bluuebunny/crossref_metadata_2025 huggingface dataset:
The interrelationship of the concentration of hydrogen ions, bicarbonate ions, carbon dioxide and calcium ions in the regulation of renal gluconeogenesis in the rat
Authors: Alleyne George A. O. , Flores Hernando , Roobol Anne
Published: August 2015

1. The interrelationship of acidosis and Ca2+on the stimulation of gluconeogenesis by rat kidney-cortex slices was studied. 2. Ca2+stimulated gluconeogenesis from glutamine, glutamate, 2-oxoglutarate, succinate, malate, pyruvate, lactate and fructose, but not from galactose. 3. The [Ca2+] needed for optimum gluconeogenesis was about 2mm, but at this concentration, acidosis, produced in vitro by a decrease of [HCO3−] in the medium at constant pCO2 or by an increase in pCO2 at constant [HCO3−], did not stimulate gluconeogenesis. 4. In the absence of Ca2+, acidosis (low [HCO3−]) stimulated gluconeogenesis from glutamine, glutamate, 2-oxoglutarate, succinate, malate, pyruvate and lactate but not from fructose or galactose. With succinate as substrate, the stimulatory effect of acidosis (low [HCO3−]) disappeared at Ca2+concentrations above 1.0mm. 5. The [HCO3−] was the most important determinant of the acidosis effect since a decrease of pH caused by an increase in pCO2 did not uniformly stimulate gluconeogenesis, whereas a decrease in [HCO3−] without a change in pH consistently stimulated glucose formation in a way similar to the stimulation produced by acidosis (low [HCO3−]) in the absence of Ca2+. 6. Acidosis in vitro inhibited the rate of decrease of activity of phosphoenolpyruvate carboxylase in slices, and Ca2+caused an increase in the activity of fructose 1-phosphate aldolase. 7. Respiratory acidosis in vitro caused an increase in the activity of phosphoenolpyruvate carboxylase in kidney cortex and an increase in gluconeogenesis from glutamine. 8. Possible points of interaction between Ca2+, H+and HCO3−with the gluconeogenic sequence are discussed.
Ethanol administration and the relationship of malonyl-coenzyme A concentrations to the rate of fatty acid synthesis in rat liver
Authors: Guynn Robert W. , Veloso Dulce , Harris Raymond L. , Lawson J. W. Randolph , Veech Richard L.
Published: August 2015

1. The effect of ethanol on liver fatty acid synthesis was studied in vivo in 24h-starved and ‘meal-fed’ rats (i.e. fed for 3h per day and not ad libitum). 2. In the fed animal3H2O was incorporated into fat at a rate of 0.46μmol of C2 units/min per g wet wt. of liver. Administration of either ethanol (3.2g/kg) or equicaloric amounts of glucose had no effect on the rate of3H2O incorporation into lipid. 3. In the 24h-starved animal, administration of the same dose of ethanol produced an increase in the rate of3H2O incorporation from 0.06 to 0.12μmol of C2 units/min per g fresh wt. after 3h whereas [malonyl-CoA] increased from 0.006 to 0.009μmol/g. Glucose given in amounts equicaloric to ethanol was significantly more lipogenic, increasing both the3H2O incorporation from 0.06 to 0.20μmol of C2 units/min per g and the malonyl-CoA content from 0.006 to 0.013 μmol/g wet wt. at 3h. 4. The decrease in the redox state of free cytoplasm NAD or NADP couples or the changes in content of citrate, glucose 6-phosphate and pyruvate of liver after ethanol administration had no measurable effect on the rate of fatty acid synthesis in vivo. 5. Under the conditions of the experiments there was no significant difference, among any of the groups, in the activity of liver fatty acid synthetase measured in vitro. A double-reciprocal plot of the rate of3H2O incorporation and the total tissue malonyl-CoA concentrations showed a striking relationship. It has been concluded that the rate of fatty acid synthesis in vivo is determined principally by the Vmax. of fatty acid synthetase and the concentration of free malonyl-CoA. 6. It has also been concluded that under the conditions of the present study, the synthesis of fatty acids de novo is unlikely to be an important factor in the increased liver lipid content associated with ethanol administration.
Induction of δ-aminolaevulinate synthetase under environmental-stress conditions
Authors: Amruthavalli E. , Ramasarma T.
Published: August 2015

1. Exposure of rats to environmental-stress conditions of hypobaria, hypoxia and cold did not alter the activity of hepatic δ-aminolaevulinate synthetase. 2. Induction of the enzyme by diethoxycarbonyldihydrocollidine was inhibited when the rats were exposed to hypobaria before or during the treatment with the drug but not after the initial phase when the process of induction was initiated. Neither increased concentration of the drug nor the time of induction had any effect on the inhibition under hypobaria. 3. A period of 12–24h of pre-exposure to hypobaria gave the maximum inhibition, and on longer exposure the inhibitory effect was decreased. 4. The inhibition was not a permanent effect and could be substantially reversed in 12h of withdrawal to ambient pressure. 5. Inhibition of induction was found under hypobaria and hypoxia, but not on exposure to cold. This suggests a specific effect of lack of O2 rather than a general effect of stress. 6. It appears possible that alteration of concentration of endogenous adenine nucleotides may control the process of diethoxycarbonyldihydrocollidine-mediated induction of δ-aminolaevulinate synthetase, since treatment with ATP, cyclic AMP or theophylline produced inhibition similar to that under hypobaria and hypoxia.
Regulation of ‘malic’ enzyme of Solanum tuberosum by metabolites
Authors: Davies D. D. , Patil K. D.
Published: August 2015

A purification of ‘malic’ enzyme from potato is described. The purified enzyme is specific for NADP and requires a bivalent cation for activity. At pH values below 7 the plot of rate versus malate concentration approximates to normal Michaelis–Menten kinetics. At pH values above 7 the plot of rate versus malate concentration is sigmoid. A number of dicarboxylic acids activate the enzyme and remove the sigmoidicity. The enzyme is inhibited by phosphate, triose phosphates and AMP. In general, effectors of the oxidative decarboxylation of malate behave in the same manner in the reductive carboxylation of pyruvate. The response of the enzyme to energy charge is reported and the physiological significance of the response to metabolites is discussed in relation to the proposed role of the enzyme in the control of pH.
A study of the properties of hybrids of oxyhaemoglobin and deoxyhaemoglobin with two porphyringlobin species
Authors: Treffry Amyra , Ainsworth Stanley
Published: August 2015

The fluorescence of porphyringlobin is quenched on adding haemoglobin to its solutions. It is suggested that this result indicates the formation of hybrids (comprising a dimer of porphyringlobin and a dimer of haemoglobin) in which quenching occurs by energy transfer from the porphyrin to the haem groups of the protein. From an analysis of fluorescence quenching, dissociation constants were calculated for the hybrids of oxy- and deoxyhaemoglobin with the fast- and slow-moving porphyringlobin species isolated by chromatography on CM-Sephadex (Treffry & Ainsworth, 1974). The values obtained are: deoxyhaemoglobin–fast-moving porphyringlobin, 0.8X10−9m; deoxyhaemoglobin–slow-moving porphyringlobin, 5X10−10m; oxyhaemoglobin–fast-moving porphyringlobin, 0.8X10−6m; oxyhaemoglobin–slow-moving porphyringlobin, 1.2X10−7m. The rates of reactions of solutions of haemoglobin and porphyringlobin, containing hybrids, with the thiol reagent 4,4′-dithiodipyridine showed that the thiol groups of the hybrids deoxyhaemoglobin–fast-moving porphyringlobin and oxyhaemoglobin–slow-moving porphyringlobin react more slowly than expected on the basis of composition alone: this result indicates that the deoxy and slow-moving conformations are the more stable, imposing themselves partially on to the fast-moving or oxy dimer of the hybrid. Also the rate of the reaction of CO with deoxyhaemoglobin is decreased when slow-moving porphyringlobin is added to its solutions: this is reflected in a movement of the oxygen equilibrium curve of such a mixture to higher oxygen partial pressures. Similar experiments with deoxyhaemoglobin solutions containing fast-moving porphyringlobin, showed an initial increase in the rate of CO uptake. Correspondingly, the oxygen equilibrium curve of the mixture showed an increased affinity for oxygen. Approximate calculations to determine the oxygen equilibria of the hybrids indicate that a functional dimer retains co-operative characteristics even when the dimer accompanying it within the tetramer has the reacted conformation.
Structural features of ring C of 20-oxo steroids and the interaction with cortisone reductase
Authors: White Ian H. , Jeffery Jonathan
Published: August 2015

Kinetic measurements were made with cortisone reductase (20-dihydrocortisone–NAD+ oxidoreductase, EC 1.1.1.53) and a series of substrates which differed in shape, size and electronic character in the region adjacent to C-11, C-14 and C-18. Structural changes at C-11 in these substrates resulted in up to 660-fold changes in the apparent Km value, up to 200-fold changes in the apparent Vmax. value and up to 800-fold changes in the ratio of these kinetic constants. It is suggested that interactions important for substrate function normally occur between the enzyme and the C ring in the region of C-11, that these interactions arise from so-called hydrophobic forces between the generally hydrophobic C ring portion of the substrate and a hydrophobic region of the enzyme, but that when the substrate contains a polar substituent in this portion of the molecule, then polar interactions with polar moieties of the enzyme can also be important. It is further suggested that the part of the enzyme that interacts with the region of C-11 in the substrate is flexible, and that substrate binding involves at least some degree of induced fit.
Cathepsin B1. A lysosomal enzyme that degrades native collagen
Authors: Burleigh Mary C. , Barrett Alan J. , Lazarus Gerald S.
Published: August 2015

1. Experiments were made to determine whether the purified lysosomal proteinases, cathepsins B1 and D, degrade acid-soluble collagen in solution, reconstituted collagen fibrils, insoluble collagen or gelatin. 2. At acid pH values cathepsin B1 released 14C-labelled peptides from collagen fibrils reconstituted at neutral pH from soluble collagen. The purified enzyme required activation by cysteine and EDTA and was inhibited by 4-chloromercuribenzoate, by the chloromethyl ketones derived from tosyl-lysine and acetyltetra-alanine and by human α2-macroglobulin. 3. Cathepsin B1 degraded collagen in solution, the pH optimum being pH4.5–5.0. The initial action was cleavage of the non-helical region containing the cross-link; this was seen as a decrease in viscosity with no change in optical rotation. The enzyme also attacked the helical region of collagen by a mechanism different from that of mammalian neutral collagenase. No discrete intermediate products of a specific size were observed in segment-long-spacing crystalloids (measured as native collagen molecules aligned with N-termini together along the long axis) or as separate peaks on gel filtration chromatography. This suggests that once an α-chain was attacked it was rapidly degraded to low-molecular-weight peptides. 4. Cathepsin B1 degraded insoluble collagen with a pH optimum below 4; this value is lower than that found for the soluble substrate, and a possible explanation is given. 5. The lysosomal carboxyl proteinase, cathepsin D, had no action on collagen or gelatin at pH3.0. Neither cathepsin B1 nor D cleaved Pz-Pro-Leu-Gly-Pro-d-Arg. 6. Cathepsin B1 activity was shown to be essential for the degradation of collagen by lysosomal extracts. 7. Cathepsin B1 may provide an alternative route for collagen breakdown in physiological and pathological situations.
The phospholipid-dependence of uridine diphosphate glucuronyltransferase. Effect of protein deficiency on the phospholipid composition and enzyme activity of rat liver microsomal fraction
Authors: Graham A. B. , Woodcock B. G. , Wood G. C.
Published: August 2015

After force-feeding a protein-free diet to male rats for 5–7 days a substantial (2.4-fold) increase in the specific activity of the liver microsomal enzyme UDP-glucuronyltransferase (EC 2.4.1.17) was observed. A similar activation of the enzyme occurred when rats were fed on a low-protein (5%, w/w, casein) diet for 60 days. Although both the short- and long-term protein-deficient diets decreased the contents of microsomal protein and phospholipid in liver tissue they did not significantly alter the ratio of these major membrane components. Protein deficiency profoundly altered the phospholipid composition of microsomal membranes. The most striking difference in microsomal phospholipid composition between control and protein-deficient rats was their content of lysophosphatides. Whereas microsomal membranes from protein-deficient rats contained significant proportions of lysophosphatidylcholine and lysophosphatidylethanolamine very little or no lysophosphatides were detected in control preparations. Pretreatment of microsomal fractions from normal rats with phospholipase A markedly increased their UDP-glucuronyltransferase activity as did their pretreatment with lysophosphatidylcholine. It is concluded that the quantities of lysophosphatides present in microsomal membranes from protein-deficient rats were sufficient to have caused the increased UDP-glucuronyltransferase activities of these preparations. Evidence is presented suggesting that these changes in microsomal phospholipid composition and UDP-glucuronyltransferase activity caused by protein deficiency reflect changes that occur in vivo. The possible physiological significance of these findings is discussed.
Porphyrins in egg shells (Short Communication)
Authors: With Torben K.
Published: August 2015

T.l.c. of esterified egg-shell porphyrin shows a mixture containing protoporphyrin with admixture of significant amounts of coproporphyrin, pentacarboxylic porphyrin and uroporphyrin and other, unidentified, porphyrins. This points to porphyrin biosynthesis taking place in the oviduct epithelium.
Chemically defined inducers of alkylsulphatases present in Pseudomonas C12B
Authors: Dodgson Kenneth S. , Fitzgerald John W. , Payne William J.
Published: August 2015

When Pseudomonas C12B is grown on nutrient broth to the stationary phase, cell extracts contain two secondary alkylsulphatases (S1 and S2) active towards potassium decan-5-yl sulphate but not towards potassium pentan-3-yl sulphate and one primary alkylsulphatase (P1) active towards sodium dodecan-1-yl sulphate (sodium dodecyl sulphate). When 10mm-sodium hexan-1-yl sulphate is included in the nutrient broth an additional primary alkylsulphatase (P2) is produced. The S1, S2, P1 and P2 enzymes are also present in extracts of cells grown on broth containing the commercial detergent Oronite, together with an additional secondary alkylsulphatase (S3) active towards pentan-3-yl sulphate as well as decan-5-yl sulphate. The P2 primary alkylsulphatase can be induced by a number of primary and secondary alkyl sulphate esters but the induction of the S3 enzyme appears to be a more specific and complex process. Studies on the ability of different fractions separated from Oronite to act as inducers suggest that the combination of a long-chain secondary alkyl sulphate(s) and a long-chain secondary alcohol(s) is responsible for the appearance of the S3 enzyme. Potassium hexadecan-2-yl sulphate or potassium tetradecan-2-yl sulphate, in combination with either hexadecan-2-ol or tetradecan-2-ol, can serve as inducers for the enzyme. Some characteristics of these specific inducer systems have been elucidated.
Isotope-dilution analysis of rate-limiting steps and pools affecting the incorporation of thymidine and deoxycytidine into cultured thymus cells
Authors: Sjostrom Dolores A. , Forsdyke D. R.
Published: August 2015

1. Rat thymus cells were incubated in homologous serum (10%) and medium 199. The effects of various quantities of thymidine or deoxycytidine (0–30μm) on the radioactive labelling of cells with the corresponding radioactive deoxynucleoside were examined. From plots of the reciprocal of the radioactivity incorporated against the total deoxynucleoside concentration (`isotope-dilution plots'), values were obtained for (a) the Vmax. of the rate-limiting step governing incorporation of the deoxynucleoside, and (b) the concentration of the pool of compounds competing with the radioactive deoxynucleoside at that rate-limiting step. From changes in these values under different experimental conditions inferences were drawn on the position and control of the rate-limiting step within intact cells. 2. Isotope-dilution plots for deoxycytidine were linear, whereas plots for thymidine were bimodal, indicating an abrupt increase in both the Vmax. and pool concentration at a critical thymidine concentration (approx. 5μm). The bimodality was removed by amethopterin. The Vmax. determined with deoxy[U-14C]cytidine was approximately equal to the sum of the Vmax. determined with deoxy[5-3H]cytidine and the Vmax. determined with [Me-3H]thymidine at thymidine concentrations above 5μm. 3. The thymidine competitor pool at thymidine concentrations above 5μm was approximately equal to the sum of the deoxycytidine competitor pool and the thymidine competitor pool at thymidine concentrations below 5μm. The pools were independent of cell concentration and dependent on serum concentration. 4. These results were explained on the following basis. Deoxycytidine in serum (16μm) is the major source of both cytosine and, by way of thymidylate synthetase, thymine, in the DNA of thymus cells. Serum deoxycytidine normally maintains a sufficient intracellular concentration of dTTP to inhibit partially the activity of thymidine kinase. When the dTTP concentration is lowered, either by decreasing the concentration of deoxycytidine or by inhibiting thymidylate synthetase, the activity of thymidine kinase increases. The activity of thymidine kinase may also be increased by concentrations of thymidine greater than 5μm, which overcome the inhibition of the enzyme by dTTP. At concentrations of thymidine below 5μm, thymidine kinase limits the rate of labelling with [Me-3H]thymidine and the radioactivity is diluted by a pool of unlabelled thymidine in serum (4μm). At thymidine concentrations greater than 5μm, the activity of DNA polymerase limits the rate of labelling and the radioactivity is diluted both by serum thymidine and, indirectly, by serum deoxycytidine.
Affinity labelling of the binding site of rabbit antibody. Evidence for the involvement of the hypervariable regions of the heavy chain
Authors: Fisher Christopher E. , Press Elizabeth M.
Published: August 2015

The binding sites of rabbit antibodies with affinity for the haptenic group 4-azido-2-nitrophenyl-lysine have been specifically labelled by photolysis of the hapten–antibody complex. The extent of covalent labelling was 0.5–0.9mol of hapten bound/mol of antibody and, by using an immunoadsorbent, antibody with 1.3mol of hapten/mol was obtained. The antibody was specifically labelled in the binding site and the ratio of labelling of heavy and light chains was in the range 3.3–5.0. The labelled heavy chains were cleaved by CNBr treatment and after reduction and alkylation of the intrachain bonds, were digested with trypsin. Evidence is presented that two regions of the heavy chain, positions 29–34 and 95–114, together contain about 80% of the label on the heavy chain; these two regions respectively include two of the hypervariable regions of rabbit heavy chain.
Structural investigations on the lignin–carbohydrate complexes of Lolium perenne
Authors: Morrison Ian M.
Published: August 2015

1. Lignin–carbohydrate complexes isolated from leaf blade, leaf sheath and stem tissue of ryegrass by extraction with dimethyl sulphoxide were examined by fractionation procedures. Although the complexes are heterogeneous, heterogeneity is shown only in the ratio of the individual monosaccharide residues and not in the ratio of lignin to carbohydrate. 2. The molecular weight of the complexes is high (≥150000), but chemical modification by alkaline hydrolysis, borohydride reduction or lead tetra-acetate oxidation does not drastically decrease it. Low-molecular-weight fragments released by alkaline treatment were shown to contain acetic acid, ferulic acid and p-coumaric acid. 3. On the basis of the chemical stability of the complexes, it is postulated that at least three types of bonding may be present between lignin and carbohydrate, namely one cleaved on borohydride reduction, another cleaved by alkali and a linkage resistant to alkali. 4. The carbohydrate portion of the complexes is composed of β-(1→4)-linked d-glucose residues (cellulose) and β-(1→4)-linked chains of xylose residues. Side chains involving arabinose and galactose residues are linked to C-3 of some of the xylose residues. 5. How the components of the complexes are held together is not certain, but it is suggested that the phenolic acids may act as cross-linking agents.
Stimulation of microsomal uridine diphosphate glucuronyltransferase by glucuronic acid derivatives
Authors: Vessey Donald A. , Zakim David
Published: August 2015

The glucuronic acid adducts of 1-naphthol, 2-naphthol and 4-methylumbelliferone activate microsomal UDP-glucuronyltransferase (EC 2.4.1.17) when the enzyme is assayed with p-nitrophenol as aglycone. Phenyl glucuronide and oestriol 3β-glucuronide also activate UDP-glucuronyltransferase. but to a lesser extent. Activation by glucuronides is not dependent on metal ions, but is blocked by prior treatment of microsomal fractions with p-chloromercuribenzoate. The kinetic mechanism of activation is concluded to be an increase in the affinity of the enzyme for UDP-glucuronic acid. Activation by 1-naphthyl glucuronide, at high concentrations of p-nitrophenol, is not affected by 1-naphthol. Apparently 1-naphthyl glucuronide activates the preparation by binding at a site that is separate from the site of glucuronidation of 1-naphthol. Further evidence for the existence of distinct effector sites for the glucuronides was provided by the finding that activation by glucuronides is inhibited competitively by aglycone glucosides. These glucosides do not inhibit the rate of glucuronidation of p-nitrophenol in the absence of glucuronide adducts, nor do they alter the rate of glucuronidation of 1-naphthol. When UDP-glucuronyltransferase is assayed with 1-naphthol as aglycone it is activated by p-nitrophenyl glucuronide, 4-methyl-umbelliferyl glucuronide and under appropriate conditions by its own glucuronide. These activations are similarly inhibited by aglycone glucosides. p-Nitrophenyl glucuronide also stimulates the rate of glucuronidation of o-aminophenol, o-aminobenzoate and bilirubin.
The interaction of α2-macroglobulin with proteinases. Binding and inhibition of mammalian collagenases and other metal proteinases
Authors: Werb Zena , Burleigh Mary C. , Barrett Alan J. , Starkey Phyllis M.
Published: August 2015

1. Experiments were performed to determine whether the specific collagenases and other metal proteinases are bound and inhibited by α2-macroglobulin, as are endopeptidases of other classes. 2. A specific collagenase from rabbit synovial cells was inhibited by human serum. The inhibition could be attributed entirely to α2-macroglobulin; α1-trypsin inhibitor was not inhibitory. α2-Macroglobulin presaturated with trypsin or cathepsin B1 did not inhibit collagenase, and pretreatment of α2-macroglobulin with collagenase prevented subsequent reaction with trypsin. The binding of collagenase by α2-macroglobulin was not reversible in gel chromatography. 3. The collagenolytic activity of several rheumatoid synovial fluids was completely inhibited by incubation of the fluids with α2-macroglobulin. 4. The collagenase of human polymorphonuclear-leucocyte granules showed time-dependent inhibition by α2-macroglobulin. 5. The collagenolytic metal proteinase of Crotalus atrox venom was inhibited by α2-macroglobulin. 6. The collagenase of Clostridium histolyticum was bound by α2-macroglobulin, and inhibited more strongly with respect to collagen than with respect to a peptide substrate. 7. Thermolysin, the metal proteinase of Bacillus thermoproteolyticus, was bound and inhibited by α2-macroglobulin. 8. It was shown by polyacrylamidegel electrophoresis of reduced α2-macroglobulin in the presence of sodium dodecyl sulphate that synovial-cell collagenase, clostridial collagenase and thermolysin cleave the quarter subunit of α2-macroglobulin near its mid-point, as do serine proteinases. 9. The results are discussed in relation to previous work, and it is concluded that the characteristics of interaction of the metal proteinases with α2-macroglobulin are the same as those of other proteinases.
Purification and properties of malyl-coenzyme A lyase from Pseudomonas AM1
Authors: Hacking A. J. , Quayle J. R.
Published: August 2015

1. Malyl-CoA lyase was purified 20-fold from extracts of methanol-grown Pseudomonas AM1. 2. Preparations of the enzyme were essentially homogeneous by electrophoretic and ultracentrifugal criteria. 3. Malyl-CoA lyase has a molecular weight of 190000 determined from sedimentation-equilibrium data. 4. Within the range of compounds tested, malyl-CoA lyase is specific for (2S)-4-malyl-CoA or glyoxylate and acetyl-CoA or propionyl-CoA. 5. A bivalent cation is essential for activity, Mg2+ or Co2+ being most effective. 6. Malyl-CoA lyase is inhibited by (2R)-4-malyl-CoA and by some buffers, but thiol-group inhibitors are without effect. 7. Optimal activity was recorded at pH7.8. 8. An equilibrium constant of 4.7×10−4m was determined for the malyl-CoA cleavage reaction. 9. The Michaelis constants for the enzyme are: 4-malyl-CoA, 6.6×10−5m; acetyl-CoA, 1.5×10−5m; glyoxylate, 1.7×10−3m; Mg2+, 1.2×10−3m.
The relationship of root-cap slimes to proteins
Authors: Wright Kenneth , Northcote D. H.
Published: August 2015

1. The patterns of incorporation of radioactivity from d-[U-14C]glucose into the pectic components of sections of sycamore roots changed so that sections nearer the tip incorporated relatively more label into arabinose and galactose compared with uronic acid. 2. Radioactive maize root-cap slime was prepared and found to contain three water-soluble component polymers which were electrophoretically (i) neutral, (ii) weakly acidic and (iii) strongly acidic at pH6.5. The neutral component was a glucan. The other components, which could be degraded by trans-elimination, consisted of an acidic backbone chain composed of galacturonic acid and glucose, attached to which were different proportions of neutral sugars. Arabinose, galactose and fucose, the main neutral sugars of the weakly and strongly acidic materials, were absent from the neutral fraction. 3. Fucose was a major sugar in maize-root slime and in a slime of similar composition synthesized by a maize callus of shoot origin. Only trace amounts were found in sycamore, pea and wheat root tips, and in pectin prepared from maize roots and coleoptiles. A high proportion of fucose is therefore a chemical characteristic of maize slime, and slime synthesis indicated a state of differentiation of the tissue. 4. The similarity between the slime and pectin is discussed; slime is a form of pectin modified in such a way as to provide a hydrated protective coating around the root tip.
Mitochondrial and cytoplasmic ribosomes. Distinguishing characteristics and a requirement for the homologous ribosomal saltextractable fraction for protein synthesis
Authors: Avadhani N. G. , Buetow D. E.
Published: August 2015

1. Mitochondrial and cytoplasmic ribosomes of Euglena gracilis differ in their total RNA and protein content. 2. Mitochondrial ribosomes dissociate to subunits at higher Mg2+concentrations than do cytoplasmic ribosomes. 3. A separable 5S RNA is obtained from cytoplasmic and chloroplast ribosomes, but not from mitochondrial ribosomes. 4. For protein-synthesizing activity with a natural mRNA, mitochondrial ribosomes use tRNA from any cell compartment and are partly active with supernatant enzymes from cytoplasm. Cytoplasmic ribosomes are partly active with enzymes and tRNA from mitochondria or chloroplasts. 5. Both mitochondrial and cytoplasmic ribosomes show high specificity for the homologous salt-extractable ribosomal fraction for protein-synthesizing activity.
The effect of nickel on secretory systems. Studies on the release of amylase, insulin and growth hormone
Authors: Dormer Robert L. , Kerbey Alan L. , McPherson Margaret , Manley Susan , Ashcroft Stephen J. H. , Schofield J. George , Randle Philip J.
Published: August 2015

The effects of Ni2+ on the release of amylase from rat parotids, insulin from mouse pancreatic islets and growth hormone from bovine pituitary slices were investigated. In all these secretory systems, Ni2+ was shown to inhibit release evoked by a variety of stimuli both physiological and pharmacological. Measurements of rates of substrate oxidation and tissue concentrations of ATP and 3′:5′-cyclic AMP suggest that this inhibitory action of Ni2+ does not arise through an effect on energy metabolism or cyclic AMP metabolism. It is concluded that although some effects of Ni2+ may involve antagonism between Ni2+ and Ca2+ in stimulus–secretion coupling, others appear to be independent of Ca2+. It is suggested that Ni2+ may block exocytosis by interfering with either secretory-granule migration or membrane fusion and microvillus formation. The possible mode of action of Ni2+ and its potential use as a tool in the study of exocytosis are discussed.
Cytotoxic effects of streptozotocin and N-nitrosomethylurea on the pancreatic B cells with special regard to the role of nicotinamide–adenine dinucleotide
Authors: Gunnarsson Rolf , Berne Christian , Hellerström Claes
Published: August 2015

The effects on the pancreatic B cell of streptozotocin and its aglucone derivative N-nitrosomethylurea were investigated in obese–hyperglycaemic mice and their lean littermates. Both streptozotocin and N-nitrosomethylurea were found to be B-cytotoxic although N-nitrosomethylurea produced less islet damage. Both substances decreased the concentrations of NAD+ in the islet cells to about 10% of the control values within 2h after injection. This NAD+ depletion was prevented by injection of nicotinamide 10min after the administration of streptozotocin or N-nitrosomethylurea. In islets taken from animals 10min after injection of streptozotocin or N-nitrosomethylurea there was no stimulatory effect of glucose on the respiration or insulin release and the oxidation of glucose was markedly decreased. Addition of nicotinamide (10mm) to the incubated islets restored glucose stimulation of both the oxygen consumption and insulin release. It is concluded that islet NAD+ depletion is probably important for the B-cytotoxin action of N-nitrosomethylurea and streptozotocin. The glucose residue in the streptozotocin molecule may potentiate the B-cytotoxic action of this drug in mice.
An electron-capture gas–liquid-chromatographic method for the determination of prostaglandin F2α in biological fluids
Authors: Wickramasinghe Asoka J. F. , Shaw Robert S.
Published: August 2015

A sensitive electron-capture gas–liquid-chromatographic method for the determination of sub-nanogram quantities of prostaglandin F2α was developed. The method is based on the sub-microgram scale conversion of the prostaglandin into the electron-capturing pentafluorobenzyl ester, and analysis of the latter as the tris-trimethylsilyl ether. The lower limit of detection was 12.5pg of the ester injected ‘on-column’ as the silylated product. The method was successfully applied to the determination of prostaglandin F2α in monkey plasma. The specificity of the analytical procedure was increased by incorporating a thin-layer chromatographic fractionation before gas–liquid chromatography. The utility of the analytical methodology developed was demonstrated by its application to the determination of plasma concentrations of intact prostaglandin F2α in a Rhesus monkey, after subcutaneous administration of a single dose of prostaglandin F2α. The electron-capture gas–liquid-chromatographic assay is compared with the radioimmunoassay and the gas–liquid-chromatographic–mass-spectrometry assay for the determination of prostaglandin F2α.
The synthesis of peptidoglycan in an autolysin-deficient mutant of Bacillus licheniformis N.C.T.C. 6346 and the effect of β-lactam antibiotics, bacitracin and vancomycin
Authors: Ward J. Barrie
Published: August 2015

The synthesis of peptidoglycan by cell-free membrane and membrane+wall preparations from an autolysin-deficient, β-lactamase-negative mutant of Bacillus licheniformis N.C.T.C. 6346 was studied. The membrane preparation synthesized un-cross-linked polymer, the formation of which was not inhibited by β-lactam antibiotics. Release of d-alanine by the action of d-alanine carboxypeptidase was inhibited variably according to the antibiotic. This inhibition was reversed by neutral hydroxylamine but not by the action of β-lactamases or by washing. Bacitracin inhibited peptidoglycan synthesis, but not the d-alanine carboxypeptidase. Examination of peptidoglycan synthesized in the presence of excess of bacitracin showed that synthesis was not restricted to the addition of one disaccharide-pentapeptide unit at each synthetic site, an average of 2–3 disaccharide-pentapeptide units being added. Peptidoglycan synthesis was three- to four-fold more sensitive to vancomycin than was the release of d-alanine by the action of the carboxypeptidase. Incorporation of newly synthesized peptidoglycan into pre-existing cell wall was studied in membrane+wall preparations. This incorporation was catalysed by a benzylpenicillin- and cephaloridine-sensitive transpeptidase. The concentrations of these antibiotics giving 50% inhibition of incorporation were almost identical with those required to inhibit growth of the bacillus. Inhibition of the transpeptidase was reversed by treatment with β-lactamase or by washing.
The characterization of myosin–product complexes and of product-release steps during the magnesium ion-dependent adenosine triphosphatase reaction
Authors: Bagshaw Clive R. , Trentham David R.
Published: August 2015

Evidence is presented that the myosin subfragment-1–ADP complex, generated by the addition of Mg2+ and ADP to subfragment 1, is an intermediate within the myosin Mg2+-dependent adenosine triphosphatase (ATPase) turnover cycle. The existence of this species as a steady-state intermediate at pH8 and 5°C is demonstrated by fluorescence measurements, but its concentration becomes too low to measure at 21°C. This arises because there is a marked temperature-dependence on the rate of the process controlling ADP dissociation from subfragment 1 (rate=1.4s-1 at 21°C, 0.07s-1 at 5°C). In the ATPase pathway this reaction is in series with a relatively temperature-insensitive process, namely an isomerization of the subfragment-1–product complex (rate=0.055s-1 at 21°C, 0.036s-1 at 5°C). By means of studies on the Pi inhibition of nucleotide-association rates, a myosin subfragment-1–Pi complex was characterized with a dissociation equilibrium constant of 1.5mm. Pi appears to bind more weakly to the myosin subfragment-1–ADP complex. The studies indicate that Pi dissociates from subfragment 1 at a rate greater than 40s-1, and substantiates the existence of a myosin-product isomerization before product release in the elementary processes of the Mg2+-dependent ATPase. In this ATPase mechanism Mg2+ associates as a complex with ATP and is released as a complex with ADP. In 0.1m-KCl at pH8 1.0mol of H+ is released/mol of subfragment 1 concomitant with the myosin-product isomerization or Pi dissociation, and 0.23 mol of H+ is released/mol of subfragment when ATP binds to the protein, but 0.23 mol of H+ is taken up again from the medium when ADP dissociates. Within experimental sensitivity no H+ is released into the medium in the step involving ATP cleavage.
The magnesium ion-dependent adenosine triphosphatase of myosin. Two-step processes of adenosine triphosphate association and adenosine diphosphate dissociation
Authors: Bagshaw Clive R. , Eccleston John F. , Eckstein Fritz , Goody Roger S. , Gutfreund Herbert , Trentham David R.
Published: August 2015

The kinetics of protein-fluorescence change when rabbit skeletal myosin subfragment 1 is mixed with ATP or adenosine 5′-(3-thiotriphosphate) in the presence of Mg2+ are incompatible with a simple bimolecular association process. A substrate-induced conformation change with ΔG0<-24kJ·mol-1 (i.e. ΔG0 could be more negative) at pH8 and 21°C is proposed as the additional step in the binding of ATP. The postulated binding mechanism is M+ATP⇌M·ATP⇌M*·ATP, where the association constant for the first step, K1, is 4.5×103m-1 at I 0.14m and the rate of isomerization is 400s-1. In the presence of Mg2+, ADP binds in a similar fashion to ATP, the rate of the conformation change also being 400s-1, but with ΔG0 for that process being -14kJ·mol-1. The effect of increasing ionic strength is to decrease K1, the kinetics of the conformation change being essentially unaltered. Alternative schemes involving a two-step binding process for ATP to subfragment 1 are possible. These are not excluded by the experimental results, although they are perhaps less likely because they imply uncharacteristically slow bimolecular association rate constants.
Comparison of the biliproteins from two strains of the thermophilic cyanophyte Synechococcus lividus
Authors: MacColl Robert , Edwards Mercedes R. , Mulks Martha H. , Berns Donald S.
Published: August 2015

C-Phycocyanins from two thermophilic strains of Synechococcus lividus that grow within different temperature ranges have been shown to be unalike. The aggregation ability of these two C-phycocyanins in sedimentation-velocity experiments varied dramatically. Surprisingly, the aggregation properties of mesophilic C-phycocyanins were found to lie between those of the two thermophilic proteins. Under identical conditions at pH7.0, one thermophilic protein (Sy I) was composed of 17S and larger aggregates, whereas the other (Sy III) was an almost homogeneous 6S aggregate. Mesophilic C-phycocyanins have a mixture of 6S, 11S and less stable 17S aggregates under these conditions. Amino acid analysis, absorption spectra, immunochemistry and fluorescence polarization all indicated differences in the composition and properties of the thermophilic proteins, which suggest that they have different modes of adaptation to very high temperatures. Allophycocyanins from the two strains of S. lividus were also purified and studied, but unlike the C-phycocyanins no major differences were found between them. Allophycocyanin was homogeneous at pH6.0, with a sedimentation coefficient of 5.54S and mol.wt. 1.03×105, as determined by sedimentation-equilibrium measurements.
The existence of an electrophilic component in the reaction catalysed by triose phosphate isomerase
Authors: Webb Martin R. , Knowles Jeremy R.
Published: August 2015

In the presence of triose phosphate isomerase, the substrate dihydroxyacetone phosphate is reduced stereoselectively by NaBH4. The reduction of enzyme-bound substrate is almost completely or completely stereoselective and occurs about one order of magnitude faster than that in free solution. This acceleration implies a polarization of the carbonyl group when dihydroxyacetone phosphate is bound.
Evaluation of glucose turnover, body mass and recycling with reversible and irreversible tracers
Authors: Katz Joseph , Rostami H. , Dunn Arnold
Published: August 2015

1. Methods are presented for the calculation of rates of synthesis or loss, mean transit time and total body pool of compounds from specific-radioactivity curves, without assuming a multicompartmental model and without fitting the data by exponential expressions. The methods apply to the steady state after either single injection or continuous infusion of a labelled compound. 2. The use of irreversible and reversible tracers and the effects of recycling of carbon on the estimations of the parameters of glucose metabolism are discussed. Methods for quantitatively determining recycling of glucose carbon by the use of glucose doubly labelled with 14C and 3H are presented.
The differential scattering of circularly polarized light by chloroplasts and evaluation of their true circular dichroism
Authors: Gregory Richard P. F. , Raps Shirley
Published: August 2015

Chloroplasts isolated from pea leaves display an intense circular dichroism in the range 600 to 720nm. Circularly polarized light is also differentially scattered by chloroplasts, and this effect can be confused with circular dichroism. By using an instrumental modification it was possible to distinguish, and record separately, the ellipticities of the transmitted light (circular dichroism) and of the scattered light in the same c.d. instrument. By means of a light-scattering apparatus, the intensity of unpolarized light scattered by chloroplasts was measured as a function of wavelength and of angle. This measurement allowed the aforementioned ellipticities to be corrected for mutual interference. At a concentration of 4μg of chlorophyll/ml (the optimum practical concentration of chloroplasts at which there was no significant interaction of scattering and absorption effects) spectra of true circular dichroism (circular differential absorption) and circular differential scattering were obtained. The former showed maxima, positive at 688nm and negative at 676nm, with an intensity Δθ′8.3m°·litre·(mg of chlorophyll)-1·cm-1. The latter had a maximum at 683nm with an intensity of +47m° with respect to the solvent baseline; this value is independent of the concentration of chloroplasts in dilute suspensions. It is suggested that the intense circular dichroism of chloroplasts reflects specific chlorophyll–chlorophyll interactions in the light-harvesting pigment. The advantages of this method for determining the c.d. of scattering suspensions over those of other investigators are discussed.
The metabolic effects of sodium dichloroacetate in the starved rat
Authors: Blackshear Perry J. , Holloway Paul A. H. , Albert K. George M. M.
Published: August 2015

1. Sodium dichloroacetate (300mg/kg body wt. per h) was infused in 24h-starved rats for 4h. 2. Blood glucose decreased significantly, an effect that had previously only been noted in diabetic animals 3. Plasma insulin concentration decreased by 63% blood lactate and pyruvate concentrations decreased by 50 and 33%, whereas concentrations of 3-hydroxybutyrate and acetoacetate increased by 81 and 73% respectively. 4. Livers were freeze-clamped at the end of the 4h infusion. There were significant decreases in hepatic [glucose], [glucose 6-phosphate], [2-phosphoglycerate], the [lactate]/[pyruvate] ratio, [citrate] and [malate], and also [alanine], [glutamate] and [glutamine], suggesting a diminished supply of gluconeogenic substrates. 5. Animals subjected to a functional hepatectomy at the end of 2h infusions showed no difference in blood-glucose disappearance but a highly significant decrease in the rate of accumulation of lactate, pyruvate, glycerol and alanine, compared with control animals. Dichloroacetate decreased ketone-body clearance. 6. After functional hepatectomy an increase in glutamine accumulation appeared to compensate for the decrease in alanine accumulation. 7. It is concluded that dichloroacetate causes hypoglycaemia by decreasing the net release of gluconeogenic precursors from extrahepatic tissues while inhibiting peripheral ketone-body uptake. 8. These findings are consistent with the activation of pyruvate dehydrogenase (EC 1.2.4.1) in rat muscle by dichloroacetate previously described by Whitehouse & Randle (1973).
Pathways of gluconeogenesis from l-serine in the neonatal rat
Authors: Snell Keith
Published: August 2015

Gluconeogenesis from amino acid precursors was increased in the perfused liver of neonatal rats compared with that of adults. Quinolinate (5mm) was less inhibitory to glucose formation from serine and hydroxyproline in the neonatal rat than in the adult, suggesting that the main route for glucose synthesis from these precursors in neonatal liver does not involve pyruvate as an intermediate.
Breakdown of phosphatidylinositol provoked by muscarinic cholinergic stimulation of rat parotid-gland fragments
Authors: Jones Lynne M. , Michell Robert H.
Published: August 2015

When rat parotid fragments that had been labelled with32P in vivo were exposed to high concentrations of acetylcholine, radioactivity was lost from phosphatidylinositol but not from other phospholipids. Simultaneously the concentration of phosphatidylinositol in the tissue decreased. If previously unlabelled tissue was incubated with32Pi an increase in incorporation of radioactivity into phosphatidylinositol was observed during this decrease in concentration. The effects of acetylcholine were blocked by atropine, but not by tubocurarine. The response to acetylcholine was rapid, with up to one-third of the tissue's phosphatidylinositol disappearing within 5min. Similar effects were evoked by stimulation with methacholine and by high concentrations of tetramethylammonium ion; these responses were also atropine-sensitive and tubocurarine-insensitive. It is concluded that the event in inositol lipid metabolism that is affected by acetylcholine stimulation is removal of the phosphorylinositol group from the molecule; this is mediated through muscarinic cholinergic receptors. This is followed by a compensatory increase in the rate of synthesis of phosphatidylinositol, which has been described in detail in the past. These observations are compared with those of previous workers and are discussed in relation to the existing hypotheses relating to the significance of stimulus-provoked phosphatidylinositol turnover.
Influence of ascorbic acid on ribosomal patterns and collagen biosynthesis in healing wounds of scorbutic guinea pigs
Authors: Harwood Richard , Grant Michael E. , Jackson David S.
Published: August 2015

Scorbutic guinea pigs were wounded and the influence of administering ascorbic acid 6 days later was studied with respect to cellular morphology, ribosomal distribution and protein synthesis. Electron-microscopic studies revealed that the dilated endoplasmic reticulum observed in the fibroblasts of scorbutic wound tissue had reverted to a normal configuration 24h after intraperitoneal injection of 100mg of ascorbate. Quantitative determination of the distribution of free and membrane-bound ribosomes indicated a significant increase in membrane-bound ribosomes in wound tissue from ascorbate-supplemented (recovery) animals. Sucrose-density-gradient centrifugation indicated a significant increase in the proportion of large membrane-bound polyribosomes in the range 300–350S and a concomitant decrease in 80S monoribosomes in the ribosome sedimentation profile of recovery tissue. Determination of the synthesis of non-diffusible [3H]hydroxyproline in scorbutic and recovery wounds showed a 3–4-fold stimulation in peptidyl-proline hydroxylation in recovery tissues. Studies carried out in which scorbutic and recovery tissues were incubated with [14C]leucine indicated that general protein synthesis, as measured by14C incorporated into non-diffusible material/μg of DNA, was unaltered by ascorbate supplementation. Similar studies of [3H]proline incorporation suggested that in recovery tissues there was a small but significant increase in [3H]proline incorporated/μg of DNA, which probably represents an increase in protocollagen synthesis. This observation correlates well with the increase seen in recovery tissues of large polyribosomes on which collagen precursor polypeptides are known to be synthesized. Preliminary characterization of the repair collagen synthesized by recovery animals showed it to be a typical Type I collagen having the chain composition (α1)2α2. The extent of glycosylation of the hydroxylysine of the newly synthesized collagen was greater than that reported for either normal guinea-pig dermal collagen or dermal scar collagen.