bluuebunny/crossref_metadata_2025 huggingface dataset:
The effect of oestradiol on the acid-soluble nucleotides of rat uterus
Authors: Oliver Janet M. , Kellie A. E.
Published: August 2015

1. Techniques have been developed to measure the concentrations of the ribonucleotides of the immature rat uterus in vivo. Tissue was frozen rapidly in liquid nitrogen, ground to a fine powder, dispersed in frozen perchloric acid and thawed slowly. Nucleotides were separated from other acid-soluble constituents on short columns of polyethyleneimine-cellulose and the mixture was resolved into individual nucleotides by two-dimensional thin-layer chromatography on polyethyl-eneimine-cellulose plates. 2. The nucleotides of immature rat uterus consisted of approximately 75% of ATP–ADP, 10–12% each of GTP–GDP and UTP–UDP and less than 2% of CTP. 3. Injection of oestradiol (5μg) promoted a linear decrease in the amounts of purine nucleotides to approximately 60% of control values in 4–5h, followed by a return to greater than control values in 8–10h. Concentrations of the pyrimidine nucleotides remained constant for 4–6h and then increased to 200% of control at 12h after hormone treatment.
The inhibition of N-acetyl-β-d-glucosaminase by the (1→4)- and (1→5)-lactones of N-acetylglucosaminic acid
Authors: Couling T. E. , Goodey R.
Published: August 2015

The inhibition of N-acetyl-β-d-glucosaminase activity by 2-acetamido-2-deoxy-d-gluconolactone was examined. Separation of the (1→4)- and (1→5)-lactones was achieved by using paper chromatography or countercurrent distribution and identification was obtained by examination of the relative stability of the components of separated material. Quantitative measurement of the two lactones was by kinetic titration or by a colorimetric method based on their reaction with hydroxylamine. It was shown that only the (1→5)-lactone acted as an inhibitor of N-acetyl-β-d-glucosaminase.
Competing addition and hydrolysis of the cytidylylcytidylyladenosine terminal residues of transfer ribonucleic acid isolated from the non-lactating bovine mammary gland
Authors: Herrington M. D. , Hawtrey A. O.
Published: August 2015

1. The enzyme fraction obtained from the pH5 enzyme of non-lactating bovine mammary gland between 40 and 100% ammonium sulphate saturation markedly inhibited the AMP-incorporating activity of rat liver nucleotide-incorporating enzyme. This inhibitory effect has been attributed to high nuclease activity which can be partially removed by adsorption of the enzyme fraction on to calcium phosphate gel. 2. The degradation action of the calcium phosphate-purified enzyme is confined mainly to the terminal trinucleotide sequence -pCpCpA of tRNA, its effect being analogous to that of venom phosphodiesterase. This enzyme is heat labile and very readily loses its degradative activity. 3. Treatment of the enzyme fraction with Macaloid results in complete removal of the phosphodiesterase, leaving an enzyme capable of incorporating AMP into tRNA. 4. Transfer RNA extracted from non-lactating bovine mammary gland in the presence of polyvinyl sulphate and Macaloid is able to accept amino acids with an efficiency 30% of that shown by lactating bovine mammary-gland tRNA isolated under identical conditions.
Bilirubin conjugates of human bile. The excretion of bilirubin as the acyl glycosides of aldobiouronic acid, pseudoaldobiouronic acid and hexuronosylhexuronic acid, with a branched-chain hexuronic acid as one of the components of hexuronosylhexuronide
Authors: Kuenzle Clive C.
Published: August 2015

Structure elucidations have been performed on the bilirubin conjugates isolated from human hepatic bile as the phenylazo derivatives. The major bilirubin conjugates are excreted, not as was formerly thought in the form of glucuronides, but as the acyl glycosides of aldobiouronic acid, pseudoaldobiouronic acid and hexuronosylhexuronic acid. The isolated aldobiouronides are proposed to have the structures of an acyl 6-O-hexopyranosyluronic acid-hexopyranoside, an acyl 4-O-hexofuranosyluronic acid-d-glucopyranoside, and an acyl 4-O-β-d-glucofuranosyluronic acid-d-glucopyranoside respectively, with the acyl radicals being those of the phenylazo derivative of bilirubin. The pseudoaldobiouronide is suggested to be the acyl 4-O-α-d-glucofuranosyl-β-d -glucopyranosiduronic acid, with the acyl radical being that of the phenylazo derivative of vinylneoxanthobilirubinic acid. The hexuronosylhexuronide presumably is the acyl 4-O-(3-C-hydroxymethylribofuranosyluronic acid)-β-d-glucopyranosiduronic acid, with the acyl radical being that of the phenylazo derivative of bilirubin. The 3-C-hydroxymethylriburonic acid, isolated as one of the components of the hexuronosylhexuronide, is the first natural branched-chain hexuronic acid to be detected, and the first branched-chain sugar ever detected in humans.
Effects of oestradiol-17β and progesterone on total and nuclear-protein synthesis in epithelial and stromal tissues of the mouse uterus, and of progesterone on the ability of these tissues to bind oestradiol-17β
Authors: Smith J. A. , Martin L. , King R. J. B. , Vértes M.
Published: August 2015

1. A method is described for separating uterine epithelium that is 80% pure and connective-tissue stroma that is 60% pure. This was used to study the effects of steroid hormones on total and nuclear-protein synthesis in these tissues. 2. Oestradiol-17β given alone produces mitoses in the epithelium but not in the stroma. It stimulated incorporation in vitro of [14C]lysine into total protein, histones and acidic nuclear proteins to a greater extent in epithelium than stroma. Incorporation into acidic nuclear proteins was most markedly stimulated, reaching four to six times the normal value 4h after treatment, and then declining rapidly. This peak was only seen in epithelial preparations. 3. After pretreatment with progesterone, oestradiol-17β has the reverse effect, producing mitoses only in stroma. Progesterone alone had no effect on the amounts or rates of incorporation of [14C]lysine into stromal nuclear proteins, but changes after oestradiol-17β treatment were similar to those seen in epithelium with oestradiol-17β alone. In the epithelium, progesterone alone depressed incorporation into histones and acidic nuclear proteins, but did not abolish the subsequent response to oestradiol-17β. With this treatment there was a rapid, large and transient increase in incorporation into epithelial total protein not seen with oestradiol-17β alone. 4. Progesterone had no qualitative effect on the distribution of specific oestrogen-binding proteins, as judged by sucrose-density-gradient centrifugation. However, progesterone treatment increased the uptake in vivo of [6,7-3H]oestradiol-17β by stroma, and it is possible that this is important although the differences were not apparent after labelling in vitro.
Non-enzymic nature of the pyridine haemochrome-cleaving activity of mammalian tissue extracts (‘haem α-methyl oxygenase’)
Authors: Colleran Emer , Carra P. Ó
Published: August 2015

1. The pyridine haemochrome-cleaving activity of extracts from mammalian liver and other tissues is shown conclusively to be entirely non-enzymic in nature and attributable to coupled oxidation with ascorbate. 2. Reduced glutathione probably contributes to the activity indirectly by continuously regenerating the ascorbate to the reduced form. 3. The cleavage shows no specificity for the α-methine bridge of pyridine haemochrome. 4. Results are presented suggesting some probable reasons for the erroneous characterization of the activity as an α-methine-specific haem-cleaving enzyme (`haem α-methenyl oxygenase') by Nakajima and co-workers (e.g. Nakajima, Takemura, Nakajima & Yamaoka, 1963; Nakajima & Gray, 1967).
Preparation and properties of creatine kinase from the breast muscle of normal and dystrophic chicken (Gallus domesticus)
Authors: Roy B. P. , Laws J. F. , Thomson A. R.
Published: August 2015

1. The purification of creatine kinase from normal and genetically dystrophic chicken breast muscle is described. Enzyme recovery was significantly lower from dystrophic muscle. 2. Both enzymes had the same number of reactive and total thiol groups and had similar specific activities and similar amino acid compositions. 3. No significant differences were observed in sedimentation, electrophoretic or kinetic properties. 4. Peptide `maps' showed no significant differences, and electrophoresis of partial acid hydrolysates of the labelled enzymes suggested that corresponding amino acid sequences around all the thiol groups were very similar. 5. The enzymes showed identical temperature stabilities. 6. No significant differences between the enzymes from normal and dystrophic muscle were observed.
The preparation and properties of yeast β-fructofuranosidase chemically attached to polystyrene
Authors: Filippusson H. , Hornby W. E.
Published: August 2015

1. A process is described for the chemical attachment of an enzyme to the surface of a polystyrene matrix. 2. By this process yeast β-fructofuranosidase was chemically attached to the surface of both polystyrene beads and polystyrene tubes. 3. The kinetics of sucrose hydrolysis by β-fructofuranosidase and polystyrene-supported β-fructofuranosidase were compared. 4. The pH–activity curve of the polystyrene-supported enzyme shows a marked difference from that of the free enzyme in solution. 5. The inhibitor dissociation constant with respect to tris is increased, whereas the inhibitor dissociation constant with respect to aniline is decreased when the enzyme is attached to polystyrene. 6. Differences between the properties of the bound and free enzyme are discussed in terms of a micro-environmental effect arising from the hydrophobic nature of the polystyrene support.
Carbonic anhydrase isoenzymes in the erythrocytes and uterus of the rabbit
Authors: McIntosh J. E. A.
Published: August 2015

1. Two forms of the zinc-containing enzyme carbonic anhydrase (EC 4.2.1.1) were isolated from rabbit erythrocytes and two forms from rabbit uterine tissue (endometrium) in the progestational stage of pregnancy (days 6–8 of gestation). Separation of the isoenzymes was achieved by ion-exchange chromatography, preparative polyacrylamide-gel electrophoresis and isoelectric focusing. A comparison was made of the general properties and kinetic behaviour of the purified isoenzymes. 2. Although indistinguishable in terms of molecular weight and zinc content the isoenzymes were very different as catalysts of the hydration of carbon dioxide. The two erythrocyte isoenzymes, found in almost equal amounts, differed more than 100-fold in specific activity. Of the two isoenzymes prepared from either endometrial or entire uterine homogenates one was kinetically indistinguishable from the erythrocyte high-activity form, whereas the other, also possessing high activity, was found only in the endometrial or uterine tissue. Present evidence suggests that the former isoenzyme originated from residual blood contaminating the tissue homogenates, and that a marked rise in the content of the latter isoenzyme accounts for the increase in rabbit endometrial carbonic anhydrase activity that previously has been observed in early pregnancy. 3. Minor forms of the erythrocyte isoenzymes, having a characteristic quantitative and electrophoretic relationship to one another, were occasionally produced during purification. 4. The actions were investigated of the inhibitors acetazolamide (5-acetamido-3,4-diazole-1-thia-2-sulphonamide), 1,1-dimethylaminonaphthalene-5-sulphonamide and ethoxyzolamide (6-ethoxybenzothiazole-2-sulphonamide) on the hydration of carbon dioxide and the hydrolysis of p-nitrophenyl acetate catalysed by the isoenzymes. 5. The low-activity erythrocyte isoenzyme was superior to the high-activity form as a catalyst of β-naphthyl acetate hydrolysis.
Carbon dioxide fixation in green sulphur bacteria
Authors: Sirevåg Reidun , Ormerod J. G.
Published: August 2015

1. About one-third of the CO2 fixed during photosynthesis by washed suspensions of Chlorobium thiosulfatophilum strain 8346 gave rise to α-oxoglutarate and branched-chain oxo acids, mainly β-methyl-α-oxovalerate. Another one-third to one-half gave rise to a polyglucose. 2. The fixation of CO2 was inhibited by fluoroacetate, increasing concentrations up to 1mm stimulating the accumulation of α-oxoglutarate and causing a decrease in the formation of the branched-chain oxo acids and polyglucose. 3. Acetate was converted into the same products as was CO2. 4. Fluoroacetate (1mm) had a negligible effect on the formation of polyglucose from acetate and caused a slight inhibition of the formation of the branched-chain oxo acids and increased accumulation of α-oxoglutarate. 5. Iodoacetate (1mm) strongly inhibited polyglucose formation from acetate and caused accumulation of pyruvate. The formation of the branched-chain oxo acids from acetate was only slightly affected by this inhibitor. 6. Pyruvate can be metabolized by this organism in the presence of a suitable electron donor whether CO2 is present or not. In the absence of CO2 pyruvate is converted into polyglucose. 7. The accumulation of oxo acids during CO2 fixation is completely inhibited by NH4+ ions. The formation of the branched-chain oxo acids is considerably decreased by the presence of isoleucine, leucine or valine, or a mixture of these. 8. CO2 fixation in two other strains of Chlorobium appears to exhibit a similar pattern to that in C. thiosulfatophilum strain 8346. 9. It is concluded that in washed suspensions, CO2 is fixed mainly by a mechanism involving the reductive carboxylic acid cycle. Acetate, the product of the cycle, is converted into polyglucose via pyruvate synthase and a reversal of glycolysis or into branched-chain oxo acids by an unknown mechanism.
Chemical properties of guinea-pig immunoglobulins γ2G and γM
Authors: Leslie R. G. Q. , Cohen S.
Published: August 2015

The isolation of guinea-pig immunoglobulins γ1G, γ2G and γM are described and methods for separating the polypeptide chains of each examined. The molecular weights, extinction coefficients and carbohydrate and amino acid compositions of the immunoglobulins and their constituent chains have been analysed. The findings provide a basis for further studies attempting to relate structural differences to distinct biological properties of guinea-pig immunoglobulins.
Isolation of some precursors of 2-methylpyrrole in the Elson–Morgan reaction
Authors: Serafini-Cessi Franca , Cessi C.
Published: August 2015

1-C-(1-Acetylacetonyl)-2-deoxy-2-(1-methyl-3-oxobut-1-enyl)amino -d-galactitol is obtained from the condensation of 2-amino-2-deoxy-d-galactose with pentane-2,4-dione in anhydrous solvent. On treatment with hot alkali it gives 2-methylpyrrole with 37% yield. By acid hydrolysis under mild conditions the compound loses the N substituent and from the resulting unstable derivative 2-methylpyrrole is obtained (52% yield). It is concluded that derivatives of aminohexoses substituted at C-1 with a dioxopentyl chain are the precursors of 2-methylpyrrole in the Cessi & Serafini-Cessi (1963) modification of the Elson–Morgan reaction. As demonstrated previously, products of condensation of aminohexoses with pentane-2,4-dione at the amino group are not converted directly into 2-methylpyrrole, but this step provides protection of the amino group during condensation at C-1.
Circular dichroism of bradykinin and related peptides
Authors: Brady A. H. , Ryan J. W. , Stewart J. M.
Published: August 2015

1. The circular dichroism of bradykinin and a number of its analogues and homologues was measured over the spectral range 200–300nm. All of the biologically active peptides showed maxima at 220nm and minima at 235nm. The spectra were independent of solvent and temperature. The vibronic transitions of phenylalanyl residues in the 250–280nm range showed no evidence of intra- or inter-molecular interactions. We take this as evidence that bradykinin and its biologically active analogues and homologues exist in solution as disordered chains. 2. None of the analogues with spectra unlike bradykinin possessed biological activity. However, peptides such as retro-bradykinin, des-6-serine-bradykinin, des-1-arginine-bradykinin and des-9-arginine-bradykinin produced spectra like that of bradykinin but were devoid of biological activity. Although we could not identify spectral features that were clearly correlated with biological activity, it appears unlikely that highly ordered peptides of the same amino acid composition as bradykinin would possess bradykinin-like effects.
A study of permeability of lysosomes to amino acids and small peptides
Authors: Lloyd J. B.
Published: August 2015

The latency of nitrocatechol sulphatase activity was measured in rat liver lysosomes before and after preincubation in 0.25m solutions of five amino acids, three dipeptides and a tripeptide. Rates of increase in ‘free’ activity were taken as an indication of rates of solute penetration into lysosomes and were correlated with the structure of each molecule studied. In general permeability was greater in solutions of pH7 than of pH5 or 6, and dipeptides entered more rapidly than amino acids or triglycine. The conclusions are in good agreement with those obtained by other methods.
Chromatographic separation of mononucleotides derived from transfer ribonucleic acids
Authors: Holness N. J. , Atfield G.
Published: August 2015

1. A method is described for fractionating alkaline and enzymic hydrolysates of ribonucleic acids and oligonucleotides on DEAE-cellulose columns by using a constant-composition eluent. 2. The relative partition coefficients of the four major and several minor nucleotides present in nucleic acid digests are given. 3. The influence of pH and of molarity of the buffer on the separation is described. 4. Relations for the quantitative determination of the major nucleotides from their absorption zone areas on the chromatographic record are derived. 5. Hydrolysis of oligonucleotides with T2 ribonuclease yields solely the nucleoside 3′-monophosphates, thereby simplifying the subsequent chromatographic separation. 6. Examples of the qualitative and quantitative analysis of oligonucleotides from enzymic hydrolysates of RNA are given.
Altered ribosomes after inhibition of Escherichia coli by rifampicin
Authors: Blundell M. R. , Wild D. G.
Published: August 2015

Addition of rifampicin to growing cells of Escherichia coli affected the ribosomes. The polyribosomes first decayed to 70S ribosomes. These later dissociated to particles distinct from ribosomal subunits. The altered ribosomes sedimented more slowly than the corresponding subunits and had lost some protein; their ribosomal RNA was intact, but they were more susceptible to degradation by ribonuclease than normal ribosomes. The addition of rifampicin to preparations of lysed cells caused no detectable changes in the ribosome fraction.
The metabolism of methoxyethylmercury salts
Authors: Daniel J. W. , Gage J. C. , Lefevre P. A.
Published: August 2015

The metabolism of methoxy[14C]ethylmercury chloride in the rat has been investigated. After a single subcutaneous dose a small proportion is excreted unchanged in urine and a larger amount in bile with some resorption from the gut. The greater part of the dose is rapidly broken down in the tissues with a half-time of about 1 day to yield ethylene and inorganic mercury. Ethylene is exhaled in the breath and the mercury migrates to the kidney and is excreted in urine. A small proportion of the dose appears as carbon dioxide in the breath and about 12% in urine as a mercury-free metabolite. It is possible that the breakdown of methoxyethylmercurychloride to ethylene and inorganic mercury is not catalysed by an enzyme system.
Effect of thyroid hormone on metabolic compartmentation in the developing rat brain
Authors: Patel A. J. , Balázs R.
Published: August 2015

1. The effects of treatment with thyroid hormone (tri-iodothyronine) and of neonatal thyroidectomy on the cerebral metabolism of [U-14C]leucine were investigated during the period of functional maturation of the rat brain extending from 9 to 25 days after birth. 2. Age-dependent changes in the labelling of brain constituents under normal conditions appear to depend on changes in the availability of blood-borne [14C]leucine resulting from differential rates of growth of body and brain; but developmental changes in the pool size of free leucine and in the rates of protein synthesis and oxidation of leucine are also involved. 3. Treatment with thyroid hormone had no significant effect on the conversion of leucine carbon into proteins and lipids; and the age-dependent changes in the concentration and specific radioactivity of leucine were similar to controls. On the other hand there was an acceleration in the conversion of leucine carbon into amino acids associated with the tricarboxylic acid cycle. These observations indicate that leucine oxidation was the process mainly affected. 4. The specific radioactivity of glutamine relative to that of glutamate was used as an index of metabolic compartmentation in brain tissue. Treatment with thyroid hormone advanced the development of metabolic compartmentation. 5. Neonatal thyroidectomy led to a marked decrease in the conversion of leucine carbon into proteins and lipids and to a significant increase in the amount of 14C combined in the amino acids associated with the tricarboxylic acid cycle. The age-dependent increase in the glutamate/glutamine specific-radioactivity ratio was strongly retarded. 6. The increased conversion of leucine carbon into cerebral amino acids applied to glutamate and aspartate, but not to glutamine and γ-aminobutyrate. This observation facilitated the understanding of the effects of thyroid deprivation on brain metabolism and provided new evidence for the allocation of morphological structures to the metabolic compartments in brain tissue. 7. In contrast with the marked effects of the thyroid state on metabolic compartmentation, it had relatively little effect on the developmental changes in the concentration of amino acids in the brain. 8. The rate of conversion of leucine carbon into the ‘cycle amino acids’ both under normal conditions and in thyroid deficiency indicated a special metabolic relationship between glutamate and aspartate on the one hand, and glutamine and γ-aminobutyrate on the other.
Studies on type II glycogenosis. Effects of cortisone derivatives on acid α-glucosidase
Authors: Bourne E. J. , Clarke K. , Pridham J. B. , Rowe J. J. M.
Published: August 2015

Cortisone causes a marked increase in the activity of liver acid α-glucosidase 2h after injection into male Wistar rats. Studies on rat liver tissue slices, isolated lysosomes and cultured skin fibroblasts have demonstrated similar elevations of acid α-glucosidase activity after incubation with cortisone. Cortisone-treated human liver tissue, obtained by needle biopsy, also shows an increase in acid α-glucosidase activity. Neutral α-glucosidase activity was not stimulated by cortisone in vivo or in liver slices.
Autolysis of Bacillus cereus cell walls and isolation of structural components
Authors: Hughes R. C.
Published: August 2015

Autolysis of Bacillus cereus N.R.R.L. 569 cell walls was accompanied by hydrolysis of the majority of the 4-O-β-N-acetylglucosaminyl-N-acetylmuramic acid linkages in mucopeptide, presumably by an endo-β-N-acetylglucosaminidase. Hydrolysis of the N-acetylmuramyl-l-alanine linkages by an amidase also occurred. Free d-alanine residues were detected in isolated cell walls and the proportion of these residues increased during autolysis, presumably due to d-alanine carboxypeptidase action. Fractionation and analysis of the products of autolysis confirmed these results. Among the products originating from mucopeptide were a disaccharide, N-acetylmuramyl-N-acetylglucosamine, and a tetrapeptide of sequence l-Ala-d-Glu-meso-Dap-d-Ala (Dap=diaminopimelate). A dimer fraction containing a d-Ala-meso-Dap cross-link was also isolated. Two polysaccharides were obtained from the products of autolysed cell walls and from walls made soluble by Chalaropsis B glycosidase. A neutral polysaccharide accounted for about 40% of the wall and contained N-acetylglucosamine, N-acetylgalactosamine and glucose. The neutral polysaccharide isolated from wall autolysates was attached to a part of the glycan moiety of mucopeptide. The molecular weight of the complex was approx. 28000. Stoicheiometric amounts of phosphorus were present, possibly in linkages between the polysaccharide and mucopeptide moieties. The second polysaccharide accounted for 12% of the wall and was very acidic. After acidic hydrolysis of the polysaccharide, glucosamine, galactosamine and unidentified acidic substances were detected. The acid polysaccharide isolated from wall autolysates contained only traces of mucopeptide constituents and no phosphorus.
Identification in skeletal muscle of a distinct extracellular pool of amino acids, and its role in protein synthesis
Authors: Hider R. C. , Fern E. B. , London D. R.
Published: August 2015

1. The kinetics of radioactive labelling of extra- and intra-cellular amino acid pools and protein of the extensor digitorum longus muscle were studied after incubations with radioactive amino acids in vitro. 2. The results indicated that an extracellular pool could be defined, the contents of which were different from those of the incubation medium. 3. It was concluded that amino acids from the extracellular pool, as defined in this study, were incorporated directly into protein.
Myocardial aminoacyl-transfer-ribonucleic acid synthetase and aminoacyl-transferring enzyme activity
Authors: Gibson K. , Harris P.
Published: August 2015

The properties of cytoplasmic aminoacyl-tRNA synthetase and aminoacyl-transferring enzymes in the myocardium were examined and methods for the assay of the activity of these enzyme systems were developed. Aminoacyl-tRNA synthetase activity was measured from the rate of incorporation of 14C-labelled amino acid into aminoacyl-tRNA. Transferase activity was measured from the rate of incorporation of amino[14C]acyl-tRNA into protein in the presence of a standard preparation of hepatic ribosomes. Aminoacyl-tRNA synthetase activity is labile once the heart has been homogenized, whereas transferase activity is stable. The source of energy for synthetase activity is ATP; that for transferase is GTP. Transferase activity was inhibited by puromycin and stimulated by dithiothreitol, whereas synthetase activity was unaffected.
Incorporation of label from [1-14C]ethanol into the glutamate–glutamine pools of rat brain in vivo
Authors: Mushahwar I K , Koeppe R. E.
Published: August 2015

Glutamate and glutamine were isolated from the brains of rats given [1-14C]ethanol by three different routes of injection with and without carrier ethanol or acetate. Without exception, the specific radioactivity of brain glutamine was 3- to 5-fold that of brain glutamate. The route of injection had no consistent effect on the results. These results indicate that small amounts of ethanol are oxidized by rat brain in the compartment responsible for the so-called ‘small metabolic pool’ of brain glutamate.
Distribution of free and antibody-bound peptide hormones in two-phase aqueous polymer systems
Authors: Desbuquois B. , Aurbach G. D.
Published: August 2015

Peptide hormones labelled with radioactive iodine were partitioned into the aqueous two-phase polymer systems developed by Albertsson (1960) and the conditions required for separation of free from antibody-bound hormone have been worked out. Hormones studied included insulin, growth hormone, parathyroid hormone and [arginine]-vasopressin. Free and antibody-bound hormones show different distribution coefficients in a number of systems tested; two systems, the dextran–polyethylene glycol and dextran sulphate–polyethylene glycol system, give optimum separation. Free hormones distribute readily into the upper phase of these systems, whereas hormone–antibody complexes, as well as uncombined antibody, are found almost completely in the lower phase. Various factors including the polymer concentration, the ionic composition of the system, the nature of the hormone and the nature of added serum protein differentially affect the distribution coefficients for free and antibody-bound hormone. These factors can be adequately controlled so as to improve separation. The two-phase partition method has been successfully applied to measure binding of labelled hormone to antibody under standard radioimmunoassay conditions. It exhibits several advantages over the method of equilibration dialysis and can be applied to the study of non-immunological interactions.
Early biochemical consequences of denervation in fast and slow skeletal muscles and their relationship to neural control over muscle differentiation
Authors: Margreth A. , Salviati G. , Di Mauro S. , Turati G.
Published: August 2015

1. One week after denervation several biochemical characteristics of the fast extensor digitorum longus and slow soleus muscles from adult rats were investigated and compared with the characteristics of the corresponding unoperated contralateral muscles. 2. After these short periods of denervation-induced atrophy, the isolated myosins showed unchanged ATPase (adenosine triphosphatase) activities, but there was the expected difference between fast and slow muscle. 3. The specific activities of several soluble enzymes and their characteristic patterns were found to be only slightly modified in both the extensor and soleus muscles after denervation, as were most of the activities measured in the isolated mitochondria. 4. The most significant modifications were in the isolated sarcoplasmic reticulum, and appeared to be specific to either slow or fast muscle. 5. Denervation of slow muscle led to a marked increase of Ca2+-transport rates, and of the specific activity of the Mg2+-activated K+-modulated Ca2+-stimulated ATPase, together with changes in the polyacrylamide-electrophoretic profiles of the microsomal membrane protein. Transformation of these several properties of slow muscle sarcoplasmic reticulum to those of fast muscle sarcoplasmic reticulum was further substantiated by electron-microscopic analysis after negative staining. Control experiments with tenotomized soleus muscle gave negative results. 6. The isolated sarcoplasmic reticulum from fast muscle showed a slight diminution of ATPase-linked Ca2+-transport activity and a selective increase of rotenone-insensitive NADH-cytochrome c reductase activity, in addition to a greater emphasis on slow-type electrophoretic components of the structural membrane protein. 7. The significance of these results in relation to specific differentiating influences from motor nerves is discussed.
Effect of cortisol on the thiol content of rat liver nuclear proteins
Authors: Doenecke D. , Beato M. , Congote L. F. , Sekeris C. E.
Published: August 2015

Administration of cortisol to normal or adrenalectomized rats leads within 15–30min to an increased thiol content of nuclear proteins, measured by the incorporation of iodo[3H]-acetate or N-[14C]ethylmaleimide or by colorimetric methods. The same effect is observed after incubation of isolated rat liver nuclei with corticosteroids. The increased thiol content of the nuclear proteins shows the same time-dependence as the stimulation of RNA synthesis by corticosteroids observed in vivo and in vitro. Amino acid analysis of the carboxymethylated proteins reveals that in the experiments in vivo most of the label is present as carboxymethylcysteine with small amounts of carboxymethyl-lysine and carboxymethylhistidine, whereas in the experiments in vitro more carboxymethyl-lysine and carboxymethylhistidine than carboxymethylcysteine are found. The increase in the content of thiol groups is due to cleavage of the disulphide bridges between the nuclear proteins. Polyacrylamide-gel electrophoresis of the acid-soluble fraction reveals that most of the iodo[3H]acetate label is incorporated into a non-histone fraction with a molecular weight of approx. 45000 whereas in the acid-insoluble fractions many protein bands are labelled.
Dopamine β-hydroxylase of bovine adrenal medullae. A rapid purification procedure
Authors: Foldes A. , Jeffrey P. L. , Preston B. N. , Austin L.
Published: August 2015

1. A rapid purification procedure for dopamine β-hydroxylase from bovine adrenal-medulla chromaffin granules is presented. The homogeneity of the purified enzyme was demonstrated by means of three independent criteria. The specific activity of the enzyme compares favourably with that obtained by more involved procedures. 2. The stability of the enzyme was investigated and storage in polypropylene tubes was found preferable to storage in glass. 3. The soluble and particulate forms of dopamine β-hydroxylase appear to be identical, since membrane-bound and membrane-enclosed forms of the enzyme exhibit similar properties as regards size, charge and amino acid composition. 4. Ca2+ was found to stimulate the release of dopamine β-hydroxylase from bovine chromaffin granules in vitro. 5. An endogenous inhibitor of the enzyme was found in the chromaffin granules. This inhibitor was not inactivated either by heating at 100°C or by pretreatment with p-chloromercuribenzoate or Cu2+ ions.
Automatic monitoring of biochemical parameters in tissue culture. Studies on synchronously growing mouse myeloma cells
Authors: Cowan N. J. , Milstein C.
Published: August 2015

An instrument is described that will maintain a population of mammalian cells at constant cell density while automatically monitoring the growth rate of the culture and the extent of precursor incorporation into a variety of cell products. The apparatus was used in an investigation of cyclic changes in the incorporation of labelled precursors into the DNA, RNA, total protein and myeloma protein synthesized in synchronous cultures of a mouse myeloma line. The incorporation of [3H]uridine into trichloroacetic acid-insoluble material reveals a slight periodicity, with maxima and minima corresponding to late S phase and the mitotic phases respectively. The incorporation of [3H]lysine into total intracellular protein also shows a slight oscillation, with maxima and minima occurring during the respective G2 and mitotic phases. Cyclical changes in the synthesis of serologically precipitable myeloma protein were found to vary somewhat according to the conditions used to synchronize the cells. In experiments conducted with 4.0mm-thymidine, maximal incorporation of label took place during S phase or early G2 phase. Experiments with 1.0mm-thymidine revealed a significantly less marked periodicity of myeloma protein synthesis.
A possible role for 5-phosphoribosyl 1-pyrophosphate in the stimulation of uterine purine nucleotide synthesis in response to oestradiol-17β
Authors: Oliver Janet M.
Published: August 2015

1. It has been reported that the rate of purine nucleotide synthesis de novo in the immature rat uterus is doubled at 6h after administration of oestradiol-17β. The present work confirms an increased incorporation of glycine and adenine into uterine nucleotides between 2 and 6h after hormone treatment and investigates the mechanism of this response. 2. Activation of regulatory enzymes is unlikely to promote increased nucleotide synthesis: the activities of 5-phosphoribosyl 1-pyrophosphate amidotransferase (EC 2.4.2.14) and adenine phosphoribosyltransferase (EC 2.4.2.7) are the same in uterine extracts from control and oestrogen-treated rats. 3. Therefore it was proposed that oestradiol might promote an increased supply of a rate-limiting substrate. The low oestrogen-sensitive rate of AMP synthesis from adenine and endogenous 5-phosphoribosyl 1-pyrophosphate in the intact uterus compared with the high, oestrogen-insensitive rate in uterine extracts supplemented with 5-phosphoribosyl 1-pyrophosphate is evidence that the supply of 5-phosphoribosyl 1-pyrophosphate limits purine nucleotide formation and may increase after hormone treatment. This proposal is supported by the decrease in AMP synthesis in the whole tissue in the presence of guanine and 7-amino-3-(β-d-ribofuranosyl)pyrazolo[3,4-d]pyrimidine (formycin). These compounds do not inhibit adenine uptake or adenine phosphoribosyltransferase activity, but they both decrease the availability of 5-phosphoribosyl 1-pyrophosphate, the former by promoting its utilization by hypoxanthine/guanine phosphoribosyltransferase (EC 2.4.2.8) and the latter by inhibiting its synthesis from ribose 5-phosphate and ATP by ribose 5-phosphate pyrophosphokinase (EC 2.7.6.1). 4. It is unlikely that the increased availability of 5-phosphoribosyl 1-pyrophosphate results from hormonal stimulation of ribose 5-phosphate formation. Methylene Blue and phenazine methosulphate both increase ribose 5-phosphate without altering the supply of 5-phosphoribosyl 1-pyrophosphate. 5. The activity of ribose 5-phosphate pyrophosphokinase is low in uterine extracts and increases rapidly in response to oestradiol. Therefore the hormonal activation of the routes of purine nucleotide synthesis both de novo and from preformed precursors may be due, at least in part, to an increased availability of the common rate-limiting substrate 5-phosphoribosyl 1-pyrophosphate, mediated by activation of ribose 5-phosphate pyrophosphokinase.
Lipoprotein particles from the Golgi apparatus of guinea-pig liver
Authors: Chapman M. John , Mills Gervase L. , Taylaur C. E.
Published: August 2015

1. A cell fraction has been isolated from guinea-pig liver and shown to be rich in Golgi apparatus by electron microscopy. The activity of UDP-d-galactose-N-acetylglucosamine galactosyltransferase was over 100-fold greater in this cell fraction than in the liver homogenate. These data support the conclusion that the fraction was enriched in Golgi apparatus. 2. The Golgi cisternae and secretory vesicles contained electron-dense particles of 10–80nm diameter. Disruption of the Golgi apparatus cell fraction released these particles, which were separated into VLD (very-low-density) and LD (low-density) species on the basis of their density. 3. The Golgi VLD particles possessed morphological, flotational, chemical and immunochemical properties which closely resembled those of the serum VLD lipoproteins from the same animals. 4. The Golgi LD particles were rich in phospholipid, containing 48.1% by weight. The chemical composition of these particles was quite distinct from that of the serum LD lipoproteins, but did, however, show some similarity to that of the serum VLD lipoproteins. A marked resemblance was noted in the chemical characteristics of the Golgi LD and VLD particles (with the exception of triglyceride content). In addition, these two species of Golgi particles possessed the same antigenic determinant. 5. The results suggest that the Golgi VLD particles are the precursors of the serum VLD lipoproteins. On the basis of similarities in gross chemical composition and in the antigenic determinant of the Golgi LD and VLD particles, we conclude that the LD particles are probably the precursors of the VLD particles. In view of the marked differences in gross chemical composition of the Golgi LD particles and serum LD lipoproteins, it appears unlikely that the LD particles are directly secreted into the serum pool.
Effects of denervation on the glycogen content and on the activities of enzymes of glucose and glycogen metabolism in rat diaphragm muscle
Authors: Turner L. V. , Manchester K. L.
Published: August 2015

1. Changes in the content and concentration of glycogen and in the activity of a number of enzymes involved in glucose and glycogen metabolism were studied in the rat hemidiaphragm after unilateral denervation. 2. After nerve section the tissue hypertrophies; this hypertrophy is said to be confined to the smaller red fibres and not to the white. 3. The total hexokinase activity increases, whereas that of total glycogen phosphorylase decreases. The specific activity of phosphorylase a, determined after Halothane anaesthesia, remains fairly constant. 4. In fed animals the denervated tissue stores less glycogen, but in the early stages its glycogen content does not fall on starvation. 5. The effect of denervation on the specific activities of several other characteristically white-fibre enzymes are not consistent with the response of glycogen phosphorylase; the increase in content of glyceraldehyde 3-phosphate dehydrogenase and lactate dehydrogenase is thought to be related to proliferation of the sarcoplasmic reticulum. 6. The ratio of lactate dehydrogenase M/H subunits increases at the height of the hypertrophy, but then declines as the mass of the tissue falls. 7. The chronology of these changes in enzyme activities suggests a multiplicity of distinct responses after nerve section not consistent with any one model, either specific fibre development or reversion to de-differentiated, foetal-type metabolism.
Uptake and release of glutamate in cerebral-cortex slices from the rat
Authors: Okamoto K. , Quastel J. H.
Published: August 2015

1. Cerebral-cortex slices from rat brain, loaded with labelled l-glutamate as a result of aerobic incubation with labelled glucose, lost less than 15% of this glutamate on subsequent incubation in the presence of unlabelled glucose and l-glutamate. This indicates that very little exchange occurs between extracellular l-glutamate and glutamate accumulated in the neurons as a result of glucose metabolism. 2. Slices, loaded with labelled l-glutamate as a result of aerobic incubation in a medium containing unlabelled glucose and labelled l-glutamate, lost more than half of this glutamate on subsequent incubation in the presence of unlabelled l-glutamate. This indicates that exchange occurs between extracellular glutamate and glutamate accumulated in brain slices as a result of its uptake from the incubation medium. 3. Evidence was obtained suggesting that only a part of the glutamate, accumulated in brain slices as a result of its uptake from an incubation medium containing both glucose and l-glutamate, entered the neurons; apparently almost all the rest entered the glia. 4. It is concluded that the slices contain a pool of glutamate, derived from glucose and located in the neurons, which is poorly exchangeable with extracellular glutamate, and another pool of glutamate, derived from extracellular glutamate and located in the glia, which is freely exchangeable with extracellular glutamate.
Glycine formation during growth of Pseudomonas AM1 on methanol and succinate
Authors: Salem A. R. , Large P. J. , Quayle J. R.
Published: August 2015

1. The mechanism of regeneration of glycine during the growth of Pseudomonas AM1 on C1 compounds has been investigated by brief incubation of bacterial suspensions with [2,3-14C2]succinate and observing the incorporation of radioactivity into various metabolites. 2. With the wild-type organism growing on methanol, radioactivity appeared rapidly in glycine and tricarboxylic acid-cycle intermediates, but there was a relatively slow labelling of serine and phosphorylated compounds. Serine became labelled predominantly in the C-2 position. 3. The proportion of radioactivity incorporated into glycine at earliest times was greatly diminished when succinate-grown cells were used. 4. Radioactivity was also incorporated from [2,3-14C2]succinate into glycine and serine by methanol-grown mutant 20S, which lacks phosphoserine phosphohydrolase. Both the glycine and serine were labelled mainly in C-2. 5. The formation of predominantly [2-14C]serine from [2,3-14C2]succinate in wild-type Pseudomonas AM1, and of [2-14C]serine and [2-14C]glycine in the mutant lacking the phosphorylated pathway from succinate to serine, is taken as strong evidence for a mechanism of glycine regeneration involving cleavage of a C4 skeleton between C-2 and C-3, rather than by a direct combination of two C1 units derived from the growth substrate. 6. The cleavage mechanism is quantitatively more significant during growth on methanol than on succinate.
The conformation of the mucopolysaccharides. Hyaluronates
Authors: Atkins E. D. T. , Phelps C. F. , Sheehan J. K.
Published: August 2015

X-ray-diffraction patterns of hyaluronate fibres from a variety of sources were obtained. Sodium hyaluronate gives well-defined patterns which index on a hexagonal unit cell with dimensions a=1.17±nm and a fibre repeat-distance of 2.85±0.03nm. A further form of sodium hyaluronate is produced by annealing at 60°C in 75% relative humidity. This stable state indexes on a hexagonal unit cell of unchanged fibre repeat-distance but with a=1.87nm. The chain conformation is a threefold helix. Analysis of these diffraction patterns led to two tentative structures for sodium hyaluronate, involving different packing of the polysaccharide chains. The significance of side-chain interaction is discussed. Hyaluronic acid produces an X-ray pattern different from that obtained with the sodium salt. The fibre repeat-distance is 1.96±0.02nm and the unit cell appears to be monoclinic. The chain conformation is a twofold helix and conformational change between free acid and monovalent salt is discussed. These findings, together with model-building experiments, are interpreted as indicating a highly ordered structure, and the physical properties of hyaluronate solutions with regard to molecular shape and polyelectrolyte behaviour are rationalized.
Biliary excretion of amphetamine and methamphetamine in the rat
Authors: Caldwell J. , Dring L. G. , Williams R. T.
Published: August 2015

1.14C-labelled amphetamine and methamphetamine were injected into rats cannulated at the bile duct under thiopentone anaesthesia and the output of their metabolites in urine and bile was determined. 2. With amphetamine, 69% of the14C was excreted in the urine and 16% in the bile in 24h. The main metabolite in bile was the glucuronide of 4-hydroxyamphetamine. The output of unchanged amphetamine was much greater in cannulated rats than in intact rats. 3. With methamphetamine, 54% of the14C appeared in the urine and 18% in the bile. The main metabolite in the bile was the glucuronide of 4-hydroxynorephedrine. The output of amphetamine, a metabolite of methamphetamine, was much greater in cannulated rats than in intact rats. 4. Evidence has been obtained for the enterohepatic circulation of certain amphetamine and methamphetamine metabolites in the rat. 5. Thiopentone anaesthesia appeared to inhibit the ring hydroxylation of amphetamine administered as such or formed as a metabolite of methamphetamine.
Determination of adenosine 3′:5′-cyclic monophosphate in cerebral tissues by saturation analysis. Assessment of a method using a binding protein from ox muscle
Authors: Weller Malcolm , Rodnight Richard , Carrera Domenico
Published: August 2015

1. The Gilman (1970) procedure for determining cyclic AMP (adenosine 3′:5′-cyclic monophosphate) by saturation analysis gave erroneous results when applied to the analysis of extracts of whole brain or preparations of membrane fragments from brain. 2. The extracts contained a non-diffusible factor, which enhanced the binding of cyclic AMP by the muscle protein fraction. 3. Extracts also contained material which inhibited binding, but net inhibition of binding was only observed when relatively concentrated extracts were analysed. 4. The error introduced by the factors modifying binding could be eliminated by incorporation of unlabelled internal standards in the unknowns. The design adopted enables a statistical estimate to be made of the standard error of a single assay. 5. The modified assay was used to determine bound cyclic AMP and adenylate cyclase activity in cerebral membrane fragments. Five preparations of synaptic membrane fragments contained less than 3.5pmol of cyclic AMP/mg of protein; a microsomal fraction from rat contained 65pmol of cyclic AMP/mg of protein.