps. The first occurring between 30 min and 1h after the start of the incubation, releases the β‐lipotropin sequence from the rest of the molecule, leaving two polypeptides with apparent molecular weights of 25000 and 27000. These two peptides are thought to have identical or closely related amino acid sequences differing only in the number of carbohydrate side chains. Analysis of the tryptic peptides of these [3H]phenylalanine‐labeled peptides showed that they contained fragments characteristic of corticotropin plus at least another fragment from the non‐corticotropin, non‐β ‐lipotropin part of the precursor molecule. Further maturation of the β‐lipotropin and the two high‐molecular‐weight forms of corticotropin occurred between 1 h and 2 h after the start of incubation. β‐Lipotropin served as a precursor for β‐endorphin while α‐melanotropin was generated by cleavage of the two high‐molecular‐weight forms of corticotropin. Peptides with apparent molecular weights of 17000 and 19000 were also recovered as major and stable end products of the maturation process. Tryptic peptide analysis showed that they were related to the portion of the precursor which does not contain either corticotropin or β‐lipotropin. When these peptides, labeled with [3H]leucine or [3H]serine, were analyzed by automated Edman degradation, it was shown that their sequence was identical in both cases to the N‐terminal sequence of the initial precursor molecule prepared during the 30‐min pulse incubation. From these data, it clearly appears that the 19000‐Mr and 17000‐Mr peptides are related to the N‐terminal fragment of the original precursor molecule.
A Flow‐Quench Apparatus for Cryoenzymic Studies
Authors: BARMAN Thomas E. , BRUN André , TRAVERS Franck
Published: March 2005

The flow‐quench method was adapted to sub‐zero conditions. Two apparatus were constructed: a rapid flow‐quench apparatus (which takes samples in the 5–300‐ms time range) and a time delay flow‐quench apparatus (0.5 s and longer). The apparatus were constructed so that the reagents are only in contact with chemically inert materials: glass, poly(tetrafluoroethylene) and poly(trifluoro‐chloroethylene).

The modified flow‐quench apparatus were used to study the initial formation of creatine phosphate by creatine kinase at –15°C in the time range 5 ms to 10 s. As at +4°C [Travers, F., Barman, T. E. and Bertrand, R. (1979) Eur. J. Biochem. 100, 149–1551, the time course of product formation was complex and consisted of three phases: a lag phase, a burst phase and the steady‐state phase.

The apparatus were also used to test chemically for reaction intermediates involving labile phosphate and phospho‐enzyme complexes on the creatine kinase reaction pathway at –15°C. Since neither type of intermediate could be detected down to 5 ms, this enzyme probably proceeds via a direct in‐line type of mechanism.
Determination of the Stability Constants of Mn2+ and Mg2+ Complexes of the Components of the NADP‐Linked Isocitrate Dehydrogenase Reaction by Electron Spin Resonance
Authors: KUCHEL Philip W. , REYNOLDS C. Hugh , DALZIEL Keith
Published: March 2005

1. The stability constants (Ks) of Mn2+ and Mg2+ complexes of isocitrate, 2‐oxoglutarate, NADP and NADPH have been estimated by using electron spin resonance to measure free Mn2+ in ligand–metal‐ion solutions.

2. The values of Ks for the Mn2+ complexes at 25°C, in triethanolamine buffer containing NaCl, pH 7.0 and ionic strength 0.15 M, are 497 M−1 for isocitrate, 39 M−1 for 2‐oxoglutarate, 467 M−1 for NADP and 943 M−1 for NADPH.

3. For the Mg2+ complexes under the same conditions, the Ks values are 357 M−1, 25 M−1, 133 M−1 and 179 M−1 respectively. The large difference between the stabilities of the isocitrate and 2‐oxoglutarate complexes is thus largely responsible for the observed variation of the apparent equilibrium constant of the NADP‐linked isocitrate dehydrogenase reaction with magnesium ion concentration.

4. NADP‐linked isocitrate dehydrogenase from bovine heart mitochondria binds Mn2+, and the stability constant of the complex is about 2.2 × 104 M−1. The formation of this complex may explain the inhibition of the enzyme‐catalysed reaction observed with Mn2+ concentrations greater than 0.2 mM in initial rate measurements.