bluuebunny/crossref_metadata_2025 huggingface dataset:
The distribution of sulphate residues in the chondroitin sulphate chain
Authors: Wasteson Åke , Lindahl Ulf
Published: August 2015

1. Electrophoresis of chondroitin sulphate, before and after partial degradation with testicular hyaluronidase, revealed charge heterogeneity of the degraded but not of the intact polymer. 2. Hyaluronidase-treated chondroitin sulphate was fractionated by gel chromatography. Two subfractions which were essentially monodisperse with regard to molecular weight (values of 8600 and 4800, respectively) were separated further by chromatography on Dowex 1. The resulting subfractions differed considerably with respect to their sulphate/disaccharide molar ratios. 3. Amino acid and neutral-sugar analyses of the Dowex 1 subfractions showed that the less sulphated fragments contained the carbohydrate–protein linkage region, whereas the high-sulphated fragments essentially lacked this constituent. It was concluded that chondroitin sulphate contains relatively less sulphate in the vicinity of the carbohydrate–protein linkage region than in the more peripheral portion of the polysaccharide chain.
Developmental aspects of uridine diphosphate galactose 4-epimerase in rat intestine
Authors: Weinstein A , Koldovský O , Segal S
Published: August 2015

Kinetic and developmental characteristics of rat intestinal UDP-galactose 4-epimerase activity have been examined. The enzyme in the adult rat had a Vmax. value 2–3 times higher than that of the newborn animal, but the Km values for the enzyme in the newborn and adult rat were the same (0.17mm). No differences in epimerase activity were found along the length of the jejuno-ileum of adult animals, but higher activity was detected in the lower portion of the villi and crypts. The specific activity of the enzyme in the newborn rat began to rise at about 17 days of age, reaching a peak at 29 days of age, and then became constant at adult values. Total epimerase activity in the newborn rat liver was 2–5 times higher than the total activity in the intestine, and total epimerase activity in the adult intestine was 3–4 times higher than the total activity in the liver. Cortisone injection did not enhance the increase of epimerase normally seen during development, but caused a decrease in activity of this enzyme in the jejunum in rats up to 17 days of age. After 17 days, cortisone treatment had no effect on epimerase activity.
The metabolism of glucose 6-phosphate by mammalian cerebral cortex in vitro
Authors: Dodd P. R. , Bradford H. F. , Chain E. B.
Published: August 2015

1. The metabolism of glucose 6-phosphate in rat cerebral-cortex slices in vitro was compared with that of glucose. It was found that a glucose 6-phosphate concentration of 25mm was required to achieve maximal oxygen uptake rates and ATP concentrations, whereas only 2mm-glucose was required. 2. When 25mm-[U-14C]glucose 6-phosphate was used as substrate, the pattern of labelling of metabolites was found to be quantitatively and qualitatively similar to the pattern found with 10mm-[U-14C]glucose, except that incorporation into [14C]lactate was decreased, and significant amounts of [14C]glucose and [14C]mannose phosphate and [14C]fructose phosphate were formed. 3. Unlabelled glucose (10mm) caused a tenfold decrease in the incorporation of 25mm-[U-14C]glucose 6-phosphate into all metabolites except [14C]glucose and [14C]mannose phosphate and [14C]fructose phosphate. In contrast, unlabelled glucose 6-phosphate (25mm) had no effect on the metabolism of 10mm-[U-14C]glucose other than to increase markedly the incorporation into, and amount of, [14C]lactate, the specific radioactivity of this compound remaining approximately the same. 4. The effect of glucose 6-phosphate in increasing lactate formation from glucose was found to occur also with a number of other phosphate esters and with inorganic phosphate. Further investigation indicated that the effect was probably due to binding of medium calcium by the phosphate moiety, thereby de-inhibiting glucose uptake. 5. Incubations carried out in a high-phosphate high-potassium medium gave a pattern of metabolism similar to that found when slices were subjected to depolarizing conditions. Tris-buffered medium gave similar results to bicarbonate-buffered saline, except that it allowed much less lactate formation from glucose. 6. Part of the glucose formed from glucose 6-phosphate was extracellular and was produced at a rate of 12μmol/h per g of tissue in Krebs tris medium when glycolysis was blocked. The amount formed was much less when 25mm-Pi or 26mm-HCO3- was present, the latter being in the absence of tris. 7. Glucose 6-phosphate also gave rise to an intracellular glucose pool, whereas no intracellular glucose was detectable when glucose was the substrate.
Effect of a modified separation procedure on the size and protein-synthetic activity of membrane-bound liver polyribosomes
Authors: Ragnotti G
Published: August 2015

1. Various subcellular fractions containing ribosomes were isolated from rat liver. 2. In the presence of [14C]leucine and Sephadex-treated cell sap the radioactivity incorporated into the synthesized protein resulting from the incubation of microsomal preparations or deoxycholate-treated polyribosomes was dependent on the amount of rRNA incubated. In contrast, when Sephadex-treated post-mitochondrial supernatant was incubated, the radioactivity incorporated into the synthesized protein was independent of the amount of rRNA incubated. 3. Microsomal preparations and membrane-bound ribosomes, prepared by the standard procedure, incorporated less [14C]leucine into protein, per mg of rRNA incubated, than free or deoxycholate-treated polyribosomes; accordingly, polyribosomes associated with the former fractions were found mainly as monomers. 4. If microsomal fractions or membrane-bound ribosomes were prepared by a simple modification of the standard procedure, i.e. by centrifugation on to a ‘cushion’ of 2m-sucrose, their protein-synthesizing activity was of the same order as that of the original post-mitochondrial supernatant, and membrane-free and deoxycholate-treated polyribosomes; in this case polyribosome profiles showed that very little degradation had occurred and compared well with those obtained for post-mitochondrial supernatant and isolated polyribosomes. 5. A method is described (Appendix) that provides a rapid and reliable assessment of the concentration of rRNA in subcellular fractions.
The effect of glycols on the renaturation of soluble collagen
Authors: Cooper D. R. , Russell A. E. , Hart G. J.
Published: August 2015

The effects of a number of related glycols and substituted glycols on the renaturation kinetics of acid-soluble calf-skin collagen have been investigated. Optical rotation recovery was monitored at a fixed temperature in the presence of perturbants and the initial rates of reaction were determined. The effects of perturbants on stability of the native protein are compared with their action in the renaturing systems. The relationship between initial recovery rates and fixed-time [α]-values is shown to be dependent upon the renaturation temperature. The influence of perturbant concentration on recovery rates is discussed in terms of present theories of the mechanism of collagen renaturation.
Effect of α-amanitin on ribonucleic acid polymerase II of rat brain nuclei and on retention of avoidance conditioning
Authors: Montanaro N. , Novello F. , Stirpe F.
Published: August 2015

1. α-Amanitin inhibits in vitro the activity of RNA polymerase II of rat brain nuclei. 2. The toxin does not pass the blood–brain barrier, but when injected intracerebrally is highly toxic for rats, and causes inhibition of RNA polymerase II of isolated brain nuclei. 3. Intracerebral injection of α-amanitin 6h before training to a passive avoidance task is followed by impaired performance of rats on retesting after 7 days, without affecting performance on retesting immediately after training.
The isolation and functional identification of a protein from the human erythrocyte ‘ghost’
Authors: Tanner M. J. A. , Gray W. R.
Published: August 2015

A protein, initially identified as a band on polyacrylamide-gel electrophoresis of erythrocyte ‘ghosts’, was isolated by selective extraction of ‘ghosts’ with EDTA solutions. The molecular weight of the polypeptide chain was estimated as 33000 and it represents approx. 5% of the membrane protein. The N-terminal sequence of the protein was established. Comparison with known protein sequences suggested that the protein might be the erythrocyte d-glyceraldehyde 3-phosphate dehydrogenase. This identification was confirmed by direct enzyme assay. It is suggested that this enzyme, which is strongly retained by erythrocyte ‘ghosts’ on haemolysis of erythrocytes, is unlikely to be an integral part of the structure of the erythrocyte membrane.
The mechanism of wall synthesis in bacteria. The organization of enzymes and isoprenoid phosphates in the membrane
Authors: Anderson R. G. , Hussey Helen , Baddiley J.
Published: August 2015

1. The synthesis of peptidoglycan and teichoic acids by cell-free preparations from Bacillus licheniformis A.T.C.C. 9945 and Bacillus subtilis N.C.T.C. 3610 has been studied under a variety of conditions. 2. It was shown that poly(glycerol phosphate) is synthesized through a lipid intermediate, and it is concluded from this and other work that all major bacterial wall polymers are formed in a similar manner through such intermediates. 3. Close interrelation between the synthesis of peptidoglycan and teichoic acids was demonstrated, and inhibition studies confirm that the polyprenol phosphate molecules participating in the synthesis of peptidoglycan are shared with the systems that synthesize teichoic acids. 4. Nucleotides for the synthesis of one polymer are inhibitory towards synthesis of the other, and these effects can be enhanced or diminished by preincubation of the enzyme system with appropriate nucleotide precursors. 5. It is concluded that the return of undecaprenol phosphate to a common pool occurs only after the completion of polymer chains, and not after each cycle in the attachment of individual repeating units. This and other observations support a model for bacterial wall synthesis in which the multi-enzyme systems for each polymer are closely aligned in the membrane, with a molecule of undecaprenol phosphate located between them in a manner that enables it to be shared. The general mechanisms of wall synthesis and its control are discussed.
Biosynthesis of the wall teichoic acid in Bacillus licheniformis
Authors: Hancock I. C. , Baddiley J.
Published: August 2015

1. The biosynthesis of the wall teichoic acid, poly(glycerol phosphate glucose), has been studied with a particulate membrane preparation from Bacillus licheniformis A.T.C.C. 9945. The precursor CDP-glycerol supplies glycerol phosphate residues, whereas UDP-glucose supplies only glucose to the repeating structure of the polymer. 2. Synthesis proceeds through polyprenol phosphate derivatives, and chemical studies and pulse-labelling techniques show that the first intermediate is the phosphodiester, glucose polyprenol monophosphate. CDP-glycerol donates a glycerol phosphate residue to this to give a second intermediate, (glycerol phosphate glucose phosphate) polyprenol. 3. The glucose residue in the lipid intermediates has the β configuration, and chain extension in the synthesis of polymer occurs by transglycosylation with inversion of anomeric configuration at two stages.
Studies on the biosynthesis of sym-homospermidine in sandal (Santalum album L.)
Authors: Kuttan Ramadasan , Radhakrishnan A. N.
Published: August 2015

The biosynthesis of the newly isolated polyamine, sym-homospermidine (NH2–[CH2]4–NH–[CH2]4 –NH2), was studied by using radioactive amino acids. Arginine was the most effective precursor, being about 10 times as active as ornithine. Unlabelled agmatine and putrescine markedly inhibited the incorporation of [14C]arginine into homospermidine. Similarly the incorporation of ornithine was inhibited by unlabelled arginine and putrescine. γ-Aminobutyraldehyde, the oxidation product of putrescine, was considered to be one of the intermediates in the biosynthesis of homospermidine. The biosynthesis may involve a Schiff-base formation of putrescine with γ-aminobutyraldehyde and subsequent reduction. A limited synthesis of spermidine also takes place under these conditions.
Isolation and properties of highly purified Halobacterium cutirubrum deoxyribonucleic acid-dependent ribonucleic acid polumerase
Authors: Louis B. Gregory , Fitt P. S.
Published: August 2015

1. The subunits α and β of Halobacterium cutirubrum DNA-dependent RNA polymerase have been purified to electrophoretic homogeneity. Both have mol.wt. 18000 and they are required in equimolar amounts for optimum activity. 2. The instability of the complete enzyme, αβ, in the absence of salt is due to the rapid inactivation of the β subunit in these conditions. 3. Nearest-neighbour analysis of the product formed on poly[d(A-T)] as template shows that the enzyme copies the latter accurately. 4. The enzyme initiates new chains with purine nucleoside triphosphates exclusively. 5. The product obtained in the standard assay conditions contains some high mol.wt. (>16S) material, but consists primarily of short chains, of average length 70–80 nucleotide units. 6. The template specificity of the complete enzyme has been studied at high and low ionic strength. Its extreme dependence on salt concentration is unrelated to the gross overall base composition of the DNA used. 7. T7 DNA is transcribed asymmetrically and the enzyme selectively copies the T7 `early' genes. 8. Preliminary amino acid analyses of α and β subunits show that their overall content of acidic, basic and neutral amino acids does not differ appreciably from that of Escherichia coli RNA polymerase.
Carnitine and brown adipose tissue metabolism in the rat during development
Authors: Hahn P. , Skala J.
Published: August 2015

1. The content of carnitine, acylcarnitine and total acid soluble carnitine in brown adipose tissue of rats increases rapidly after birth, attaining a peak on about day 10 and then decreases. Similar changes with age were found for carnitine acetyltransferase activity in mitochondria from brown adipose tissue and heart. The activity of this enzyme in brain and in liver is much smaller, but also increases postnatally. 2. The activity of carnitine palmitoyltransferase in brown adipose tissue, however, decreases after birth then increases later in life. 3. Exposure of 18-day-old rats to the cold for 20 days leads to an increase in carnitine content in brown adipose tissue and raises the activity of carnitine acetyltransferase. The activity of carnitine palmitoyltransferase is not affected by cold adaptation.
The isolation of glycoproteins from bovine achilles tendon and their interaction with collagen
Authors: Anderson J. C. , Jackson D. S.
Published: August 2015

Two glycoprotein fractions, A and B, were isolated from bovine achilles tendon. Glycoprotein A was prepared from a 0.2m-sodium chloride extract and glycoprotein B was isolated from a 3m-magnesium chloride extract. They were free from serum proteins. Glycoprotein A was essentially free of collagen, but glycoprotein B contained about 8% collagen. Both glycoproteins gave several bands on isoelectric focusing. This technique was also used to demonstrate that both glycoprotein fractions interacted strongly with acid-soluble calf skin tropocollagen. It is concluded that these fractions are true components of tendon, and that they may have some function in the macromolecular organization of the tissue.
Thiol reduction of human α2-macroglobulin. The subunit structure
Authors: Jones J. M. , Creeth J. M. , Kekwick R. A.
Published: August 2015

1. Human α2-macroglobulin was prepared from a fraction obtained during the large-scale separation of normal human plasma proteins for clinical use. 2. Sedimentation-equilibrium measurements indicated a molecular weight of 725000. A value of 18.1S was obtained for s020,w. 3. The dissociation that occurs in the pH range 4.5–2.5 and in the region of neutrality in urea-containing solutions is consistent with a dimeric structure of the molecule. 4. The effects of the thiol reagents mercaptoethanol, mercaptoethylamine and N-acetylcysteine were investigated over a range of experimental conditions. Distinct components having sedimentation coefficients of 15, 12 and 8.5S were identified. 5. Conditions were found under which limited reduction with thiol liberated a subunit with a molecular weight approximately one-quarter of that of the intact molecule. This subunit retains the serological specificity of the whole molecule.
Immunochemical studies on human calcitonin M leading to information on the shape of the molecule
Authors: Byfield P. G. H. , Clark M. B. , Turner K. , Foster G. V. , MacIntyre I.
Published: August 2015

1. Two antisera were obtained from a single rabbit. Both are highly specific for human calcitonin M but react with different parts of the amino acid sequence. 2. The different sequences that react with the antibodies of the two antisera were located. The first antiserum reacts at two sites in the molecule, one in the sequence residues 11–18, probably with residue 17 as the immunodominant group, and another on either side of the 28–29 peptide bond. The second antiserum, harvested 9 months later, reacts principally at one site bridging the 28–29 peptide bond. 3. A consideration of the properties of the hormone's binding sites and of data relating biological activity to structure enables some conclusions to be drawn with regard to the shape of the molecule. It appears that the peptide chain is folded to bring N- and C-termini closer together and that there is non-covalent interaction between regions in the chain near both termini. One of these is located near residue 8.
Multiple forms of choline acetyltransferase in several species demonstrated by isoelectric focusing
Authors: Malthe-Sørenssen D. , Fonnum F.
Published: August 2015

1. The behaviour of choline acetyltransferase from pigeon, guinea-pig, rat and cat brain on isoelectric focusing was studied. 2. Choline acetyltransferase from pigeon and guinea-pig brain showed single peaks with isoelectric points at pH6.6 and 6.8 respectively. Only one molecular form of the enzyme was therefore detected in these species. 3. Three peaks of choline acetyltransferase activities with isoelectric points 7.3–7.6, 7.7–7.9 and 8.3 were obtained with enzyme preparations from rat brain. 4. The separate identities of each of the three forms were confirmed by refocusing. 5. Choline acetyltransferase activity from a high-speed supernatant of rat brain homogenate was distributed similarly to a partially purified enzyme preparation from rat brain in the isoelectric gradient. 6. The enzyme activities from cat brain were separated into two distinct peaks with isoelectric points 7.0 and 8.4, and a possible third peak with isoelectric point 7.6. 7. The two main peaks showed considerable differences in stability on storage, and their identities were confirmed by refocusing. 8. The distribution of the enzyme activities was unaltered by isoelectric focusing in the presence of 3m-urea. 9. The apparent Km for choline of choline acetyltransferase from rat, cat and guinea-pig brain was 0.8mm, whereas for the pigeon enzyme it was 0.4mm.
Some properties of pyruvate and 2-oxoglutarate oxidation by blowfly flight-muscle mitochondria
Authors: Hansford R. G.
Published: August 2015

1. High rates of state 3 pyruvate oxidation are dependent on high concentrations of inorganic phosphate and a predominance of ADP in the intramitochondrial pool of adenine nucleotides. The latter requirement is most marked at alkaline pH values, where ATP is profoundly inhibitory. 2. Addition of CaCl2 during state 4, state 3 (Chance & Williams, 1955) or uncoupled pyruvate oxidation causes a marked inhibition in the rate of oxygen uptake when low concentrations of mitochondria are employed, but may lead to an enhancement of state 4 oxygen uptake when very high concentrations of mitochondria are used. 3. These properties are consistent with the kinetics of the NAD-linked isocitrate dehydrogenase (EC 1.1.1.41) from this tissue, which is activated by isocitrate, citrate, ADP, phosphate and H+ ions, and inhibited by ATP, NADH and Ca2+. 4. Studies of the redox state of NAD and cytochrome c show that addition of ADP during pyruvate oxidation causes a slight reduction, whereas addition during glycerol phosphate oxidation causes a `classical' oxidation. Nevertheless, it is concluded that pyruvate oxidation is probably limited by the respiratory chain in state 4 and by the NAD-linked isocitrate dehydrogenase in state 3. 5. The oxidation of 2-oxoglutarate by swollen mitochondria is also stimulated by high concentrations of ADP and phosphate, and is not uncoupled by arsenate.
Molecular properties of lupin and serradella leghaemoglobins
Authors: Broughton W. J. , Dilworth M. J. , Godfrey C. A.
Published: August 2015

1. Leghaemoglobins were extracted from the root nodules of lupin (Lupinus luteus L.) and serradella (Ornithopus sativus Brot.) plants and fractionated into different leghaemoglobin components on DEAE-cellulose–acetate columns. 2. The first two fractions eluted from columns loaded with either lupin or serradella leghaemoglobins were in the Fe3+ oxidation state. 3. These components have protohaem IX as the prosthetic group and glycine as the N-terminal amino acid. 4. Other properties are: lupin component I, pI5.08, molecular weight 19000; lupin component II, pI5.13, molecular weight 20600; serradella component I, pI5.00, molecular weight 17500; serradella component II, pI5.05, molecular weight 19100. 5. Leghaemoglobins are thus heterogeneous with respect to size and charge.
Glycerylphosphorylcholine phosphodiesterase in rat liver. Subcellular distribution and localization in plasma membranes
Authors: Lloyd-Davies Katherine A. , Michell Robert H. , Coleman Roger
Published: August 2015

1. A simple new assay for glycerylphosphorylcholine phosphodiesterase is described, in which radioactive glycerylphosphorylcholine is used as substrate and the reaction products are separated by adsorption on an anion-exchange resin. 2. Rat liver subcellular fractions contained both particulate (58%) and soluble (42%) glycerylphosphorylcholine phosphodiesterase. Both activities released free choline from glycerylphosphorylcholine. 3. The particulate glycerylphosphorylcholine phosphodiesterase was recovered mainly in the nuclear and microsomal fractions and showed a distribution similar to those of 5′-nucleotidase and alkaline phosphodiesterase I, both of which are constituents of the liver plasma membrane. 4. During purification of plasma membranes glycerylphosphorylcholine phosphodiesterase, 5′-nucleotidase and alkaline phosphodiesterase I showed largely similar behaviour, indicating that glycerylphosphorylcholine phosphodiesterase is also localized in liver plasma membranes. Slight differences in the distributions of these three enzymes in density-gradient separations are discussed in relation to the possibility that they are unevenly distributed on different areas of the cell surface. 5. The differences between glycerylphosphorylcholine phosphodiesterase and alkaline phosphodiesterase I indicate that these two activities are not functions of a single enzyme. 6. The glycerylphosphorylcholine phosphodiesterase of liver plasma membranes has a pH optimum of 8.5 and a Km for glycerylphosphorylcholine of 0.95mm. It is inhibited by EDTA and fully reactivated by a variety of bivalent cations (and Fe3+).
Metabolism of fatty acids by bovine spermatozoa
Authors: Neill A. R. , Masters C. J.
Published: August 2015

The incorporation of 14C-labelled myristic, palmitic, stearic, oleic and linoleic acids in vitro into the lipids of bovine spermatozoa was measured at intervals from 2min to 2h. All acids were rapidly incorporated into diglycerides, myristic acid being metabolized to the greatest extent. Whereas the low incorporation of acids into total phospholipids reflected the relative stability of the major phospholipid fractions in sperm, the minor phospholipids, particularly phosphatidylinositol, showed comparatively high metabolic activity. Although, in general, saturated acids were incorporated more actively than unsaturated substrates, stearic acid was poorly incorporated into all lipids except phosphatidylinositol. In regard to fatty acid composition of sperm lipids it was notable that diglycerides contained myristic acid as the major component, and this acid was also a prominent moiety of phosphatidylinositol. Docosahexaenoic acid was the principal fatty acid of the major phospholipid classes. These findings have been discussed in relation to the role of lipids in the metabolism of spermatozoa.
A decreased aminoacyl-transfer-ribonucleic acid-binding capacity of 40S ribosomal subunits resulting from hypophysectomy of the rat
Authors: Barden N. , Korner A.
Published: August 2015

A technique that permitted the reversible dissociation of rat liver ribosomes was used to study the difference in protein-synthetic activity between liver ribosomes of normal and hypophysectomized rats. Ribosomal subunits of sedimentation coefficients 38S and 58S were produced from ferritin-free ribosomes by treatment with 0.8m-KCl at 30°C. These recombined to give 76S monomers, which were as active as untreated ribosomes in incorporating phenylalanine in the presence of poly(U). Subunits from normal and hypophysectomized rats were recombined in all possible combinations and the ability of the hybrid ribosomes to catalyse polyphenylalanine synthesis was measured. The results show that the defect in ribosomes of hypophysectomized rats lies only in the small ribosomal subunit. The 40S but not the 60S subunit of rat liver ribosomes bound poly(U). The only requirement for the reaction was Mg2+, the optimum concentration of which was 5mm. No apparent difference was seen between the poly(U)-binding abilities of 40S ribosomal subunits from normal or hypophysectomized rats. Phenylalanyl-tRNA was bound by 40S ribosomal subunits in the presence of poly(U) by either enzymic or non-enzymic reactions. Non-enzymic binding required a Mg2+ concentration in excess of 5mm and increased linearly with increasing Mg2+ concentrations up to 20mm. At a Mg2+ concentration of 5mm, GTP and either a 40–70%-saturated-(NH4)2SO4 fraction of pH5.2 supernatant or partially purified aminotransferase I was necessary for binding of aminoacyl-tRNA. Hypophysectomy of rats resulted in a decreased binding of aminoacyl-tRNA by 40S ribosomal subunits.
Disequilibrium in the malate dehydrogenase reaction in rat liver mitochondria in vivo
Authors: Heath D. F. , Phillips J. C.
Published: August 2015

1. When [2-14C]pyruvate is injected into rats the C3-position of liver glutamate becomes more heavily labelled than the C2-position, thus establishing that oxaloacetate and fumarate are not in equilibrium in rat liver mitochondria in vivo. The amount of disequilibrium was shown to be simply related to the value that the C3-label/C2-label ratio would have were no label recycled. This ratio, z, was calculated for post-absorptive rats in environmental temperatures of 20° and 30°C from determinations of the distribution of label within glutamate 1, 3 and 10min after intravenous injection of [2-14C]pyruvate. The values of z (best estimate and range) were 1.65 (1.60–1.69) in rats at 20°C and 2.43 (2.23–2.63) in rats at 30°C. These values of z imply the following rates of interconversion in mitochondria of fumarate and oxaloacetate (in terms of the oxaloacetate→citrate flux, R) in rats at 20°C: [Formula: see text] and in rats at 30°C: [Formula: see text] 2. The kinetic parameters of malate dehydrogenase and fumarate hydratase and the intramitochondrial concentrations of NAD+ and NADH under (as far as could be judged) conditions in vivo were collated. From them and the best estimates of R now available were calculated the rates of interconversion of fumarate, malate and oxaloacetate required to give the found values of z. These rates showed that the fumarate hydratase reaction was nearly in equilibrium, but that the malate dehydrogenase reaction was considerably out of equilibrium. The calculations also led to the following conclusions. 3. In livers of rats at 20° and 30°C mitochondrial malate concentrations were respectively about 5 and 1.5 times mean cellular concentrations. 4. Mitochondrial oxaloacetate concentrations were less than 0.2 of the mean cellular concentrations. They were also only 0.65 and 0.55 of the equilibrium concentrations for the malate dehydrogenase reaction in rats at 20° and 30°C respectively. 5. Malate dehydrogenase activity was low because of the very low oxaloacetate concentrations in the mitochondria and the very small fraction of the enzyme complexed with NAD+, i.e. in each direction one substrate concentration was very sub-optimal.
Metabolism of [4-14C]oestradiol by oestrogen-induced uterine peroxidase
Authors: Lyttle C. R. , Jellinck P. H.
Published: August 2015

1. An enzyme that catalyses the metabolism and binding of [4-14C]oestradiol to protein and to other high-molecular-weight substances in the presence of H2O2 was shown to be absent from the uteri of immature rats and to be induced by physiological doses of oestrogen or pregnant-mare-serum gonadotrophin. 2. The pH optimum, stability to heat and other characteristics of the uterine enzyme system as well as its subcellular distribution were determined. 3. The increase in the ability of uterine preparations to convert [4-14C]oestradiol into water-soluble products as a result of oestrogen treatment was accompanied by an increase in peroxidase and NADH oxidase activities and was inhibited by actinomycin D and cycloheximide. 4. The results support the proposal that the increase in peroxidase activity after oestrogen treatment might be part of an adaptive response of the uterus permitting it to bind and inactivate oestrogens and thus limit the duration of their effect upon this target tissue.
The use of acid alumina and Sephadex LH-20 for the separation and characterization of ethanol-soluble peptides produced by Bacillus brevis
Authors: Bartley I. M. , Hodgson B. , Walker J. S. , Holme G.
Published: August 2015

Cell extracts from Bacillus brevis (A.T.C.C. 10068), grown with various media, incorporated certain 14C-labelled amino acids that are normally components of tyrothricin into material that was extracted by ethanol from the precipitate formed by adding acid. When this material was separated by paper and silica-gel thin-layer chromatography and paper electrophoresis 14C was located in those regions that also contained gramicidin and tyrocidine. From a study of the properties of the system responsible for the incorporation it was deduced that non-tyrothricin materials were present. It was shown that the methods normally used to characterize tyrothricin do not adequately distinguish between tyrothricin and non-tyrothricin materials. However, a method for separating these materials was devised. This involved elution with ethanol from columns of acid alumina followed by gel filtration on Sephadex LH-20 with dimethylformamide–water solvent. The behaviour of gramicidin and tyrocidine on the Sephadex LH-20 column was examined, and it was concluded that the separation was not caused simply by gel filtration of unassociated molecules. Also, tyrocidine molecules with different amino acid compositions seemed to have different affinities for the Sephadex LH-20 column.
Glucose metabolism in the developing rat. Studies in vivo
Authors: Vernon Richard G. , Walker Deryck G.
Published: August 2015

1. The specific radioactivity of plasma d-glucose and the incorporation of 14C into plasma l-lactate, liver glycogen and skeletal-muscle glycogen was measured as a function of time after the intraperitoneal injection of d-[6-14C]glucose and d-[6-3H]glucose into newborn, 2-, 10- and 30-day-old rats. 2. The log of the specific radioactivity of both plasma d-[6-14C]- and d-[6-3H]-glucose of the 2-, 10- and 30-day-old rats decreased linearly with time for at least 60min after injection of labelled glucose. The specific radioactivity of both plasma d-[6-14C]- and d-[6-3H]-glucose of the newborn rat remained constant for at least 75min after injection. 3. The glucose turnover rate of the 30-day-old rat was significantly greater than (approximately twice) that of the 2- and 10-day-old rats. The relative size of both the glucose pool and the glucose space decreased with age. Less than 10% of the glucose utilized in the 2-, 10- and 30-day-old rats was recycled via the Cori cycle. 4. The results are discussed in relationship to the availability of dietary glucose and other factors that may influence glucose metabolism in the developing rat.
Gluconeogenesis from lactate in the developing rat. Studies in vivo
Authors: Vernon Richard G. , Walker Deryck G.
Published: August 2015

1. The specific radioactivity of plasma l-lactate and the incorporation of 14C into plasma d-glucose, liver glycogen and skeletal-muscle glycogen were measured as a function of time after the intraperitoneal injection of l-[U-14C]lactate into 2-, 10- and 30-day-old rats. 2. Between 15 and 60min after the injection of the l-[U-14C]lactate, the specific radioactivity of plasma lactate decreased with a half-life of 20–33min in animals at all three ages. 3. At all times after injection examined, the specific radioactivity of plasma glucose of the 2- and 10-day-old rats was at least fourfold greater than that of the 30-day-old rats. 4. Although 14C was incorporated into liver glycogen the amount incorporated was always less than 5% of that present in plasma glucose. 5. The results are discussed with reference to the factors that may influence the rate of incorporation of 14C into plasma glucose, and it is concluded that the rate of gluconeogenesis in the 2- and 10-day-old suckling rat is at least twice that of the weaned 30-day-old animal.
The effect of propionate on the metabolism of pyruvate and lactate in the perfused rat liver
Authors: Chan T. M. , Freedland R. A.
Published: August 2015

1. Rates of gluconeogenesis in the perfused rat liver from propionate, l-lactate, pyruvate and the combination of propionate with either lactate or pyruvate were measured. Less than additive rates were obtained with either propionate plus lactate or propionate plus pyruvate. 2. The uptake of pyruvate plus lactate from the perfusion medium was decreased more seriously when propionate was present with lactate than with pyruvate. 3. The use of [2-14C]pyruvate in the presence of propionate showed that the decreased disappearance of pyruvate plus lactate did not result in their formation from propionate. 4. The addition of sodium butyrate to the perfusion medium caused an inhibition of gluconeogenesis from propionate and stimulated gluconeogenesis and uptake of pyruvate and lactate. 5. The observations are consistent with there being a sparing effect of propionate on lactate and pyruvate metabolism.
The intermediary role of 5-pregnene-3β,20β-diol in the biosynthesis of 16-unsaturated C19 steroids in boar testis
Authors: Loke K. H. , Gower D. B.
Published: August 2015

1. The possible involvement of 5-pregnene-3β,20β-diol in 16-unsaturated C19 steroid biosynthesis has been investigated. 2. 5,16-Androstadien-3β-ol (andien-β) formation from [4-14C]pregnenolone (3β-hydroxy-5-pregnen-20-one), 5-pregnene-3β,20α-diol and 5-pregnene-3β,20β-diol was studied in homogenates of boar testis and the mean yields obtained were 25.6, 2.7 and 16.0% respectively. 3. Short-term kinetic studies with pregnenolone and 5-pregnene-3β,20β-diol separately and together suggested that the latter compound might be an intermediate in the biosynthesis of andien-β. 4. In agreement with this interpretation, radioactive 5-pregnene-3β,20β-diol has been isolated during andien-β biosynthesis from [4-14C]pregnenolone in the presence of NADPH, more radioactivity being trapped under limiting conditions of andien-β formation with NADH present as cofactor. 5. Further, 5-pregnene-3β,20β-diol and andien-β have been shown to inhibit the formation of the 16-unsaturated C19 steroid from [4-14C]pregnenolone, the yield of radioactive 5-pregnene-3β,20β-diol increasing in the presence of added unlabelled andien-β. 6. It is concluded that there may be two pathways leading to 16-unsaturated C19 steroid formation from pregnenolone, one of these involving 5-pregnene-3β,20β-diol as an intermediate. Possible mechanisms are presented and discussed.
The relationship between δ-aminolaevulinate synthetase induction and the concentrations of cytochrome P-450 and catalase in rat liver
Authors: Rao M. R. Satyanarayana , Malathi K. , Padmanaban G.
Published: August 2015

The porphyrinogenic drug 2-allyl-2-isopropylacetamide causes the degradation of microsomal cytochrome P-450 and inhibits the synthesis of catalase in rat liver. The inhibition of catalase synthesis follows the induction of δ-aminolaevulinate synthetase and the consequent overproduction of haem. The allylisopropylacetamide-mediated breakdown of cytochrome P-450 is a rapid event and has a reciprocal relationship to the pattern of δ-aminolaevulinate synthetase induction. Breakdown of cytochrome P-450 appears to be one of the conditions leading to the ‘derepression’ of δ-aminolaevulinate synthetase.
The metabolism of testosterone and dihydrotestosterone in an androgen-dependent tumour. A possible correlation between dihydrotestosterone and tumour growth in vivo
Authors: Bruchovsky Nicholas
Published: August 2015

The effects of dihydrotestosterone (17β-hydroxy-5α-androstan-3-one) and testosterone on the growth of the androgen-dependent Shionogi SC-115 tumour in mice have been compared and the metabolites in the tumour arising from each steroid have been identified. After the transfer of SC-115 tumour cells to castrated male mice, treatment of the recipients with dihydrotestosterone produced a striking proliferative response that enabled earlier tumour detection and led to a higher tumour incidence than obtained with testosterone. At short intervals after the intravenous injection of 200μCi of [1,2-3H]testosterone the amounts of radioactivity in tumour, muscle and seminal vesicles were almost equal. The metabolism of [1,2-3H]testosterone in tumour and muscle was slight in comparison with the extensive metabolism in seminal vesciles. Whereas up to 7% of the total neutral steroid recovered from whole tumour tissue and isolated nuclei was in the form of [1,2-3H]dihydrotestosterone, the amount of this compound in the corresponding preparations from seminal vesciles was several times greater. When the metabolism of [1,2-3H]dihydrotestosterone in tumour tissue was studied, it was found that more than 60% of the total neutral steroid in both cytoplasm and nuclei consisted of [1,2-3H]dihydrotestosterone. Thus much higher intracellular concentrations of dihydrotestosterone occurred with the administration of this steroid than with testosterone. Tumour cell proliferation was suppressed by oestradiol and the amount of androgen in nuclei was significantly decreased by high doses of this hormone.
Differential inhibition of mammalian ribonucleic acid polymerases by an exotoxin from Bacillus thuringiensis. The direct observation of nucleoplasmic ribonucleic acid polymerase activity in intact nuclei
Authors: Beebee T. , Korner A. , Bond R. P. M.
Published: August 2015

The effects of the exotoxin from Bacillus thuringiensis on DNA-dependent RNA polymerases from rat liver were examined. The exotoxin inhibits all RNA polymerase activity at both low and high ionic strength in intact nuclei, and soluble enzymes are similarly affected. This inhibition is relieved by ATP. Dephosphorylated exotoxin did not inhibit the soluble enzymes. Nucleolar and nucleoplasmic RNA polymerases respond to different concentration ranges of exotoxin, and the compound can be used in intact nuclei to isolate the nucleoplasmic activity.
The expression of catechol oxidase activity during the hydroxylation of p-coumaric acid by spinach-beet phenolase
Authors: Vaughan P. F. T. , Butt V. S.
Published: August 2015

1. The conditions under which oxygen consumption in excess of that required for the hydroxylation of p-coumaric acid to caffeic acid, catalysed by spinach-beet phenolase, can be suppressed, have been examined. 2. With dimethyltetrahydropteridine as electron donor, oxygen uptake was exactly equivalent to the caffeic acid produced, provided that p-coumaric acid was in excess, but with excess of reductant, oxygen uptake caused by the further oxidation of caffeic acid was also observed. 3. With equal concentrations of ascorbate and p-coumaric acid, equivalent oxygen uptake and caffeic acid production was found only in the first stages of the reaction, whereas with NADH substituted for ascorbate, oxygen uptake was in excess throughout. 4. When ascorbate was used, the period of the reaction over which this equivalence was found was decreased at high reaction rates and not observed at all with aged enzyme preparations; equivalence was restored by adding bovine serum albumin to these aged preparations. 5. Equivalence between oxygen consumption and caffeic acid production was observed with NADH, if small quantities of dimethyltetrahydropteridine were also added. 6. It is concluded that hydroxylation proceeds without the concomitant production of caffeic acid only if the enzyme is stabilized for hydroxylation by p-coumaric acid and the reductant, and is protected from attack by o-quinones.
Isolation and characterization of 17β-hydroxy steroid dehydrogenase from human erythrocytes
Authors: Mulder E. , Lamers-Stahlhofen G. J. M. , Van Der Molen H. J.
Published: August 2015

1. The 17β-hydroxy steroid dehydrogenase was solubilized during haemolysis of erythrocytes and was isolated from the membrane-free haemolysate. Membrane preparations isolated in different ways did not contain 17β-hydroxy steroid dehydrogenase activity. The 17β-hydroxy steroid dehydrogenase activity in the haemolysate was concentrated by repeated ammonium sulphate precipitation and gel filtration on Sephadex G-150. The 17β-hydroxy steroid dehydrogenase activity of the purified preparation per unit weight of protein was 350–3000 times higher than the activity of the crude erythrocyte haemolysate. The 20α-hydroxy steroid dehydrogenase activity was lost during this purification procedure. 2. The 17β-hydroxy steroid dehydrogenase was NADP-dependent and had a pH optimum for conversion of testosterone between 8.5 and 10. For the molecular weight of the enzyme a value of 64000 was calculated from Sephadex chromatography results. 3. p-Chloromercuribenzoate inhibited the enzymic activity. The oxidative activity of the enzyme for the 17β-hydroxyl group was only partly inhibited when a large excess of 17-oxo steroids was added. The catalysing activity of the enzyme was influenced by the NADP+/NADPH ratio. The oxidation of the 17β-hydroxyl group in the presence of NADP+ proceeded faster than the reduction of the 17-oxo group with NADPH. When both reduced and oxidized cofactors were present the oxidation of the 17β-hydroxyl group was inhibited to a considerable extent. 4. The enzyme had a broad substrate specificity and not only catalysed the conversion of androstanes with a 17β-hydroxyl group, or 17-oxo group, but also the conversion oestradiol⇆oestrone. In addition the steroid conjugates dehydroepiandrosterone sulphate and oestrone sulphate were also converted. There were no indications that more than one 17β-hydroxy steroid dehydrogenase was present in the partially purified preparation.
The metabolism of sodium cortisone 27-[35S]-sulphate in the rat
Authors: Gatehouse P. W. , Roy A. B. , Dodgson K. S. , Powell G. M. , Lloyd A. G. , Olavesen A. H.
Published: August 2015

1. The metabolism of sodium cortisone 21-[35S]sulphate was investigated in rats. 2. Quantitative and qualitative experiments showed that substantial amounts of 35SO42- appeared in the urine of free-ranging rats receiving the ester. 3. Whole-body radioautograms indicated considerable biliary elimination of 35S and also pointed to the liver as the site of metabolism. 4. When female rats with bile-duct cannulae received sodium cortisone 21-[35S]sulphate approx. 70% of the dose appeared in the bile as a doubly conjugated steroid (metabolite I). This metabolite was identified as 3α-(β-d-glucopyranuronosido)- 17α-hydroxy-21-[35S]sulpho-oxy-5α-pregnane-11,20-dione. 5. When metabolite I was administered to a rat with a bile-duct cannula 90% of the dose appeared in the bile unchanged. After the administration (intraperitoneally or orally) of metabolite I to free-ranging rats considerable amounts of 35SO42- appeared in the urine. 6. The route by which 35SO42- might be produced from cortisone [35S]sulphate in free-ranging animals is discussed.
Collagen heterogeneity within different growth regions of long bones of rachitic and nonrachitic chicks
Authors: Toole Bryan P. , Kang Andrew H. , Trelstad Robert L. , Gross Jerome
Published: August 2015

The different anatomical regions involved in osteogenesis in the chick long bone have been examined for heterogeneities in collagen structure that might relate to the mechanism of ossification. Experimentally induced lathyrism was employed to enhance collagen solubility, and vitamin D deficiency to allow accumulation of osteoid, the precursor of bone matrix. The extractable lathyritic collagens of the cartilaginous and osseous regions of growing long bones from rachitic and non-rachitic chicks were examined for α-chain type and amino acid composition. In both groups of animals the growth plate and cartilaginous regions of the epiphysis gave collagen molecules of the constitution [α1(II)]3, whereas the ossifying regions contained [α1(I)]2 α2. The degree of hydroxylation of the lysine moieties was increased by approximately 50% in the α1(I)-chain and α2-chain of rachitic bone collagen. Since uncalcified osteoid is greatly enriched in rachitic bone, it is concluded that the collagen of osteoid has the configuration [α1(I)]2 α2, similar to that of bone matrix, but has an elevated hydroxylysine content. The possible relationship of this difference to the mechanism of calcification is discussed.
The oligosaccharide units of rabbit immunoglobulin G. Multiple carbohydrate attachment sites
Authors: Fanger M. W. , Smyth D. G.
Published: August 2015

The carbohydrate structure of rabbit immunoglobulin G isolated from pooled sera was investigated. Amino sugar analysis of fragments of the molecule allowed three oligosaccharides to be located at separate sites on the H-chain. The corresponding glycopeptides were isolated. The average composition of the C1-oligosaccharide was 2 glucosamine, 1 mannose and 2 galactose residues. It appeared to be present in approx. 15% of the H-chains; the carbohydrate was coupled through the amide group of asparagine in a peptide containing asparagine, glycine and threonine within the Fd fragment of the molecule. The average composition of the C2-oligosaccharide was 1 galactosamine, 1 galactose and either 1 or 2 sialic acid residues. It was present on approx. 40% of the H-chains and was attached glycosidically to the OH group of threonine in a peptide Ser-Lys-Pro-Thr-Cys-Pro-Pro-Glu-Leu in the hinge region of the molecule. The average composition of the C3-oligosaccharide was 5 glucosamine, 2 galactose, 5 mannose, 1 fucose and 1 sialic acid residue. It appeared to be present in all the H-chains and was linked through the amide group of asparagine in a peptide Gln-Gln-Phe-Asn-Ser-Thr-Ile-Arg within the Fc fragment of the molecule.