bluuebunny/crossref_metadata_2025_split huggingface dataset:
Purification and Physicochemical Properties of Fatty Acid Synthetase and Acetyl‐CoA Carboxylase from Lactating Rabbit Mammary Gland
Authors: HARDIE D. Grahame , COHEN Philip 
Published: March 2005

Fatty acid synthetase was purified 13‐fold from lactating rabbit mammary glands by a procedure which involved chromatography on DEAE‐cellulose, ammonium sulphate precipitation and gel filtration on Sepharose 4B. The preparation was completed within two days and over 100 mg of enzyme was isolated from 100–150g of mammary tissue, which represented a yield of over 40%. The preparation was homogeneous by the criteria of polyacrylamide gel electrophoresis and ultracentrifugal analysis. The sedimentation constant, s20, w was 13.3S, the absorption coefficient, A280nm1%, measured refractometrically was 10.0 ± 0.1, and the amino acid composition was determined.The subunit molecular weight determined by gel electrophoresis in the presence of sodium dodecyl sulphate was 252 000 ± 6 000, and the molecular weight of the native enzyme measured by sedimentation equilibrium was 515 000. These experiments indicate that at the concentrations which exist in mammary tissue (2–4 mg/ml) fatty acid synthetase is a dimer. The purified enzyme did however show a tendency to dissociate to a monomeric 9‐9S species on storage for several days or following exposure to a low ionic strength buffer at pH 8.3. There was only a small quantity of alkali labile phosphate (0.2 molecules per subunit) bound covalently to the purified enzyme.Acetyl‐CoA carboxylase was purified 300‐fold in a 50% yield within 24h by ammonium sulphate and polyethylene glycol precipitations [Hardie, D. G. and Cohen, P. (1978) FEBS Lett. 91, 1–7]. The preparation was in a state approaching homogeneity as judged by polyacrylamide gel electrophoresis, gel filtration on Sepharose 4B and ultracentrifugal analysis. The sedimentation constant, s20, w, was 50.5S, the absorption index, A280nm1%, was 14.5 ± 0.7, and the amino acid composition was determined.The subunit molecular weight of acetyl‐CoA carboxylase determined by gel electrophoresis in the presence of sodium dodecyl sulphate was identical to that of fatty acid synthetase (252 000) as shown by electrophoresis of a mixture of the two proteins. The preparations also contained two minor components of molecular weight 235 000 and 225 000, which appear to be derived from the major species of mol. wt 252 000. A large amount of phosphate (3.2 molecules per subunit) was found to be bound covalently to the purified enzyme.The properties of fatty acid synthetase and acetyl‐CoA carboxylase are compared to those obtained by other workers.
The Mechanism of Substrate Activation of Pyruvate Decarboxylase: A First Approach
Authors: HÜBNER Gerhard , WEIDHASE Rosemarie , SCHELLENBERGER Alfred 
Published: March 2005

The sigmoidal shape of the curve for v/[S], characteristic of pyruvate decarboxylase, indicates that the catalytic activity of this enzyme is regulated by the substrate. The enzyme, which is inactive in the absence of its substrate, is activated not only by 2‐oxo acids but also by 2‐oxo acid amides, which cannot act as a substrate of the enzyme. Whilst the dissociation constant of the enzyme‐activator complex depends on the electrophilic nature of the carbonyl group, the catalytic activity reached at saturation concentrations of the activator species is indepedent of the structure of the activator molecules.The mechanism of activation which proceeds via two reversible steps could be evaluated exactly by stopped‐flow techniques. The kinetic parameters of the activation and deactivation reaction were estimated and the validity of the equations derived which describe the activation kinetics could be proved by comparing them with the measured data.Using glyoxylic acid as an irreversibly binding active‐site marker, it could be shown that addition of the substrate to the enzyme‐bound thiamin diphosphate is the step of the catalytic mechanism whose rate is controlled by the substrate (activator) molecule.
β‐Adrenergic Receptors in Brown‐Adipose Tissue. Characterization and Alteratios during Acclimation of Rats to Cold
Authors: BUKOWIECKI Ludwik , FOLLEA Nicole , VALLIERES Julien , LEBLANC Jacques 
Published: March 2005

The capacity of brown adipose tissue to respond calorigenically to catecholamines increases markedly during cold‐acclimation of adult rats. To investigate this phenomenon, we have used the potent radioactive ligand (‐)‐[3H]dihydroalprenolol to directly estimate the number, the density and the affinity of β‐adrenergic receptors in brown adipose tissue membranes from control, cold‐exposed and cold‐acclimated rats.
Binding of (−)‐[3H]dihydroalprenolol to unfractionated membranes was rapid, stable, saturable and reversible. It displayed the affinity, specificity and stereoselectivity expected of binding to adenylate cyclase‐coupled β‐adrenergic receptors. β‐Adrenergic agonists competed for binding sites with an order of potency typical of the β1 subtype of adrenergic receptors: (−)‐Isoproterenol > (−)‐norepinephrine ≥ (−)‐epinephrine. Binding exhibited a remarkable stereoselectivity, the (−)‐isomers of β‐adrenergic agonists and antagonists being 34 to 280 times more potent than the (+)‐isomers in competing for (−)‐[3H]dihydroalprenolol binding sites.
Total interscapular brown adipose tissue of the adult, warm‐acclimated rat contained 1.12 ± 0.08 pmol of (−)‐[3H]dihydroalprenolol binding sites. During cold‐acclimation, growth of the tissue was accompanied by a 4–5‐fold increase in the total number of recptor sites. However, this increase did not keep pace with the increse in brown adipose tissue cellularity (as estimated by total tissue DNA content), resulting in a 40–50% reduction in receptor density. The decrease in receptor density was associated with cold‐exposure rather than with cold‐acclimation.
The affinity of (−)‐[3H]dihydroalprenolol receptor sites was not significantly altered by cold‐acclimation.
The results of this study are interpreted as indicating that catecholamines released via activation of the sympathetic nervous system regulate both the density and the number of their own receptors in brown adipose tissue of cold‐exposed animals. The development of the hyperadrenergic response of this thermogenic tissue during cold‐acclimation may result from a marked organ hyperplasia associated with an increased number of β‐adrenergic receptor sites and cannot be explained by alterations in receptor density or affinity.
Chemical and Spectral Properties of Putidamonooxin, the Iron‐Containing and Acid‐Labile‐Sulfur‐Containing Monooxygenase of a 4‐Methoxybenzoate O‐Demethylase from Pseudomonas putida
Authors: BERNHARDT Frithjof‐Hans , HEYMANN Eberhard , TRAYLOR Patricia S. 
Published: March 2005

Gel chromatography indicates that putidamonooxin has a molecular weight of about 126000. On the other hand, the amino acid composition and the iron‐to‐protein ratio point to a minimal molecular weight of 33000 and 31000 respectively. On sodium dodecylsulfate/polyacrylamide gel electrophoresis the enzyme migrated as a homogeneous band corresponding to a molecular weight of about 40000. The number of spots found in the tryptic peptide map of the carboxymethylated and digested enzyme indicates that putidamonooxin is composed of three or four identical subunits. After covalent cross‐linking of the subunits with dimethyl suberimidate and subsequent dodecylsulfate electrophoresis the main bands were in the molecular weight range of 40000, 87000 and 124000. These findings lead us to propose that putidamonooxin is either a trimer or tetramer.The amino acid composition of putidamonooxin and related data calculated from this are given. The isoelectric point was shown by isoelectric focusing to be at pH 4.7. Low‐temperature optical spectra of the reduced and oxidized enzyme as well as of three different putidamonooxin substrate complexes are given together with those recorded at 10°C. Enzyme · substrate binding spectra are observed with the oxidized putidamonooxin but not with the reduced enzyme. For the oxidized putidamonooxin a molar absorption coefficient at 455 nm of 14.7 mM−1 cm−1 was determined. Ks values of putidamonooxin towards different substrates and substrate analogues (i.e. tight couplers, partial uncouplers and uncouplers) are presented and possible reasons for the difference between the Ks values here obtained and the previously reported Km values are discussed.
The 50‐S Ribosomal Subunit Inhibits Functional Interaction between the 30‐S Subunit and Phage RNA
Authors: ZIPORI Pnina , BOSCH Leendert , VAN DIEIJEN Gerbrand , VAN DER HOFSTAD Gerard A. J. M. 
Published: March 2005

Vacant 30‐S subunits treated with the 7‐S immunoglobulin G fraction of sera from rabbits, immunized against ribosomal protein S1, (anti‐S1) lose their capacity to bind phage RNA. However, upon interaction of the subunits with MS2 phage RNA at 37°C, complexes between the 30‐S subunits and MS2 RNA (30‐S · MS2‐RNA) are obtained, which do not dissociate under the effect of anti‐S1.The capacity of such complexes to carry out protein synthesis in the presence of anti‐S1 has been examined. It is found that only a 30‐S · MS2‐RNA complex which is formed in the presence of initiation factor IF‐3 and in the absence of 50‐S subunits is resistant to anti‐S1 throughout the translation process. In contrast, when a complex formed under similar conditions but in the presence of 50‐S subunits is tested in this way, it is found to be sensitive to anti‐S1.The inhibition derived from 50‐S subunits is relieved if IF‐2 and tRNAMetf are present, in addition to IF‐3, during the formation of the ribosome · mRNA complex.It is concluded that the 50‐S subunit suppresses the potential of the 30‐S subunit to attach properly to phage RNA and induces the formation of an immature 30‐S · MS2‐RNA complex. Possible mechanisms by which the 50‐S particle inhibits the formation of the mature 30‐S · MS2‐RNA complex are discussed.The data are consistent with the view that displacement of the 50‐S ribosomal subunit is essential for initiation of protein synthesis on natural messengers.
N‐(2‐Oxoacyl)amino Acids and Nitriles as Final Products of Dipeptide Chlorination Mediated by the Myeloperoxidase/H2O2/Cl− System
Authors: STELMASZYŃSKA Teresa , ZGLICZYNSKI Jan M. 
Published: March 2005

The chlorination of dipeptides by the myeloperoxidase/H2O2/Cl− system takes place at the N‐terminal amino group, whereas no chlorination of the amide nitrogen of the peptide bond can be observed. The N‐terminal amino group is chlorinated to N‐monochloroamine or/and N‐dichloroamine. N‐Monochloropeptides were the main products at higher pH values, at lower pH a mixture of N‐monochloropeptides andN‐dichloropeptides was formed owing to the dismutation of N‐monochloroamine to N‐dichloroamine. N‐Monochloropeptides decompose, yielding NH3 and the corresponding N‐(2‐oxoacyl)amino acids. N‐Dichlorodipeptides decompose faster but to nitriles and the free C‐terminal amino acids. N‐Dichloroglycyl‐amino acid decomposes through a relatively stable intermediate (cyano‐formylamino acid) to hydrogen cyanide, cyanogen chloride and the free C‐terminal amino acid.Insulin chlorination also yields N‐terminal glycyl and phenylalanyl N‐monochloro derivatives, which deaminate to glyoxylyl and phenylpyruvyl residues.
Effect of Various Nucleotides on Folding and Enzymic Properties of a Synthetic 63‐Residue Analog of Ribonuclease A and Natural Ribonuclease A
Authors: GUTTE Bernd 
Published: March 2005

When a reduced synthetic 63‐residue analog of ribonuclease A (RNase A) was reoxidized in the presence of 2′‐CMP, a competitive inhibitor of RNase A, the overall activity after removal of the inhibitor was lower than that of the same analog reoxidized in the absence of 2′‐CMP. However, reoxidation in the presence of 2′‐CMP diminished the activity toward poly(U) more strongly than that toward poly(C).To investigate the influence of substrate analogs on folding and enzymic properties of the natural enzyme, reduced RNase A was reoxidized in the presence of 3′‐CMP, 3′‐UMP, adenosine 2′:3′‐monophosphate (2′:3′‐AMP) or guanosine 2′:3′‐monophosphate (2′:3′‐GMP). The activities of ribonuclease reoxidized in the presence of 3′‐CMP were essentially unaltered whereas RNase A refolded in the presence of 3′‐UMP had 150% activity toward 2′:3′‐UMP and RNase A refolded in the presence of 2′:3′‐AMP had 170% activity toward poly(A) as compared with the activity of untreated natural RNase A against these substrates.Kinetic studies using uridine 2′:3′‐monophosphate (2′:3′‐UMP) as substrate showed that the maximum velocity of the reaction catalyzed by RNase A which had been reoxidized in the presence of 3′‐UMP was twice that found with untreated native RNase A as enzyme. Comparison of the Michaelis constants indicated that refolding of RNase A in the presence of 3′‐UMP did not increase the affinity for 2′:3′‐UMP.The conformational isomers of RNase A formed through reoxidation in the presence of various nucleotides were not very stable. Thus, raising the temperature to 40°C lowered the activity against poly(A) from 170% to 115%. However, it cannot be excluded that conformational isomers of this kind are also formed during the biosynthesis of RNase A.
Ascorbate Oxidase from Cucurbita pepo medullosa
Authors: MARCHESINI Augusto , KRONECK Peter M. H. 
Published: March 2005

Ascorbate oxidase has been isolated from the green squash Cucurbita pepo medullosa by a new purification method. Furthermore a low‐molecular‐weight copper protein containing one type‐1 copper/20000 Mr could be separated during the purification of the oxidase. The six‐step procedure developed improved the yield of ascorbate oxidase by a factor of 2.5. The method is well reproducible and a constant value of 8 Cu (7.95 ± 0.1/140000 Mr) has been established. By ultracentrifugal and electrophoretic criteria the enzyme preparations have been found to be homogeneous. They exhibited a specific activity of 3930 ± 50 units/mg protein or 1088 ± 15 units/μ copper.
The pure enzyme is characterized by the following optical purity indices: A280/A610= 25 ± 0.5, A330/A610= 0.65 ± 0.05 and A610/A500= 7.0 ± 0.25. The molar absorption coefficient of the characteristic absorption maximum at 610 nm (oxidized minus reduced) amounts to 9700 M−1 cm−1.
Computer simulations of the electron paramagnetic resonance (EPR) spectra of the oxidized enzyme reveal the following parameters: for the type‐1 (blue) copper gz= 2.227, gy= 2.058, gx= 2.036; Az= 5.0 mT, Ay=Ax= 0.5 mT, for the type‐2 (non‐blue) copper g‖= 2.242, g⊥, = 2.053 A‖= 19.0mT, A⊥= 0.5 mT. Out of the eight copper atoms present in the oxidase four are detectable by EPR. Of these, three belong to the type‐1 class, and one to the type‐2 class, as demonstrated by computer simulations of the EPR spectra.
To achieve full reduction of the enzyme, as measured by bleaching of the blue chromophore, four equivalents of L‐ascorbate or reductate must be added in the absence of molecular oxygen. Upon reduction of the enzyme the fluorescence at 330 nm (λexmax= 295 nm) is enhanced by a factor of 1.5 to 1.75. The reduced enzyme is readily reoxidized by dioxygen, ferricyanide or hydrogen peroxide. It binds two molecules of hydrogen peroxide in the oxidized state (1/type‐3 Cu pair), which can be monitored by a characteristic increase of the absorbance around 310nm(Δɛ= 1000 ± 50 M−1 em−1). Corresponding changes in EPR and fluorescence spectra have not been detected.
Transfer of the 1‐pro‐R and the 1‐pro‐S Hydrogen Atoms of Ethanol into Krebs‐Cycle and Related Acids in vivo
Authors: BLOMBERG Sverker , CRONHOLM Tomas 
Published: March 2005

A method for gas chromatographic/mass spectrometric analysis of low‐molecular‐weight organic acids in liver tissue is described. The method involves formation of methoximes of oxo acids, preventing exchange of hydrogen by enolization. The acids are separated as methyl esters by gas chromatography on an SP‐1000 capillary column. The origin of fragment ions in the mass spectrometric analysis was studied by analysis of deuterated analogs.Lactate, pyruvate, 3‐hydroxybutyrate, fumarate, succinate, malate, 2‐oxoglutarate and citrate were analyzed in freeze‐clamped livers of rats given [1, 1‐2H]ethanol, (1R)‐[1‐2H]ethanol and (1S)‐[1‐2H]ethanol. No deuterium was incorporated in succinate and the oxo acids analyzed. The incorporation of deuterium from [1, 1‐2H2]ethanol in one position of malate and fumarate was about the same as in lactate (18–23% of the atoms), but the deuterium in lactate originated to a larger extent from the 1‐pro‐R position than in malate and fumarate. This indicates lack of equilibration of the NADH pools utilized by cytosolic malate dehydrogenase and lactate dehydrogenase, and that the former pool is more closely related to cytosolic acetaldehyde oxidation. 3‐Hydroxybutyrate incorporated the 1‐pro‐S hydrogen of ethanol to a larger extent than the 1‐pro‐R hydrogen. This is in agreement with the presence of 3‐hydroxybutyrate dehydrogenase and aldehyde dehydrogenase in the mitochondrial compartment. Hydrogen in citrate appears to be derived from C‐1 of ethanol both via oxaloacetate and via intramitochondrial isocitrate dehydrogenase.
Desialized Low‐Density Lipoprotein Regulates Cholesterol Metabolism in Receptor‐Deficient Fibroblasts
Authors: FILIPOVIC Ivan , BUDDECKE Eckhart 
Published: March 2005

Binding of homologous low density lipoproteins (LDL) to specific cell‐membrane receptors initiates regulation of cholesterol metabolism in normal human fibroblasts, but minor regulatory effects are observed in receptor‐deficient cells. After enzymic removal of sialic acid residues, however, the desialized LDL aquires the ability to regulate cholesterol biosynthesis and cholesterol esterification in receptor‐deficient cells as effective as native LDL in normal cells. This is indicated by a suppression of hydroxymethylglutaryl‐CoA reductase activity, by a reduced incorporation of [14C]acetate into cholesterol and by a stimulation of the [3H]oleate incorporation into cholesterol esters in both normal and receptor deficient cells. Competitive binding and internalisation experiments revealed that the action of desialized LDL in normal and receptor‐deficient cells is receptor‐specific.The results suggest that desialisation of LDL facilitates its binding to cell membrane receptors, its internalisation and subsequent regulation of cellular cholesterol metabolism, this effect becoming significant in fibroblasts with reduced number of LDL receptors.
Exchange of ADP, ATP and 1:N6‐Ethenoadenosine 5′‐Triphosphate at G‐Actin
Authors: ENGEL Jürgen , NEIDL Cornelia 
Published: March 2005

Nucleotide exchange was monitored by the strong increase of fluorescence accompanying binding of 1:N6‐ethanoadenosine 5′‐triphosphate (ɛATP) to G‐actin. ɛATP · G‐actin was titrated by ADP and a relative binding constant KADP/ɛATP= 0.03 was obtained. With KɛATP/ATP= 0.19, the physiologically important relative binding constant KADP/ATP= 0.0057 was calculated. From kinetic measurements of the exchange reaction a rate constant of ADP dissociation k−= 8 × 10−4 s−1 was derived which was compared with the corresponding values for ATP (8 × 10−4 S−1) and eATP (4 × 10−3 S−1) (all values in 20 mM Tris · HC1 buffer, pH 8.2 containing 0.8 mM CaCl2 at21°C).The exchange of ADP versus ATP is an important step in the steady‐state equilibrium of actin polymerization. Since polymerization of ATP · G‐actin to ADP · F‐actin is irreversible, depolymerization can take place only by dissociation of ADP · G‐actin followed by nucleotide exchange. It was found from a comparison of k‐ with the rate constants of the polymerization steps that the ratio of ADP · G‐actin to ATP · G‐actin is much higher in the steady state than at equilibrium.
Metabolic Carbohydrate Labelling of Glycoproteins from Mitogen‐Stimulated Mouse Lymphocytes
Authors: VAN EIJK Ronald V. W. , ROSENFELDER Günter , MÜHLRADT Peter F. 
Published: March 2005

CBA/J mouse lymphocytes from thymus, spleen and lymph nodes were separated into fractions enriched in either T (thymus‐derived) or B (bone‐marrow‐derived) cells. The cells were stimulated with ‘T cell mitogens’ (concanavalin A or phytohemagglutinin), ‘B cell mitogens’ (lipopolysaccharide or lipoprotein), and pokeweed mitogen to incorporate tritiated mannose, galactose or fucose. Mitogen‐induced incorporation of carbohydrates into acid‐precipitable material proved to be specific for the cell population.Carbohydrate‐labelled cells were extracted with dodecyl sulfate buffer and the extracts subjected to polyacrylamide gel electrophoresis and subsequent autoradiography. Depending on the carbohydrate used for labelling several glycoproteins specific for activated T cells (GP 200, GP 170, GP 125) and activated B cells (GP 210, GP 85 and GP 75) were noted. Also T or B cell subpopulations could be distinguished by their glycoprotein patterns. From these patterns the following conclusions were derived.a) Concanavalin‐A‐reactive T cells from thymus, spleen and lymph nodes comprise different subpopulations.b) Concanavalin A and phytohemagglutinin stimulate the same T cell subpopulations in spleen and lymph nodes.c) Lipopolysaccharide and lipoprotein stimulate different B cell subpopulations of the spleen.d) Lipopolysaccharide stimulated or lipoprotein‐stimulated B cells from lymph nodes differ from those in the spleen in that the latter show a markedly higher rate of labelling for GP 75, suggesting a higher rate of immunoglobulin M (IgM) synthesis.
Purification and Properties of Strictosidine Synthase, the Key Enzyme in Indole Alkaloid Formation
Authors: TREIMER Johannes F. , ZENK Meinhart H. 
Published: March 2005

A new enzyme, strictosidine synthase, which catalyzes the synthesis of 3‐α(S)‐strictosidine from tryptamine and secologanin was isolated from the soluble protein extract of Catharanthus roseus cell suspension cultures and was purified approximately 50‐fold by ammonium sulfate fractionation, column chromatography on DEAE‐cellulose, Ultrogel AcA34 and isoelectric focusing. The apparent molecular weight of the enzyme was 34000. The pH optimum was 6.8, apparent Km values for tryptamine and secologanin were 2.3 mM and 3.4 mM respectively for the enzyme to synthesize strictosidine. Strictosidine synthase shows high substrate specificity. No apparent cofactor requirement could be demonstrated. Of several enzyme inhibitors tested, only p‐chloromercuribenzoate inhibited the enzyme. The enzyme was relatively stable and could be stored at −20°C for periods of up to 1 year without appreciable loss of catalytic activity. The enzyme was demonstrated to occur in suspension cultures of 15 different species belonging to 9 different genera of the indole‐alkaloidproducing subfamily Plumerioideae of the Apocynaceae family.This enzyme is responsible for the synthesis of strictosidine the key intermediate in the formation of the majority of monoterpenoid indole alkaloids occurring in the plant kingdom.