bluuebunny/crossref_metadata_2025_split huggingface dataset:
On the Catabolism of Deoxyribonucleosides in Cells and Cell Extracts of Escherichia coli
Authors: Munch‐Petersen A. 
Published: March 2005

Extracts of Escherichia coli cells differ considerably from whole cells in their deoxyribosidecatabolizing activities.Whole cells catalyze an efficient transfer of the deoxyribosyl group from thymine to adenine, whereas phosphorolysis of the deoxyribonucleosides proceeds more slowly. The nucleoside purine pyrimidine deoxyribosyltransferase activity is shown to depend on the presence in the cells of thymidine phosphorylase as well as purine nucleoside phosphorylase.Cell extracts show a considerable loss of the nucleoside purine pyrimidine deoxyribosyltransferase activity, but catalyze the phosphorolysis of the deoxyribonucleosides much more efficiently than whole cells.Whole cells readily catabolize the deoxyribose moiety of thymidine while cell extracts have partially lost this capacity. The loss seems to be due to a destruction of deoxyribomutase activity during sonic treatment of the cells, while thymidine phosphorylase and deoxyriboaldolase activities are resistant to this treatment.All the above mentioned enzymes, involved in catabolism of deoxyribonucleosides, are released in the medium when the cells are subjected to osmotic shock‐treatment. They are all induced by growth of the culture in the presence of deoxyribonucleosides.These observations have led to the assumption that the enzymes concerned with breakdown of deoxyribonucleosides in E. coli structurally and functionally constitute a complex, located at or near the cell membrane, and depend on the intact structure of the cells for proper function. Uptake of exogenous thymine into cellular DNA is probably controlled by this complex of enzymes, since the cellular pool of deoxyribosyl groups, necessary for the formation of thymidine from thymine, may be regulated through an interplay between these enzymes.
The Reaction of Fatty Acyl Thiolesters with Proteins
Authors: Marsh J. B. 
Published: March 2005

The enzyme fraction precipitated from rat liver cell sap at pH 5 catalyzes the formation of fatty acyl coenzyme A thiolesters. During the incubation, about 3% of the labeled fatty acid appears covalently bound to protein. The fatty acyl CoA esters were the intermediates in the reaction. The covalent linkage of the fatty acid to the protein was not enzymatic, since labeled palmityl CoA reacted directly with protein. Proof for the covalent nature of the binding was obtained in three ways: (a) the label was not removed by a series of solvent extractions which removed free fatty acid or fatty acyl CoA, (b) labeled palmityl peptides were isolated from peptic digests of labeled protein, (c) peptides isolated from protein labeled with [1‐14C]palmitate were not decarboxylated by azide in the Schmidt reaction.Covalent labeling of the pH 5 enzyme protein was extensive only with fatty acids above 10 carbon atoms. Acetyl CoA gave less than 10% of the reaction given by palmityl CoA. These results suggested a preliminary binding of the hydrocarbon tail of the CoA esters with hydrophobic regions of the protein.Evidence for the involvement of protein hydroxyl and amino groups in the reaction was obtained. Reduction of pronase peptides of protein labeled with palmitic acid with LiAlH4 gave rise to labeled cetyl alcohol. SH Groups were not involved as judged by the failure of neutral hydroxylamine to remove the label and by the fact that gamma globulin, which has no free SH groups, was capable of reacting. Acid or alkaline hydrolysis at 100° removed less than half of the label, suggesting the presence of amide bonds. Blockage of amino groups by forming the picryl derivative of the protein greatly reduced the extent of the reaction.
Structural and Functional Aspects of Ribosome Interaction with Endoplasmic Reticulum Membranes
Authors: Comorosan S. , Lugojan P. 
Published: March 2005

The different biochemical components of endoplasmic reticulum membranes (phospholipids, phosphoproteins and RNA) were investigated as to their ability to interact with the ribosomal protein and RNA.
Light‐scattering determinations, viscosity measurements and gel‐filtration techniques were employed.
A complex between a fraction of ribosomal protein (dialyzed against urea) and a phosphoprotein fraction from endoplasmic reticulum membranes, and a complex between a fraction of ribosomal protein (dialyzed against water) and two different phosphoprotein fractions from endoplasmic reticulum membranes, were obtained. No complexes could be detected between the other components under investigation. Likewise no complexes could be detected in the control experiments with lecithin, pepsin and cytochrome c, tested with ribosomal protein fractions and endoplasmic reticulum membranes phosphoproteins.
These results suggest an additional new mechanism concerning the interaction of ribosomes with the endoplasmic reticulum membranes.
Protonated Polynucleotide Structures
Authors: Courtois Y. , Fromageot P. , Guschlbauer W. 
Published: March 2005

The change of the optical rotatory dispersion spectra as a function of pH of DNAs of different base composition and the synthetic polynucleotides poly d(A–T) and poly (dG) · poly (dC) has been studied.
It is shown that the acid titration of DNA when followed by optical rotatory dispersion is considerably more complex than it appears from ultraviolet absorption data. The gradual appearance of a peak at 260–270 mμ between pH 3 and 4 before acid denaturation, indicates a change of conformation of the guanosine residues in this pH region.
DNA methylated in N‐7 of guanine does not show this inversion of Cotton effects. It is concluded that this position plays an important role in the acid titration of DNA.
Protonation on N‐7 of guanine, rotation of guanine out of the helix, reversion into the syn‐position, pairing in Hoogsteen manner and thus sharing the proton with N‐1 on cytosine is suggested as a possible interpretation of these results.
From the data presented it is concluded that poly (dG) · poly (dC) has a conformation and/or structure different from that of DNA (B‐form). In analogy with published data on nucleo‐sides, it is suggested that guanosine is in the syn‐conformation. Three possible structures seem to be feasible: (a) Watson‐Crick pairing with parallel strands; (b) Hoogsteen pairing with anti‐parallel strands; (c) Watson‐Crick pairing with antiparallel strands in a left handed helix.
The Dissociation into Subunits of Aspartate Aminotransferase from Pig Heart Muscle
Authors: Banks B. E. C. , Doonan S. , Gauldie J. , Lawrence A. J. , Vernon C. A. 
Published: March 2005

Ultracentrifugation studies show that, at concentrations of about 0.1 mM the aspartate aminotransferase of pig heart muscle does not dissociate into subunits either in 6 M guanidine hydrochloride, pH 7.5, or 8 M urea solution, pH 7.5. On prolonged treatment (8 weeks, 0°) with urea, however, complete dissociation occurs and the measured value of the molecular weight drops to approx. 40,000. The protein so obtained is enzymically inactive and migrates on electrophoresis in starch gel at pH 8.0 as a single, fast‐moving band.Gel filtration experiments show that the holoenzyme (pyridoxal form) partly dissociates into two subunits at a concentration of about 3 nM. The specific activity of the enzyme does not change in this region; the monomer and dimer have, therefore, the same catalytic activity per mole of cofactor.The apoenzyme is unstable at high dilution and its dissociation has not been directly demonstrated by gel filtration. However, indirect evidence obtained from a study of the kinetics of its recombination with cofactor suggests that dissociation occurs at a concentration of about 50 nM.
The Interconversion of Liver Glycogen Synthetase a and b in vitro
Authors: de Wulf H. , Hers H. G. 
Published: March 2005

The activation of liver glycogen synthetase b into a in vitro is greatly accelerated by caffeine and theophylline, and is inhibited by fluoride, EDTA, glycogen, Pi and sulfate. The inhibition by fluoride and EDTA is partially released by an excess of magnesium but not by the methyl‐xanthines. This activation is also suppressed when ATP is added to the homogenate or when the concentration of endogenous ATP is maintained at a high level by shaking the preparation in the air.The inactivation of synthetase a into b in vitro can be studied after its previous activation performed either in vitro at 20° or in vivo by treatment of the animals with glucose or with glucocorticoids. The inactivation occurs only in the presence of exogenous or endogenous ATP and is accelerated by 3′,5′‐cyclic AMP. It is inhibited by glucose 6‐phosphate, even in the presence of the cyclic AMP, and by glycogen only in the absence of the nucleotide. A half maximal inactivation is obtained in the presence of 0.2 μM 3′,5′‐AMP.All these findings are in agreement with the hypothesis that the activation of liver glycogen synthetase is catalyzed by a specific phosphatase whereas its inactivation is a phosphorylation at the expense of ATP under the action of a specific kinase.
The Preparation and Some Properties of Bromelain Covalently Attached to O‐(Carboxymethyl)‐Cellulose
Authors: Wharton C. W. , Crook E. M. , Brocklehurst K. 
Published: March 2005

Purified bromelain has been attached to CM‐cellulose by reaction with its acid azide derivative to yield insoluble preparations which contain 6.5–11.0 mg of bromelain/100 mg of product.The result of an extensive washing procedure strongly suggests that this protein is attached chemically to the CM‐cellulose.The apparent catalytic constant for the catalysis of the hydrolysis of α‐N‐benzoyl‐l‐arginine ethyl ester (BzArgOEt) at pH 7.0, I= 0.2 and 25.0° by CM‐cellulose‐bromelain is 52% of the corresponding value for the bromelain‐catalysed hydrolysis when the enzyme concentration is estimated by protein analysis and 77% of the value for the bromelain‐catalysed hydrolysis when the enzyme concentration is estimated by analysis of the thiol content of CM‐cellulose‐bromelain.The Michaelis constant for the CM‐cellulose‐bromelain‐catalysed hydrolysis of BzArgOEt is about an order of magnitude lower than the corresponding value for the bromelain‐catalysed hydrolysis at low ionic strength and approaches the value for the bromelain‐catalysed hydrolysis at high ionic strength.The attachment of bromelain to CM‐cellulose protects the enzyme from oxidation at 4° but offers little or no protection from heat denaturation.
The Nature of the Perturbation of the Michaelis Constant of the Bromelain‐Catalysed Hydrolysis of α‐N‐Benzoyl‐l‐Arginine Ethyl Ester Consequent upon Attachment of Bromelain to O‐(Carboxymethyl)‐Cellulose
Authors: Wharton C. W. , Crook E. M. , Brocklehurst K. 
Published: March 2005

The apparent Michaelis parameters for the catalysis of the hydrolysis of α‐N‐benzoyl‐l‐arginine ethyl ester (BzArgOEt) at pH 7.0 and 25.0° by bromelain and by CM‐cellulose‐bromelain have been measured over a wide range of ionic strength.An equation has been derived which relates the apparent Km to the ionic strength of the assay medium for the catalysis of a reaction involving a charged substrate by an enzyme attached to a solid matrix which carries a charge opposite in sign to that of the substrate.This equation has been shown to describe the change observed in the apparent Km for the catalysis of the hydrolysis of BzArgOEt by CM‐cellulose‐bromelain consequent upon changing the ionic strength of the assay medium. This finding supports the interpretation of the perturbation of the Km of the free enzyme catalysis by attachment of the enzyme to CM‐cellulose as mainly the result of charge‐charge interactions.The effect of changing the ionic strength on Vmax for the catalysis by bromelain and by CM‐cellulose‐bromelain is discussed briefly and compared with the ionic strength effect on the rate of non‐enzymic hydrolysis of BzArgOEt.
Activation, Inhibition, and pH‐Dependence of the Hydrolysis of α‐N‐Benzoyl‐l‐Arginine Ethyl Ester Catalyzed by Kallikrein from Porcine Pancreas
Authors: Fiedler F. , Werle E. 
Published: March 2005

Kallikrein from porcine pancreas is inhibited by heavy metal ions and activated by sulfhydryl compounds or chelating agents. This activation is interpreted as a reversal of heavy metal inhibition. From the experiments the conclusion is drawn that kallikrein is neither a “sulfhydryl protease” nor a metal‐dependent protease.The pH dependency of the hydrolysis of α‐N‐benzoyl‐l‐arginine ethyl ester by kallikrein at low substrate concentrations indicates three basic groups with pK 7.9 and 5.7 in the free enzyme and pK 6.9 in the enzyme‐substrate complex which participate in catalysis. This pH‐dependence resembles trypsin‐ and especially cholinesterase‐catalyzed hydrolyses. From the most alkaline pK value the participation of an amino group and from the other pK values the participation of a histidine residue in kallikrein‐catalyzed hydrolysis is inferred.
Oxidative Metabolism in Cells Isolated from Brown Adipose Tissue
Authors: Prusiner S. B. , Cannon B. , Ching Te May , Lindberg O. 
Published: March 2005

Non‐stimulated brown fat cells exhibit a respiration controlled by availability of phosphate acceptor systems.
Cells stimulated by nor‐epinephrine gradually lose the respiratory control as demonstrated by a diminishing response to carbonyl cyanide p‐trifluoromethoxyphenylhydrazone (FCCP).
The initial phase of increased respiration upon norepinephrine addition is characterized by an increased availability of activated fatty acids.
A mechanism cannot be envisaged which could utilize all the ATP which would be produced by a tightly coupled oxidative phosphorylation system.
A fine metabolic control must exist which regulates a balance between ATP production for fatty acid activation and a loosening of coupling between electron transport and phosphorylation.
3α‐Hydroxysteroid: NAD Oxidoreductase Activity in Crystalline Preparations of 20β‐Hydroxysteroid: NAD Oxidoreductase
Authors: Pocklington T. , Jeffery J. 
Published: March 2005

Crystalline 20β‐hydroxysteroid: NAD oxidoreductase preparations from Streptomyces hydrogenans were shown to have 3α‐hydroxysteroid: NAD oxidoreductase activity. Compounds of both the 5α‐androstane and 5α‐pregnane series served as substrates.Among the compounds tested, no evidence was found of 3β‐hydroxysteroid: NAD oxidoreductase activity. The 3α‐hydroxysteroid: NAD oxidoreductase activity did not extend to compounds having 5β‐H, a 4,5 double bond, a 5,10 double bond, or 1,2 and 4,5 double bonds together. In the 5α‐H series, a 1,2 double bond prevented the compound from serving as a substrate.Substituents at C‐11, ‐16 and ‐17 altered the kinetic constants for the 3α‐hydroxysteroid: NAD oxidoreductase reaction, and in the case of an 11β‐hydroxy or 11‐oxo group the effects were similar to the effects reported for these groups for 20β‐hydroxysteroid: NAD oxidoreductase activity.Kinetic data were consistent with the view that two types of 3α‐hydroxysteroid: NAD oxidoreductase were present which involved different active centres, neither of which was the active centre for the 20β‐hydroxysteroid: NAD oxidoreductase activity. It is not yet established whether these enzymic activities are associated with the same or different molecules. The 3α‐hydroxysteroid: NAD oxidoreductase activities do not correspond to those of previously characterised enzymes (3α‐hydroxysteroid dehydrogenase and α‐hydroxycholanate dehydrogenase.
Kinetic Evidence for the Existence of α‐Globin Pool in β‐Thalassemic Reticulocytes
Authors: Bargellest A. , Pontremoli S. , Menini C. , Conconi F. , Melloni E. 
Published: March 2005

The kinetics of incorporation of 3H‐labeled amino acids into α, β, and γ globin has been determined, following incubation of intact reticulocytes, in stroma‐free hemolysates and in purified Hb F and Hb A from homozygous β‐thalassemic subjects.The labeled globins present in the cytoplasm as well as in purified Hb F and Hb A at various incubation times, were separated by CM‐cellulose chromatography, and the ratios of [3H]α to [3H]counterpart globins were determined.The results obtained show that in the initial stages of incubation, the ratios of [3H]α to [3H]‐counterpart globins are higher than 1 in the total lysate and in purified hemoglobins; these ratios, after long periods of incubation, decrease to values below 1.These findings suggest that the excess of newly synthesized α molecules flows very rapidly from the polyribosomes to the hemoglobins, and then, more slowly, to a “stromal”α pool.A model is presented, showing the most likely mechanism for the overall process.
On the Mechanism of Na+‐ and K+‐Stimulated Hydrolysis of Adenosine Triphosphate
Authors: Schoner W. , Beusch R. , Kremer R. 
Published: March 2005

The nucleotide specificity of membrane‐bound Na+‐ and K+‐activated ATPase from ox brain was compared with that of the Na+‐dependent phosphorylation reaction of ATPase protein. [32P]Phosphate is incorporated into enzyme protein from terminally‐labelled [32P]nucleotide triphosphates (ATP, ITP, GTP), a process dependent on the presence of Na+. Subsequent addition of K+ ions to the test system is followed by a rapid decrease in incorporated radioactivity. Enzyme‐bound phosphate exchanges its label rapidly with adenine nucleotides and slowly with inosine and guanosine nucleotides.ATP, ITP, and GTP are substrates of Na+‐ and K+‐activated ATPase. The ratio of the reaction rates is: ATP: ITP: GTP = 27: 2:1. An almost identical activation pattern of nucleotide triphosphate hydrolysis by Na+ ions in the presence of saturating concentrations of K+ is found with all substrates (Km= 10 mM NaCl). On the other hand, the optimal stimulation by K+ at saturating concentrations of Na+ is variable: hydrolysis of ATP is increased 10 times, that of ITP 4 times and that of GTP 1.2 times.ADP inhibits both ATP and ITP hydrolysis. Only high concentrations of IDP (50 mM) inhibit ATP hydrolysis, whereas low concentrations of IDP inhibit ITP hydrolysis.Identical nucleotide triphosphate specificities were found for Na+‐ and K+‐activated ATPase from ox brain and from human red blood cells.It is concluded that all nucleotide triphosphates investigated are hydrolyzed via the same acceptor group. The Na+‐dependent phosphorylation reaction of ATPase protein is considered as part of the overall reaction of membrane‐bound Na+‐ and K+‐activated ATPase.
Structure and Function of Carbamoylphosphate Synthase
Authors: Guthöhrlein G. , Knappe J. 
Published: March 2005

Carbamoylphosphate synthase of rat liver has been purified to a homogeneous state. Its molecular weight is about 250,000.When the catalytic activity is measured at 10°, using an optical method, an activation phase is detectable, which could be assigned to the N‐acetyl‐l‐glutamate induced transition of the enzyme from an inactive conformation to the active conformation. The half‐time of the process is about 1 min at 10 mM acetyl‐glutamate. The specific effect of the cofactor was found to be the labilization of the inactive conformation (as opposed to a stabilization of the active conformation).At low temperatures the enzyme dissociates reversibly into probably two identical subunits. The dissociation seems to occur at the stage of the active conformation. ATP was found to hold back the dissociation by specifically stabilizing the active conformation.
Purification and some Properties of Leucyl‐tRNA Synthetase from Bacillus stearothermophilus
Authors: Vanhumbeeck J. , Lurquin P. 
Published: March 2005

The leucyl‐tRNA synthetase from Bacillus stearothermophilus has been purified 350‐fold. The enzyme is stable when stored at 4° in the presence of sulfhydryl protecting compounds.The Km values for leucine and ATP are respectively 240 and 0.84 μM. The ATP‐PPi exchange and the aminoacylation reaction satisfy the Arrhenius law up to 45°.After thermal denaturation the enzyme has been partially renatured.
The pH 6 Acetolactate‐Forming Enzyme from Aerobacter aerogenes
Authors: Tveit B. , Störmer F. C. 
Published: March 2005

Crystalline pH 6 acetolactate‐forming enzyme from Aerobacter aerogenes contains 4 thiol groups per enzyme molecule, determined with 5,5′‐dithiobis(2‐nitrobenzoic acid) (DTNB) in 50 mM sodium dodecyl sulfate, or acetate.In the presence of 50 mM pyruvate, propionate, as well as acetamide, 3 thiol groups are accessible for titration with DTNB. In the presence of phenylpyruvate, 2.3 thiol groups reacted.The apparent Michaelis kinetics, obtained in the presence of acetate, is not altered after 1–3 thiol groups are allowed to react with DTNB.
The pH 6 Acetolactate‐Forming Enzyme from Aerobacter aerogenes
Authors: Störmer F. C. , Solberg Y. , Hovig T. 
Published: March 2005

The diffusion and sedimentation constants of the pH 6 acetolactate‐forming enzyme, determined by analytical ultracentrifugation, were used to calculate a molecular weight of 200 000 for the enzyme.The amino acid composition of the enzyme has been worked out, and 3 molecules of cocarboxylase, and 6 atoms of phosphorus were found to be tightly bound to one molecule of the enzyme. In addition, the effect of divalent cations was studied, results indicated that the enzyme has a requirement for manganese. An absolute requirement for cocarboxylase and manganese was not established.Two crystalline forms of the enzyme were studied by electron microscopy using negative staining technique. The plate‐shaped crystals were composed of rows of particles measuring approximately 100 × 60 Å. These particles consisted of sub‐units, and in some areas a helical organization of the crystal could be seen. The molecular organization was different in the two different crystalline forms of the enzyme.
On the Biosynthesis of Cyclopenin and Cyclopenol, Benzodiazepine Alkaloids from Penicillium cyclopium Westling
Authors: Nover L. , Luckner M. 
Published: March 2005

By feeding of isotope labelled compounds to cultures of Penicillium cyclopium Westling and degradation of the cyclopenin (3‐benzyl‐3,10‐epoxy‐4‐methyl‐2,3,4,5‐tetrahydro‐1 H‐1,4‐benzodiazepine‐2,5‐dione) and cyclopenol [3‐(m‐hydroxy‐benzyl)‐3,10‐epoxy‐4‐methyl‐2,3,4,5‐tetrahydro‐1 H‐1,4‐benzodiazepine‐2,5‐dione] formed the biosynthesis of these alkaloids was investigated:

The carbon skeletons of both alkaloids originate from all the C‐atoms of anthranilic acid and l‐phenylalanine and from the methyl group of methionine.
N(1) and N(4) of the diazepine ring derive from all the nitrogen atoms of anthranilic acid and l‐phenylalanine respectively.
For cyclopenol, of several phenylpropane compounds tested, only l‐phenylalanine is a direct precursor. Thus, the introduction of the m‐hydroxy group by a mixed functional oxygenase as one of the later steps of its biosynthesis appears very likely.
A Quantitative Study of the Biosynthesis of Cytochrome c
Authors: Kadenbach B. 
Published: March 2005

Sucrose‐density gradient experiments have shown it impossible to isolate microsomes from rat liver homogenate free from contaminated mitochondrial cytochrome c.
The incorporation of l‐[14C]lysine into cytochrome c from rat liver mitochondria and microsomes in vivo has been studied. The kinetics of the specific radioactivities of free l‐lysine, that of microsomal cytochrome c and that of mitochondrial cytochrome c show precursor‐product relationships.
From the kinetics of radioactivity of free l‐lysine and microsomal cytochrome c, the true pool‐size of the latter was calculated to be 1.02 nmoles/g microsomal protein, that is about 1/270 of total rat liver cytochrome c.
The turnover‐time of cytochrome c in the endoplasmic reticulum was found to be 102 min. From this data a turnover‐time of total rat liver cytochrome c of 19 days and a half‐life of 13.2 days was calculated.