bluuebunny/crossref_metadata_2025 huggingface dataset:
The Effects of Thyroxine on Hepatic Gluconeogenesis and Ketogenesis in Dairy Cows
Authors: Heitzman Raymond J. , Hibbitt Kenneth G. , Mather Ian
Published: March 2005

Four daily injections of l‐thyroxine were given to healthy cows in early lactation. Blood and liver samples were taken during the course of the experiment and assayed for the following metabolites and enzymes: glucose, glycogen, free fatty acids, lactate, pyruvate, 3‐hydroxybutyrate, acetoacetate, citrate, 2‐oxoglutarate, phosphoenolpyruvate, 2‐phosphoglycerate, 3‐phosphoglycerate, malate, NAD+, NADH, glucose‐6‐phosphatase, pyruvate kinase, phosphopyruvate carboxylase and α‐glycerolphosphate dehydrogenase.

The changes in the liver enzyme activities and metabolite levels following the thyroxine injections indicate an increase in the rate of gluconeogenesis and ketogenesis.

Out of a group of 12 cows only 2 animals showed the early clinical signs of ketosis, otherwise no other unusual clinical signs were observed. Thyroxine elevated the milk yields of the cows, although this increase was not maintained in the animal showing the more severe signs of ketosis.
Influence of Ring Substituents on the Action of Phenols on Some Dehydrogenases, Phosphokinases and the Soluble ATPase from Mitochondria
Authors: Stockdale Martin , Selwyn Michael J.
Published: March 2005

The effects of phenols on the isolated mitochondrial ATP phosphohydrolase (ATPase) have been compared with their effects on several dehydrogenases, phosphokinases, catalase and acid phosphatase. 2,4‐Dinitrophenol and pentachlorophenol inhibit several of these enzymes at phenol concentrations similar to those which inhibit the ATPase. Stimulation of enzyme activity, by certain phenols which have a para‐substituent, is observed with the ATPase but not with any of the other enzymes.

The concentrations of phenols required to inhibit lactate dehydrogenase, hexokinase and the ATPase, and to produce maximal stimulation of the ATPase have been measured. These biological effects have been correlated with constants which describe the effect of ring substituents on the hydrophobic bonding and electronic properties of the phenols.

The kinetics of the inhibition of lactate dehydrogenase and hexokinase by phenols have been investigated. These inhibitions are competitive with the adenine‐containing substrate and are second order in phenol concentration. When the adenine in NADH is replaced by hypoxanthine the inhibition becomes first order in phenol concentration. When ATP is replaced by ITP as the substrate for the ATPase the inhibition by phenols changes from second to first order.

It is concluded that the effects of phenols on the coupling factor ATPase are not related to their ability to uncouple oxidative phosphorylation. The observations suggest that phenols bind to sites in enzymes which normally bind an adenine residue in a substrate.
Calcium and the Exportable Protein in Rat Parotid Gland
Authors: Wallach David , Schramm Michael
Published: March 2005

Most of the calcium (>80%) present in rat parotid gland slices was secreted concomitantly with the exportable protein. Detailed kinetics showed that secretion induced by catecholamines and by N6 monobutyryl adenosine 3′:5′‐monophosphate proceeded at an approximately constant ratio of calcium to amylase (60 nmoles calcium/mg exported protein). It was calculated that the calcium which is tightly bound to amylase could account for only 11% of the exported calcium.

Among the subcellular fractions of the gland, the secretory granules showed the highest calcium/protein ratio which was identical with that found for the material secreted by the slices. Both amylase and calcium were sedimented in a crude granule fraction to the extent of 50% of the total present in the homogenate of the gland.

Various treatment that caused release of amylase from the crude secretory granule fraction also caused release of calcium to a similar extent. The calcium in secretory granules was not readily exchangeable when the granules were suspended in a medium containing 45Ca. The possible role of calcium in the aggregation of the exportable proteins in the secretory granules is discussed in view of the information on the presence of relatively high concentrations of bivalent cations in some other gland systems.
l‐Threonine Deaminase of Rhodospirillum rubrum
Authors: Feldberg Ross S. , Datta Prasanta
Published: March 2005

A procedure for the purification of threonine deaminase from Rhodospirillum rubrum is described. The 2200‐fold purified enzyme was judged to be pure by disc‐gel electrophoresis and ultracentrifugation.

Sedimentation velocity centrifugation yielded an s°20,w value of 8.1 S and a D20,w of 4.65 × 10−7 cm2× sec−1. Assuming a partial specific volume of 0.74 g/ml the molecular weight of 164000 was calculated from the Svedberg equation. However, from calibrated Sephadex G‐200 columns, a D20,w of 4.15 × 10−7 cm2× sec−1 was obtained and using this equation, the molecular weight was calculated to be 180000.

Electrophoresis of the enzyme on polyacrylamide gels in the presence of sodium dodecyl sulfate revealed that the native enzyme was composed of four subunits, each of about 46000 molecular weight. In addition to the native tetrameric molecule, an active octameric species was detected in polyacrylamide gel electrophoresis.

The spectrum of the pure enzyme showed absorption maxima at 279 nm and 412 nm, characteristic of pyridoxal 5′‐phosphate‐containing enzymes. Analysis of the cofactor content revealed the presence of 4.2 moles of pyridoxal phosphate per 180000 g of protein.

The enzyme displayed a normal Michaelis‐Menten substrate saturation curve in the absence of isoleucine. High concentrations of isoleucine inhibited the enzyme activity somewhat only at low levels of the substrate; the inhibition was weak as compared to all other biosynthetic threonine deaminases. No other amino acid or any nucleotide tested produced any significant activation or inhibition.

In assays at pH values greater than 8.4, a transient build up of a 245 nm absorbing species was observed. It is suggested that this species corresponds to one of the two intermediates of the deamination reaction, α‐aminocrotonate or α‐iminobutyrate, and that at high pH the rate‐limiting step in the reaction is the hydrolysis of the intermediate to the final products, 2‐oxobutyrate and ammonia.
Transcription of Mitochondrial DNA in vitro from Neurospora crassa
Authors: Schäfer Klaus P. , Bugge Gisela , Grandi Maria , KÜNTZEL Hans
Published: March 2005

Intact mitochondrial DNA from Neurospora crassa (linear molecules of contour length 25–26 μm) has been transcribed in vitro with RNA polymerase from Escherichia coli.

At low concentrations (6 μg/ml) mitochondrial DNA is transcribed as efficiently as DNA from E. coli or calf thymus. The transcription product has the low G + C content characteristic of mitochondrial RNA.

After formation of an initial complex between enzyme and mitochondrial DNA only a limited number of bound enzymes can start RNA synthesis in the presence of rifampicin. The sigma factor is required to confer rifampicin resistance.

By comparing the initial rate of rifampicin resistant transcription of bacteriophage and mitochondrial DNA under saturating enzyme concentrations, the number of specific binding sites per mitochondrial genome (molecular weight 50 to 60 × 106) has been estimated to be 8 to 9. The transcription product synthesized in the presence of rifampicin has been characterized by competition‐hybridization and by RNAase digestion in solution. About 50% of the sequences hybridizing with mitochondrial DNA are homologous to RNA isolated from intact mitochondria; 35% of the product consists of rapidly renaturing double‐stranded regions characteristic of ribosomal RNA, and 5% of slowly renaturing double strands.

25% of the sequences synthesized in vitro form double strands only after annealing for 16 h at 62 °C but do not contain sequences complementary to stable mitochondrial RNA synthesized in vivo.

From these results it is concluded that bacterial RNA polymerase recognizes mitochondrial DNA sequences resembling bacteriophage initiation sites, and that enzymes bound to binding sites conferring rifampicin resistance transcribe asymmetrically cistrons coding for mitochondrial RNA.
Enzyme Reactions in Polymer Media
Authors: Laurent Torvard C.
Published: March 2005

The chemical potential of a compound often increases when a neutral polymer is added to the solution. This has been interpreted as an exclusion of the compound from part of the solvent. The present paper describes how the exclusion influences some enzyme reactions. A theoretical prediction based on the Michaelis‐Menten treatment is presented. Three enzyme systems have been examined experimentally, i.e.: (a) the degradation of hyaluronic acid by hyaluronate lyase in the presence of polyethylene glycol; (b) the lactate dehydrogenase reaction in dextran solution; (c) the cleavage of benzoyl dl‐arginine p‐nitroanilide by trypsin and its inhibition by serum albumin in dextran solution. In all cases, the presence of the polymer produced a decrease of the apparent Km value for the substrates and of the apparent Km value for the substrates and of the apparent Ki value for the inhibitor. The magnitude of this decrease correspond with that calculated theoretically. The effects were moderate in dextran solutions as concentrated as 0.20–0.25 g/ml.
Studies on the Nature of the “Microfibrillar” Component of Elastic Fibers
Authors: Robert Barbara , Szigeti Marthe , Derouette Jean‐Claude , Robert Ladislas , Bouissou Hubert , Fabre Marie‐Thérèse
Published: March 2005

Elastin was prepared from calf ligamentum nuchae with several methods using the following solvents for the extraction of extraneous (non‐elastin) proteins: 0.1 N NaOH at 100 °C, 88% formic acid at 45 °C, 70% trichloracetic acid at +4 °C, 5 M guanidine HCl with a thiol reagent and autoclaving in water. All elastin samples contained small amounts of carbohydrate. Electron microscopy showed the presence in all samples of electron‐translucent lamellae and osmiophilic uranyl‐lead stainable “microfibrillar” elements.

The osmiophilic “microfibrils” decreased strongly in the elastin preparations after extraction with 8 M urea–0.1 M 2‐mercaptoethanol, 5 M guanidine–0.05 M dithiothreitol or 0.1 M Na2SO3. These solvents extracted a significant proportion of the hexose content of the elastin samples with only 6–7% of their protein content. Similar results were obtained with chymotrypsin digestion although this enzyme acts less selectively than the above mentioned solvents and degrades elastin also.

When 125I‐labelled elastin was used for extraction with 8 M urea–0.1 M mercaptoethanol the extracted glycopeptides had a significantly higher specific activity than the residual elastin. These results suggest that the microfibrillar elements are derived from or are identical with the structural glycoproteins which were extracted from elastin‐rich tissues by identical procedures [12].

The presence of glycoprotein in varying proportions in purified elastin preparations can explain the variation of its amino acid content (ratio of polar to non‐polar amino acids) with age or in certain pathological conditions (atheromatosis, emphysema) and some of its immunochemical properties (cross reactions with structural glycoproteins).
Purification and Properties of a Catechol‐O‐Methyltransferase of Human Liver
Authors: Ball Peter , Knuppen Rudolf , Breuer Heinz
Published: March 2005

An S‐adenosylmethionine: catechol‐O‐methyltransferase occurs in the 105000 ×g supernatant of human liver. As compared with the homogenate, the enzyme was purified 380‐fold by adjusting the 24000 ×g supernatant to pH 5, fractionation with (NH4)2SO4, gel filtration on Sephadex G‐200 and G‐100 columns and ion‐exchange chromatography on CM‐Sephadex and DEAE‐cellulose columns. The purified enzyme preparation was homogeneous when subjected to further chromatography on Sephadex G‐100 and DEAE‐cellulose, disc electrophoresis on polyacrylamide gel at pH 9.5 and analytical ultracentrifugation. The enzyme exhibited a s20, w of 3.6 S with a molecular weight of 29000. The final preparation of catechol‐O‐methyltransferase was very labile, but could be stabilised by the addition of EDTA and MgCl2 in equimolar concentrations. The activity of the enzyme was assayed in the presence of cysteine and MgCl2 with 2‐hydroxy‐oestradiol‐17β and radioactive S‐adenosylmethionine as substrates. The metabolites of 2‐hydroxyoestradiol‐17β formed during incubation were identical with 2‐methoxyoestradiol‐17β and 2‐hydroxyoestradiol‐17β 3‐methyl ether. Throughout the purification procedure, the ratio of the 2‐ and 3‐methyl ethers, respectively, remained constant at 2.1.
Equilibrium of the ATP:Glutamine Synthetase Adenylyltransferase Reaction
Authors: Wohlhueter Robert M.
Published: March 2005

The activity of glutamine synthetase from Escherichia coli is regulated by an enzymecatalyzed adenylylation of a tyrosyl residue. The equilibrium of the adenylyl‐group transfer has been measured. Apparent ΔG°=−;1.8 kcal/mole (Keq= 19.6 at 25 °C, 10 mM MgSO4), implying that ΔG° for the hydrolysis of the tyrosyl‐O‐AMP bond is about 10 kcal/mole. Apparent enthalpy of the transfer reaction was estimated from the temperature dependence of the equilibrium constant; ΔH°app=−1.7 kcal/mole, ΔS°app∼ 0 cal deg−1 mole−1.

Solutions for the distribution of the various ionic forms of ATP and pyrophosphate (protonated and magnesium‐complexed) present in the reaction mixtures have been obtained by computer. A model reaction written with MgATP, magnesium pyrophosphate, and the subunit of glutamine synthetase as the thermodynamic reactants provides the best interpretation of equilibrium data at various conditions of magnesium and hydrogen‐ion concentrations.
The Protection by 70 S Ribosomes of N‐Acyl‐aminoacyl‐tRNA against Cleavage by Peptidyl‐tRNA Hydrolase and its Use to Assay Ribosomal Association
Authors: Vogel Zvi , Vogel Tikvah , Zamir Ada , Elson David
Published: March 2005

Free N‐carbobenzyloxy‐phenylalanyl‐tRNA is rapidly cleaved by the enzyme peptidyl‐tRNA hydrolase. When bound to a 30 S ribosome in the presence of poly(U), the substrate is hydrolyzed as rapidly as when free. The addition of 50 S ribosomal subunits to form the 70 S ribosomal binding complex protects the bound substrate from the enzyme. The degree of protection depends on the state of activity of the 50 S subunit. Under conditions favoring the formation of 70 S ribosomes, active 50 S subunits (capable of catalyzing the peptidyl transferase reaction) afford complete protection at one mole per mole of 30 S subunits. Inactive 50 S subunits also protect the substrate, but less effectively.

The protection of bound substrate can be used to measure the amount of 70 S ribosomal binding complex formed and the rate of its formation. Using highly active hydrolase preparations, we have developed a rapid assay procedure and present several examples of its use. Under conditions favoring the association of ribosomal subunits, the formation of the 70 S complex may reach completion within a few seconds.
The Structure of Nucleohistone
Authors: Henson Patricia , Walker Ian O.
Published: March 2005

The viscosity of histone‐depleted nucleohistone has been measured at high and low ionic strength. Native nucleohistone at low ionic strength and f1‐depleted nucleohistone at both low and high ionic strength all have the same intrinsic viscosity of 10.0 dl/g. This implies that these molecules are not flexible but behave in solution as rigid particles. On the assumption that they are rods they have a weight‐average length of 1160 ± 120 nm and diameter 5.6 ± 0.3 nm. The DNA is compressed in the rod to give an increase in mass per unit length for the DNA of 5.0 ± 0.5.

By contrast the intrinsic viscosity of nucleohistone depleted of f1, f2a2 and 75% of F2b and F3, varies with ionic strength in the manner typical of a flexible polyelectrolyte. The shapes of the sedimentation boundaries measured at high ionic strength of depleted nucleohistone show that the dissociation of histone from DNA takes place so that all molecules are depleted to the same extent. It thus has a conformation intermediate between that of native nucleohistone and DNA.

Polyacrylamide gel electrophoresis of histones removed by 1.0 M NaCl using a technique capable of resolving all five main histone fractions, shows that if one fraction only is responsible for supercoiling nucleohistone it must be f2a2.
Effects of 3,3′,5‐Triiodo‐l‐thyronine Administration on the Embden‐Meyerhof Pathway in the Kidney Cortex of the Rat
Authors: Meldolesi Maria F.
Published: March 2005

The amount of lactate and pyruvate production by kidney cortex homogenates (cell sap and microsomal fraction) from all intermediates of the glycolytic pathway has been investigated in normal and 3,3′,5‐triiodo‐l‐thyronine‐treated rats.

In normal animals the lactate and pyruvate production increases steadily from glucose (154 nmoles × mg protein−1× h−1) through phosphoenolpyruvate (31.850 nmoles × mg protein−1 h−1).

The addition in vitro of triiodo‐l‐thyronine to kidney cortex homogenates has no significant effect on lactate production.

In the animals treated for 5 days with 1 mg/kg body weight and per diem of triiodo‐l‐thyronine, the lactate and pyruvate formation from various intermediates of the Embden‐Meyerhof pathway is significantly (P < 0.01) increased: from glucose 225% of the control value, from glucose 6‐phosphate and fructose 6‐phosphate, 192%, from fructose 1,6‐bisphosphate and glyceraldehyde 3‐phosphate, 132%, and from 3‐phosphoglycerate and 2‐phosphoglycerate, 120%. No significant increase in lactate and pyruvate production has been observed using either phosphoenolpyruvate or pyruvate as substrates.
Studies on the Mode of Action of Oestradiol on Chicken Oviduct
Authors: Cox Ronald F. , Catlin Graham H. , Carey Norman H.
Published: March 2005

Macromolecules which bind oestradiol‐17,β after incubation in vitro of oviduct tissue from immature chickens have been studied.

Analysis of cytoplasmic extracts and salt‐extracts of nuclei on sucrose density gradients demonstrate the presence of 10 S and 7 S components in cytoplasm and nucleus respectively which bind oestradiol‐17β. The cytoplasmic component was not dissociated by 0.5 M KCl. Neither of these components was present in extracts isolated from chicken spleen, nor in chicken plasma.

Kinetics of binding of oestradiol to cytoplasmic and nuclear components were studied at 37°C. The rate of formation of cytoplasmic complexes was biphasic; it accumulated rapidly at fist (0–1 h) and more slowly at later times (1–4 h). Nuclear complex accumulated gradually between 0–4 h.

Precipitation of oestradiol‐bound cytoplasmic complex with protamine sulphate was used to estimate the number of binding sites in the cytoplasmic extract and the dissociation constant of the complex. Values obtained were 2.6 pmoles/mg protein and 9.3 pM, respectively. Binding sites of correspondingly high affinity for oestradiol‐17β were not detected in chicken liver cytoplasm.

Radioactivity associated with both 10 S and 7 S complexes was identified as almost entirely (98%) oestradiol‐17β. Nucleases had little effect on the integrity of the complexes; pronase destroyed the complexes.

Competition studies with unlabelled steroids were carried out to determine their relative affinity for both cytoplasmic and nuclear components as compared with oestradiol–17β. Both macromolecules exhibited high affinity for diethylstilboestrol (approximately 100% competition). Progesterone, testosterone and oestrone showed less than 11%, competition for the nuclear component. These steroids exhibited 34%, 8%, and 20% competition respectively for the cytoplasmic component. Oestradiol‐17α does not compete for either receptor.

The ultrastructural changes occurring in immature chicken oviduct epithelial cells after brief oestrogen treatment in vivo are described. However, 10S and 7S complexes have not yet been detected after administration of oestradiol‐17βin vivo.
The Influence of the Dimerisation on the Stoichiometry of the Active Center in Ferredoxin from Clostridium pasteurianum
Authors: Gersonde Klaus , Trittelvitz Eberhard , Schlaak Heinz‐Eberhard , Stabel Hans‐Henning
Published: March 2005

Determinations of the molcular weight by aid of gel filtration demonstrate that ferredoxin from Clostridium psteurianum can exist as well in a monomeric (Mr= 6000) as in a dimeric form (Mr= 12000). The dimerisation takes place in the presence of oxygen. Using exactly anaerobic conditions pure monomeric material can be prepared; using aerobic conditions, however, pure dimeric material or a mixture of monomers, dimers, and apoferredoxins of higher molecular weights appear.

Comparing the analytical data of the active center one finds eight atoms of iron and labile sulfur in monomeric ferredoxin, whereas four atoms of iron and labile sulfur are found in the sub‐unit of dimeric ferredoxin.

Results of the determination of the Hg‐content in mersalyl‐apoferredoxin prepared from a dimeric sample and of the titration of free SH‐groups in monomers and dimers can be explained assuming that four cysteinyl residues per subunit participate in disulfide bridges in dimeric ferredoxin. That means, dimerisation is accompanied by a loss of iron and labile sulfur and therefore by a decrease of the absorption coefficient ɛ390= 30000 M−1× cm−1 to E390= 15000 M−1× cm−1 related to one subunit with the molecular weight of 6000. The ratio of absorbances A390: A280 seems to remain unchanged.

In the phosphoroclastic system the biological activity of the dimeric ferredoxin decreases by a factor of two referring to one subunit.
Incorporation in vitro of [14C]Leucine into the Mitochondrial Protein of Lucilia cuprina
Authors: Williams Keith L. , Birt L. Michael
Published: March 2005

The basic requirements for incorporation in vitro of [14C]leucine into sterile mitochondria isolated from thoracic flight muscles of newly emerged Lucilia cuprina (the Australian sheep blowfly) have been studied in detail. Particular attention has been given to the influence of bacterial contamination on the incorporation.

The incubation medium developed has the following composition: (final concentrations) 18 mM MgSO4; 1 mM EDTA; 10 mM potassium phosphate; 50 μM each of 19 amino acids (excluding leucine); 65 mM KCl; 10mM HEPES (N‐2‐hydroxyethylpiperazine‐N′‐2‐ethane sulfonic acid) buffer; pH optimum7.4 (ATP‐generating system) or 8.0 (dl‐α‐glycerophosphate as substrate); and either ATP‐generating system (4 mM ATP, 5 mM phosphoenolpyruvate, 20 μg pyruvate kinase) or substrate‐supported system (20 mM dl‐α‐glycerophosphate + 4 mM ADP). This medium is similar to that used for investigation of amino acid incorporation by mitochondria from the tobacco hornworm, but different from that used with locust mitochondria, especially in having a higher Mg2+ content, higher pH optimum and lower amino acid mixture content.
Properties of 2‐Decylcitrate Synthase from Penicillium spiculisporum Lehman
Authors: Måhlén Anders
Published: March 2005

2‐Decylcitrate synthase is a mould enzyme, which condenses lauroyl‐CoA with oxalacetate. It with utilize acyl‐CoA of chain‐length less than C12 and also 11‐formamidoundecanoyl‐CoA but not lauroyl‐pantetheine. Its kinetic behaviour with respect to lauroyl‐CoA does not follow the theory of Michaelis‐Menten at concentrations that can be studied, and there is strong substrate inhibition. Km for lauroyl‐CoA is less than 0.1 μM. Km for oxalacetate is 35 μM at 21 μM lauroyl‐CoA and Km for 11‐formamidoundecanoyl‐CoA is 30 μM at 330 μM oxalacetate. The enzyme forms a very strong complex with palmitoyl‐CoA, lauroyl‐CoA and decanoyl‐CoA. It is inhibited by palmitoyl‐CoA, acetyl‐CoA, CoASSCoA and by SH reagents in the absence of lauroyl‐CoA. One SH group in a molecule of molecular weight 90000 is required for enzymatic activity. The enzyme consists of peptide chains each of molecular weight 45000. A purification method for acyl‐CoA using Sephadex G‐15 is given.
The Transport of Citric‐Acid‐Cycle Intermediates in Rat‐Liver Mitochondria
Authors: Papa Sergio , Lofrumento Nicola E. , Kanduc Darja , Paradies Giuseppe , Quagliariello Ernesto
Published: March 2005

The electrical nature of the malate‐citrate, malate‐Pi and malate‐2‐oxoglutarate exchange diffusion reactions in rat liver mitochondria has been studied. The exchanges have been followed in the presence of respiratory inhibitors and oligomycin to suppress energy supply.

The study of the effect of ionophores and uncouplers on the exchange‐diffusion reactions indicates that the citratein‐malateout and the malatein‐Pi,out exchanges are stimulated by inducing with nigericin or with valinomycin plus carbonyl cyanide p‐trifluoromethoxy phenylhydrazone (FCCP) electroneutral influx of H+. These exchanges are unaffected by inducing electrogenic efflux of K+ from mitochondria with valinomycin. The malatein‐citrateout exchange appears to be inhibited by electroneutral influx of H+.

Measurement of the extra‐ and intramitochondrial pH shows that the malate‐citrate exchange diffusion is accompanied by translocation of protons, across the mitochondrial membrane, in the same direction as citrate and the Pi‐malate exchange by proton translocation in the direction of malate. Evidence is presented that the proton translocation is a direct consequence of the anion exchange.

It is demonstrated that the H+: citrate and the H+: Pi ratios determined experimentally for the malate‐citrate and malate‐Pi exchanges practically coincide with the theoretical ratios expected for electroneutral malate2‐citrate2‐ and malate2‐HPO:2‐4 exchanges.

The malate‐2‐oxoglutarate exchange is not affected by ionophores and/or uncouplers neither it is accompanied by proton translocation across the mitochondrial membrane.

It is concluded that the malate‐citrate, malate‐Pi and malate‐2‐oxoglutarate exchange diffusion reactions are electroneutral and that the proton translocation accompanying the malate‐citrate and malate‐Pi exchanges is determined by the ionization state of malic, citric and phosphoric acid.
Studies on Lichen Enzymes
Authors: Schultz Joachim , Mosbach Klaus
Published: March 2005

From the lichen Lasallia pustulata a new enzyme, “orsellinate depside hydrolase”, capable of catalyzing the hydrolysis of certain depsides to corresponding resorcylic acid monomers, has been isolated.

The enzyme is extremely substrate‐specific; only depsides based on orsellinic acid (2,4‐di‐hydroxy‐6‐methyl‐benzoic acid) alone are acceptable. Phenyl benzoate and m‐digallic acid are not hydrolysed. Purification of the enzyme 135‐fold has been achieved with a final specific activity of 1300 U/mg of protein. The enzyme has a molecular weight of 42000 as determined by dodecylsulphate‐polyacrylamide gel electrophoresis. Only a single sharp peak was obtained by analytical ultracentrifugation. Electrofocussing of the hydrolase in polyacrylamide gel resolved the protein into four distinct enzymatically‐active bands. A Km of 56 μM has been determined using the depside lecanoric acid as substrate. The enzyme has been shown to be unusually stable to heat. On treatment of the enzyme for 22 h with 5 mM diisopropylfluorophosphate 75% of the activity was lost, typical of a serine esterase.

A spectrophotometric method suitable for the assay of orsellinate depside hydrolase is described.
Fatty Acid Synthetase from Pig Liver
Authors: Dutler Hans , Kull Arthur , Mislin Roger
Published: March 2005

Experimental evidence and theoretical considerations are presented which show that the pure enzyme with oxidoreductase activity for alicyclic ketones which was isolated from pig liver is a fatty acid synthetase. The enzyme exhibits fatty acid synthetase activity with the natural substrates, acetyl‐ and malonyl‐CoA, and when incubated with 14C‐labeled acetyl‐CoA, yields [14C]‐palmitate and [14C]stearate. The oxidoreductase activity for alicyclic ketones and the fatty acid synthetase activity of the enzyme could not be separated by chromatography on two different column materials. Kinetic data obtained with the model substrate, S‐acetoacetyl‐N‐acetyl‐cysteamine, as well as stereospecificity considerations support the view that it is the 3‐oxoacyl‐acyl‐carrier protein reductase component of the fatty acid synthetase complex which is responsible for the oxidoreductase activity toward alicyclic ketones.
Studies on Deoxyribonucleoprotein Structure
Authors: Ilyin Yurii V. , Varshavsky Alexander Ya. , Mickelsaar Ustav N. , Georgiev Georgii P.
Published: March 2005

The addition of tRNA to chromatin gel (initial insoluble deoxyribonucleoprotein preparation) in the presence of 1 mM MgCl2 or of 40 mM NaCl results in the transfer of the very lysine‐rich histone (F1) and of some nonhistone proteins to the tRNA, whereas all other histones remain in the deoxyribonucleoprotein. This result provides a new general method for the mild removal of histone F1 from the chromatin gel. The addition of tRNA to chromatin gel in the absence of Mg2+ and Na+ results not only in the rapid removal of histone F1 and of some nonhistone proteins from the chromatin gel, but also in the slow transfer of histones F2a2 and F2b to the tRNA. A similar pattern of histone redistribution is observed when the tRNA (or DNA) is added to the soluble deoxyribonucleoprotein preparation obtained by mechanical disruption of the chromatin gel.

There is no protein redistribution in the above‐mentioned soluble deoxyribonucleoprotein preparation (in the absence of exogeneous free polynucleotides).

The addition of free DNA or tRNA to the soluble deoxyribonucleoprotein preparation, obtained by treatment of the chromatin gel with 2 M urea, allows one to remove quantitatively histones F1, F2a2 and F2b from the deoxyribonucleoprotein, leaving the initial distribution of the arginine‐rich histones (F2a1 and F3) on the endogeneous DNA unchanged.

The redistribution of proteins (and in particular of histones) occurs in the soluble deoxyribonucleoprotein preparation obtained by urea treatment even in the absence of any exogeneous polynucleotides suggesting that conservation of the initial distribution of proteins in this soluble deoxyribonucleoprotein preparation is not complete.
The Purification and Properties of the NAD‐Linked Aldehyde Dehydrogenase from Pig Brain
Authors: Duncan R. Julian S. , Tipton Keith F.
Published: March 2005

The NAD‐linked aldehyde dehydrogenase from pig brain has been purified some 90‐fold over extracts of acetone powders of whole brain. This preparation of enzyme is unstable, and a less pure, more stable preparation has been used to investigate some of the properties of the enzyme.

Inhibition of the enzyme by the monoamine oxidase inhibitors 2‐phenylethylhydrazine and 2‐phenyl‐1‐methylethylhydrazine is reported.

Ethanol and reserpine have both been found not to inhibit aldehyde dehydrogenase.

On the basis of inhibition by o‐iodosobenzoate and phenylarsenious oxide it is proposed that the enzyme has two juxtaposed sulphydryl groups at or near the active site.

Irreversible inhibition by certain conjugated, unsaturated aldehydes is reported.

Non‐enzymic reactions of NAD+ with indole‐3‐acetaldehyde and phenylacetaldehyde are described. The reaction products are spectrally similar to NADH, but by enzymic criteria are not NADH. It is postulated that the products are the adducts between the aldehydes and the nicotinamide moiety of the coenzyme.
Interaction between Arginase and Ornithine Carbamoyltransferase in Saccharomyces cerevisiae
Authors: Messenguy Francine , Penninckx Michel , Wiame Jean‐Marie
Published: March 2005

The inhibition of ornithine carbamoyltransferase by arginase in Saccharomyces cerevisiae, which is under the control of arginine and ornithine, involves a regulatory site on the ornithine carbamoyltransferase distinct from its catalytic site. This regulatory site is responsible for the partial inhibition of ornithine carbamoyltransferase by excess of ornithine. The strong inhibition of ornithine carbamoyltransferase by arginase in the presence of arginine requires the integrity of the mechanism which leads to partial inhibition by excess of ornithine.
Studies on the Attachment of the Bovine J Blood‐Group Substance at the Erythrocyte Membrane
Authors: Schröffel Jaroslav , Thiele Otto Wolfgang , Koch Johannes
Published: March 2005

By means of quantitative studies of the J activity of bovine intact erythrocytes and of erythrocyte lipids it is concluded that all of the J activity of J‐containing cells is due to a lipid and that all J substance is present on the erythrocyte surface and thus available for J antibody. No J substance seems to be buried in the depth of the membrane.

During the process of hemolysis and subsequent washings bovine erythrocytes release a considerable portion of their membrane constituents in a “soluble” form. In order to prevent a disruption of ghosts a small amount of MgCl2 must be added to the hemolyzing mixture. We observed a loss of more than 30% of the original J activity along with about 15% of various stroma constituents unless MgCl2 had been added to the hemolyzing mixture.

By treatment of bovine stroma (prepared in the presence of added MgCl2) with organic solvents of increasing polarity two lipid fractions, “loosely” and “strongly” bound lipids, can be obtained. The J activity was found only in the “strongly” bound fraction along with the majority of glycolipids.

By treatment of bovine J‐cell ghosts with hypertonic saline part of the stroma constituents, including the J substance, is solubilized. It is concluded that the J substance, though secondarily absorbed from the serum onto the erythrocyte surface, is fully integrated to the other membrane constituents.
Synthetic Polynucleotides
Authors: Roychoudhury Ranajit , Kössel Hans
Published: March 2005

The use of chemically synthesized hexathymidylate as primer for terminal deoxynucleotidyl transferase in the presence of a ribonucleoside triphosphate and Mg2+ results in a limited addition of ribonucleotides at the 3′‐end of the primer. The incorporation is absolutely dependent upon the presence of Mg2+ and primer, and the pH optimum is similar to that observed for deoxynucleotide incorporation.

The priming efficiency decreases when oligothymidylates of smaller chain length are used according to the order: hexa > penta > tetra > tri. The incorporation into the trinucleotide is very small.

Depending upon the primer: triphosphate ratio, the reaction mixture after incubation contains either a mono‐ or a diaddition product, or both and such products can be synthesized under controlled conditions. The upper limit of this terminal addition has been found to be up to two nucleotide units. A ribonucleotide to ribonucleotide linkage is formed in the case of the diaddition product.

The ribonucleotide terminated hexathymidylates are capable of accepting dAMP residues from dATP in the presence of terminal deoxynucleotidyl transferase to give rise to a polymeric product whereby the newly synthesized polymer remains covalently linked to the original hexathymidylate by a ribonucleotide linkage.

Due to the presence of this ribonucleotide linkage, the polymeric product can be cleaved from the primer by alkaline hydrolysis, resulting in a separation of the primer sequences from the product sequences. The use of ribonucleotide terminated oligodeoxynucleotides thus provides a valuable tool for enzymic synthesis of polydeoxynucleotides, whereby the material used as primer (necessary for an enzyme catalyzed reaction) can be removed from the product.
Replication of Bacteriophage M13
Authors: Kluge Friedrich , Staudenbauer Walter L. , Hofschneider Peter Hans
Published: March 2005

After infection of Escherichia coli K12 strains with the single stranded DNA bacteriophage M13, between 85–95% of the intracellular parental DNA molecules are found attached to the host membrane.

This attachment is not irreversible. Even in singly infected cells up to 30% of the membrane bound parental DNA molecules are released from the membrane site and transferred to progeny phages during the first hour of infection.

The turnover of the membrane‐bound parental DNA depends strongly on the host system studied, indicating that this process may reflect primarily a property of the host.
Chemical Structure and Biochemical Activity of Cord Factor Analogs
Authors: Kato Masahiko , Asselineau Jean
Published: March 2005

By using mycolic acids isolated from virulent tubercle bacillus, two glycolipids, methyl 6‐mycoloyl‐α‐d‐glucopyranoside and 6,6′‐dimycoloyl sucrose, have been prepared. Their toxicity for mice, expressed as LD50, was respectively 187 and 161 μg, compared to 50 μg for 6,6‐dimycoloyl‐α‐d‐trehalose (cord factor). In mitochondria preincubated with these glycolipids, there was a decrease of the P/O ratio in the presence of succinate or pyruvate + malate as substrate, a functional damage at coupling site II, and a stimulation of ATPase activity, to a lesser extent than when mitochondria were preincubated with natural cord factor. In contrast to 6,6′‐dimycoloyl sucrose (and cord factor), methyl 6‐mycoloyl‐α‐d‐glucoside failed to affect the transfer of electrons in the NADH pathway.
The J Blood‐Group Substance of Cattle
Authors: Schröffel Jaroslav , Radaš Antonín , Thiele Otto Wolfgang , Koch Johannes
Published: March 2005

In cattle with J blood‐group substance both on erythrocytes and dissolved in serum, J activity was found in the total lipids extracted from erythrocytes, but not in the proteins precipitated by lipid extraction and not in the mucoproteins prepared from erythrocytes. Serum from these animals, however, does not only exhibit J activity of its total lipid, but also of its mucoproteins and of its residue after lipid extraction. This serum, therefore, contains the J substance in at least two different forms, as a lipoprotein and as a mucoprotein. Only the J lipoprotein is able to coat bovine erythrocytes during a postnatal period. It is suggested that hydrophobic groups are responsible for the attachment of the J substance to the erythrocytes.

In cattle with J substance only in serum, coating of erythrocytes is missing. This is apparently due to the finding that the serum from these animals contains J mucoproteins, but virtually no J lipoprotein. Since the determinant of the J lipoprotein has been found in the glycosphingolipid fraction the genetically fixed difference between animals which have J substance on erythrocytes and in serum and those which have it only in serum seems to consist in the inability of the latter animals to synthesize a glycosphingolipid containing the sequence of sugars which is responsible for J specificity.
The Isolation of Peroxisomes, Mitochondria and Chloroplasts from Leaves of Spinach Beet (Beta vulgaris L. ssp. vulgaris)
Authors: Parish Roger W.
Published: March 2005

Attempts to isolate peroxisomes, mitochondria and broken chloroplasts from leaves of Beta vulgaris using classical sucrose homogenising media and gradients gave poor results.

A homogenising medium consisting of 0.5 M sorbitol and 2.5%, Ficoll in 0.05 M Tris‐HCl buffer, pH 7.4 (SF medium) greatly increased catalase (peroxisome) sedimentation.

Density gradients made from mixtures of SF medium and sucrose (SFS gradients) gave good separation of the three organelles. Best results were obtained with the pellet sedimenting between 1000 ×g (20 min) and 6500 ×g (20 min).

The majority of catalase, glycolate oxidase and NADH‐glyoxylate reductase activity in the gradient was restricted to the peroxisomes.

Electron microscopy of the gradient fractions showed the isolated peroxisomes were intact and had retained the appearance they had in vivo.

The isolation methods described should prove useful with other plant tissue where isolation of cell fractions has hitherto been difficult.
Functional Non‐Identity of Subunits and Isolation of Active Dimers of d‐Glyceraldehyde‐3‐phosphate Dehydrogenase
Authors: Ovádi Judit , Telegdi Marianna , Batke József , Keleti Tamás
Published: March 2005

Maleylation of about four groups per tetramer of NAD‐free glyceraldehyde‐3‐phosphate dehydrogenase or of enzyme. NAD2 complex inactivates the enzyme. However, enzyme. NAD4 complex is inactivated only by maleylation of about 24 groups per tetramer. This shows that the presence of two moles of the relatively loosely bound coenzyme in addition to the firmly bound ones protects the tetramer against inactivation. Up to the maleylation of about 12 groups per tetramer NAD‐free enzyme, the reactive SH‐groups do not react with maleic anhydride. Maleylation of the enzyme · NAD4 complex up to the same extent does not affect the Racker band characteristic of the enzyme · coenzyme complex and the coenzyme remains firmly bound to the enzyme. However, after maleylation of NAD‐free tetrameric enzyme only two NAD molecules can be rebound to the inactive enzyme.

Four moles of ATP can be bound tightly to the NAD‐free enzyme. Two of them can be bound within a few minutes, whereas the further two only after prolonged incubation. Binding of two moles of ATP to the NAD‐free tetrameric enzyme in the absence of 2‐mercaptoethanol results in the irreversible inactivation of the enzyme. In the presence of 2‐mercaptoethanol the inhibition is reversible and of mixed type between ATP and NAD.

The above data provide evidence for the functional non‐identity and interaction of subunits.

The dissociation of tetrameric enzyme is increased by ATP or maleic anhydride and this process is irreversible in the absence of 2‐mercaptoethanol. An enzymically active dimeric form of the ATP‐treated enzyme has been isolated in the presence of 2‐mercaptoethanol. ATP treatment causes only a slight change in the steric structure of the protein as measured by difference spectrophotometry, showing that the dissociation into dimers is not accompanied necessarily by the unfolding of the molecule.

Interaction was observed between active and inactive enzyme molecules, which suggests the dissociation of oligomer and recombination of subunits.

In solution, the enzyme exists as a tetramer‐dimer equilibrium mixture and both forms are enzymically active.
The Smooth Lipopolysaccharide Character of 1,4,(5),12 and 1,9,12 Transductants Formed as Hybrids between Groups B and D of Salmonella
Authors: Nurminen Marjatta , Hellerqvist Carl G. , Valtonen Ville V. , Mäkelä P. Helena
Published: March 2005

The structure of the cell wall lipopolysaccharide (or O antigen) in hybrids between Salmonella O groups B and D was studied. We compared transductants of S. typhimurium (group B) with either the original rfbB gene cluster or the rfbD gene cluster of S. enteritidis (group D). Methylation analysis of delipidized lipopolysaccharides and of a material obtained after acetylation of all free hydroxyl groups in the lipopolysaccharides have provided the essential information in this study.

The O‐antigenic repeating units of the transductants with rfbB were found to be the same as those in wild type group B organisms, and those of the transductants with rfbD the same as those in wild type group D organisms. This similarity concerns the sugar units, their mode of linkage and the anomeric configurations. The degree of polymerization of the O side‐chain was the same in both types of transductants, and all the core “stubs” were substituted by O‐specific side‐chains.

As an unexpected finding we discovered a new sort of O‐acetylation in the lipopolysaccharide of the transductants. Approximately 25%, of the terminal d‐glucose residues in the O‐specific side‐chains were O‐acetylated at C‐4 in all those transductants (1,9,12 of group d‐type, 1,4,12 of group B‐type) that did not have the antigenic factor 5 and the 2‐O‐acetylabequose that corresponds to it. In the lipopolysaccharide of the 5+ strain (1,4,5,12 of group B‐type) abequose was O‐acetylated at C‐2 to about 60%, and the non‐reducing terminal d‐glucose units were unacetylated as had been the case in the wild type parent.
Inhibition of Phosphoglycerate Kinase by Products and Product Homologues
Authors: Larsson‐Raźnikiewicz Märtha , Arvidsson Lars
Published: March 2005

This paper gives a presentation of ADP and AMP inhibition of phosphoglycerate kinase with MgATP2− and 3‐phospho‐d‐glycerate as substrates at high and low Mg2+ concentrations and pH 7.8.

The enzyme seems to contain at least two nucleotide binding sites, one presumably binding to MgATP2− and the other to ADP3−. The ADP3− binding site might bind MgADP1− also. AMP2− competes for the same form of the enzyme, probably the same site, as MgATP2−. ADP3− and MgADP1− are competive inhibitors and AMP2− is a non‐competitive inhibitor of 3‐P‐glycerate. Values of the inhibition constant, Ki, for ADP3− at low Mg2+ level and MgADP1− at high Mg2+ level are 0.2 and 0.02 mM, respectively. The latter value is about ten times less than the expected Michaelis constant for corresponding substrate in the reverse reaction. Ki for AMP is about 2.0 mM at both low and high Mg2+ concentrations but the inhibition is stronger at a high than at a low Mg2+ level, probably caused by conformational and/or other differences of the enzyme at these two metal ion concentrations.

The main catalytic reaction suits a pattern that is consistent with a rapid equilibrium random mechanism.
Enhanced Stability of Uridine Kinase from Mouse Leukemic Cells Resistant to 5‐Azacytidine
Authors: Veselý Jiří , Čihák Alois , Šorm František
Published: March 2005

The specific activity of uridine kinase purified 100‐fold from mouse leukemic cells made resistant to 5‐azacytidine in vivo is diminished 50% in comparison with the same enzyme from the parental cell line. The enzyme has been found to be considerably more stable towards heating and p‐chloromercuribenzoate. The inhibition of both enzymes by cytidine 5′‐triphosphate was not modified following partial heat inactivation, while the treatment with p‐chloromercuribenzoate resulted in an enhanced feedback inhibition especially pronounced in uridine kinase from 5‐azacytidine‐sensitive cells. These findings are discussed in relation to conformational and/or structural changes of the enzyme molecules.
Human Placental Choline Acetyltransferase
Authors: Morris David , Grewaal Darshan S.
Published: March 2005

A radiometric procedure for estimation of choline acetyltransferase using [3H]acetylphosphate, CoA and phosphate acetyltransferase as a source of [3H]acetyl‐CoA is described.

Analogues of choline in which the hydroxyl group is replaced by chlorine are weak competitive inhibitors of choline acetyltransferase. The results indicate that the hydroxyl group of choline is important in the binding of this substrate.

Haloacetylcholines are potent inhibitors of choline acetyltransferase. The inhibition is shortlived and may be correlated with the short half‐life of the inhibitors under the conditions used.

Isotopic exchange between [14C]choline and acetylcholine is completely dependent on the presence of CoA and indicates that a sequential rather than a “ping‐pong” mechanism is operating. The exchange is strongly inhibited by chloroacetylcholine.
The Substrate Activation in Some Pyridine Nucleotide Linked Enzymic Reactions
Authors: Watkinson Ian A. , Wilton David C. , Rahimtula Anver D. , Akhtar M. Muhammad
Published: March 2005

The conversion of desmosterol into cholesterol was carried out in the presence of either tritiated water or [43H2]NADPH. The side chain fragment consisting of carbon atoms 23–27 of cholesterol was obtained through the combination of biological and chemical techniques. Selective degradation of the fragment led to the conclusion that in the saturation of the 24,25‐double bond of desmosterol a hydrogen atom from the medium is added to C‐24 and another from the 4‐position of NADPH to C‐25. These results in conjunction with similar studies previously reported on the saturation of other C═C in steroid biosynthesis are interpreted in terms of a general mechanism. It is suggested that the first crucial event in the pyridine nucleotide linked reduction is the activation of the substrate through protonation to give an electron deficient species which in the next step is neutralized by the addition of a hydride from NAD(P)H to furnish the product.