bluuebunny/crossref_metadata_2025_split huggingface dataset:
Hepatic Microsomal Ethanol Oxidation
Authors: Thurman Ronald G. , Ley Heinz Georg , Scholz Roland 
Published: March 2005

Microsomes from rat liver form hydrogen peroxide in the presence of an NADPH‐generating system in proportion to protein concentrations as determined by three independent methods: ferrithiocyanate, cytochrome c peroxidase, and scopoletin fluorescence. Maximal rates observed were about 15 μmol H2O2/g microsomal protein per minute. The oxygen concentration for half‐maximal rates was 50 μM. It is suggested that NADPH‐dependent hydrogen peroxide formation in microsomes is mainly due to NADPH oxidase; however, partial inhibition by carbon monoxide suggests that about one third arises from the autoxidation of cytochrome P‐450.Similarities exist between microsomal acetaldehyde production from ethanol (i.e. the microsomal ethanol‐oxidizing system of Lieber and DeCarli [4]) and hydrogen peroxide formation: viz. requirement for NADPH and oxygen, identical oxygen concentrations for halfmaximal rates, and sensitivity to carbon monoxide. Microsomal acetaldehyde production in the presence of either an NADPH‐ or an H2O2‐generating system exhibits identical characteristics as follows: (a) ethanol concentration for half‐maximal rates (i.e. 12 mM); (b) dependency of maximal rates on rates of hydrogen peroxide formation; (c) competitive inhibition by peroxidatic substrates for catalase, e.g. formate (half‐maximal effect: 150 μM); (d) inhibition by catalase inhibitors, e.g. azide (half‐maximal effect: 50 μM), with identical azide insensitive rates; (e) diminished acetaldehyde production in microsomes from rats pretreated with aminotriazole or pyrazole with identical residual rates. Moreover, NADPH‐dependent acetaldehyde production is suppressed in the presence of an active H2O2‐utilizing system.Thus, it is concluded that the NADPH‐dependent microsomal ethanol‐oxidizing system of Lieber and DeCarli [4] is due to a hydrogen peroxide formation from NADPH and a subsequent peroxidation of ethanol by contaminating catalase. The data indicate that the existence of a unique system in addition to the peroxidatic reaction of catalase as postulated recently [4] is highly doubtful.
Oxygen‐Dependent Cleavage of the Vinyl‐Ether Linkage of Plasmalogen
Authors: Yavin Ephraim , Gatt Shimon 
Published: March 2005

The vinyl‐ether linkage of plasmalogen was cleaved by rat brain homogenates; practically all activity was localized in the soluble fraction of the homogenate.
The cleavage of the vinyl‐ether linkage was tightly coupled to the reduction of molecular oxygen; about 3.5–4 mol oxygen were reduced for each mol vinyl‐ether cleaved. Both the reduction of oxygen and the cleavage of vinyl‐ether ceased if the reaction was run in an atmosphere of nitrogen or if antioxidants such as N,N‐diphenyl‐p‐phenylenediamine, butylated hydroxytoluene or Quercetin were used, in the presence of oxygen. Either reaction was also inhibited by the addition of the following chelators: EDTA, citrate, dipyridyl, desferal and o‐phenanthroline.
A detergent such as sodium deoxycholate was required for optimal activity. The reaction (i.e., vinyl‐ether cleavage or oxygen uptake) was directly proportional to protein concentration and time and had an optimal pH at about 7.3. When reaction rates were plotted against substrate concentration, hyperbolic curves of the Michaelis‐Menten type were obtained.
The active component of rat brain supernatant was dialyzable, was not inactivated when heated for 10 min at 100 °C and did not precipitate in a saturated ammonium sulfate solution. This suggests that cleavage of the vinyl‐ether linkage of plasmalogen is catalyzed by a thermostable, low‐molecular‐weight, non‐protein component of rat brain supernatant.
Galactosyltransferases in Human Submaxillary Glands and Stomach Mucosa Associated with the Biosynthesis of Blood Group B Specific Glycoproteins
Authors: Poretz Ronald D. , Watkins Winifred M. 
Published: March 2005

The immunodominant group of the blood group B determinant is a terminal non‐reducing α‐D‐galactosyl residue; however β‐D‐galactosyl residues are present in B‐active glycoproteins within the interior of the carbohydrate chains and some incomplete chains probably end with terminal non‐reducing β‐D‐galactosyl units. Partially fractionated homogenates of human gastric mucosa and submaxillary glands were used to detect and characterise the α‐ and β‐galactosyltransferases involved in the transfer of D‐galactose to endogenous, as well as exogenous, macromolecular acceptors.α‐D‐Galactosyltransferases were demonstrated in tissues from blood group B and AB donors that transferred D‐galactose in α‐linkage to H‐active glycoproteins. This activity was not detectable in tissues from group A or O donors. The enzymes were membrane‐bound but partial solubilisation was achieved with Triton X‐100 or digitonin. The products of galactosyl transfer were characterised by precipitation with immunological reagents, β‐D‐Galactosyltransferases that used H‐active glycoproteins as acceptors were detected in tissue preparations from donors of all ABO blood groups.The transferases were activated by Mn2+ ions and incorporation of D‐galactose into endogeneous B‐substance was stimulated by uridine diphosphate N‐acetylglucosamine and guanosine diphosphate L‐fucose.
Kinetik der Dissoziation von Alkylisocyanidkomplexen monomerer und oligomerer Hämoglobine
Authors: Blanck Jürgen , Ruckpaul Klaus , Scheler Werner , Jung Friedrich 
Published: March 2005

The dissociation of isocyanide complexes of human adult and fetal haemoglobin, of the Hb of carp and cheironomus, and of the horse myoglobin has been investigated by means of replacing the R‐NC ligand by CO. Furthermore the dissociation of Hb‐A complexes induced by dilution has been studied.Ligand replacement on Mb occurs homogeneously. Associated Hb however exhibit biphasic reactions. This behaviour is believed to depend on subunit differences in reactivity. No ligand specifity could be observed.The dissociation rate constant k decreases in the sequence Mb > Hb‐F ≅ Hb‐A ≅ carp Hb > cheironomus Hb. The only small variation of k confirms that the species differences in the equilibrium constant K are mainly attributable to the variation of the respective association velocity constants k′.Compared with Hb‐A, carp‐ and especially cheironomus‐Hb exhibit an enhanced decrease of k with increased ligand size, this being probably due to interactions between apolar protein side chains in the haem pocket and the respective ligand.Additional dilution relaxation experiments show that the dissociation rate constant k decreases with increasing percentage R‐NC‐saturation of Hb; this confirms the dissociation reaction to be the kinetic basis of haem haem interaction.In the presence of allosteric effectors (inositole hexaphosphate) again the decreased equilibrium constants of the R‐NC complexes are mainly due to lower k' values, while k varies to a minor extent.
An Improved Purification of Cell‐Bound Pullulanase from Aerobacter aerogenes
Authors: Mercier Christiane , Frantz B. May , Whelan William J. 
Published: March 2005

This communication describes a method of preparing crystalline Aerobacter aerogenes pullulanase free from α‐amylase. The preparation is suitable for structural studies of starch and glycogen. Two forms of pullulanase have been observed, differing in molecular weight the proportions varying according to the conditions of growth of the organism. The crystalline enzyme is that of lower molecular weight, concentration‐dependent values of 108000 to 145000 being found.The action of the enzyme on polysaccharide substrates has been compared. The rate of action on branched substrates is most rapid when the outer chains have been shortened by α‐ or β‐amylase. Under comparable conditions, action on rabbit liver glycogen is almost negligible.
The Primary Structure of a Monoclonal Human λ‐Type Immunoglobulin L‐Chain of Subgroup II (Bence‐Jones Protein NEI)
Authors: Garver Fred A. , Hilschmann Norbert 
Published: March 2005

Protein NEI, which has a molecular weight of 23 500 and contains 216 amino acids, was isolated from the urine of a patient with multiple myeloma by salt‐precipitation and purified by ion‐exchange chromatography, block electrophoresis and gel filtration. 23 tryptic peptides have been isolated from the aminoethylated protein and 13 chymotryptic peptides from the carboxymethylated protein by ion‐exchange chromatography. Sequence studies have mainly been carried out with the tryptic peptides; the chymotryptic peptides have been used for overlaps in the variable region, since the alignment of the tryptic peptides by homology was not unambiguous.Protein NEI belongs to subgroup II of the λ‐chains. It has a series of subgroup specific exchanges and a subgroup specific deletion at position 96. Protein NEI differs from all other λ‐chains of subgroup II by possessing a sixth cysteine residue. This additional cysteine residue at position 88 is so far unique, as it is adjacent to an invariant cysteine at position 87. Protein NEI also contains a carbohydrate moiety attached to an aspartic acid or asparagine (Asx) residue at position 93, one of the hypermutable areas of the variable part.Furthermore we conclude by comparison with other λ‐chains that all completely elucidated λ‐chains can be ordered into four subgroups. The subgroups and linkage groups within subgroups are in complete agreement with an evolutionary origin of antibody variability and are incompatible with somatic hypermutation models.
Molecular Characterization of Pullulanase from Aerobacter aerogenes
Authors: Eisele Bernhard , Rasched Ihab R. , Wallenfels Kurt 
Published: March 2005

By means of ultracentrifugal studies and acrylamide gel electrophoresis in the presence of sodium dodecylsulfate, pullulanase of Aerobacter aerogenes was shown to be a single polypeptide chain molecule with a molecular weight of 143000. Cyanogen bromide cleavage yields 18 to 20 individual bands in the electrophoresis. Also the amino acid analysis suggests a chemical unit with a minimum molecular weight of 110000 to 120000.Previous results, indicating lower molecular weights and a subunit structure of the enzyme are explained by partial proteolytie degradation of the enzyme. This is shown by dodecyl sulfate electrophoresis under different conditions.The shape factors are calculated from the hydrodynamic data and indicate an oblate rotational ellipsoid.
Kinetic Study of the Activation Process of β‐Galactosidase from Escherichia coli by Mg2+
Authors: Tenu Jean‐Pierre , Viratelle Odile M. , Yon Jeannine 
Published: March 2005

The action of Mg2+ ions on β‐galactosidase activity has been studied under strictly controlled conditions with different substrates. The activation effect does not depend on the substrate.In the absence of Mg2+ ions, a residual activity has been found and the catalytic properties of Mg2+‐free enzyme have been determined.The activation process induced by Mg2+ is a slow process, the rate of which depends on the Mg2+ concentration. The deactivation process obtained by adding EDTA is also a slow process. Activation by Mg2+ is not a cooperative process. The apparent dissociation constant of the Mg2+ enzyme complexe is rather small, 0.65 ± 0.05 μM.The kinetics of activation and deactivation have been studied with phenyl galactoside as substrate.The results indicate that activation by Mg2+ operates through a binding with free enzyme. This binding involves at least two steps: the first and more rapid one is a single metal‐protein binding, the following slow step probably involves a conformational change of the enzyme induced by the metal binding.
Preparation and Characterization of Fragments from Yeast tRNAphe
Authors: Harbers Klaus , Thiebe Rainer , Zachau Hans G. 
Published: March 2005

The preparation of 13 fragments from yeast tRNAPhe is described. The fragments were prepared by partial digestion with T1‐, T2‐, N1‐, or pancreatic RNAase, by endonuclease and subsequent exonuclease digestion and by a combination of a chemical chain scission with endoor exonuclease degradation. In order to obtain high yields of fragments, in each case several conditions of digestion were investigated by analytical disc electrophoresis. The fragments were purified by column chromatography and preparative gel electrophoresis. They were characterized by their mobility in disc electrophoresis and by analysis of the mono‐ and oligonucleotides after complete digestion with T1‐RNAase.In the presence of MgCl2 three regions of tRNAPhe seem to be particularly exposed to an attack by endonucleases. These regions are the anticodon loop, the dihydrouridine loop and the nucleotides at the 3′‐end of the molecule. The T‐Ψ‐C loop of tRNAPhe seems to be less exposed. Fragments resulting from a cleavage in this region could be obtained in relatively good yield by digestion with N1‐RNAase or T1‐RNAase, the latter one in the presence of ethidium bromide instead of MgCl2.The specificity of the various nucleases towards yeast tRNAPhe is briefly discussed.
Stereochemical Course of Decarboxylation of S‐Malate on Malic Enzyme
Authors: Clifford Kenneth H. , Cornforth John W. , Donninger Cyril , Mallaby Richard 
Published: March 2005

A preparation of malic enzyme was made from chicken liver.
The enzyme was incubated, in tritiated buffer containing NADP, with 2S, 3S‐[2,3‐2H2]malate. A third malate, 2S, 3R‐[3‐3H1]malate, was incubated with the enzyme, NADP, and unlabelled buffer. Conditions were chosen so that the initially formed pyruvate was converted into lactate, which was isolated.
The lactate from each of the three incubations was oxidized chemically to acetate, which was purified.
Each specimen of acetate was assayed for chirality by the previously established method of conversion into S‐malate by condensation with glyoxylate on malate synthase, and measuring the fractional loss of tritium on incubation of the S‐malate with fumarate hydratase.
In this way, the acetate derived from 2S, 3R‐[3‐2H1]malate was shown to have S chirality and the acetate derived from 2S, 3S‐[2,3‐2H2]malate was shown to have R chirality. As expected, the acetate from 2S, 3R‐[3‐3H1]malate was found non‐chiral.
It is deduced that when S malate is decarboxylated on malic enzyme, carboxyl is replaced by hydrogen without net change of configuration. This confirms a result obtained by Rose in 1970 by different methods.
Multiple Phases in the Reduction of Xanthine Oxidase by Substrates
Authors: Swann John C. , Beay Robert C. 
Published: March 2005

A sytematic study has been made using electron paramagnetic resonance spectroscopy of the effects of enzyme concentration and relative proportions of active and inactivated enzyme on the anaerobic reduction of xanthine oxidase at pH 8.2 by the substrates xanthine, purine and NADH. Changes in intensity of two molybdenum (V) signals (earlier designated Rapid and Slow) and iron‐sulphur and flavin radical signals were monitored over the time‐range 0.1 sec to 2 days. A few measurements with the inhibitory substrate allopurinol were also made.
Reduction can occur in as many as four phases, all of which are clearly seen with xanthine as substrate. The Rapid type of signals from active enzyme first appear in a time comparable to the turnover time (phase I), then decrease in intensity over a somewhat longer time (phase II). This is followed by a further increase in intensity over periods of up to a few hours (phase III), to levels higher than those attained in phase I. Finally with a half‐life of several hours (phase IV) the Slow signal, which is known to be derived from inactivated xanthine oxidase, appears also. Fewer phases can be clearly distinguished with the other substrates.
In accordance with other work, results are interpreted in terms of redox equilibria among different enzyme species with molybdenum signal intensity controlled by interconversions between the paramagnetic Mo(V) and the diamagnetic Mo(IV) and Mo(VI) oxidation states. The observed kinetics indicate that Mo(V) produced in the catalytic phase I is “over reduced” in phase II to Mo(IV) in a bimolecular reaction in which inactivated enzyme is not involved and for which the reducing agent may possibly be a substrate radical. This Mo(IV) of active xanthine oxidase is then slowly reoxidised to Mo(V) by inactivated enzyme, which remains inert in the catalytic phase but undergoes reduction in the slower phases. The reducing equivalents appear in the inactivated form, first in the flavin and iron‐sulphur and then in the molybdenum. However, with NADH, where catalytic reduction is known to occur via flavin instead of via molybdenum, both active and inactivated enzymes are reduced simultaneously, thus confirming that the difference between these enzyme forms is minimal.
Function of Met‐tRNAMetf and Met‐tRNAMet in Peptide‐Chain Elongation in Cell‐Free Systems from Mouse‐Liver and Ascites‐Tumor Cells
Authors: Drews Jürgen , Grasmuk Hans , Weil Rudolf 
Published: March 2005

The two methionine‐accepting tRNA species from mouse ascites tumor cells were investigated with respect to their function in peptide chain elongation, using a mouse liver polysome system and a T‐factor dependent ribosomal‐binding assay. In support of results published previously it was found that in a non‐initiating cell‐free system Met‐tRNAMetf inserts methionine into internal positions of nascent peptide chains, even in the presence of Met‐tRNAMet.Correspondingly, T factor from ascites tumor cells stimulated the binding of both methionine‐accepting tRNAs to washed ribosomes. When Met‐tRNAMet and Met‐tRNAMetf were introduced together into the ribosomal binding assay, a strong preference for the T‐factor‐induced attachment of Met‐tRNAMet became visible. Formylated initiator tRNA was not recognized by T factor Whereas the ribosomal binding of formylated or unformylated initiator tRNA promoted by initiation factors functioned equally well with poly(A,U,G) and the triplet ApUpG, the T‐factor‐induced attachment of both Met‐tRNAMet species required the presence of the polynucleotide.Competition experiments with high concentration of Val‐tRNAVal and Met‐tRNAMetf revealed the occurrence of a partial mutual interference of these two tRNA species during chain elongation.The magnitude of this effect, however, was not sufficient to attribute the function of Met‐tRNAMetf in chain elongation to miscoding. No competition was observed between Val‐tRNAVal and Met‐tRNAMet.
Active and Inactive Forms of Pyruvate Dehydrogenase in Rat Liver
Authors: Wieland Otto H. , Patzelt Christoph , Löffler Georg 
Published: March 2005

Active form and total activity of pyruvate dehydrogenase were measured in rat liver homogenates. The activity obtained immediately after homogenization was considered to represent the active form, i.e. dephospho pyruvate dehydrogenase, originally present in the liver. Enzyme activity found after incubation of the homogenate with Mg2+ and partially purified pyruvate dehydrogenase phosphatase from pig heart, whereby inactive dehydrogenase is dephosphorylated to give the active form, was regarded as total pyruvate dehydrogenase activity. In the livers from normal fed rats, the active form accounts for only one sixth of total pyruvate dehydrogenase activity. This differs from other tissues such as heart muscle, kidney, brain and adipose tissue where about two thirds of total pyruvate dehydrogenase are present as the active form (Wieland et al., 1971; Siess et al., 1971).To elucidate the possible role of pyruvate dehydrogenase interconversion in the regulation of pyruvate metabolism in liver, the activity of active and total pyruvate dehydrogenase were determined in livers of rats subjected to various metabolic conditions. After fasting and refeeding with glucose as well as after treatment with nicotinic acid or insulin there were significant changes of active dehydrogenase activity without gross alterations of total activity. In general, metabolic states associated with decreased plasma fatty acids resulted in an increase of active pyruvate dehydrogenase whereas a rise in plasma fatty acids led to a lowering. It is concluded that in liver, similar to heart muscle and kidney (Wieland et al., 1971), fatty acids play an important role in the control of pyruvate dehydrogenase interconversion. The significance of this mechanism for the regulation of pyruvate metabolism in liver relative to the feedback control of pyruvate dehydrogenase by acetyl‐CoA is discussed.
Studies on there‐Citrate Synthase Reaction
Authors: Gottschalk Gerhard , Dittbrenner Susanne , Lenz Helmut , Eggerer Hermann 
Published: March 2005

re‐Citrate synthase catalyzes the hydrolysis ofR‐citryl‐CoA andR‐malyl‐CoA. TheSdiastereomers are not attacked. The stereospecificity of the hydrolase activity is an agreement with the stereochemical course of citrate synthesis as catalyzed by this enzyme. The cooperative action of 2‐oxoglutarate and metal ions (Co2+) induces maximum acetyl‐CoA enolase activity ofre‐citrate synthase.R‐Malate is not effective in this reaction. SinceS‐malate is the best inducer of the enolase activity ofsi‐citrate synthase this suggests differences in the architecture of the active sites of these enzymes.Whenre‐citrate synthase is incubated with citrate and coenzyme A in tritiated water, tritium is incorporated into citrate. This indicates that there‐citrate synthase reaction is reversible.
Occurrence and Particle Character of Aminoacyl‐tRNA Synthetases in the Post‐Microsomal Fraction from Rat Liver
Authors: Vennegoor Claudia , Bloemendal Hans 
Published: March 2005

A post‐microsomal fraction (Fraction X) was isolated from cell sap from rat liver by centri‐fugation at 105000 ×g for 15 h. The activity of a number of aminoacyl‐tRNA synthetases was considerably higher in the resulting pellet than in the supernatant. These enzymes (glutaminyl‐, isoleucyl‐, leucyl‐, lysyl‐, and methionyl‐tRNA synthetase) were purified by gel filtration on Sephadex G‐200, chromatography on DEAE‐Sephadex A‐50 and on hydroxyapatite. As compared with crude cell sap a 120‐ to 170‐fold purification was achieved.In all purification steps, including additional centrifugation on sucrose gradients or isoelectric focusing, the major peak of activity of the five enzymes coincided.In the electron microscope circular and rectangular particles, 11–14 nm in size, could be visualized. From these findings we suggest that in rat liver cells, if not in all animal cells, part of the aminoacyl‐tRNA synthetases occur in a particulate state.