bluuebunny/crossref_metadata_2025_split huggingface dataset:
The Cholesterol Side‐Chain Cleavage Enzyme System in Mitochondria of Human Term Placenta
Authors: Mason James I. , Boyd George S. 
Published: March 2005

The cholesterol side‐chain cleavage enzyme system of human term placental mitochondria has been studied. Native mitochondria were capable of converting [4‐14C]cholesterol to [4‐14C]‐progesterone in the presence of NADPH and oxygen.
Disruption of mitochondrial structure resulted in an inhibition of the enzymic activity. However, active enzyme could be extracted from the disrupted mitochondria by high speed centrifugation, the endogenous inhibitor remaining associated with the particulate material.
The nature of the endogenous inhibitor has been determined. It appears to involve the mitochondrial phospholipid, especially lecithin. Investigations with authentic phospholipids confirm these studies.
Separation of the enzyme system from the bulk of the mitochondrial material permitted investigation of the characteristics of the enzyme.
Light reversibility studies of the carbon monoxide inhibition of the enzymic activity showed the involvement of cytochrome P450 in the enzyme system.
Fractionation of the enzyme using gel exclusion and ion—exchange chromatography revealed the involvement of a heam iron protein (presumably cytochrome P450), a component having NADPH‐diaphorase activity (flavoprotein) and a non‐haem iron ‘protein in the enzyme system.
On the Role of Quinones in Bacterial Electron Transport
Authors: Kröger Achim , Dadák Vladimír , Klingenberg Martin , Diemer Friederike 
Published: March 2005

The individual roles of ubiquinone and menaquinone in the electron transport of Proteus rettgeri have been investigated and their functional positions with respect to the cytochromes and dehydrogenases elucidated.

A method has been developed for the parallel determination of both ubiquinone and menaquinone in the nanomole range in the same extract.
Ubiquinone and menaquinone are not separated by various methods of cell disintegration and remain associated with the cytochromes.
The relative contents of ubiquinone and menaquinone are independent of the hydrogen source and depend on the redox acceptor offered in the growth medium. As in Escherichia coli, ubiquinone is preferentially formed in cutures with maximal oxygen supply and menaquinone in anaerobic cultures. The activity of fumarate reduction by NADH and by formate parallels the content of menaquinone.
On extraction of the quinones the electron transport is inhibited. Ubiquinone reactivates the respiration with succinate, NADH and formate and menaquinone restores the fumarate reduction. In anaerobically grown P. rettegri the respiration with NADH and formate is partly restored by menaquinone, too.
In the anaerobic states, ubiquinone is reduced with succinate, NADH and formate, and menaquinone with NADH and formate only. Ubiquinone is partly oxidized in the steady state of respiration and completely reduced in the steady state of fumarate reduction, where menaquinone is partly oxidized.
2‐n‐Heptyl‐4‐hydroxyquinoline‐N‐oxide inhibits the respiration of succinate, NADH and formate at a common site located on the oxygen side of ubiquinone.
Two b‐type cytochromes (besides cytochrome o) are differentialed: (1) Cytochrome bF is situated on the substrate side of 2‐n‐heptyl‐4‐hydroxyquinoline‐N‐oxide action and is reduced by formate only. (2) Cytochrome b1 is located on the oxygen side of this action and is reducible by all substrates.
Nicotinamide Mononucleotide Determination of its Enzymatic Formation in vitro and its Physiological Rôle for the Biosynthesis of Nicotinamide‐Adenine Dinucleotide in Mice
Authors: Streffer Christian , Beneš Jiri 
Published: March 2005

A method is described to measure the activity of the NMN pyrophosphorylase (NMN: pyrophosphate phosphoribosyltransferase) in crude extracts of mouse tissues. The homogenate is incubated with 5‐phosphoribose‐1‐pyrophosphate, ATP, Mg2+ and [14C]nicotinamide in the presence of unlabelled NMN. Optimal conditions for the enzymatic assay were determined.
The NMN pyrophosphorylase activity was measured in several organs of mice by this method. The observed activites were found to be considerably higher than reported in the literature.
The formation of 14C‐labelled pyridine nucleotides in mouse liver after intraperitoneal injection of [14C]nicotinamide was studied. After the application of a high nicotinamide dose (1 mmole/kg) the biosynthesis of NAD was mainly via nicotinate. However, after the injection of a small nicotinamide dose within the physiological range the pathway via NMN is utilized to a large extent. Under these conditions a partial inhibition of the metabolic pathway of nicotinate to NAD has no influence on the NAD formation from nicotinamide. It is concluded that NMN is the physiological intermediate for NAD biosynthesis from nicotinamide.
Essential Fatty Acid Deficiency and the Activity of the Sarcoplasmic Calcium Pump
Authors: Seiler Dieter , Hasselbach Wilhelm 
Published: March 2005

The fatty acid composition of the lipids of the sarcoplasmic membranes isolated from rats is changed when the animals are kept on a diet free of essential fatty acids. The fatty acids of the oleic acid family are increased and the fatty acids of the linoleic acid family are decreased.The calcium permeability of the modified membranes is reduced giving rise to a higher rate of calcium uptake, a reduction of the extra ATPase activity and a higher calcium concentrating ability.
Energy Transduction in Mitochondrial Fragments
Authors: Azzi Angelo , Fabbro Antonio , Santato Mario , Gherardini Pier Luigi 
Published: March 2005

Atebrin and acridine orange fluorescence and absorbance characteristics are sensitive to their environment.Anions, such as iodide, nitrate or chloride strongly decrease acridines fluorescence, while sulfate or acetate are much less effective.Energization by succinate or ATP of rat liver mitochondrial fragments produces a fluorescence decrease of atebrin, only in the presence of iodide, nitrate or chloride. In the presence of sulfate or acetate or in the absence of anions, no changes are detected.Binding changes of acridines are also observed in association with energy conservation in mitochondrial fragments in the presence of iodide, nitrate or chloride.These data are consistent with a model in which the membrane of mitochondrial fragments, becoming more positive than the medium when energized, creates local electrolyte concentration changes affecting fluorescence, absorbance and binding properties of acridine dyes.
Mechanism of Microbial Transformation of Cholesterol into Coprostanol
Authors: Björkhem Ingemar , Gustafsson Jan‐Åke 
Published: March 2005

The conversion of cholesterol into coprostanol by cecal contents from rats under anaerobic conditions was studied using deuterated water, [4β‐3H,4‐14C]‐ and [3α‐3H,4‐14C]cholesterol. On the basis of the following experimental findings, it was concluded that the conversion of cholesterol into coprostanol proceeds to at least 50% by means of the intermediate formation of Δ4‐cholestenone.

Conversion of [3α‐3H,4‐14C]cholesterol into coprostanol occurred with loss of 50% of the tritium. The tritium retained in coprostanol was located in the C‐3α position. However, part of the tritium retained might have been introduced in a reductive step following an initial oxidative step. Evidence for this possibility was provided by the finding that coprostanol formed from [4‐14C]cholesterol in the presence of [3α‐3H]β‐sitosterol contained significant amounts of tritium in the C‐3α position.
After conversion of part of [3α‐3H,4‐14C]cholesterol into coprostanol, unchanged cholesterol had a higher 3H/14C ratio than the cholesterol added to the incubation mixture, indicating that oxidation of the 3β‐hydroxyl group is at least partly rate limiting in the over‐all conversion of cholesterol into coprostanol.
After incubation of cholesterol in the presence of deuterated water, the coprostanol isolated had a deuterium content of about 1.4 atoms at C‐2, C‐3 and C‐4, about 0.5 atoms at C‐5 and about 0.1 atoms at C‐6, indicating extensive oxido‐reduction at C‐3 during or after conversion of cholesterol into coprostanol.
Coprostanol isolated after incubation of [4β‐3H,4‐14C]cholesterol retained 60% of the tritium. The main part of this tritium had been transferred to the C‐6 position, showing that the conversion of cholesterol into coprostanol involves isomerization of the Δ5 double bond to a Δ4 double bond.
Incubation of Δ4‐[4‐14C]cholestenone resulted in efficient formation of coprostanol.
Cold Inactivation of l‐Threonine Deaminase from Rhodospirillum rubrum
Authors: Feldberg Ross S. , Datta Prasanta 
Published: March 2005

Threonine deaminase from Rhodospirillum rubrum is subject to reversible cold inactivation at low protein concentrations. The inactivation appears to involve a dissociation of the native tetrameric protein to dimers. The standard free energy of dissociation at 0 °C was + 7.8 kcal/mole, while ΔS was—3.2 kcal/mole and ΔS was—41 entropy units. These values suggest that hydrophobic interactions are important in the maintenance of the tetrameric structure.The labilization of the protein by low concentrations of urea and the protection by high salt concentrations also indicate the importance of hydrophobic bonds in the protein. However, the effects of pH and low salt concentrations on the cold inactivation suggest that ionic bonds do play some role in subunit interactions.The normal allosteric modifier for threonine deaminase, l‐isoleucine, protected the enzyme against cold inactivation. Although the R. rubrum enzyme is virtually insensitive to isoleucine inhibition, it is clear that this ligand can induce profound changes in the protein structure.
Bovine Luteinizing Hormone Study of the Primary Structure around the Carbohydrate Attachment Sites of the Luteinizing Hormone α‐Subunit
Authors: Maghuin‐Rogister Guy C. , Hennen Georges P. 
Published: March 2005

The α‐subunit of bovine luteinizing hormone is electrophoretically heterogeneous. Two subfractions of this subunit, designated LHαA and LHαB, were isolated and found homogeneous by electrophoresis.
Bovine LHαA was cleaved by both cyanogen bromide and trypsin. Two distinct groups of homologous glycopetides were defined. The first group corresponds to the carboxy‐terminal portion of the subunit. The sequence accounting for the major peptide population is the following: Gly‐Asx‐Val‐Arg‐GlxVal‐Glx‐Asx‐HisSer‐ Thr‐Glx‐Cys‐His‐SerHis‐Cys‐Thr‐Cys‐Tyr‐Tyr‐His‐Lys‐Ser‐ COOH. Ambiguities in the amino‐terminal sequence are observed at the fifth, eighth and thirteenth position.Another population of peptides lacks three (or four) amino acids at the carboxy‐terminal end.The second group of glycopeptides corresponds to an internal portion of the subunit. The most abundant peptide population exhibits the following sequence: Asx‐Thr‐Thr‐Ser‐Ala‐Glx‐Cys‐Ile‐Cys‐Val‐Ala‐Lys‐COOH. A variant population exhibits the sequence Asx‐Ile‐Thr‐Ser‐Ala‐.
Two sites are proposed for the attachment of the polysaccharide prosthetic groups, i.e. the tripeptide Asx‐HisSer‐Thr and the tripeptide Asx‐IleThr‐Thr, corresponding to the carboxy‐terminal portion and to the inner part of the subunit, respectively.
Within both groups of glycopeptides, a relationship was observed between the type of residue × occupying the α position at the carbonyl side of Asx in the tripeptide sequence Asx‐X‐Thr and the composition of the polysaccharide side chain.
Our data document the degree of homology existing between LHα and the common type of subunit present in the thyroid‐stimulating hormone molecule (i.e. the α‐subunit).
Effects of Ring Substituents on the Activity of Phenols as Inhibitors and Uncouplers of Mitochondrial Respiration
Authors: Stockdale Martin , Selwyn Michael J. 
Published: March 2005

The effects of substituents on the activity of phenols as uncouplers and inhibitors of mitochondrial respiration have been examined. These activities have been correlated with constants describing the effects of the substituents on the electronic and hydrophobic‐bonding properties of the phenol. The coefficients in these correlation equations have been compared with each other and with the coefficients in similar correlations for the effects of phenols on the solubilised ATPase from mitochondria, and their activity in mediating proton conduction across artificial lipid membranes and as inhibitors of a variety of enzymes.Differences in the coefficients in the correlation equations for uncoupling and inhibition of respiration indicate that these depend on different properties of the phenol and hence that they are separate effects and that inhibition of substrate accumulation is not a satisfactory explanation for the inhibition of respiration.High concentrations of uncouplers induce swelling of mitochondria. In the case of 2,4‐dibromophenol, or dicoumarol, this occurs at concentrations similar to those producing inhibition of respiration. With other phenols inhibition of respiration is observed at concentrations lower than those required to induce swelling.The observations reported here support the hypothesis that phenols uncouple by mediating proton conduction across the inner mitochondrial membrane and that, in general, the inhibition of respiration by high concentrations of phenols is due to direct action on a protein component of the respiratory chain.
Studies on Ferricytochrome c
Authors: Wilson Michael T. , Greenwood Colin 
Published: March 2005

A model is proposed to explain the biphasic reduction of ferricytochrome c at alkaline pH assuming an equilibrium distribution between a reducible and non‐reducible conformer. An analysis of this model is given in an appendix to this paper.
Protein denaturants, increasing pH and temperature are found to disturb the conformer equilibrium in favour of the irreducible form. The thermodynamic parameters governing this equilibrium have been determined.
Kinetic parameters defined in the model were measured under a variety of conditions.
The kinetic and equilibrium data strongly suggests that only the reducible conformer possesses the 695 nm band.
Possible origins of the 695 nm band are discussed.
Relation of Toxicity and Conformation of Phallotoxins as Revealed by Optical Rotatory Dispersion Studies
Authors: Faulstich Heinz , Wieland Theodor 
Published: March 2005

Natural variants and chemically modified derivatives of phalloidin have been found or prepared which show all degrees of toxicity. High toxicity persists if only three of the side chains are modified. Acetylation of two of the four OH groups of phalloidin leads to less toxic products, the triacetyl derivative is nontoxic. On tosylation toxicity disappeared after reaction of only two OH groups. Alkylation of the indole nitrogen leads to equally or less toxic (N‐methy), ‐ethyl, carbamoylmethyl, ‐tert.butyl) or nontoxic (N‐propyl, ‐pentyl) products. Toxicity is also completely absent if only one of the two rings of the molecule is intact.In optical rotatory dispersion spectra all of the toxic compounds show in aqueous solution two cotton effects centered around 300 nm and 248 nm, whereas in nearly all of the nontoxic derivatives at least one of the effects is missing. Triacetylphalloidin and tribromophalloidin, both nontoxic, also exhibit “toxic” curves in water. Their conformation, however, changes on going to organic solvents like methanol or acetonitril as observed by optical rotatory dispersion studies. Thus, stability of the “aqueous” conformation in a lipophilic environment is apparently the second prerequisite for a toxic action of phallopeptides.
Mutations habilitant Escherichia colià croître sur acides gras moyens
Authors: Vanderwinkel Edgard , de Vlieghere Monique , Vande Meerssche Jacques 
Published: March 2005

New spontaneous mutants of Escherichia coli able to use butyrate or valerate as carbon source have been studied for their growth, adaptation lag and the level of enzymes which may participate in the utilisation of these compounds.Each mutant acquires the simultaneous constitutivity for uptake of these fatty acids, CoA transferase and high thiolase activities. This mutation designated as butR does not affect the regulation of the β‐oxidation nor that of the glyoxylate cycle.Induction or mutation for constitutivity (oleR) of β‐oxidation act multiplicatively on the level of thiolase derepressed by the butR mutation.If the presence of CoA transferase, thiolase and C4–C5 fatty acids entry system is a prerequisite of the growth ability on these fatty acids, the genetic constitutivities (and not the preinduction) of the β‐oxidation (oleR) and particularly of the glyoxylic cycle (aceD) greatly increase the growth capacity. This implies that not only the C, and C, fatty acyl derivatives are very poor inducers of β‐oxidation, as already reported by others, but that some products of their catabolism repress the glyoxylic cycle.The acquisition of the new growth capacity is thus accomplished by spontaneous mutations affecting regulatory processes; one of these discloses activities which are cryptic in the normal parental strains; the physiological significance of these enzymatic potentialities in wild strains and the mechanism of the interaction between two different regulatory units brought to light here are some of the open issues.
Isolation and Properties of tRNA Nucleotidyl Transferase from Yeast
Authors: Sternbach Hans , von der Haar Friedrich , Schlimme Eckhard , Gaertner Erika , Cramer Friedrich 
Published: March 2005

tRNA nucleotidyl transferase has been purified 5000‐fold from commercial bakers' yeast. The isolation procedure includes ammonium sulfate fractionation and chromatography on DEAE‐cellulose, CM‐cellulose, Biogel and phosphocellulose.
The enzyme is homogeneous on analytical centrifugation and by sodium dodecylsulfate gel electrophoresis.
The molecular weight of the native and denatured enzyme Mc=Or is 71000 as determined by ultracentrifuge measurements and 70000 as determined by sodium dodecylsulfate gel electrophoresis.
On micro isoelectric focusing the enzyme shows an isoelectric point at about pH 7.5.
The enzyme catalyses the incorporation of ATP and CTP into the 3′‐end of tRNA, and, in the presence of pyrophosphate, the pyrophosphorolyses of adenylic and cytidylic acid residues from the 3′‐end of tRNA.
Mise en évidence et analyse des ribosomes et des RNA associés aux mitochondries de cellules de mammifères
Authors: Eladari Marthe‐Elisabeth , Galibert Francis , Boiron Michel 
Published: March 2005

Mitochondrial fraction prepared from mammalian cells (rat liver) by high and slow speed centrifugation and purified by sedimentation in an isopycnic sucrose gradient are shown to contain ribosomes.These ribosomes have a sedimentation coefficient of 83 S (measured in the absence of KCl) and a buoyant density of 1.54 g/cm3 as compared to 80S and 1.55 g/cm3 for cytoplasmic ribosomes.The sedimentation behaviour of mitochondrial ribosomes varies as a function of KCl concentration. The dissociation of cytoplasmic and mitochondrial ribosomes into sub‐units begins towards 0.1 M KCl and is complete in 1 M KCl. The sedimentation coefficient of the intact ribosomes and their dissociated units fall as the concentration of KCl is increased between 0 and 1 M.RNA prepared from rat liver mitochondrial fraction contains a low molecular weight component and two high molecular weight species whose sedimentation coefficients 26 S and 17 S correspond to molecular masses of 1.51 × 106 and 0.61 × 106 daltons. These mitochondrial RNA can be separated by sucrose gradient sedimentation from cytoplasmic ribosomal RNA species sedimenting at 28 S and 18 S.Mitochondrial RNA were also characterized by acrylamide gel electrophoresis. In 2.50% gels, the electrophoretic mobility of high molecular weight mitochondrial RNA was found to be slightly higher than that of their cytoplasmic counterparts. Moreover, the RNA corresponding to the smaller units was separated into two distinct peaks.Electrophoresis in 8% acrylamide gels revealved 5 S and 4 S RNA with a migration pattern similar to that of cytoplasmic 5 S and transfer RNA but present in different amounts and additional RNA with an electrophoretic mobility of 0.31 and 0.85 if that of 4 S RNA is equalled to 1.
Studies on the Biosynthesis of NAD from Nicotinamide and on the Intracellular Pyridine Nucleotide Cycle in Isolated Perfused Rat Liver
Authors: Keller Jürgen , Liersch Michael , Grunicke Hans 
Published: March 2005

Isolated perfused rat liver incorporates nicotinamide at a concentration of 10 μM into NADvianicotinamide mononucleotide. A deamidation of nicotinamide could not be demonstrated.Evidence is presented which indicates that the exchange reaction catalyzed by NAD glycohydrolase is not involved in the uptake of nicotinamide into NAD.At the concentrations used, the rate of the incorporation of nicotinamide into NAD is about 40% of the incorporation rate observed with nicotinic acid.Results obtained after perfusion with [14C]nicotinic acid support the concept of an intracellular pyridine nucleotide cycle. However, in contrast to the general opinion, our results demonstrate that in rat liver nicotinamide generated by NAD breakdown is not deamidated but reutilizedvianicotinamide mononucleotide.After perfusion with 0.1 mM [14C]nicotinamide for 2.5 h, less than 2% of the total radioactivity is excreted into the bile. The same value is obtained after perfusion with 50 μM [14C]nicotinic acid. From these results it is concluded that the excretion of nicotinamide by the liver and the subsequent deamidation within the intestinal tract which have been considered important in the utilization of nicotinamide by the liver, are without biological relevance at normal nicotinamide concentrations.
Synthetic Polynucleotides
Authors: Kössel Hans , Roychoudhury Ranajit 
Published: March 2005

The enzyme terminal deoxynucleotidyl transferase catalyzes the addition of one or two riboadenylic acid residues to deoxyoligonucleotides. This reaction allows the specific labelling of deoxyoligonucleotides at the 3′‐ends if [α‐32P]ATP is used as substrate.In order to eliminate the product of the addition of two riboadenylic acid residues the total reaction mixtures after incubation with terminal transferase are treated with alkali and subsequently with phosphatase. Deoxyoligonucleotidyl [32P]pAr is then obtained as the sole reaction product. Digestion with spleen phosphodiesterase yields the deoxynucleotide‐3′‐[32P]monophosphates corresponding to the 3′‐ends of the deoxyoligonucleotides.The riboadenosyl residues at the 3′‐termini of the monoaddition products also can be eliminated by treatment with periodate and cyclohexylamine whereupon deoxyoligonucleotides specifically labelled at the 3′‐termini with [32P] phosphomonoester groups are obtained.The sensitivity of the method and the faithfulness in the identification od the 3′‐terminal deoxynucleoside residues could be tested using as little as 0.1 A260 units (5 μg) of oligodeoxynucleotides of specific base sequences.