bluuebunny/crossref_metadata_2025_split huggingface dataset:
Differential Incorportion of 5‐Azapyrimidines into the RNA of Phage f2 and of Bacterial Host
Authors: DoskoČIL Jiří , ŠOrm František 
Published: March 2005

Particles of phage f2 formed in the presence of 5‐azacytidine have nearly normal sedimentation coefficient and buoyant density, but their infectivity with respect to populations of normal phage is reduced. RNA extracted from these particles is partly degraded to small fragments and has very low template activity.Incorporation studies in vivo indicate that phage RNA‐replicase can utilize both 5‐azacytidine and 5‐azauridine triphosphates for the synthesis of RNA. Phage RNA, however, contains much less 5‐azacytosine than either replicative‐from RNA or host RNA; apparently some secondary mechanism is operative, tending to eliminate plus‐strands of RNA with high content of of 5‐azacytosine from entering mature phage particles.Analyses of replicative‐form RNA formed in cultures treated with 5‐azacytidine show that this analogue is incorported mostly instead of cytosine, but may also replace uracil quite frequently, apparently by incorrect base pairing.
Relationships between the 3α‐and 20β‐Hydroxysteroid
Authors: Gibb William , Jeffery Jonathan 
Published: March 2005

Crystalline cortisone reductase prepared from Strephtomyces hydrogenans has 3α‐ and 20β‐hydroxysteroid: NAD oxidoreductase activity. The total rate of reaction for mixtures of 17 α, 21‐dihydroxypregn‐4‐ene‐3,11,20‐trione with 5α‐androstan‐3,16‐dione, and of 17β‐hydroxy‐1α‐methyl‐5α‐androstan‐3‐ one with either of these compounds indicated that these substrates do not react independently. 3β‐Hydroxy‐5α‐androstan‐16‐one inhibited the reduction of both 17α, 21‐dihydroxypregn‐4‐ene‐3,11,20‐trione and 5α‐androstane‐3,16‐dione.20β‐Hydroxypregn‐4‐en‐3‐one also inhibited the reduction of both these substrates. The findings allow the view that 3α‐ and 20β‐activities may depend upon a single active centre with complex binding characteristics.
Metabolic Instability of Myelin Protein and Proteolipid Fractions
Authors: Marks Neville , Mela Paula , D'monte Brian 
Published: March 2005

Turnover of protein components of myelin in vivo was shown to be comparable to that of other brain proteins. Myelin was purified, to remove soluble contamination, by water shock (fraction M2) and centrifugation on sucrose and CsCl gradients. These methods gave two fractions, one of which consisted of characteristic myelin membranes free of visible contamination (M3), and the second consisted of fragmented membranes and vesicles (M4).Incorporation following intracisternal injection of [14C]lysine occurred at later time periods in myelin compared to mitochondria, probably as a result of different rates for penetration of precursor. The half‐lives for the different categories of protein and proteolipid components prepared by Triton X‐100 acetate procedures were markedly heterogeneous. In myelin, the highest incorporation occurred in the soluble basic proteins with half‐lives of 14‐21 days (fraction III), followed by the interface (Folch‐Lees, fraction II) material with half‐lives of 12–24 days, and the insoluble proteins (Wolfgram, fraction III) with half‐lives of 28–32 days. The half‐lives varied with the pulse period used (15–360 min) and give evidence of both short‐lived and long‐lived proteins. Heterogeneity of turnover was supported by the complex disc‐gel patterns found with electrophoresis on acrylamide using a phenol‐urea solvent system for solubilization. In comparable mitochondrial fractions, incorporation was highest in the interface material (half‐life 6–4 days), followed by insoluble proteins (half‐life 3–6 days), and lowest in the soluble (brain) fraction (half‐life 6–26 days).Considerable hydrolase activity was observed in crude fractions and in hypotonic myelin fractions, particularly acid and neutral proteinases, aminopeptidase, and monoacyl and dipeptidyl arylamidases. It appears that hydrolases associated with the sheath could be involved in turnover, but in most cases are not associated with the highly purified membranes prepared in sucrose and CsCl gradients. Purification resulted in an increased specific activity of an aminopeptidase hydrolysing Leu‐Gly‐Gly as the substrate, and of ribonucleoside‐2′ : 3′‐cyclic‐phosphate esterase. It is concluded that these two hydrolases are intrinsic components of the myelin sheath membranes.
Regulation of δ‐Aminolevulinic Acid Synthetase by Drugs and Steroids in vivo and in Isolated Perfused Rat Liver
Authors: Bock Karl W. , Krauss Eva , FrÖHLING Wolfgang 
Published: March 2005

δ‐Aminolevulinic‐acid‐synthetase induction by drugs depends upon the nutritional state of the animal. In allylisopropylacetamide‐treated animals the enzyme induction is reduced in fed compared with starved rats whereas after phenobarbital treatment an induction of the enzyme seen in starved animals is not observed in fed rats. δ‐Aminolevulinic‐acid‐synthetase induction is studied in relation to the synthesis of cytochrome P450.In the isolated rat liver perfused with either rat blood or washed bovine erythrocytes and Eagle minimal essential medium, δ‐aminolevulinic acid synthetase decreases sharply. This decrease can be prevented by the continuous addition of dexamethasone (9α‐fluoro‐16α‐methyl‐pregna‐1,4‐diene‐11β,17α,21‐triol‐3,20‐dione) or hydrocortisone (pregn‐4‐ene‐11β,17α,21‐triol‐3,20‐dione) but not by testosterone (androst‐4‐ene‐17β‐ol‐3‐one) or etiocholanolone (5β‐androstan‐3α‐ol‐17‐one). δ‐Aminolevulinic acid synthetase cannot be significantly induced by allylisopropyl‐acetamide unless corticoids are added to the perfusion medium. The results indicate that the synthesis of heme and, consequently, hemoproteins is controlled by corticoids or their metabolites in adult rat liver.
Electron Flow and Cholesterol‐Side‐Chain Cleavage in Ovarian Mitochondria
Authors: Robinson John , Stevenson Patricia M. 
Published: March 2005

The supply of electrons for the hydroxylation reactions associated with cholesterol side‐chain cleavage has been studied in intact porcine luteal mitochondria. The work was based on an assay of the NADPH‐dependent side‐chain‐cleavage reaction, the activity of the mitochondrial enzyme complex being expressed as the percentage of [4‐14C]cholesterol converted to the products, [4‐14C]pregnenolone and [4‐14C]progesterone, in 1 h. The reaction could best be supported by intermediates of the tricarboxylic acid cycle, although NADPH, and to a lesser degree NADH, would also support some activity.Investigations of the ADP:O ratios of the substrates which could support cholesterol side‐chain cleavage showed that they were lower than the ADP:O ratios found for the same substrates in liver mitochondria from the same animal; respiratory‐control ratios were also lower in the ovarian preparation. In the adrenal cortex, another steroid‐hydroxylating tissue, similar results obtained by other workers have been interpreted as indicating that the two oxidative chains, the first terminating in cytochrome oxidase and the second in cytochrome P450, are connected by an energy‐dependent transhydrogenase. In the ovary this explanation may be true for succinate, but it does not satisfy the results obtained with substrates which reduce NAD(P)+. Although spectrophotometric assays showed that there was some energy‐linked transhydrogenase, as well as non‐energy‐linked transhydrogenase activity in ovarian mitochondria, all the evidence obtained by investigating cholesterol side‐chain cleavage supported by NADH and the NAD+‐specific 2‐oxoglutarate dehydrogenase indicated that in ovary hydrogen transfer from NADH to the cholesterol‐hydroxylation site was not dependent on energy. In contrast, inhibitors of NADH oxidase markedly stimulated NADH‐supported side‐chain cleavage. Energy was required for the transfer of electrons from succinate to the site of cholesterol side‐chain cleavage.
Pyridine‐Nucleotide Transhydrogenase
Authors: van den Broek Hendrikus W. J. , Veeger Cornelis 
Published: March 2005

Anaerobic titration experiments of transhydrogenase from Azotobacter vinelandii with NADPH and NADH in the presence of NADase from Neurospora crassa show that two moles of reductant are required to reduce one mole of enzyme‐bound flavin. These observations have been confirmed by titrations with sodium dithionite. No stable 2‐equivalent reduced intermediate can be detected.
Differences found in the titration of the flavoprotein with dithionite, NADPH in the absence and presence of NADP+, and NADH in the absence and presence of NADP+ and NAD+ indicate that NADP+ and NAD+ interact in a different manner with the enzyme. From the results obtained it is concluded that NADP+ has a higher affinity for the oxidised enzyme, while NAD+ has a higher affinity for the reduced form of the enzyme. The results also indicate that the level of reduction of the enzyme‐bound flavin is determined by the ratio of reduced and oxidised pyridine nucleotides.
Assuming an unknown reducible group X will exist next to the flavin, a reduction scheme is proposed in which the enzyme is reduced to the 2‐equivalent reduced state, followed by dissociation of the oxidised pyridine nucleotide and internal transfer of electrons to the reducible group X. Reduction to the 4‐equivalent reduced state is supposed to be the last step which is not followed by dissociation of the product. Titration experiments of the dithionite‐, NADH‐ or NADPH‐reduced enzyme with NADP+ and photoreduction experiments support the proposed reductive mechanism.
Liver Acetyl‐Coenzyme‐A Carboxylase
Authors: Hashimoto Takashi , Isano Hiroshi , Iritani Nobuko , Numa Shosaku 
Published: March 2005

The mechanism of the liver acetyl‐coenzyme‐A carboxylase reaction was studied by kinetic experiments. Studies on inhibition by kynurenate, a novel dead‐end inhibitor binding to the uncarboxylated form of the enzyme, as well as malonyl‐CoA‐[14C]acetyl‐CoA exchange studies further substaintiated our previous conclusions that the reaction proceeds through the ordered bi‐bi uni‐uni ping‐pong mechanism, and that the site of action of citrate, an allosteric activator, lies predominantly on the carboxylated form of the enzyme. The complex patterns produced by malonyl‐CoA inhibition were accounted for by mixed product and dead‐end inhibition; malonyl‐CoA was found to form a dead‐end complex with the inactive (or less active) species of the carboxylated form of the enzyme.On the other hand, the dissociation constant of the uncarboxylated enzyme · citrate complex was estimated to be 3–14 μM in terms of [14C]citrate binding, protection against avidin inactivation and shift in the protomer‐polymer equilibrium. This value is about 103‐fold smaller than the dissociation constant of the carboxylated enzyme · citrate complex found by kinetic studies, i.e. 3–6 μM, so that the effect of citrate on the uncarboxylated form of the enzyme does not manifest itself upon kinetic analysis of the carboxylase‐catalyzed reactions. These findings indicate that carboxylation of the biotinyl prosthetic group gives rise to a conformational change at the citrate site, resulting in a marked decrease in the affinity of the enzyme for the activator. This further supports our previously proposed concept that the main role of citrate in the carboxylase catalysis is to keep the carboxylated form of the enzyme in active conformation by shifting the equilibrium between the active and inactive (or less active) species of this enzyme form.
Stereochemistry of the re‐Citrate‐Synthase Reaction
Authors: Wunderwald Peter , Buckel Wolfgang , Lenz Helmut , Buschmeier Verena , Eggerer Hermann , Gottschalk Gerhard , Cornforth John W. , Redmond John W. , Mallaby Richard 
Published: March 2005

R‐Acetate and S‐acetate, as the coenzyme‐A esters, were converted into citrates on recitrate synthase.
The citrate were assayed for chirality at the 2‐position (a) by incubation with aconitate hydratase (aconitase) and (b) by cleavage to malate by citrate lyase combined with malate dehydrogenase, followed by incubation of the isolated malate with fumarate hydratase (fumarase).
In each case, the citrate (or malate) derived from R‐acetate retained most of its tritium, and the citrate (or malate) derived from S‐acetate lost most of its tritium, on incubation with the hydratase.
It was concluded that if a normal intramolecular deuterium‐isotope effect is operative, the condensation of oxaloacetate with acetyl‐coenzyme A on re‐citrate synthase proceeds with inversion of configuration at the methyl group.
Studies of the Biosynthesis of the O9 Antigen from Escherichia coli O9:K30(A):H12
Authors: Fitzgerald‐Chandler Donna K. , Jann Klaus 
Published: March 2005

The O9 antigen from Escherichia coli O9:K30 (A):H12 contains mannose (90%) and glucose (10%). Incorporation of [14C]mannose from guanosine diphosphate [14C]mannose (GDP‐[14C]Man) into acetic‐acid precipitates has been studied with a preparation from cells ground with alumina. The time‐course of mannose incorporation was biphasic (rapid reaction for 2–5 min followed by a slower, linear reaction for 5–20 min) and the incorporation was stimulated by uridine diphosphate glucose (UDP‐Glc) during the early part of the reaction. The reactions (mannose incorporation after 2 min and 15 min, ± UDP‐Glc) were nevertheless proportional to protein concentration. [14C]Glucose was incorporated into acetic‐acid precipitates from UDP‐[14C]Glc and this reaction was slightly stimulated by GDP‐Man. Radioactivity from GDP‐[14C]Man was incorporated into butanol extracts and the early part of the reaction (2–5 min) was markedly stimulated by UDP‐Glc. Bacitracin did not inhibit the incorporation of mannose into acetic‐acid precipitates in the early part of the reaction, but did inhibit incorporation about 20% between 5 and 30 min.Larger‐scale incubation mixtures were extracted with 45% aqueous phenol. The product was of high molecular weight as indicated by Sephadex G‐100 chromatography and yielded a single radioactive peak corresponding to mannose after total hydrolysis and paper chromatography. This high‐molecular‐weight product precipitated with anti‐O9 antiserum in an antigen‐binding assay, indicating that it was serologically active. Thus, it is likely that the O9 antigen of E. coli O9: K30(A):H12 is synthesized from GDP‐Man and UDP‐Glc through a butanol‐extractable (lipid‐linked) intermediate. Reactions appear to be analogous to those described for lipopolysaccharide biosynthesis in Salmonella.
Métabolisme phospholipidique des hématies nucléées
Authors: Soula Georges , Souillard Cécilia , Gard Catherine , Douste‐Blazy Louis 
Published: March 2005

The in vitro incorporation of [32P]orthophosphate into chicken‐erythrocyte phospholipids has been studied by means of one‐ and two‐dimensional thin‐layer chromatography as a time‐related function: from 10 min to 6 h.Labelled phosphatidic acid appears after 10 min and represents about 90% of the total labelled phospholipids. Sphingomyelin turnover is as low as in mammal erythrocytes whereas glycero‐phospholipids' turnover is high. The specific activity of phosphatidylserine is ten times higher than that of phosphatidylethanolamine; phosphatidylserine decarboxylase seems to be absent. In contrast with the metabolism of non‐nucleated erythrocytes, the [32P]orthophosphate is incorporated into phosphatidylglycerol, the turnover of which is important.These data should agree with a de novo biosynthesis of phospholipids in nucleated erythrocytes, but many studies on mammal erythrocytes have demonstrated that there are essentially exchange reactions of phospholipids with plasma phospholipids and eventual acylation of plasma lysophospholpids.
Oxygen‐Affinity Studies of Avian Hemoglobins
Authors: Vandecasserie Christian , Schnek Arthur Georges , Léonis José 
Published: March 2005

The oxygenation of hemoglobin is under the control of the regulatory function of organic phosphates.The one in mammals corresponds to 2,3‐bisphosphoglycerate but in birds and turtles appears to be inositol hexakisphosphate.The latter component decreases the oxygen affinity of the two chicken hemoglobin compounds (the minor and the major) and the lone pigeon hemoglobin component.The Bohr effects have also been determined for the two hemoglobin samples.As has already been observed by electrophoresis and by chromatography, pigeon hemoglobin seems to behave more like that of the major chicken component by disclosing analogous log p50 values, i.e. the partial pressure of oxygen corresponding to a 50% load of the hemoglobin.
An Association between the Kinetic and Electrophoretic Properties of Human Purine‐Nucleoside‐Phosphorylase Isozymes
Authors: Turner Bryan M. , Fisher Rachel A. , Harris Harry 
Published: March 2005

Human purine‐nucleoside phosphorylase has been studied in extracts of red‐blood cells, cultured fibroblasts and cultured‐lymphocytoid‐cell lines. The enzyme from red cells has been found to contain at least seven electrophoretically distinct isozymes while the enzyme from the cultured cells gives a single electrophoretic band corresponding to the slowest band of the red‐cell enzyme.
Human erythrocytes have been separated into groups of different mean‐ages by densitygradient centrifugation; nucleoside phosphorylase from the youngest cells was found to give only four or five electrophoretic bands of which the slowest was the most intensely staining. There was a progressive increase in number and intensity of the faster bands as the mean age of the red‐cell fraction increased. An age‐related decline in the total activity of the enzyme was also observed.
The Lineweaver‐Burk plot for human‐erythrocyte purine‐nucleoside phosphorylase assayed in a red‐cell haemolysate with inosine as the variable substrate showed a downward curvature at inosine concentrations above about 0.2 mM. When a form of the enzyme giving only a single electrophoretic band was used the Lineweaver‐Burk plots were found to be completely linear. Generation of secondary isozymes in vitro resulted in a change in this linear plot to the curved form shown by the red‐cell enzyme.
It is concluded that the secondary isozymes generated as a result of ageing in vivo or in vitro differ in kinetic behaviour from the primary isozyme. This may account for the observed curvature of the Lineweaver‐Burk plot at high inosine concentrations.
Substrate Properties of Yeast tRNAPhe Oxidized and Reduced at the 3′‐Terminal Ribose
Authors: von der Haar Friedrich , Schlimme Eckhard , Gómez‐Guillen Manuel , Cramer Friedrich 
Published: March 2005

Native yeast tRNAPhe and this tRNAPhe with the 3′‐terminal AMP removed were oxidized by NaIO4 and subsequently reduced by NaBH4. The following investigations were undertaken with these substrates: aminoacylation in borate buffer and measurement of the life‐time of the aminoacylated oxidized an reduced tRNA, hydrolysis with snake‐venom phosphodiesterase and pyrophosphorolysis with tRNA nucleotidyl transferase.Whereas the oxidized and reduced yeast tRNAPhe is a good substrate for the enzymes investigated, the corresponding tRNAPhe with the 3′‐terminal AMP removed is a very poor, if any, substrate in these reactions. From the data obtained and from proton‐magnetic‐resonance investigations with oxidized and reduced AMP and ATP it is concluded that the removal of the C2′—C3′ bond and introduction of two hydrogen atoms instead distorts the original ribose conformation at the 3′‐terminus. Under the assumption that this distortion is different for C75 and A76 the difference in reactivity between the two oxidized and reduced tRNAs can be explained.
Iodine, Hemin and Heminester as Oxidants in a Synthesis of ATP from ADP and Pi Mediated by Thiols and Disulfides
Authors: Bäuerlein Edmund , Klingenfuss Marlies , Wieland Theodor 
Published: March 2005

The conversion of oxidation energy into the energy‐rich phosphoryl linkage of ATP has been demonstrated in a system consisting of the tertiary‐butylammonium salts of ADP and Pi in pyridine and utilising several mercapto carboxylic acids or their disulfides as mediators and iodine or hemin or hemindimethyl ester as oxidants.Iodine with several thiols in 1:1 molar proportions produces relatively poor yields of ATP (1.5–5.4%) which can be considerably increased (10–15%) when the corresponding disulfides are used, whereas a thioether (N‐acetylmethionine) had no effect. This led to the conclusion that disulfides are better substrates and accordingly much higher amounts of ATP were formed from all thiols on using a two‐fold amount of oxidant (up to 25.6%). Taking this into account a mechanism has been formulated in which the formation of a phosphorylating species, RS‐OPO3H2, is assumed to be formed from disulfides.Hemin and (less efficiently) its dimethyl ester used as oxidants also give rise to the formation of ATP in our system, but only under aerobic conditions. Here optimal yields (up to 16.7%) were obtained with thioglycolate or its disulfide as mediators, a thioether (N‐acetylmethionine) being almost ineffective, and no difference in efficiency between thiol acid and disulfide was observed at a hemin: sulfur ratio of 1:1. The yields depend in some way on the hemin: S ratio, lower values giving higher amounts of ATP on oxidation of the mercapto compound, but not of its disulfide. Therefore a reaction mechanism different from that of iodine must be presumed. The formation of the same phosphorylating species as above has been assumed to occur preferently with a thiol and to a lesser extent with a disulfide. The role of oxygen is still rather obscure, for it can not be determined whether it is the terminal electron acceptor or electron carrier. A catalytic participation of hemin could not be demonstrated, perhaps on account of the slow autoxidation rate of the ferrous heme iron.
The pH‐Dependence of Rates of Individual Steps in Ficin Catalysis
Authors: Hollaway Michael R. , Antonini Eraldo , Brunori Maurizio 
Published: March 2005

The kinetics of the ficin‐catalysed hydrolysis of benzoylglycine p‐nitrophenyl ester have been studied at different pH values with widely varying initial concentrations of enzyme and substrate. The time‐courses of proton release and p‐nitrophenol release were followed.
The failure to observe a lag period for p‐nitrophenol release employing a stopped‐flow spectrophotometer leads to a minimum value for the rate constant for the formation of the E · S complex of 5 × 105 M−1 sec−1. Evidence is presented for the formation of E · S being a pseudo‐equilibrium process.
The results are consistent with a three‐step mechanism involving an acyl‐enzyme intermediate. Time‐courses of reactions with [E0] ∼ [S0] were independent of wavelength in the range 291 to 400 nm so that it is unlikely that an oxazolinone is formed as an intermediate.
The pH‐dependencies have been determined for the dissociation constant of the E · S complex (Ks) as well as for the individual rate constants associated with the catalytic steps (k+2 and k+3).
pH profiles of k+2/Ks implicate ionising groups of pKa values 4.3 and 8.58 in the free enzyme (25 °C, I= 0.1).
The pH dependency of k+2 (measured directly at pH values below neutrality) implicates an ionising group in the catalytic step with pKa∼ 5.8. The change in the value of Ks with pH is in accord with this being identical to the group with pK 4.3 in the free enzyme.
The value of k+3 does not vary significantly between pH 3.9 and 6.6, from which it is deduced that the group of pKa∼ 5.8 in the E · S complex undergoes a downward shift in pKa by at least 2 units on formation of the acyl‐enzyme intermediate. The release of protons concomitant with acyl‐enzyme formation (pH 5 and [E0] ∼ [S0]) is consistent with such a pKa shift.
At neutral pH values the k+3 step is rate limiting in catalysis whereas at pH 3.9 k+2 becomes rate limiting. This change in rate‐limiting step with pH illustrates the danger in the assumption that kcatversus pH profiles for thiol protease action on substrates with good leaving groups are equivalent to k+3versus pH profiles.