bluuebunny/crossref_metadata_2025 huggingface dataset:
Effect of prostaglandin E1 alone and in combination with theophylline or aspirin on collagen-induced platelet aggregation and on platelet nucleotides including adenosine 3′:5′-cyclic monophosphate
Authors: Ball G. , Brereton G. G. , Fulwood Mary , Ireland D. M. , Yates Patricia
Published: August 2015

1. Human platelet nucleotides were labelled by incubating platelet-rich plasma with [U-14C]adenine. With such platelets, the effects of prostaglandin E1, theophylline and aspirin were determined on collagen-induced platelet aggregation and release of platelet ATP and ADP. Intracellular changes of platelet radioactive nucleotides, particularly 3′:5′-cyclic AMP, were also determined both with and without collagen treatment. 2. Prostaglandin E1, theophylline and aspirin inhibited collagen-induced aggregation of platelets in a dose-dependent manner. Collagen-induced release of ATP and ADP and breakdown of radioactive ATP were also inhibited in a dose-dependent manner. 3. Prostaglandin E1 stimulated the formation of platelet radioactive 3′:5′-cyclic AMP in a dose-dependent manner. With a given dose of prostaglandin E1, maximum formation of radioactive 3′:5′-cyclic AMP occurred by 10–30s and thereafter the concentrations declined. The degree of inhibition of aggregation produced by prostaglandin E1, however, increased with its time of incubation in platelet-rich plasma before addition of collagen, so that there was an inverse relationship between the radioactive 3′:5′-cyclic AMP concentration measured at the time of collagen addition and the subsequent degree of inhibition of aggregation obtained. 4. Neither theophylline nor aspirin at a concentration in platelet-rich plasma of 1.7mm altered platelet radioactive 3′:5′-cyclic AMP contents. In the presence of prostaglandin E1, theophylline increased the concentration of radioactive 3′:5′-cyclic AMP over that noted with prostaglandin E1 alone, but aspirin did not. 5. Mixtures of prostaglandin E1 and theophylline had a synergistic effect on inhibition of platelet aggregation. The same was true to a lesser extent with mixtures of prostaglandin E1 and aspirin. Such mixtures also inhibited collagen-induced release of platelet ATP and ADP and breakdown of platelet radioactive ATP. 6. Certain concentrations of either theophylline or aspirin and mixtures of small concentrations of prostaglandin E1 with either theophylline or aspirin caused little or no increase of radioactive 3′:5′-cyclic AMP at the time of collagen addition, but inhibited aggregation to a marked degree, whereas higher concentrations of prostaglandin E1 alone caused a much greater increase of radioactive 3′:5′-cyclic AMP at the time of collagen addition but inhibited aggregation to a lesser extent. With these compounds there does not appear to be a correlation between these parameters.
The effect of Trypan Blue, suramin and aurothiomalate on the breakdown of 125I-labelled albumin within rat liver lysosomes
Authors: Davies Malcolm , Lloyd J. B. , Beck F.
Published: August 2015

1. A fraction enriched in lysosomes was prepared by centrifugation from the livers of rats that had been injected 0.5h before death with 125I-labelled albumin. When suspended in sucrose-protected buffer, pH7.4, and incubated at 22°C for 2h, the particles progressively released iodotyrosine into the medium. Albumin digestion did not occur if the particles were subjected to treatments known to break lysosomes or if particles from uninjected rats were incubated in medium containing 125I-labelled albumin. It is concluded that the observed production of iodotyrosine results from protein hydrolysis within intact heterolysosomes. 2. Particles from rats pre-treated with Trypan Blue, suramin or aurothiomalate released iodotyrosine more slowly than controls. Since these compounds are enzyme inhibitors that concentrate in liver lysosomes after administration in vivo, their effect is ascribed to intralysosomal inhibition of proteolysis. The doses used did not decrease endocytosis of albumin into liver or cause increased lysosome breakage during incubation, thus allowing some alternative explanations of the decreased proteolysis to be eliminated. Particulate carbon, a non-inhibitor that also concentrates in lysosomes, did not affect albumin hydrolysis.
A method for measuring the content of a specific messenger ribonucleic acid in Escherichia coli
Authors: Bishop J. O. , Irving M. I.
Published: August 2015

A method is described for measuring the porportion of a specific messenger RNA in the total RNA extracted from pulse-labelled cells. A model system consisting of total ribosomal RNA and Escherichia coli DNA is used to validate the method and to define the conditions under which it can be used.
The diffusion capsule, a novel device for the addition of a solute at a constant rate to a liquid medium. Its application to metabolic regulation
Authors: Pirt S. J.
Published: August 2015

The diffusion capsule consists of a cylindrical container that can be completely filled with a solution and sealed with a small semi-permeable membrane at one end. In use, the capsule is immersed in an agitated liquid. Experiments on concentrated solutions in the capsule showed that, contrary to diffusion theory, the rate of diffusion of solute (sugars or amino acids) out of the capsule remained virtually constant until about 65% of the solute had diffused out of the capsule. Thus the device has been used to maintain constant material feed rates for periods exceeding 30h. The capsule is a simple and compact substitute for a pump and is superior to a pump for small feed rates in many applications. The capsule greatly extends the scope of the shake-flask culture technique for micro-organisms in that substrate-limited growth, possibly the aspect of greatest interest, is readily achieved simply by dropping in the flask a capsule containing the substrate. Diffusion feed should facilitate study of the metabolism of toxic substrates: also it is likely to provide an improved means for supplying a pulse of tracer to a culture.
The metabolism of cyclohexanol by Nocardia globerula CL1
Authors: Norris D. B. , Trudgill P. W.
Published: August 2015

1. Nocardia globerula CL1, isolated by enrichment on cyclohexanol and grown with it as carbon source, oxidized it with a Qo2 of 39μl/h per mg dry wt. and the overall consumption of 2.2μmol of oxygen/mol of substrate. Cyclohexanone, 2-hydroxycyclohexan-1-one dimer and cyclohexane-1,2-dione were oxidized with Qo2 values similar to that for cyclohexanol whereas ∈-caprolactone and 6-hydroxycaproate were oxidized very slowly and adipate not all. 2. Disrupted cell suspensions could not be shown to catalyse the conversion of cyclohexanol into cyclohexanone. 3. A cyclohexanol-induced cyclohexanone oxygenase (specific activity 0.55μmol of NADPH oxidized/min per mg of protein) catalysed the consumption of 1mol of NADPH and 1mol of O2 in the presence of 1mol of cyclohexanone. NADPH oxidation did not occur under anaerobic conditions. The only detected reaction product with 25000g supernatant was 6-hydroxycaproate. 4. Extracts of cyclohexanol-grown cells contained a lactone hydrolase (specific activity 15.6μmol hydrolysed/min per mg of protein), which converted ∈-caprolactone into 6-hydroxycaproate. 5. Incubation of 6-hydroxycaproate with 25000g supernatant in the presence of NAD+ resulted in NAD+ reduction under anaerobic conditions, oxygen consumption under aerobic conditions and the conversion of 6-hydroxycaproate into adipate. 6. Cyclohexanone oxygenase fractions devoid of ∈-caprolactone hydrolase catalysed the stoicheiometric formation of ∈-caprolactone from cyclohexanone in the presence of excess of NADPH. 7. The reaction sequence for the oxidation of cyclohexanone by N. globerula CL1 is: cyclohexanol → cyclohexanone → ∈-caprolactone → 6-hydroxycaproate → adipate. 8. It is suggested that the adipate may be further dissimilated by β-oxidation.
Purification of ribonuclease T1 by diethylaminoethylcellulose chromatography
Authors: Fields Robert , Dixon Henry B. F. , Law Gillian R. , Yui Chie
Published: August 2015

A procedure is described for isolating the enzyme ribonuclease T1 from Takadiastase, an extract of the mould Aspergillus oryzae. It involves an initial concentration of the enzyme by adsorption on DEAE-cellulose followed by gradient elution. Later the enzyme is chromatographed on the same adsorbent with an eluent of constant composition. Yields of 350–380mg of ribonuclease T1 from 500g of Takadiastase were obtained.
The reaction of aralkyl sulphate esters with glutathione catalysed by rat liver preparations
Authors: Gillham B.
Published: August 2015

1. Rat liver supernatant preparations catalyse the reactions of some aralkyl sulphate esters with GSH to yield S-aralkylglutathione derivatives. 2. A glutathione S-transferase that catalyses these reactions has been purified 16-fold. 3. The purified enzyme preparation catalyses the release of sulphate ions from benzyl sulphate, 1-menaphthyl (naphth-1-ylmethyl) sulphate and phenanthr-9-ylmethyl sulphate only in the presence of GSH. It does not cause the release of sulphate ions from prop-1-yl sulphate, l-serine O-sulphate, phenyl sulphate or oestrone 3-sulphate even when GSH is added. 4. The stability and specificity of the enzyme and its response to inhibitors and to changes of pH were studied. 5. The activity of the preparation was compared with the activities of glutathione S-transferases described previously.
Incorporation of l-[14C]leucine into eġġ proteins by liver slices from cod
Authors: Plack P. A. , Fraser N. W.
Published: August 2015

1. Liver slices from cod (Gadus morhua L.) were incubated with l-[14C]leucine and the incorporation of label into total protein, precipitated with trichloroacetic acid, and into egg proteins, precipitated with an antibody after addition of carrier egg proteins, was measured. 2. Liver slices from immature male or female cod, and from male fish with developing testes, did not incorporate significant amounts of l-[14C]leucine into egg proteins, whereas with slices from female cod with developing ovaries the rate of incorporation into egg proteins was 8% of the rate of incorporation into total protein. 3. Liver slices from immature male or female fish that had received an intramuscular injection of oestradiol benzoate (1mg/kg) 5–8 days previously incorporated l-[14C]leucine into egg proteins at about 26% of the rate of incorporation into total protein. 4. Incorporation into total protein and into egg proteins was inhibited by puromycin, and 1.2 and 0.13μg of puromycin/mg of tissue protein, respectively, gave 50% inhibition.
New derivatives of argininosuccinic acid in the urine of a patient with argininosuccinicaciduria
Authors: Lee C. R. , Pollitt R. J.
Published: August 2015

The urine of a patient with argininosuccinicaciduria contains several previously undescribed compounds that can be derived chemically from argininosuccinic anhydride (I). One of the major compounds is 8-carboxy-2-carboxymethyl-3-oxo-2,3,5,6,7,8-hexa-hydro-1H-imidazo [1,2-a][1,3]diazepine, which exists as two diastereoisomers that are readily interconvertible at pH5. The corresponding ring-opened monocyclic diazepines are also present, as well as traces of the isomeric hexahydroimidazodiazepines that are formally related to argininosuccinic anhydride (II). The formation and stereochemistry of these compounds are discussed.
Protein–polysaccharide biosynthesis. Membrane-bound saccharides
Authors: Derge J. G. , Davidson E. A.
Published: August 2015

Membrane-bound saccharides obtained from ear cartilage from rabbits recovering from papain injection were examined for glycosyl acceptor properties, alkali lability, molecular size and degree of sulphation. Although considerable glucuronosyl transfer from UDP-glucuronic acid could be demonstrated, less than one-third could be identified as representing chondroitin linkage formation; there was little or no effect of molecular size on acceptor properties. Approximately one-half of the membrane-associated chondroitin-like saccharide chains are solubilized by alkali, whereas one-half require proteolysis for solubilization. These fractions are analytically indistinguishable and both contain xylose and galactose as well as uronic acid and sulphated N-acetyl-galactosamine. All fragments that were examined contained stoicheiometric amounts of axial sulphate ester, strongly suggesting a close relationship between chain extension and sulphation.
Distribution of creatine, guanidinoacetate and the enzymes for their biosynthesis in the animal kingdom. Implications for phylogeny
Authors: Van Pilsum John F. , Stephens Grover C. , Taylor Dorris
Published: August 2015

1. The distribution of creatine and the creatine-synthesizing enzymes in the animal kingdom has been investigated. Creatine was found in tissues of all vertebrates examined, and in various invertebrates from phyla Annelida, Echinodermata, Hemichordata and Chordata, subphylum Cephalochordata. The activities of the creatine-synthesizing enzymes, arginine–glycine transamidinase and guanidinoacetate methylpherase, were not detected in the hagfish or in any of the invertebrates, including those in which creatine was found, with the exception that transamidinase activities were detected in the amphioxus and salt water clam; however, these activities are considered to be artifacts for reasons mentioned in the text. Additional evidence that the hagfish and various creatine-containing invertebrates could not synthesize creatine was the observation that these animals did not convert one or the other of the likely precursors of creatine (arginine and glycine) into creatine, in vivo. Further, the inability of these animals to synthesize creatine is correlated with the observations that all animals tested were able to abstract creatine from their aqueous environment. 2. The activities of the creatine-synthesizing enzymes were detected in the sea lamprey and in all but a few of the other vertebrates examined. Neither activity could be detected in the sharks and rays (cartilaginous fish), buffalo fish (bony fish) or the snapping turtle. Transamidinase or guanidinoacetate methylpherase activity could not be found in the salamander or garter snake, respectively. 3. The results obtained with the lamprey are in direct contrast with those obtained with the hagfish (both subphylum Agnatha, class Cyclostomata). The lamprey had the ability to synthesize creatine and did not abstract creatine from lake water. The hagfish did not have any apparent ability to synthesize creatine and did abstract creatine from sea water. The present report thus supports the theory that the myxinoid (hagfish) and petromyzoid (lamprey) agnathans are only distantly related. 4. The lack of creatine-synthesizing enzyme activities in the cartilaginous fishes may have phylogenetic significance, but may also be explained by the availability of creatine in the diet of these animals. The lack of one or both enzyme activities in vertebrates other than the hagfish and the cartilaginous fish is suggested to be the result of creatine in the diet.
Separation of two forms of rabbit metallothionein by isoelectric focusing
Authors: Nordberg G F , Nordberg M. , Piscator M. , Vesterberg O.
Published: August 2015

Rabbits were given repeated injections of cadmium chloride. Cadmium- and zinc-containing protein fractions were obtained from the livers of these animals by precipitation procedures and Sephadex G-75 chromatography. The protein thus obtained showed several characteristics similar to those of the earlier described protein metallothionein. Further separation by isoelectric focusing showed two main protein peaks with isoelectric points at 3.9 and 4.5 respectively. Amino acid analysis of these two forms showed similar content of most amino acids [residues per cent.: cysteine (28%), aspartate (8%), threonine (5–6%), serine (12%), glycine (7%), alanine (13%), methionine (2%), isoleucine (2%)] but with a small difference in content of lysine (12 and 13% respectively), proline (9 and 5% respectively) and glutamate (2 and 4% respectively). The two forms of the protein both contained cadmium, but only the one with pI4.5 contained also significant amounts of zinc.
Elementary processes of the magnesium ion-dependent adenosine triphosphatase activity of heavy meromysin. A transient kinetic approach to the study of kinases and adenosine triphosphatases and a colorimetric inorganic phosphate assay in situ
Authors: Trentham D. R. , Bardsley R. G. , Eccleston J. F. , Weeds A. G.
Published: August 2015

Transient kinetic studies of Mg2+-dependent heavy-meromyosin ATPase (adenosine triphosphatase) were done by monitoring the release of both ADP and Pi into the reaction medium by using linked assay systems. The release of Pi was monitored by its quantitative transfer to ADP, with concomitant reduction of NAD+in the presence of d-glyceraldehyde 3-phosphate, d-glyceraldehyde 3-phosphate dehydrogenase and phosphoglycerate kinase. The dissociation rates of the products, ADP and Pi, from heavy meromyosin were shown to be faster than the rate-controlling process, which occurs after the initial bond cleavage of ATP. The chromophoric ATP analogue, 6-mercapto-9-β-d-ribofuranosylpurine 5′-triphosphate (thioATP) was used as a substrate and spectral changes associated with a single turnover of heavy meromyosin could be assigned to elementary processes of the mechanism. It was shown that the dissociation rate of thioADP was not the rate-controlling process of the thioATPase, whose catalytic-centre activity was 7.6 times that of the ATPase at pH8. The dissociation rate of ADP from heavy meromyosin was measured by using thioATP as displacing agent and was found to be 2.3s-1, which is about 50 times the catalytic-centre activity of the ATPase at pH8. Transient kinetic studies with chromophoric adenosine phosphate analogues have general application for kinases and ATPases both in characterizing the chemical states of the intermediates and in delineating the elementary processes of the enzyme mechanism.
The effect of cytochrome c and its ‘dimer’ on electron transfer and energy transformation
Authors: Shur-Perek T. , Avi-Dor Y.
Published: August 2015

A preparation that contained cytochrome c, mainly in the form of its ‘dimer’, was studied and compared with native cytochrome c with respect to its ability to support electron transfer and energy transformation in cytochrome c-depleted rat liver mitochondria. When the depleted mitochondria were titrated with either cytochrome c or the ‘dimer’, the extent of coupling between respiration and phosphorylation was enhanced, as manifested by an increase in the P/O ratio. The ‘dimer’ was relatively ineffective as an electron carrier in the respiratory system, but it was as effective as cytochrome c in reconstitution of oxidative phosphorylation in depleted mitochondria. Addition of ‘dimer’ to the depleted mitochondria, in the presence of a low, non-saturating concentration of cytochrome c, increased the P/O ratio without concomitant stimulation of respiration. Both cytochrome c and the ‘dimer’ stimulated spontaneous swelling and electron transport-driven proton translocation in depleted mitochondria. The pattern of action of cytochrome c and its ‘dimer’ is in accord with the assumption that they affect an early step in energy conservation.
Transfer ribonucleic acid methylases of bone. Studies on vitamin A and D deficiency
Authors: Bradford David S. , Hacker Bruce , Clark Irwin
Published: August 2015

Methods were devised for the assay of tRNA methylases of rat bone. The activities of bone tRNA methylases are similar to those from other mammalian tissues. However, unlike reports on liver methylases, no inhibitors were found in the supernatant fraction from pH5 precipitate of bone extracts. The effects of vitamins A and D on the methylation of tRNA by cell-free extracts of rat bone were studied. Deficiency of either vitamin resulted in a decrease in the rate and extent of tRNA methylation, whereas the administration of vitamin A to hypovitaminotic-A rats and vitamin D to hypovitaminotic-D rats increased the rate and extent of tRNA methylation. These effects appear to be apart from changes in ribonuclease activity or in concentrations of calcium or magnesium. No evidence of inhibitors of tRNA methylases was found in bone extracts from vitamin-deficient rats nor of activators in bone extracts from deficient rats given vitamin A or D. The pattern of tRNA methylation under conditions of vitamin A or D deficiency was not changed, suggesting a generalized cellular deficiency. It was of significance to find that the specificity for methylation of specific bases in tRNA was different after the administration of vitamin A as contrasted with the effects of vitamin D. The possible significance of tRNA methylation to the biochemical action of the vitamins on bone is discussed.
The fractionation of phosphatidylinositol into molecular species by thin-layer chromatography on silver nitrate-impregnated silica gel
Authors: Luthra M. G. , Sheltawy A.
Published: August 2015

1. Two methods for the fractionation of phosphatidylinositol into molecular species were developed. In addition to preserving the fatty acid moiety of the molecule, the first method preserves the phosphorus, and the second preserves both the phosphorus and inositol ring. 2. In the first method, phosphatidylinositol was oxidized with periodate and the products reacted with diazomethane. I.r. examination showed that the main product was identical with dimethylphosphatidic acid. Fractionation to molecular species was carried out on thin layers impregnated with silver nitrate. The fatty acid composition of the species was determined by gas–liquid chromatography, and their distribution in lamb liver phosphatidylinositol was studied by a method using [3H]methanol. 3. In the second method, phosphatidylinositol was acetylated under mild reaction conditions. The major product was the triacetylated derivative of this phospholipid. This was reacted with diazomethane and the methylated–triacetylated phosphatidylinositol was fractionated into molecular species on silver nitrate-impregnated thin layers. Solvent mixtures containing acetone and distilled chloroform were found most suitable for this purpose. The fatty acid composition of the molecular species was determined by g.l.c., and their distribution in lamb liver phosphatidylinositol was studied by a method using [1-14C]acetic anhydride during the acetylation reaction. 4. Results from both methods agree fairly well. The most predominant species of lamb liver phosphatidylinositol is the monoenoic (60%) followed by the tetraenoic (17%). The di- and tri-enoic species existed as minor components.
3-Hydroxybutyrate dehydrogenase in tissues from normal and ketonaemic sheep
Authors: Watson Helen R. , Lindsay D. B.
Published: August 2015

1. In liver, rumen epithelium and kidney cortex of the sheep, a dehydrogenase active against dl-3-hydroxybutyrate occurred in both the cytosol and particulate fractions of the tissues. In brain, heart, skeletal and smooth muscles, the enzyme occurred only in the particulate fraction. 2. Enzyme activity in the cytoplasmic fraction of liver and rumen epithelium was similar with either d(-)-3-hydroxybutyrate or dl-3-hydroxbutyrate, but was less with acetoacetate as the substrate. The cytosol fraction of kidney cortex showed very little activity with d(-)-3-hydroxybutyrate, confirming that most of the activity with dl-3-hydroxybutyrate was with the l(+) isomer in this tissue. 3. 3-Hydroxybutyrate dehydrogenase activities in the cytosol and particulate fractions of liver, rumen epithelium and kidney cortex and in the particulate fraction of brain tissue were not stimulated by phosphatidylcholine, unlike the enzyme in sheep muscle and in tissues of other species. 4. The activity of 3-hydroxybutyrate dehydrogenase was not increased significantly in any of the tissues of ketonaemic sheep. 5. Comparison of rates of 3-hydroxybutyrate production in vivo with the enzyme activity in ketogenic tissue suggested that in sheep the maximum rate of production might be limited by this activity.
Reaction of dihydropyrrolizines with deoxyribonucleic acids in vitro
Authors: White I. N. H. , Mattocks A. R.
Published: August 2015

1. Thermal denaturation profiles of Escherichia coli DNA pretreated with monocrotaline pyrrole in vitro showed no difference from control DNA samples during heating. A substantial increase in the degree of renaturation during cooling of the pretreated DNA samples was observed. The degree of renaturation was dependent on the concentration of pyrrole used. 2. When rat liver DNA was pretreated with monocrotaline pyrrole there was a greater degree of renaturation after heat treatment than was found with E. coli DNA. 3. Equilibrium-density-gradient centrifugation in alkaline caesium chloride showed that DNA pretreated with monocrotaline pyrrole and heat denatured, renatured to a greater extent on quenching in ice than did untreated control DNA. The degree of renaturation was similar whether the initial treatment of the DNA with pyrrole was for 1 or for 15min. The reaction also appeared to be independent of pH between 5.5 and 9.0. 4. Retrorsine pyrrole was as effective as monocrotaline pyrrole in cross-linking the DNA, but monocrotaline pyrrole exposed to water for 5min before the addition of the DNA was ineffective. Pretreatment of E. coli DNA with synthetic bis-hydroxymethylpyrrole esters also caused renaturation after heat treatment. Monoesters were ineffective. 5. After treatment of rats with retrorsine, no cross-linking of the liver DNA could be demonstrated.
The binding of ethyl isocyanide to ferroperoxidase
Authors: Phelps Charles , Antonini Eraldo , Brunori Maurizio
Published: August 2015

The equilibrium and kinetics of ethyl isocyanide binding to ferroperoxidase were studied. At pH9.1 the results of both studies are consistent with a single-process model with an affinity constant of 95m−1 and combination and dissociation constants of 2.2×103m−1·s−1 and 23s−1 respectively. Ethyl isocyanide is not bound significantly at pH values lower than 6.0, and in this behaviour and the pH-dependence of the affinity constant, similarities exist between isocyanide and cyanide binding. The enthalpy of the process measured by equilibrium methods is −59kJ/mol (−14kcal/mol). At pH values below 9, the ethyl isocyanide adduct changes in a slow time-dependent manner, giving rise to a new species. These changes are reversible on increasing the pH. The results are discussed in relation to other known information about ligand binding to ferroperoxidase and to myoglobin.
The cellular production of hydrogen peroxide
Authors: Boveris Alberto , Oshino Nozomu , Chance Britton
Published: August 2015

1. The enzyme–substrate complex of yeast cytochrome c peroxidase is used as a sensitive, specific and accurate spectrophotometric H2O2 indicator. 2. The cytochrome c peroxidase assay is suitable for use with subcellular fractions from tissue homogenates as well as with pure enzyme systems to measure H2O2 generation. 3. Mitochondrial substrates entering the respiratory chain on the substrate side of the antimycin A-sensitive site support the mitochondrial generation of H2O2. Succinate, the most effective substrate, yields H2O2 at a rate of 0.5nmol/min per mg of protein in state 4. H2O2 generation is decreased in the state 4→state 3 transition. 4. In the combined mitochondrial–peroxisomal fraction of rat liver the changes in the mitochondrial generation of H2O2 modulated by substrate, ADP and antimycin A are followed by parallel changes in the saturation of the intraperoxisomal catalase intermediate. 5. Peroxisomes supplemented with uric acid generate extraperoxisomal H2O2 at a rate (8.6–16.4nmol/min per mg of protein) that corresponds to 42–61% of the rate of uric acid oxidation. Addition of azide increases these H2O2 rates by a factor of 1.4–1.7. 6. The concentration of cytosolic uric acid is shown to vary during the isolation of the cellular fractions. 7. Microsomal fractions produce H2O2 (up to 1.7nmol/min per mg of protein) at a ratio of 0.71–0.86mol of H2O2/mol of NADP+ during the oxidation of NADPH. H2O2 is also generated (6–25%) during the microsomal oxidation of NADH (0.06–0.025mol of H2O2/mol of NAD+). 8. Estimation of the rates of production of H2O2 under physiological conditions can be made on the basis of the rates with the isolated fractions. The tentative value of 90nmol of H2O2/min per g of liver at 22°C serves as a crude approximation to evaluate the biochemical impact of H2O2 on cellular metabolism.
Effects of denervation on the activities of some tricarboxylic acid-cycle-associated dehydrogenases and adenine-metabolizing enzymes in rat diaphragm muscle
Authors: Turner L. V. , Manchester K. L.
Published: August 2015

1. The activity of several tricarboxylic acid-cycle-associated dehydrogenases, adenine-metabolizing enzymes and glutathione reductase and the content of myoglobin were measured in rat diaphragm muscle after unilateral nerve section. 2. Consistent with morphological disintegration of the mitochondria there was a rapid diminution in activity of NAD- and NADP-linked isocitrate dehydrogenase, malate dehydrogenase and glutamate dehydrogenase. 3. Creatine phosphokinase and adenylate kinase, by contrast, showed little change in activity; adenylate deaminase and glutathione reductase activities increased during the hypertrophic phase. The concentration of myoglobin at first declined, then increased again. 4. The distribution of enzymes between the left and right hemidiaphragms was found not to be uniform. 5. Activities of adenine-metabolizing enzymes in the diaphragm were as great as in white muscle. It is suggested that their reputedly lower activities in red muscle properly refer to muscle containing a high proportion of intermediate fibres, which is not the case with diaphragm. 6. The possible causes of the transient hypertrophy after nerve section are discussed.
Metabolism of apigenin and related compounds in the rat. Metabolite formation in vivo and by the intestinal microflora in vitro
Authors: Griffiths L. A. , Smith G. E.
Published: August 2015

1. The metabolism of a group of flavonoid compounds related in structure to apigenin (4′,5,7-trihydroxyflavone) and including apigenin, apiin, naringin, phlorrhizin, acacetin, kaempferol, robinin, chrysin, tectochrysin and 4′,7-dihydroxyflavone, was studied both in vivo after oral administration to the rat, and in vitro in cultures of micro-organisms derived from the intestine of the rat. 2. The rat intestinal microflora is capable of effecting degradation of flavonoid compounds to metabolites observed in the urine after oral administration of the specific flavonoid. 3. All compounds possessing free 5- and 7-hydroxyl groups in the A ring and a free 4′-hydroxyl group in the B ring gave rise to ring-fission products, which included 4′-hydroxyphenylacyl derivatives. 4. On anaerobic incubation in a thioglycollate medium, intestinal micro-organisms can effect flavonoid-ring fission, cleavage of glycosidic bonds and the reduction of double bonds in the side chains of certain metabolites. 5. Two flavonoids (chrysin and tectochrysin) undergo hydroxylation in the 4′-position in vivo but not during incubation with the intestinal microflora in vitro. 6. Observations on the metabolism of other compounds substituted in the 4′-position, e.g. epiafzelechin, pelargonin and the isoflavones, genistein, biochanin A, daidzein and formononetin, by the intestinal microflora of the rat are also reported.
16-Unsaturated C19 3-oxo steroids as metabolic intermediates in boar testis
Authors: Brophy P. J. , Gower D. B.
Published: August 2015

1. The formation of the two 16-unsaturated alcohols 5α-androst-16-en-3α-ol and 5α-androst-16-en-3β-ol from [5α-3H]5α-androst-16-en-3-one has been demonstrated in boar testis homogenates. 2. The optimum yield (23%) of the 3α-alcohol was obtained in the presence of NADPH, whereas that for the 3β-alcohol (74%) was obtained when NADH was the added cofactor. 3. The two alcohols were not interconvertible. 4. Prolonged storage of boar testis tissue at −20°C abolished the ability to form all androst-16-enes except androsta-4,16-dien-3-one from [4-14C]progesterone. 5. The production of 5α-androst-16-en-3-one and the two alcohols from [7α-3H]androsta-4,16-dien-3-one only occurred when fresh tissue was used, whereas reduction of [5α-3H]5α-androst-16-en-3-one was unaffected by storage of testis at −20°C. 6. NADPH was the preferred cofactor for the reduction of androsta-4,16-dien-3-one. 7. The previously established conversion of androsta-5,16-dien-3β-ol into androsta-4,16-dien-3-one was shown to be reversible, NADH and NADPH being equally effective cofactors. 8. Pathways of biosynthesis of 5α-androst-16-en-3α- and 3β-ols, with the C19 3-oxo steroids as intermediates, are presented.
Phospholipid synthesis in HeLa cells exposed to immunoglobulin G and complement
Authors: Güttler Flemming
Published: August 2015

1. HeLa cells were cultured in the presence of heterologous immunoglobulin G and guinea-pig serum together with [32P]phosphate. 2. Incorporation of [32P]phosphate was significantly stimulated by anti-HeLa immunoglobulin G and complement-sufficient serum compared with immunoglobulin G from unimmunized rabbits and complement. Within 2.5h heat-inactivated guinea-pig serum and anti-HeLa immunoglobulin G stimulated [32P]phosphate incorporation to the same extent as heat-inactivated complement and immunoglobulin G from unimmunized rabbits. 3. Compared with cells exposed to immunoglobulin G from unimmunized rabbits together with complement, anti-HeLa immunoglobulin G with complement increased the phospholipid content of HeLa cells twofold within 5h of incubation. 4. Exposure of HeLa cells to anti-HeLa immunoglobulin G and complement for 5–22h resulted in a twofold increase in the net accumulation of [32P]phosphate in sphingomyelin and phosphatidylcholine and a 50% increase in the net accumulation of [32P]phosphate in phosphatidylethanolamine, compared with cultures exposed to immunoglobulin G from unimmunized rabbits and complement. 5. A transient accumulation of 32P-labelled lysophosphoglycerides in HeLa cells exposed to antibody and complement was detected, confirming previous findings (Güttler & Clausen, 1969b). 6. The stimulation of [32P]phosphate turnover occurred in cells filling up their cytoplasma with vacuoles. This supports the suggestion that the accumulation of phospholipid in these cells may be concerned with the synthesis and function of cytomembranes.
Comparison of mammalian mitochondrial ribosomal ribonucleic acid from different species
Authors: Mitra R. S. , Bartoov B. , Monahan J. , Freeman K. B.
Published: August 2015

Mitochondrial ribosomal RNA species from mouse L cells, rat liver, rat hepatoma, hamster BHK-21 cells and human KB cells were examined by electrophoresis on polyacrylamide–agarose gels and sedimentation in sucrose density gradients. The SE (electrophoretic mobility) and S values of mitochondrial rRNA of all species were highly dependent on temperature and ionic strength of the medium; the SE values increased and the S values decreased with an increase in temperature at a low ionic strength. At an ionic strength of 0.3 at 23–25°C or an ionic strength of 0.01 at 3–4°C the S and SE values were almost the same being about 16.2–18.0 and 12.3–13.6 for human and mouse mitochondrial rRNA. The molecular weights under these conditions were calculated to be 3.8×105–4.3×105 and 5.9×105–6.8×105, depending on the technique used. At 25°C in buffers of low ionic strength mouse mitochondrial rRNA species had a lower electrophoretic mobility than those of human and hamster. Under these conditions the smaller mitochondrial rRNA species of hamster had a lower electrophoretic mobility than that of human but the larger component had an identical mobility. Mouse and rat mitochondrial rRNA species had identical electrophoretic mobilities. Complex differences between human and mouse mitochondrial rRNA species were observed on sedimentation in sucrose density gradients under various conditions of temperature and ionic strength. Mouse L-cell mitochondrial rRNA was eluted after cytoplasmic rRNA on a column of methylated albumin–kieselguhr.
The formation of pyrrolid-2-one-5-carboxylic acid at the N-terminus of immunoglobulin G heavy chain
Authors: Stott D. I. , Munro A. J.
Published: August 2015

We propose that pyrrolid-2-one-5-carboxyl-tRNA is not involved in the initiation of protein synthesis in eukaryotic cells and that the N-terminal pyrrolid-2-one-5-carboxylic acid group of an IgG (immunoglobulin G) (that secreted by the mouse plasmacytoma Adj PC5) is formed by the enzymic cyclization of the N-terminal glutamine of the heavy chain of the completed IgG molecule and that the cyclization takes place inside the cell. We base these conclusions on the following evidence. (1) Pyrrolidonecarboxyl-tRNA was not found in incorporation experiments with rat liver preparations and [U-14C]-pyrrolidonecarboxylic acid, glutamic acid and glutamine, even though an incorporation extent of less than 2% of the total products could have been detected. (2) Double-labelling experiments showed that less than 8% of the nascent peptides of heavy chains (those obtained by precipitation by the antibody to Fc fragment) began with pyrrolidonecarboxylic acid. (3) Further double-labelling experiments showed that 60–66% of the heavy chains of the completed intracellular IgG molecule began with pyrrolidonecarboxylic acid after both 1 and 5h of labelling. (4) The IgG, after secretion by plasmacytoma Adj PC5, was found to have the sequence [unk]Glu- Val-Gln-Leu- at the N-termini of the heavy chains.
Inducible phosphoenolpyruvate-dependent hexose phosphotransferase activities in Escherichia coli
Authors: Kornberg H. L. , Reeves Richard E.
Published: August 2015

1. A method is described for measuring the rate of phosphoenolpyruvate-dependent phosphotransferase activity for a variety of hexoses in toluene-treated suspensions of Escherichia coli. 2. The specific activities of the phosphotransferases that catalyse the phosphorylation of hexoses are greatly affected by the carbon source for growth. 3. In all strains of E. coli tested, fructose phosphotransferase activity is induced by growth on fructose. 4. Strains of E. coli differ greatly in the rate at which they phosphorylate glucose, but all strains possess at least a low glucose phosphotransferase activity under any tested condition of growth. Glucose phosphotransferase activity is further induced by growth on glucose; this does not occur in a mutant that lacks the ability to take up methyl α-d-[14C]glucopyranoside and hence grows poorly on glucose. 5. When growing on fructose, two strains of E. coli synthesize the inducible glucose phosphotransferase system gratuitously, and to specific activities higher than observed during growth on glucose. A phosphotransferase catalysing the phosphorylation of mannose is similarly induced.
The chemistry of the collagen cross-links. A convenient synthesis of the reduction products of several collagen cross-links and cross-link precursors
Authors: Davis Norman R. , Bailey Allen J.
Published: August 2015

New synthetic routes to the reduction products of several collagen cross-links and cross-link precursors are described. By the use of these routes hydroxynorleucine, 5,6-dihydroxynorleucine, hydroxylysinonorleucine and hydroxylysinohydroxynorleucine can be synthesized via one common synthetic intermediate. The synthetic routes provide a convenient source of these unusual amino acids, as well as confirming the structure of hydroxylysinohydroxynorleucine and the other lysine-derived residues found in borohydride-reduced collagens.
A method for obtaining linear reciprocal plots with caeruloplasmin and its application in a study of the kinetic parameters of caeruloplasmin substrates
Authors: Young S. N. , Curzon G.
Published: August 2015

1. The curved plots of 1/v against 1/[S] obtained when caeruloplasmin oxidizes NN-dimethyl-p-phenylenediamine were investigated. The first free-radical oxidation product of caeruloplasmin oxidation of NN-dimethyl-p-phenylenediamine is required for curvature, as straight-line plots were obtained when activities were measured either before appreciable free-radical product had appeared or in the presence of ascorbate, which reduced it back to NN-dimethyl-p-phenylenediamine. 2. In the presence of ascorbate linear reciprocal-plots were obtained with all of the 37 substrates tested. Vmax. values varied over only an eightfold range and those for the 20 p-amino compounds over only a twofold range. Km values, however, varied over a 104-fold range. The small range of Vmax. values indicates that the rate-limiting step in caeruloplasmin action is relatively independent of the nature of the substrate. Km values suggest that substrates bind primarily by ring electrons, although certain side-chain groups increased the Km in a manner unrelated to likely changes of ring-electron densities. A mechanism involving repulsion between negative charges on the substrate and the enzyme was supported by the variation of the Km of 5-hydroxyindol-3-ylacetic acid with pH.
The activity of deoxyribonucleic acid polymerase in some species of algae
Authors: Schönherr O. Th. , Keir H. M.
Published: August 2015

1. The activities of DNA polymerase preparations from the algae Euglena gracilis, Chlamydomonas reinhardtii, Chlorella pyrenoidosa, Anabaena variabilis and Anacystis nidulans were measured. The blue–green algae Anabaena and Anacystis contain a 5–20-fold higher activity of the enzyme than do the green algae. DNA polymerases from the blue–green algae show a pH optimum of 9 and prefer a relatively low Mg2+concentration (1–3mm). DNA polymerases from the green algae, however, display a pH optimum between 7.5 and 8.5 and an optimum Mg2+concentration of 8mm. With all algae, a higher polymerase activity was obtained with denatured salmon sperm DNA as template than with native DNA. All four deoxyribonucleoside 5′-triphosphates must be present for full activity of the polymerases. 2. With one exception, the deoxyribonuclease activities in the preparations, measured under conditions of the DNA polymerase assay, are low compared with corresponding preparations from Escherichia coli. Chlamydomonas extracts contain a high deoxyribonuclease activity. 3. After purification on columns of DEAE-cellulose, the polymerase activity was linear over a wide range of protein concentrations, except for Chlamydomonas preparations, where the observed deviation from linearity was probably attributable to the high nuclease activity. 4. DNA polymerases from all these algae bind strongly to DNA–cellulose; 6–40-fold purifications of the enzyme were obtained by chromatography on columns of DNA–cellulose. 5. The partially purified polymerases of Euglena and Anacystis are heat-labile but become much more heat-stable when tested in the presence of DNA.
Protein synthesis in the liver of rats injected with cholesteryl 14-methylhexadecanoate
Authors: Komárková E. , Hradec J.
Published: August 2015

1. Rats were injected intraperitoneally with cholesteryl 14-methylhexadecanoate and killed after various intervals of time up to 3 days; ribosomes and cell sap were isolated from their liver tissue. These fractions were tested for their ability to participate in protein synthesis. 2. Protein synthesis in complete systems containing ribosomes, cell sap and all necessary cofactors was significantly enhanced at 12 and 72h after the injection and significantly inhibited at 24h. At early times after injection isolated ribosomes had a slightly enhanced ability to bind nRNA. Peptide-elongation processes (i.e. binding of aminoacyl-tRNA to ribosomes, peptidyl transfer and polyphenylalanine synthesis) showed significant stimulation or inhibition depending on the time after injection of the ester. 3. A correlation was found between the ability of cell sap to stimulate polyphenylalanine synthesis and the relative cholesteryl 14-methylhexadecanoate content in the postmicrosomal supernatant at different time-intervals after administration of the ester. No significant changes were found in its content in the whole liver tissue. 4. Since the injected ester has previously been shown to accumulate in some enzymic fractions, the changes in its relative content may represent a regulatory mechanism modulating the rate of protein synthesis.
Structures of bilirubin conjugates synthesized in vitro from bilirubin and uridine diphosphate glucuronic acid, uridine diphosphate glucose or uridine diphosphate xylose by preparations from rat liver
Authors: Fevery J. , Leroy P. , Van De Vijver M. , Heirwegh K. P. M.
Published: August 2015

1. In incubation mixtures containing digitonin-activated or untreated preparations from rat liver, albumin-solubilized bilirubin as the acceptor substrate and (a) UDP-glucuronic acid, (b) UDP-glucose or (c) UDP-xylose as the sugar donor, formation of the following ester glycosides was demonstrated: with (a), bilirubin β-d-monoglucuronoside, with (b), bilirubin β-d-monoglucoside and with (c), bilirubin monoxyloside or mixtures of the mono-and di-xyloside. 2. With UDP-glucuronic acid prolonged incubation and variation of the composition of the incubation mixtures yielded equimolar amounts of azodipyrrole (I) and azodipyrrole β-d-monoglucuronoside (II) after treatment of the incubation mixtures with the diazonium salt of ethyl anthranilate. The azo-derivatives were identified by t.l.c. by reference to known compounds and by the following chemical tests. After ammonolysis the conjugated azo-derivative (II) yielded d-glucuronic acid and the carboxylic acid amide of azodipyrrole, indicating transfer of a glucuronic acid residue to the carboxylic acid groups of bilirubin. The β-d-configuration of the sugar moiety and binding at C-1 were demonstrated by enzymic hydrolysis tests. 3. Analogous evidence established the structure of the reaction product obtained with UDP-glucose as the sugar donor, as bilirubin β-d-monoglucoside. 4. With UDP-xylose as the sugar donor xylosyl transfer to the carboxylic acid groups of bilirubin with attachment at C-1 was demonstrated in an analogous way. A β-d-configuration is considered very likely, but requires confirmation. 5. Monoxyloside formation was predominant at pH7.4, whereas at decreasing pH values increasing fractions of the substrate were converted into the dixyloside. Prolonged incubation, low concentrations of bilirubin and high concentrations of UDP-xylose favoured diconjugate formation. The available evidence supports the synthesis sequence: bilirubin → bilirubin monoxyloside → bilirubin dixyloside.
Biosynthesis of willardiine and isowillardiine in germinating pea seeds and seedlings
Authors: Ashworth T. S. , Brown E. G. , Roberts F. M.
Published: August 2015

The synthesis of the pyrimidinyl amino acids willardiine and isowillardiine was studied in vivo and in vitro. Uracil derivatives stimulate the biosynthesis of both compounds; the free base is the most effective. Significant incorporation of [2-14C]uracil and [6-14C]orotate into willardiine and isowillardiine was found. Incorporation of [6-14C]orotate was substantially decreased in the presence of uracil, and to a lesser extent by uridine and UMP. [3-14C]Serine was incorporated into the alanine side chain of the two uracilylalanines but not into the ring. The effect of a number of uracil analogues and inhibitors of pyrimidine metabolism was examined. Some were shown to stimulate the biosynthesis; the most noticeable effects were obtained with 6-azauracil and 2-thiouracil. Attempts to obtain extracts capable of synthesizing the uracilylalanines from uracil and serine were unsuccessful, but weak activity was observed when serine was replaced by O-acetylserine.