bluuebunny/crossref_metadata_2025 huggingface dataset:
Increased degradation rates of protein synthesized in hepatoma cells in the presence of amino acid analogues
Authors: Knowles S E , Gunn J M , Hanson R W , Ballard F J
Published: August 2015

1. Reuber H35 hepatoma cells incorporate the arginine analogue canavanine into cell protein when arginine is omitted from the incubation medium. 2. By labelling arginine-containing proteins with (14-C)leucine and then canavanine-containing proteins with (3-H)leucine in the same cells, it is possible to measure the degradation of both types of protein during a subsequent ‘chase’ period. With this technique it has been shown that canavanine-containing proteins are degraded at a rate severalfold greater than normal proteins. Comparable results were found when 6-fluorotryptophan was used as an analogue to tryptophan. 3. Control experiments in which the labelling order was reversed or where the animo acid and its analogue were incubated in separate cell cultures support the conclusion that abberrant proteins are rapidly degraded in vivo.
A mannosyl-carrier lipid of bovine adrenal meddulla and rat parotid
Authors: White D A , Waechter C J
Published: August 2015

1. The transfer of mannose from GDP-(U-14-C)mannose into endogenous acceptors of bovine adrenal medullla and rat parotid was studied. The rapidly labelled product, a glycolipid, was partially purified and characterized. 2. It was stable to mild alkaline hydrolysis but yielded (14-C)mannose on mild acid hydrolysis. It co-chromatographed with mannosyl phosphoryl dolichol in four t.l.c. systems and on DEAE-cellulose acetate. Addition of dolichol phosphate or a dolichol phosphate-enriched fraction prepared from pig liver stimulated mannolipid synthesis. 3. The formation of mammolipid appeared reversible, since addition of GDP to a system synthesizing the mannolipid caused a rapid loss of label from the mannolipid. UDP-N-acetylglucosamine did not inhibit mannolipid synthesis except at high concentrations (2 mM), even though in the absence of GDP-mannose, N-acetylglucosamine was incorporated into a lipid having the properties of a glycosylated polyprenyl phosphate. 4. Mannose from GDP-mannose was also incorporated into two other acceptors, (2y being insoluble in chloroform-methanol (2:1, v/v) but soluble in choloroform-methanol-water (10:10:3, by vol.) and (ii) protein. These are formed much more slowly than the mannolipid. 5. Exogenous mannolipid served as a mannose donor for acceptors (i) and (ii), and it is suggested that transfer of mannose from GDP-mannose to mannosylated protein occurs via two intermediates, the mannolipid and acceptor (i).
The effects of diet, lipolysis and limb ischaemia on the distribution of plasma tryptophan in the rat
Authors: Stoner H B , Cunningham V J , Elson P M , Hunt A
Published: August 2015

A non-linear relationship between the plasma non-esterified fatty acid concentration and the percentage of free plasma tryptophan was found in rats in different nutritional states, although non-esterified fatty acids are not the only factors determining the percentage of free tryptophan. This relationship was not seen in rats injured by limb ischaemia. The effect of drugs causing rapid increases in the plasma non-esterified fatty acid concentration was also studied. Isoprenaline decreased the total plasma tryptophan concentration. Dichloroisoprenaline caused a sustained increase in the plasma non-esterified fatty acid concentration which was accompanied by an increase in the concentration of free plasma tryptophan and followed by a fall in the concentration of total tryptophan. The loss of tryptophan from the plasma was attributed to an altered distribution of tryptophan in the extracellular space rather than to increased metabolism. This interpretation was supported by determinations of the irreversible disposal rate of plasma tryptophan which in uninjured rats was unaffected by the concentration of free plasma tryptophan. In the injured rats this rate was unaltered during limb ischaemia but was decreased after removal of the tourniquets; increased competition for tissue entry by other neutral amino acids and the fall in body temperature could be factors in this fall.
A kinetic study of sulphate transport in rat liver mitochondria
Authors: Crompton M , Palmieri F , Capano M , Quagliariello E
Published: August 2015

The kinetics of sulphate uptake catalysed by the dicarboxylate carrier were measured. The Km value for sulphate is about 0.3 mM. A completely competitive relation exists between the influxes of sulphate and malonate, whereas the relation between sulphate and phosphate is of the mixed type. The inhibition of sulphate influx by mersalyl and bathophenanthroline is similar to that of malonate influx and different from the inhibition of phosphate influx. It is considered that sulphate and malonate probably bind to the same locus on the carrier, whereas phosphate occupies a different site. The possible implications of this conclusion are discussed.
Differential effects of lipid depletion on membrane sodium-plus-potassium ion-dependent adenosine triphosphatase and potassium ion-dependent phosphatase
Authors: Wheeler K P , Walker J A
Published: August 2015

The phospholipid-dependence of the (Na-++K-+)-dependent ATPase (adenosine triphosphatase) (EC 3.6.1.3) and associated K-+-dependent phosphatase activity (EC 3.6.1.7) have been compared. Unlike the (Na-++K-+)-dependent ATPase activities, the K-+-dependent phosphatase activities of a number of different preparations were not closely correlated with their total phospholipid contents. After partial lipid depletion with a single extraction in Lubrol W the residual ATPase and phosphatase activities were correlated, but their magnitudes were quite different: on average only about 5% of the former remained compared with 50% of the latter. A similar differential effect on these activities was found after extraction with deoxycholate. In contrast with the ATPase, consistent restoration of the phosphatase activity of Lubrol-extracted enzymes by added exogenous phospholipids was not observed. We conclude that, although the K-+-dependent phosphatase may be lipid-dependent, the lipid requirement must be different from that of the complete ATPase system, and this difference should help investigations of their relationship.
The effect of zinc on the activity and fluorescence of carbonic anhydrase holoenzymes
Authors: Hesketh T R , Flanagan M T
Published: August 2015

The addition of Zn2+ to human carbonic anhydrase B holoenzyme was shown to enhance the protein fluorescence, and this enhancement was correlated with the inhibition of the p-nitrophenyl acetate esterase activity. The affinity for the inhibitory Zn2+ was increased when the ionic inhibitors, acetate or chloride, were added, suggesting that the inhibitory Zn2+-binding site is within the region of the protein that undergoes an anion-induced conformational change. A similar fluorescence enhancement was observed when Zn2+ was added to human carbonic anhydrase C and to bovine carbonic anhydrase, demonstrating that the binding site is not a thiol group. Circular-dichroism studies showed that the C isoenzyme but not the B isoenzyme underwent a major conformational change in the presence of Zn2+. A mechanism for the Zn2+-induced fluorescence enhancement was suggested on the basis of studies with simple compounds.
Activation of membrane-bound high-affinity calcium ion-sensitive adenosine triphosphatase of human erythrocytes by bivalent metal ions
Authors: Pfleger H , Wolf H U
Published: August 2015

The Ca2+-sensitive ATPase (adenosine triphosphatase) of human erythrocyte membranes is activated, not only by Ca2+ ions, but also by a series of other bivalent metal ions including Sr2+, Ba2+, Mn2+, Ni2+, Co2+, Cd2+, Cu2+, Zn2+ and Pb2+. The degree of activation is dependent on the radius of the ion rather than on its nature, in contrast with the dissociation constant of the enzyme--metal ion complex.
Hydrodynamic changes accompanying the loss of metal ions from concanavalin A.
Authors: Sawyer H W , Dabscheck R , Nott P R , Selinger B K , Kuntz I D
Published: August 2015

The hydrodynamic changes which accompany the dissociation of metal ions, from concanavalin A at acid pH are a result of charge effects rather than of dissociation of metal ions as such. Measurements of the rotational relaxation time are discussed in terms of the hydration of the protein and its polymeric heterogeneity.
Rates of degradation and synthesis of glycosidases de novo during growth and differentiation of Dictyostelium discoideum
Authors: Every D , Ashworth J M
Published: August 2015

1. Injection of a purified preparation of β-N-acetylglucosaminidase from the spent growth medium of myxamoebae of Dictyostelium discoideum into rabbits gave rise to an antibody preparation containing both anti-α-glucosidase and anti-β-acetylglucosaminidase activities. 2. These two activities were shown to reside in different immunoglobulin molecules and it was concluded that the β-N-acetylglucosaminidase preparation contained trace amounts of highly antigenic α-glucosidase. 3. A single precipitin band having β-N-acetylglucosaminidase activity was formed in Ouchterlony plates when this antibody preparation was tested against extracts obtained from differentiated cells or from myxamoebae grown either axenically or on bacteria. 4. The antibody preparation was used to show that both β-N-acetylglucosaminidase and α-glucosidase molecules are synthesized de novo from isotopically labelled amino acids during both the growth and differentiation phases of the life cycle and to show that neither of these proteins is significantly degraded during the growth phase or during the first 9h of differentiation. 5. The rates of accumulation of these assayable enzyme activities are thus equal to their rates of synthesis during growth and early differentiation. 6. The factors regulating cellular enzyme activity during the life cycle of D. discoideum are discussed.
Thymidine transport in phytohaemagglutinin-stimulated pig lymphocytes
Authors: Barlow S D , Ord M G
Published: August 2015

Thymidine and uridine transporters in peripheral pig lymphocytes have structural features in common, but are not identical. Accelerated entry of [3H]thymidine begins 12h after the addition of phytohaemagglutinin. The increased thymidine uptake into the cells is characterized by an increase in Vmax. Without alteration of the apparent Km(0.6+/-0.08muM). Thymidine kinase activity is increased 12h after stimulation. Both the increased thymidine uptake and the increased thymidine kinase activity are inhibited in cultures incubated with puromycin: rates of degradation of the two systems are unchanged after phytohaemagglutinin addition, and indicate similar half-lives of about 2h. Thymidine kinase is rate-limiting for thymidine entry up to 18h after phytohaemagglutinin addition; increase in its synthesis is detectable about 6h before net incorporation of thymidine into DNA is significantly promoted.
A comparison of the activity of phosphatidylinositol phosphodiesterase against substrate in dispersions and as monolayers at the air-water interface
Authors: Quinn P J , Barenholz Y
Published: August 2015

The activity of phosphatidylinositol phosphodiesterase, purified from rat brain, against substrate in three forms, (a) multibilayer liposomes, (b) single bilayer vesicles of phosphatidylinositol and (c) phosphatidylinositol oriented as monolayers at the air-water interface, was examined. The reaction rate was similar against the two substrate dispersions prepared with the same phospholipid concentration, although there was a large difference in substrate surface area available to the enzyme, and this similarity could not be accounted for by any differences in the microviscosity of the hydrocarbon region of the phospholipid bilayers. The reaction showed apparent zero-order reaction kinetics until about 10% of the substrate had been degraded, whereupon the rate decreased. The reaction against monolayers of phosphatidylinositol was linear throughout the entire digestion of the film, provided that more than 0.25 mg of enzyme was present in the subphase. The pH optimum was 6.6. Bivalent ions)Ca2+, Mg2+, Co2+, Ni2+ and Mn2+) facilitated enzyme penetration into substrate monolayers, but the enzyme was only activated by Ca2+ (optimal concentration, 1mM) and to a lesser extent by Mg2+. The reaction rate was independent of initial surface pressures of less than about 22mN·m-1 but at higher pressures the rate decreased. This decrease could be prevented by the addition of 10mol of octadecylamine/90mol of phosphatidylinositol to the substrate monolayer; the amine did not increase the rate of reaction in films of less than 22mN·m-1.
Synthesis of thiol-containing analogues of puromycin and a study of their interaction with N-acetylphenylalanyl-transfer ribonucleic acid on ribosomes to form thioesters
Authors: Gooch John , Hawtrey Arthur O.
Published: August 2015

1. The thiol-containing analogue of puromycin, 6-dimethylamino-9-{1′-[3′-(2″-mercapto-3″-phenylpropionamido)-3′-deoxy-β-d-ribofuranosyl]}purine (XVII) in which the primary amino group of the antibiotic is replaced with a thiol grouping, was synthesized chemically (compound XVII is abbreviated to thiopuromycin). 2. Thiopuromycin (XVII) was found to be active in releasing N-[3H]acetylphenylalanine from its tRNA carrier as the thioester, N-acetylphenylalanylthiopuromycin (XIX) in the Escherichia coli ribosomal system. The reaction product (XIX) was synthesized chemically from thiopuromycin and N-acetylphenylalanine and found to be stable to hydrolysis in the standard incubation medium at pH7.6. dl-Phenyl-lactylpuromycin (XXI), the hydroxy analogue of puromycin, was also synthesized chemically and shown to release N-acetylphenylalanine from its tRNA carrier in the E. coli ribosomal system, thus confirming the previous results of Fahnestock et al. [Biochemistry (1970) 9, 2477–2483]. 3. In marked contrast with the results obtained in the E. coli system, both thiopuromycin (XVII) and hydroxypuromycin (XXI) were found to be inactive in releasing N-acetylphenylalanine from its tRNA carrier in the rat liver ribosomal system.
The amino acid sequence of cytochrome f from the brown alga Alaria esculenta (L.) Grev
Authors: Laycock M V
Published: August 2015

Cytochrome f was isolated from the brown alga Alaria esculenta and the amino acid sequence was determined. The native haemoprotein has a molecular weight of 9800 and consists of a single polypeptide chain of 86 amino acid residues with a haem group bonded to cysteine residues at positions 14 and 17. The N-terminus is not acetylated and no methylated lysines were found. Sequences of three other algal cytochromes f were compared with that of Alaria and 22 out of 92 positions were common to the four sequences. One-half of these conserved sites occur between positions 49 and 63. Detailed evidence for the amino acid sequence of Alaria cytochrome has been deposited as Supplementary Publication SUP 50048 (6 pages) at the British Library (Lending Division), Boston Spa, Wetherby, West Yorkshire LS23 7BQ, U.K., from whom copies can be obtained on the terms indicated in Biochem. J. (1975) 145, 5.
Reduction of disulphide bonds in proteins mixed disulphides catalysed by a thioltransferase in rat liver cytosol
Authors: Mannervik B , Axelsson K
Published: August 2015

The reduction of mixed disulphides of some proteins and GSH [Protein(-SSG)n] is accomplished with GSH as a reductant and a thioltransferase from rat liver as a catalyst, thus: See article. The spontaneous reaction is negligible in comparison with the enzymic reaction in vivo, and any direct reduction with glutathione reductase is not detectable with the substrates used. The reduction is only indirectly dependent on NADPH, which is required for the regeneration of GSH from GSSG. Other protein disulphides apparently are reduced via analogous GSH-dependent reactions
Regulatory state of ribosomal genes and physiological changes in the concentration of free ribonucleic acid polymerase in Escherichia coli
Authors: Bremer H , Dalbow D G
Published: August 2015

The concept of promoter efficiency is introduced as frequency of RNA chain initiation at a given promoter normalized to the intracellular concentration of free (but functional) RNA polymerase. Previous observations from this laboratory on the synthesis of ribosomes and β-galactosidase are used to show that during a nutritional shift-up from succinate minimal to glucose-amino acids medium (3-fold increase in steady-state growth rate) the concentration of free (active) RNA polymerase decreases to one-quarter of the pre-shift value and the promoter efficiencies of the genes for ribosomal RNA and ribosomal proteins increase 9- and 6-fold respectively. This extent of control of ribosomal genes is much greater than expected on the basis of the increase in the rate of ribosome synthesis (3-fold).
Influence of anti-oestrogens on the specific binding in vitro of (3H)oestradiol by cytosol of rat mammary tumours and human breast carcinomata
Authors: Powell-Jones W , Jenner D A , Blamey R W , Davies P , Griffiths K
Published: August 2015

The anti-oestrogenic potential of two nitrogen-mustard-containing compounds, I.C.I. 79792 and I.C.I. 85966, was studied. I.C.I. 85966 usually did not decrease specific binding of [3H]oestradiol by breast-tumour cytoplasmic proteins. I.C.I. 79792 decreased specific [3H]oestradiol binding, but not to the same extent as similar concentrations of I.C.I. 46474, diethylstilboestrol or dibutyldihydrostilboestrol.
Characteristics of aspartate deamination by the puring nucleotide cycle in the cytosol fraction of rat liver
Authors: Moss K M , McGivan J D
Published: August 2015

1. The component reactions of the puring nucleotide cycle were studied in cytosol extracts of rat liver. 2. AMP deaminase was strongly activated by ATP and analogues of ATP. 3. IMP was converted into ATP by a system requiring the presence of aspartate, GTP and a nucleoside triphosphate-regenerating system. 4. Under appropriate conditions, NH3 was produced from aspartate. 5. From the rates at which these reactions occur it is concluded that the purine nucleotide cycle may have sufficient activity to be a major pathway of amino acid deamination in liver.
Glutamate uptake by chick retina
Authors: Tunnicliff G
Published: August 2015

The uptake of glutamate was found to be via a single high-affinity transport mechanism with Km values of 35 and 95 μm for chick-embryo and mature chick retina respectively. These data contrast with the uptake of γ-aminobutyratewhich in the same tissue has previously been shown to display two kinetically distinct mechanisms in the embryo, but a single low-affinity process in the mature retina.
Studies on the role of insulin in the regulation of glyceride synthesis in rat epididymal adipose tissue
Authors: Sooranna S R , Saggerson E D
Published: August 2015

1. When rat isolated fat-cells were incubated with fructose and palmitate, insulin significantly stimulated glyceride synthesis as measured by either [14C]fructose incorporation into the glycerol moiety or of [3H]palmitate incorporation into the acyl moiety of tissue glycerides. Under certain conditions the effect of insulin on glyceride synthesis was greater than the effect of insulin on fructose uptake. 2. In the presence of palmitate, insulin slightly stimulated (a) [14C]pyruvate incorporation into glyceride glycerol of fat-cells and (b) 3H2O incorporation into glyceride glycerol of incubated fat-pads. 3. At low extracellular total concentrations of fatty acids (in the presence of albumin), insulin stimulated [14C]fructose, [14C]pyruvate and 3H2O incorporation into fat-cell fatty acids. Increasing the extracellular fatty acid concentration greatly inhibited fatty acid synthesis from these precursors and also greatly decreased the extent of apparent stimulation of fatty acid synthesis by insulin. 4. These results are discussed in relation to the suggestion [A.P. Halestrap & R.M Denton (1974) Biochem. J. 142, 365-377] that the tissue may contain a specific acyl-binding protein which is subject to regulation. It is suggested that an insulin-sensitive enzyme component of the glyceride-synthesis process may play such a role.
Hepatic albumin and urea synthesis: The mathematical modelling of the dynamics of [14C]carbonate-derived guanidine-labelled arginine in the isolated perfused rat liver
Authors: Tavill A S , Nadkarni D , Metcalfe J , Black E , Hoffenberg R , Carson E R
Published: August 2015

A mathematical model was constructed to define the dynamics of incorporation of radioactivity into urea carbon and the guanidine carbon of arginine in plasma albumin after the rapid intraportal-venous administration of Na214CO3 in the isolated perfused rat liver. 2. The model was formulated in terms of compartmental analysis and additional experiments were designed to provide further information on subsystem dynamics and to discriminate between alternative model structures. 3. Evidence for the rapid-time-constant of labelling of intracellular arginine was provided by precursor-product analysis of precursor [14C]carboante and product [14C]urea in the perfusate. 4. Compartmental analysis of the dynamics of newly synthesized urea was based on the fate of exogenous [13C]urea, endogenous [14C]urea and the accumulation of [12C]urea in perfusate water, confirming the early completion of urea carbon labelling, the absence of continuing synthesis of labelled urea, and the presence of a small intrahepatic urea-delay pool. 5. Analysis of the perfusate dynamics of endogenously synthesized and exogenously administered [6-14C]arginine indicated that although the capacity for extrahepatic formation of [14C]-urea exists, little or no arginine formed within the intrahepatic urea cycle was transported out of the liver. However, the presence of a rapidly turning-over intrahepatic arginine pool was confirmed. 6. On the basis of these subsystem analyses it was possible to offer feasible estimations for the parameters of the mathematical model. However, it was not possible to stimulate the form and magnitude of the dynamics of newly synthesized labelled urea and albumin which were simultaneously observed after administration of [14C]carbonate on the basis of a preliminary model which postulated that both products were derived from a single hepatic pool of [16-14C]arginine. On the other hand these observed dynamics could be satisfied to a two-compartment arginine model, which also provided an explanation for discrepancies observed between albumin synthesis measured radioisotopically and immunologically. This was based on a relative overestimation of [14C]urea specific radioactivity resulting from the rapid dynamics of [14C]carbonate and the [14C]urea subsystem relative to the labelled albumin subsystem. The effects of arginine compartmentalization could be minimized in the model by minor slowing of the rate of [14C]carbonate turnover or by constant infusion of [14C]carbonate, both of which permitted valid determination of albumin-synthesis rates.
The investigation of substrate-induced changes in subunit interactions in glyceraldehyde 3-phosphate dehydrogenases by measurement of the kinetics and thermodynamics of subunit exchange
Authors: Osborne H H , Hollaway M R
Published: August 2015

An investigation was made of changes in subunit interactions in glyceraldehyde 3-phosphate dehydrogenase on binding NAD+, NADH and other substrates by using the previously developed method of measurement of rates and extent of subunit exchange between the rabbit enzyme (R4), yeast enzyme (Y4) and rabbit-yeast hybrid (R2Y2) [Osborne & Hollaway (1974) Biochem. J. 143, 651-662]. The free energy of activation for the conversion of tetramer into dimer for the rabbit enzyme (R4 leads to 2R2) is increased by at least 12kJ/mol in the presence of NAD+. This increase is interpreted in terms of an NAD+-induced ‘tightening’ of the tetrameric structure probably involving increased interaction at the subunit interfaces across the QR plane of the molecule [see Buehner et al. (1974) J. Mol. Biol. 82, 563-585]. This tightening of the structure only occurs on binding the third NAD+ molecule to a given enzyme molecule. Conversely, binding of NADH causes a decrease in the free energy of activation for the R4 leads to 2R2 and Y4 leads to 2Y2 conversions by at least 10kJ/mol. This is interpreted as a NADH-induced ‘loosening’ of the structures arising from decreased interactions across the subunit interfaces involving the QR dissociation plane. In the presence of NADH the increase in the rate of subunit exchange is such that it is not possible to separate the hybrid from the other species if electrophoresis is carried out with NADH in the separation media. In the presence of a mixture of NADH and NAD+ the effect of NAD+ on subunit exchange is dominant. The results are discussed in terms of the known co-operativty between binding sites in glyceraldehyde 3-phosphate dehydrogenases.
The reaction of Pseudomonas aeruginosa cytochrome c oxidase with carbon monoxide
Authors: Parr S R , Wilson M T , Greenwood C
Published: August 2015

The binding of CO to ascorbate-reduced Pseudomonas cytochrome oxidase was investigated by static-titration, stopped-flow and flash-photolytic techniques. Static-titration data indicated that the binding process was non-stoicheiometric, with a Hill number of 1.44. Stopped-flow kinetics obtained on the binding of CO to reduced Pseudomonas cytochrome oxidase were biphasic in form; the faster rate exhibited a linear dependence on CO concentration with a second-order rate constant of 2 × 10(4) M-1-s-1, whereas the slower reaction rapidly reached a pseudo-first-order rate limit at approx. 1s-1. The relative proportions of the two phases observed in stopped-flow experiments also showed a dependency on CO concentration, the slower phase increasing as the CO concentration decreased. The kinetics of CO recombination after flash-photolytic dissociation of the reduced Pseudomonas cytochrome oxidase-CO complex were also biphasic in character, both phases showing a linear pseudo-first-order rate dependence on CO concentration. The second-order rate constants were determined as 3.6 × 10(4)M-1-s-1 and 1.6 × 10(4)M-1-s-1 respectively. Again the relative proportions of the two phases varied with CO concentration, the slower phase predominating at low CO concentrations. CO dissociation from the enzyme-CO complex measured in the presence of O2 and NO indicated the presence of two rates, of the order of 0.03s-1 and 0.15s-1. When sodium dithionite was used as a reducing agent for the Pseudomonas cytochrome oxidase, the CO-combination kinetics observed by both stopped flow and flash photolysis were extremely complex and not able to be simply analysed.
S-adenosylmethionine decarboxylase from baker's yeast
Authors: Pösö H , Sinervirta R , Jänne J
Published: August 2015

1. S-Adenosyl-L-methionine decarboxylase (S-adenosyl-L-methionine carboxy-lyase, EC 4.1.1.50) was purified more than 1100-fold from extracts of Saccharomyces cerevisiae by affinity chromatography on columns of Sepharose containing covalently bound methylglyoxal bis(guanylhydrazone) (1,1′[(methylethanediylidene)dinitrilo]diguanidine) [Pegg, (1974) Biochem J. 141, 581-583]. The final preparation appeared to be homogeneous on polyacrylamide-gel electrophoresis at pH 8.4. 2. S-Adenosylmethionine decarboxylase activity was completely separated from spermidine synthase activity [5′-deoxyadenosyl-(5′),3-aminopropyl-(1),methylsulphonium-salt-putrescine 3-aminopropyltransferase, EC 2.5.1.16] during the purification procedure. 3. Adenosylmethionine decarboxylase activity from crude extracts of baker's yeast was stimulated by putrescine, 1,3-diamino-propane, cadaverine (1,5-diaminopentane) and spermidine; however, the purified enzyme, although still stimulated by the diamines, was completely insensitive to spermidine. 4. Adenosylmethionine decarboxylase has an apparent Km value of 0.09 mM for adenosylmethionine in the presence of saturating concentrations of putrescine. The omission of putrescine resulted in a five-fold increase in the apparent Km value for adenosylmethionine. 5. The apparent Ka value for putrescine, as the activator of the reaction, was 0.012 mM. 6. Methylglyoxal bis(guanylhydrazone) and S-methyladenosylhomocysteamine (decarboxylated adenosylmethionine) were powerful inhibitors of the enzyme. 7. Adenosylmethionine decarboxylase from baker's yeast was inhibited by a number of conventional carbonyl reagents, but in no case could the inhibition be reversed with exogenous pyridoxal 5′-phosphate.
Some enzymes present in the walls of mesophyll cells of tobacco leaves
Authors: Yung K H , Northcote D H
Published: August 2015

Cell-wall enzymes were assayed by the difference between enzyme activities in the whole cell and the protoplast. Both peroxidase (85.2%) and acid phosphatase (21.9%) were located in the wall. However, malate dehydrogenase was found only in the protoplast. A study of the time-course of the release of peroxidase and malate dehydrogenase into the incubation medium from cells either treated with cellulase or untreated, also indicated that peroxidase and not malate dehydrogenase was located in the wall. Only two anodic isoenzymes of peroxidase were present in the cell wall. These were more negatively charged than those of horseradish peroxidase.
Protein chromatography on adsorbents with hydrophobic and ionic groups. Some properties of N-(3-carboxypropionyl)aminodecyl-sepharose and its interaction with wheat-germ aspartate transcarbamoylase
Authors: Yon R J , Simmonds R J
Published: August 2015

1. The charge state of two derivatives of Sepharose prepared by the CNBr activation method were studied by acid-base titration and by ion-exchange chromatography. Dodecyl-Sepharose exhibited cationic groups (21mumol/ml of settled gel; pKa=9.6) that were tentatively assigned to the coupling isourea group. 2. CPAD-Sepharose [N-(3-carboxypropionyl)aminodecyl-Sepharose] has anionic (carboxyl) groups (pKa=4.5) and cationic groups (pKa=9.6) in roughly equal concentrations (e coupling group. CPAD-Sepharose is slightly negatively charged at pH 7.0 and substantially negatively charged at pH 8.5. 3. The pKa values of dodecyl-Sepharose and CPAD-Sepharose are unaffected by a 100-fold increase in the concentration of KCl. 4. CPAD-Sepharose has considerable affinity for wheat-germ aspartate transcarbamoylase at pH 8.5 when the adsorbent and enzyme are both negatively charged. The interaction involves the C10 chain but is relatively moderate compared with C10 chains associated only with positive charge. 5. Desorption of the enzyme adsorbed to CPAD-Sepharose can be achieved by raising the pH to increase the electrostatic repulsion, or by introducing the detergent sodium deoxycholate. Acetone and butan-1-ol also weaken the adsorption at pH 8.5. 6. High concentrations of sodium acetate or sodium phosphate induced the enzyme to bind more tightly to CPAD-Sepharose. 7. These results are discussed in terms of a ‘repulsion-controlled’ model or hydrophobic chromatography.
The purification and properties of pig spleen phosphofructokinase
Authors: Hickman P E , Weidemann M J
Published: August 2015

Pig spleen phosphofructokinase has been purified 800-fold with a yield of 17%. Two isoenzymes that appear to be kinetically identical can be separated by DEAE-cellulose column chromatography. In common with the enzyme from other mammalian sources, the spleen enzyme has a pH optimum of 8.2. At pH 7.0 it displays sigmoidal kinetics with respect to fructose 6-phosphate concentration but its co-operative behaviour is very dependent on pH, protein concentration and the concentration of MgATP. MgGTP and MgITP can replace MgATP as phosphate donors but, unlike MgATP, these nucleotides do not cause significant inhibition. Mn2+ and Co2+ (as the metal ion-ATP complexes) act as cofactors and in the free form are far more inhibitory than free Mg2+. The spleen enzyme responds to a wide variety of potential effector molecules: ADP, AMP, cyclic AMP, aspartate, NH4+, fructose 6-phosphate, fructose 1,6-diphosphate and Pi all act as either activators or protectors, whereas Mg-ATP, Mg2+, citrate, phosphoenol-pyruvate and the phosphoglucerates are inhibitors.
A reporter group delivery system with both absolute and selective specificity for thiol groups and an improved fluorescent probe containing the 7-nitrobenzo-2-oxa-1,3-diazole moiety
Authors: Stuchbury T , Shipton M , Norris R , Malthouse J P G , Brocklehurst K , Herbert J A L , Suschitzky H
Published: August 2015

1. 4-(N-2-Aminoethyl2′-pyridyl disulphide)-7-nitrobenzo-2-oxa-1,3-diazole (compound I) was synthesized and evaluated as a fluorescent labelling reagent for thiol groups. 2. The design of compound (I) as one example of a general type of reporter group delivery reagent (2-pyridyl-S-S-X, where X contains an environmentally sensitive spectroscopic probe) is discussed. 3. The electronic absorption spectrum of compound (I) was determined over a wide range of pH and the spectral changes that accompany its reaction with low-molecular-weight thiols, e.g. L-cysteine, and with papain (EC 3.4.22.2) and bovine serum albumin are discussed. 4. A new value of epsilon343 for 2-thiopyridone (Py-2-SH) was determined as 8.08 × 10(3) +/- 0.08 × 10(3)M-1-cm-1. 5. Spectral analysis of the reactions of compound (I) with L-cysteine and with papain (in the pH range 3.5-8.0) showed that even under equimolar conditions the reaction (thiol-disulphide interchange to release Py-2-SH) is essentially stoicheimoetric and probably proceeds by specific attack at the sulphur atom distal from the pyridyl ring of compound (I). 6. The fluorescence-emission spectra of compound (I) and of the products of its reaction with papain and with ficin (EC 3.4.22.3) were determined. Compound (I) is highly fluorescent in aqueous solution. Excitation within the intense visible absorption band (lambda max. 481 nm, epsilon max. 2.52 × 10(4)M-1-cm-1) provides green fluorescence with an emission maximum at 540 nm. Both papain and ficin labelled by reaction with compound (I) are characterized by fluorescence-emission maxima (535 nm and 530 nm respectively) of even higher intensity. The fluorescence emission of the product of the reaction of papain with compound (I) was shown to be 25 times more intense than that of the product of the reaction of papain with 4-chloro-7-nitrobenzo-2-oxa-1,3-diazole (Nbd chloride). 7. The second-order rate constants (k2) for the reactions of compound (I) and of Nbd chloride with GSH, papain, albumin, ficin, 2-benzimidazolylmethanethiol and 2-benzimidazolylethanethiol were determined at 25.0 degrees C and various pH values. At pH4 the values of k2(compound I)/k2(Nbd chloride) are: GSH, 288; albumin, 36; papain 3 × 10(3); ficin, 3 × 10(4). 8. The pH-k2 profiles for the reactions of compound (I) and of Nbd chloride with the two 2-benzimidazolylalkanethiols were determined. Of the four profiles only that for the reaction of compound (I) with 2-benzimidazolylmethanethiol is characterized by a striking rate maximum in acidic media.
The structure of keratan sulphates from various sources
Authors: Choi H U , Meyer K
Published: August 2015

Quantitative structural comparisons were made between keratan sulphates isolated from various sources, namely pig nucleus pulposus, bovine cornea, and the costal cartilages of children, a young adult with Marfan syndrome and of old human autopsies. In human costal cartilage the amount of keratan sulphate increases markedly with age, although total mucopolysaccharide decreases to some extent, concomitant with a decrease in chondroitin 4-sulphate and an increase in chondroitin 6-sulphate. Comparison of molecular weights estimated by gel chromatography with those calculated from the molar ratio of galactose to mannose indicates that keratan sulphates of human costal cartilages of children and of a young adult with Marfan syndrome, and of pig nucleus pulposus, contain one mannose residue per chain, whereas keratan sulphates of old human costal cartilage and of bovine cornea contain one to two, and two, per chain respectively. After mild acid-catalysed desulphation of pig nucleus pulposus keratan sulphate, approx. 12% of the mucopolysaccharide aggregates irreversibly once the water is removed from the polysaccharide. The following conclusions have been drawn from a methylation analysis of keratan sulphates of various sources, aided by g.l.c.-mass spectrometry. (1) Fucose and N-acetylneuraminic acid are non-reducing terminal residues and the sialic acid is linked to the 3-position of galactose residues. (2) Pig nucleus pulposus keratan sulphate has approximately 4 non-reducing terminal groups per molecule and appears to be slightly less branched than the costal-cartilage keratan sulphate of children. The branching in human costal-cartilage keratan sulphates decreases with age. Bovine corneal keratan sulphate appears to be unbranched. (3) Mannose residues are linked by 3 different substituents in human costal-cartilage and bovine corneal keratan sulphates, and by two different substituents in pig nucleus pulposus keratan sulphate. (4) The sulphate ester groups are all on the 6-position of N-acetyl-glucosamine and galactose residues. The degree of sulphation increases with age in costal keratan sulphates with the increase mainly of the galactose 6-sulphate residues.
Behaviour of tritium-labelled isopenicillin N and 6-aminopenicillanic acid as potential penicillin precursors in an extract of Penicillum chrysogenum
Authors: Fawcett P A , Usher J J , Abraham E P
Published: August 2015

1. 3H was incorporated into solvent-soluble penicillin from isopenicillin N and 6-aminopenicillanic acid 3H-labelled in the 2β-methyl group when the labelled compounds were incubated with a crude extract of Penicillum chrysogenum. 2. With a soluble protein fraction of the extract incorporation from isopenicillin N occurred on addition of phenyl-acetyl-CoA. 3. Labelled benzylpenicillin was isolated after incubation of the crude extract with phenylacetyl-CoA and isopenicillin and the addition of unlabelled benzylpenicillin as a carrier. 4. No incorporation of 3H into solvent-soluble penicillin was detected on incubation of these extracts with penicillin N.
The thermochemical characterization of sodium dithionite, flavin mononucleotide, flavin-adenine dinucleotide and methyl and benzyl viologens as low-potential reductants for biological systems
Authors: Watts G D , Burns A
Published: August 2015

The heat of reaction (ΔH) of Fe(CN)63−, Methyl Viologen, FMN and FAD with S2O42− in aqueous buffer solutions was measured calorimetrically. In addition ΔH values for reduction of Fe(CN)63-, FMN and FAD by reduced Methyl Viologen were determined. The resulting calorimetric data and corresponding E0′ values were combined to yield thermodynamic data for these simple reducing agents in a form useful for applications to biological reactions. Thermodynamic data for the reduction of spinach ferredoxin are also presented.
The induction of enzyme activity in the endosperm of germinating castor-bean seeds
Authors: Marriott K M , Northcote D H
Published: August 2015

1. Endosperm extracts were prepared at various times during germination from intact castor-bean seeds and from seeds from which the embryos had been removed. The sterilized seeds were incubated either on solid water agar or on agar containing 0.3 mM-gibberellic acid. 2. Isocitrate lyase and 3-hydroxyacyl-CoA dehydrogenase had very low activities in the mature seeds, but increased 44-fold and 27-fold respectively during germination. In contrast, the extracts of mature seeds had considerable acid and alkaline lipase activity and this only increased two- to three-fold during the incubation period. 3. Incubation of the seeds with gibberellic acid accelerated the rate of appearance of isocitrate lyase and 3-hydroxyacyl-CoA dehydrogenase. It also increased the total activity attained. However, the application of hormone had, in comparison, little effect on the development of lipase activity. 4. The removal of the embryo had little influence on the development of enzyme activity in the endosperm tissue; only with isocitrate lyase was a decrease in activity observed in the absence of the embryo.
Purification and characterization of guinea-pig intestinal brush borders
Authors: Andersen K , Lippe G V , Morkrid L , Schjonsby H
Published: August 2015

The purification of guinea-pig intestinal brush borders by a rapid sucrose-gradient-centrifugation step is reported. A 29-fold increase in the maltase/DNA quotient indicates considerable purification of the brush borders from nuclei. The biological activity of the brush borders was well preserved, as demonstrated by a high recovery of human gastric-juice-mediated uptake of 57Co-labelled vitamin B-12; homogeneity and purity were confirmed by scanning electron microscopy. Both the morphological appearance and biological activity were unchanged after prolonged storage in glycerol.
Effects of cholecalciferol on the translocation of calcium by non-everted ileum in vitro
Authors: Holdsworth Eric S. , Jordan John E. , Keenan Ellen
Published: August 2015

An apparatus is described that allows perfusion of a non-everted segment of intestine in vitro and the study of the accumulation of substances within the mucosal cells. The translocation of Ca2+ by rachitic-chick ileum and the effect of pretreatment with cholecalciferol was investigated, with the following conclusions. (1) Entry of Ca2+ across the microvilli into mucosal cells is by diffusion; it does not require metabolic energy or the presence of any other inorganic ions. (2) Pretreatment of the chick with cholecalciferol causes increased permeability of the microvillus to Ca2+ in both directions (lumen to cell, cell to lumen). The increased transport brought about by cholecalciferol in vivo can be partially mimicked by sodium dodecyl sulphate added in vitro. (3) The sign and the magnitude of the electrical potential difference prevailing across the ileum does not influence Ca2+ transport. (4) Exit of Ca2+ from the mucosal cell is temperature-sensitive, requires metabolic energy and Na+. (5) Pretreatment with cholecalciferol caused increased movement of Ca2+ out of the cell across the basement membranes. This effect of cholecalciferol given in vivo could be markedly increased by the presence of dicyclohexylcarbodi-imide in the perfusion fluid. These observations suggested that cholecalciferol increased Ca2+ entry (and exit) at the mucosal surface and also caused Ca2+ to be more available to the pump at the serosal surface.
Folate metabolism in the rat liver during regeneration after partial hepatectomy
Authors: Barbiroli Bruno , Bovina Carla , Tolomelli Brunella , Marchetti Mario
Published: August 2015

1. Folate metabolism was studied during the early phases of liver regeneration after partial hepatectomy in rats accustomed to eating during the first 8h of a daily 12h dark period. 2. The content of 5-CH3–H4folate was drastically decreased during the first hours of regeneration. 3. The total HCO–H4folate coenzymes showed a constant decrease during the first 3 days of regeneration, and a continuous interconversion between 5-HCO–H4folate and 10-HCO–H4folate. 4. 10-HCO–H4folate synthetase, serine hydroxymethyl-transferase and 5,10-CH2–H4folate dehydrogenase activities were relatively low during the first hours after the operation, and increased only several hours later. 5. The increase in enzyme activities showed a stepwise pattern, apparently due to an interaction between the regeneration process and the controlled feeding schedules.
Precocious development of glucuronidating and hydroxylating enzymes in chick embryos treated with pituitary grafts
Authors: Wishart Graham J. , Dutton Geoffrey J.
Published: August 2015

1. Initiation of precocious development of UDP-glucuronyltransferase by an endogenous factor is reported for the first time. 2. This development occurs in chick embryo liver and kidney after grafting of the cephalic lobe of chicken pars-distalis pituitary tissue on to the chorioallantoic membrane, and in liver results in a rise in the enzyme activity from virtually zero to ‘adult’ values. Aniline hydroxylase also precociously develops in the liver of grafted embryos, its activity rising from one-third to the full adult value. Specific activities of glucose 6-phosphatase, cytochrome P-450 and NADPH–cytochrome c reductase did not significantly change. 3. The response of the transferase does not require the presence of host pituitary gland nor, apart from 1 day's necessary initiation, the presence of the graft itself. 4. The host becomes competent to respond on the 14th day of incubation; response continues for at least 3 days after removal of the graft, and for 2 days in the isolated liver. Grafting of embryonic pars distalis younger than 17 days does not evoke a response in the host liver. 5. Secretion of the pituitary factor increases suddenly some 24–48h before the naturally developing surge in liver UDP-glucuronyltransferase activity and may be responsible for initiating this rise in vivo. 6. The factor is probably not a growth or luteinizing hormone; its nature and the likelihood of a secondary hormone acting directly on the liver are discussed.
A difference in the incorporation of 14C into hippurate glycine from dl-[2-14C]glutamate and dl-[5-14C]glutamate in guinea pigs
Authors: Rowsell Kathleen V. , Ali Tahsin A. J. , Rowsell Edward V.
Published: August 2015

The specific radioactivity of urinary hippurate glycine was determined after injecting guinea pigs with benzoate and either dl-[2-14C]glutamate or dl-[5-14C]glutamate. The isotope dilution factor for the formation of [14C]glycine was significantly greater (30%) with C-2 labelled glutamate. With either form of labelled glutamate the hippurate glycine was largely carboxyl-group labelled. The observations suggest a route for the incorporation of glutamate carbon into glycine that involves C-5 but not C-2. A hypothesis for glycine biosynthesis from l-glutamate is advanced, consistent with these findings, that includes conversion of l-glutamate to 4-hydroxy-2-oxoglutarate, the scission of the latter to glyoxylate and pyruvate, and the formation of glycine by transamination.
Treatment of immature mice with gonadotrophins. The influence of mouse age on the response of ovarian alkaline phosphatase activities to gonadotrophins
Authors: Bramley Thomas A.
Published: August 2015

Treatment of mice aged 23–25 days with chorionic gonadotrophin induced large amounts of an ovarian alkaline phosphatase activity (phosphatase Ib) kinetically distinct from that of untreated ovaries (phosphatase I). The activities of alkaline phosphatase I and Ib varied with age in untreated mice. Phosphatase Ib appeared when serum luteinizing hormone concentrations increased (days 4–10 and days 35–45), and disappeared when concentrations were low (days 11–35). Injection of human chorionic gonadotrophin induced progressively larger amounts of phosphatase Ib activity between day 19 and day 29. However, gonadotrophin treatment failed to induce this activity on days 10–18 and 30–35. Nevertheless, during the latter period, human chorionic gonadotrophin induced especially large increases in uterine weight. Treatment at different ages with sheep luteinizing hormone plus human pituitary follicle-stimulating hormone induced a pattern of response identical with that induced by human chorionic gonadotrophin, although sheep luteinizing hormone alone was ineffective before 35 days. In contrast, human luteinizing hormone induced a response in the absence of exogenous follicle-stimulating hormone.