bluuebunny/crossref_metadata_2025 huggingface dataset:
Effect of 3-mercaptopicolinic acid on gluconeogenesis and gluconeogenic metabolite concentrations in the isolated perfused rat liver
Authors: Goodman M N
Published: August 2015

3-Mercaptopicolinic acid inhibited gluconeogenesis from lactate and alanine, but not dihydroxyacetone, in the perfused rat liver. Hepatic metabolite concentrations suggested that gluconeogenesis was inhibited at phosphoenolpyruvate carboxykinase. The compound is very effective at low concentrations, and seems an ideal agent for use in studying metabolic regulation involving gluconeogenesis and anaplerotic mechanisms.
Glucocorticoids and the regulation of phosphoenolpyruvate carboxykinase (guanosine triphosphate) in the rat
Authors: Gunn J M , Hanson R W , Meyuhas O , Reshef L , Ballard F J
Published: August 2015

The effect glucocorticoids on the synthesis and degradation of phosphoenolpyruvate carboxykinase (GTP)(EC4.1.1.32) in rat liver and kidney in vivo was studied immunochemically. The glucocorticoid analogue triamcinolone (9α-fluoro-11β, 21-dihydroxy-16α,17α-isopropylidenedioxypregna-1,4-diene-3,20-dione) increased the synthesis rate of the kidney enzyme in starved animals. Both triamcinolone and cortisol decreased the synthesis rate of hepatic phosphoenolpyruvate carboxykinase (GTP) in fed and starved rats, but were without effect on the degradation rate of the enzyme. This effect of triamcinolone in liver was reversed by injection of dibutyryl cyclic AMP. However, in diabetic animals glucocorticoids increased the synthesis rate of hepatic phosphoenolpyruvate carboxykinase (GTP). Triamcinolone administration to starved rats in vivo is shown to cause an increase in the portal blood concentrations of insulin and glucose. Since the physiological de-inducer of liver phosphoenolpyruvate carboxykinase (GTP) is insulin, this is the probable cause of the decrease in the synthesis rate of the hepatic enzyme noted when glucocorticoids are administered to non-diabetic animals.
Pectin synthesis during the wall regeneration of plasmolysed tobacco leaf cells
Authors: Boffey S A , Northcote D H
Published: August 2015

1. Discs of tobacco leaf lamina were floated on media containing D-[U-14C]glucose. Glucose uptake was mainly through the cut edges, and diffusion through the veins and intracellular spaces was slow. 2. Radioactivity was detected in all polysaccharide fractions extracted from the discs, including those associated with the wall. 3. Plasmolysis in sorbitol or KCL decreased the incorporation of radioactive material into all fractions. 4. Incorporation of arabinose into pectins was increased or unaffected by plasmolysis, but the incorporations of other sugars were decreased. Removal of the lower epidermis did not affect this result. 5. Seperate mechanisms for arabinan and polygalacturonorhamman syntheses must exist, and these must differ in their responses to the physiochemical, structural and organizational changes that accompany plasmolysis.
The autotrophic growth of Micrococcus denitrificans on Methanol
Authors: Cox R B , Quayle J R
Published: August 2015

Ribulose bisphosphate carboxylase is present at a high specific activity in extracts of methanol-grown Microccus denitrificans. Enzymic and physiological evidence indicates that, during growth on methanol, the ribulose bisphosphate cycle is the route of carbon assimilation.
A rapid NAD+-linked assay for microsomal uridine diphosphate glucuronyltransferase of rat liver and some observations on substrate specificity of the enzyme
Authors: Mulder G J , van Doorn A B
Published: August 2015

1. A new and rapid continuous assay of rat liver microsomal UDP-glucuronyltransferase (EC 2.4.1.17) has been developed. It is based on measurement of UDP production from UDP-glucuronate during the glucuronidation reaction; UDP production was continuously measured by coupling it to the conversion of NADH into NAD+ through pyruvate kinase and lactate dehydrogenase. This assay is independent of the acceptor substrate used; several findings confirm its applicability. 2. The glucuronidation rate of a series of phenol derivatives was determined with this assay, by using a Triton X-100-activated microsomal preparation as enzyme source. Conjugation of a series of nitrophenol derivatives was also investigated by the ‘classical’ assay (measurement of disappearance of the yellow colour of the nitrophenol during glucuronidation). The substrate with the highest conversion rate was 3-methyl-2-nitrophenol. 3. Both electron releasing and electron withdrawing ring substituents increased the glucuronidation rate of the phenol derivatives, as compared with phenol. 4. Lipid solubility seems important for determining the conversion rate: poorly lipid-soluble substrates were glucuronidated only at a low rate and high lipid solubility seems to be a prerequisite for high conversion rate. Glucuronidation of poorly lipid-soluble compounds may be limited by diffusion. 5. The consequences of these findings for the interpretation of studies on heterogeneity of the enzyme are discussed.
The separation of bovine brain β-N-acetyl-D-hexosaminidases. Abnormal gel-filtration behaviour of β-N-acetyl-D-glucosaminidase C
Authors: Overdijk B , van der Kroef W M J , Veltkamp W A , Hooghwinkel G J M
Published: August 2015

Bovine brain tissue was extracted and the 50 000g supernatant was separated by electrophoresis, DEAE-Sephadex chromatography and gel filtration on Sephadex G-200 and Bio-Gel P-200. The electrophoretic separation showed that the β-N-acetyl-D-hexosaminidases (hexosaminidases) of bovine brain tissue were composed of four different fractions. Two fractions (A and B) exerted both glucosaminidase and galactosaminidase activity, a third fraction (C) showed only glucosaminidase activity, whereas a fourth form (D) with specificity towards the galactosaminide moiety was found to be present. DEAE-Sephadex chromatography at pH 7.0 showed that the B form was eluted with the void volume, whereas the A and D forms could be eluted in one peak by raising that salt concentration. The C form could not be detected in the eluate. Gel filtration on Sephadex G-200 showed that the B, A and D forms had almost equal molecular weights. In this case also the C form could not be detected in the column eluates. Gel filtration on Bio-Gel P-200 revealed that the C form was eluted with the void volume.
Reversible modification of pig heart mitochondrial malate dehydrogenase by pyridoxal 5′-phosphate
Authors: Chen S S , Engel P C
Published: August 2015

1. Pig heart mitochondrial malate dehydrogenase incubated with pyridoxal 5′-phosphate at pH 8.0 and 25 degrees C gradually loses activity. Such inactivation can be largely reversed by dialysis or by addition of L-lysine or L-cysteine, and can be made permanent by NaBH4 reduction. 2. Modification of malate dehydrogenase with pyridoxal 5′-phosphate at 35 degrees C involves two phases, an initial inactivation which is reversible and a slower irreversible second stage. 3. The initial reaction between pyridoxal 5′-phosphate and malate dehydrogenase appears to involve reversible formation of a Schiff base with the epsilon-amino group of a lysine residue. 4. Inactivation of malate dehydrogenase by pyridoxal 5′-phosphate at 10 degrees C involves only the reversible reaction. 5. At 10 degrees C repeated cycles of treatment with pyridoxal 5′-phosphate and NaBH4 reduction lead to a stepwise decline in residual activity. 6. Apparent Km values for malate and NAD+ are unaltered in the partially inactivated enzyme. 7. NAD+ and NADH give only partial protection against pyridoxal 5′-phosphate inactivation. Substrates give no effect.
Radioimmunoassay of colchicine
Authors: Boudene C , Duprey F , Bohuon C
Published: August 2015

Antibodies to cholchicine were raised in rabbits after injection of N-desacetylthiocolchicine conjugated to bovine serum albumin. Antisera diluted 1:100 bound 50% of 1.6ng of [3H]colchicine. Cross-reaction of the antisera with colchicine derivatives and other drugs was measured and a radioimmunoassay of colchicine in biological fluids was proposed.
The comparative specificity of three oestradiol-binding proteins. Rat α-foetoprotein, rat liver 17β-hydroxy steroid dehydrogenase and anti-(oestradiol-6-carboxymethyloxime-bovine serum albumin) antiserum
Authors: Laurant C , de Lauzon S D , Cittanova N , Nunez E , Jayle M F
Published: August 2015

1. The specificity of 3 oestradiol-binding proteins was studied. Two of these proteins are naturally occurring (rat α-foetoprotein and rat liver microsomal 17β-hydroxy steroid dehydrogenase) and the third is an artificially induced model, anti-(oestradiol-6-carboxymethyloxime-bovine serum albumin) γ-globulins. 2. A specific binding procedure for each protein model permitted a determination of its affinity for oestradiol and for 30 other steroids. 3. The results obtained have brought to light the different areas of the steroid molecule that are important for its recognition by each of the three proteins. The two naturally occurring proteins (α-foetoprotein and 17β-hydroxy steroid dehydrogenase) recognize the edge of the steroid defined by C-4, C-6, C-8 and C-15. On the other hand, the γ-globulins recognize the opposite edge, i.e. that defined by C-2, C-10, C-11 and C-17. 4. Diethylstilboestrol, whose structure is analogous to that of a steroid, is only recognized by the two naturally occurring proteins.
Intramolecular electron transport in human ferroxidase (caeruloplasmin)
Authors: De Ley M , Osaki S
Published: August 2015

The oxidation of reduced human ferroxidase by molecular O2 was studied in a stopped-flow spectrophotometer. It was shown that the two type 1 copper atoms behave differently in the absence of iron. The effect of iron on the kinetic parameters was investiagted. A working model for intramolecular electron transport in the enzyme is proposed.
The effects of glucose and of potassium ions on the interconversion of the two forms of glycogen phosphorylase and of glycogen synthetase in isolated rat liver preparations
Authors: Hue L , Bontemps F , Hers H
Published: August 2015

1. In the isolated perfused rat liver, increasing glucose concentration from 5.5 to 55 mm in the perfusion medium caused a sequential inactivation of glycogen phosphorylase and activation of glycogen synthetase. The latter change was preceded by a lag period which corresponded to the time required to inactivate the major part of the phosphorylase. 2. The same sequence of events was observed in isolated rat hepatocytes incubated at 37°C. In this preparation, the rate of phosphorylase inactivation was greatly increased by increasing the concentration of glucose and/or of K+ ions in the external medium. The same agents also caused the activation of glycogen synthetase, but this effect was secondary to the inactivation of phosphorylase. 3. In both types of preparations, the rate of synthetase activation was modulated by the residual amount of phosphorylase a that remained after the initial phase of rapid inactivation and was independent of glucose concentration. 4. In isolated hepatocytes, the rate of conversion of glucose into glycogen was propotional to the activity of synthetase a in the preparation. This conversion was preceded by a lag period which could be shortened by increasing either glucose or K+ concentration in the medium. The incorporation of labelled glucose into glycogen was simultaneous with a glycogenolytic process which could not be attributed to the activity of phosphorylase a.
Initiation factors in protein synthesis by free and membrane-bound polyribosomes of liver and hepatoma
Authors: Murty C N , Verney E , Sidransky H
Published: August 2015

The activity of initiation factors obtained from free and membrane-bound polyribosomes of liver and of transplantable H5123 hepatoma of rats was investigated by using an assay of protein synthesis in vitro in which poly (U)-directed polyphenylalanine synthesis was measured. Initiation factors of membrane-bound polyribosomes prepared by using the anionic detergent deoxycholate exhibited less activity in incorporating [14C]phenylalanyltRNA into polypetides than did initiation factors of free polyribosomes. However, when membrane-bound polyribosomes were prepared after using the non-ionic detergent Triton X-100, no significant differences in activities in polyphenylalanine synthesis were observed between the initiation factors of free and membrane-bound polyribosomes. These results suggest that Triton X-100 is preferable to deoxycholate in the isolation of of initiation factors from polyribosomes. Initiation factors, prepared by using Triton X-100, of free polyribosomes of hepatoma exhibited greater activity in the stimulation of polyphenylalanine synthesis than did the initiation factors of free or membrane-bound polyribosomes of host livers or of membrane-bound polyribosomes of hepatomas.
The measurement of protein turnover by density labelling
Authors: Boudet Alain , Humphrey Thomas J. , Davies David D.
Published: August 2015

A method for measuring the rate of protein degradation in plant tissue is described. The method uses density labelling to avoid difficulties associated with compartmentation and recycling of amino acids. Although the technique cannot be readily adapted to measure the rate of degradation of single proteins, it avoids difficulties of interpretation due to enzyme activation or inactivation. Values for the half-life of Lemna minor protein obtained by this method are compared with values obtained by a number of other methods. To obtain satisfactory results it was necessary to improve the method of isopycnic centrifugation in CsCl gradients. A considerable improvement was achieved by using KBr gradients, and the advantages of using KBr rather than CsCl for the separation of density-labelled protein are discussed.
Short Communications. Changes in lectin-induced deoxyribonucleic acid synthesis in cultures of chick-embryo fibroblasts at various stages of development
Authors: Roguet Roland , Bourrillon Roland
Published: August 2015

Concanavalin A and Robinia pseudoacacia lectin decreased [3H]thymidine incorporation into acid-insoluble material of fibroblasts cultured from 6–10-day chick embryos. In contrast, these lectins stimulated [3H]thymidine incorporation in cells from 16-day embryos. These effects are due to neither [3H]thymidine permeability modification nor toxicity of the lectins. The specificity of lectin action was proved by blocking experiments with α-methyl mannopyranoside and with anti-(Robinia lectin) serum.
Some aspects of fatty acid oxidation in isolated fat-cell mitochondria from rat
Authors: Harper R D , Saggerson E D
Published: August 2015

Mitochondrial were prepared from fat-cells isolated from rat epididymal adipose tissues of fed and 48 h-starved rats to study some aspects of fatty acid oxidation in this tissue. The data were compared with values obtained in parallel experiments with liver mitochondria that were prepared and incubated under identical conditions. 2. In the presence of malonate, fluorocitrate and arsenite, malate, but not pyruvate-bicarbonate, facilitated palmitoyl-group oxidation in both types of mitochondria. In the presence of malate, fat-cell mitochondria exhibited slightly higher rates of palmitoylcarnitine oxidation than liver. Rates of octanoylcarnitine oxidation were similar in liver and fat-cell mitochondria. Uncoupling stimulated acylcarnitine oxidation in liver, but not in fat-cell mitochondria. Oxidation of palmitoyl- and octanoyl-carnitine was partially additive in fat-cell but not in liver mitochondria. Starvation for 48 h significantly decreased both palmitoylcarnitine oxidation and latent carnitine palmitoyltransferase activity in fat-cell mitochondria. Starvation increased latent carnitine palmitoyltransferase activity in liver mitochondria but did not alter palmitoylcarnitine oxidation. These results suggested that palmitoylcarnitine oxidation in fat-cell but not in liver mitochondria may be limited by carnitine palmitoyltransferase 2 activity. 3. Fat-cell mitochondria also differed from liver mitochondria in exhibiting considerably lower rates of carnitine-dependent oxidation of palmitoyl-CoA or palmitate, suggesting that carnitine palmitoyltransferase 1 activity may severely rate-limit palmitoyl-CoA oxidation in adipose tissue.
Adenosine deaminase of cultured brain cells
Authors: Trams Eberhard G. , Lauter Carl J.
Published: August 2015

Two types of adenosine deaminase (EC 3.5.4.4) were found in cultured cells of central-nervous-system origin. The predominant and more active enzyme was obtained in soluble form from the cytosol of mouse neuroblastoma (N-18), neonatal hamster astrocytes (NN), human oligodendroglioma (HOL) and human astrocytoma (Cox Clone). Particulate adenosine deaminase was probably associated with the plasma membrane. When radioactive adenosine was added to superfusates of monolayer cultures it was rapidly converted into inosine and hypoxanthine. The metabolic conversion required adenosine uptake by the cells, a probable transition through the intracellular ATP pool(s) and a rapid excretion into the superfusate of the catabolic products. We discuss the evidence that points to adenosine and its derivatives as neurohumoral modulators of central-nervous-system function.
Carbohydrate composition of lymphocyte plasma membrane from pig mesenteric lymph node
Authors: Snary D , Allen A K , Faulkes R A , Crumpton M J
Published: August 2015

Pig lymphocyte plasma membrane isolated from mesenteric lymph node contained 69 mug of carbohydrate/mg dry wt., which was made up of neutral sugar, amino sugar and sialic acid in the molar proportions 5:1.7:1. The neutral sugar comprised fucose, ribose, mannose, glucose, galactose and inositol (molar proportions 2:9:11:15:26:1), and the amino sugar glucosamine and galactosamine (molar ratio 2:1). The ribose was most probably derived from RNA. All of the fucose and mannose and almost all of the glucosamine were associated with the membrane protein whereas the membrane lipid contained all of the inositol. The remaining sugars were distributed in various ratios between the protein and lipid fractions.
The theory of the deoxyribonucleic acid - ribonucleic acid hybridization reaction
Authors: Thomou H , Katsanos N A
Published: August 2015

A general equation is derived describing data of DNA-RNA hybridization in the presence of a competing self-annealing reaction of RNA. The well known double-reciprocal relation and the Scatchard equation are shown to be limiting cases of this general equation. Some new hybridization data at various temperatures are presented and analysed by using the new equation. The results can only be explained if we assume that the behavior of DNA towards single RNA molecules is the same as that towards the annealed form, (RNA12. The variation of the equilibrium constant of the hybridization reaction with temperature is small, indicating a small heat of reaction. The maximum amount of hybridized RNA at equilibrium appears to be independent of temperature.
A method for the direct demonstration of the lectin-binding components of the human erythrocyte membrane
Authors: Tanner M J A , Anstee D J
Published: August 2015

1. A method which allows the characterization of lectin-binding components is described. This method should be useful in defining the nature and heterogeneity of these components in cell membranes. 2. The method, which we have used on erythrocyte “ghosts”, involves the fixation of “ghost” components after sodium dodecyl sulphate/polyacrylamide-gel electrophoresis and incubation with purified 125I-labelled lectins. 3. Each of the four lectins used shows an individual pattern of reactivity towards “ghosts” components. Band 3, the major membrane-penetrating glycoprotein, is bound by the lectins from Ricinus communis and Phaseolus vulgaris (phytohaemagglutinin) and by concanavalin A. The major erythrocyte sialoglycoprotein is bound by the lectins from R. communis, P. vulgaris and Maclura aurantiaca. 4. Three of the lectins displays binding for other membrane components, some of which are not demonstratable by conventional protein- and carbohydrate-staining techniques.
Studies by electron-paramagnetic-resonance spectroscopy on the mechanism of action of xanthine dehydrogenase from Veillonella alcalescens
Authors: Dalton H , Lowe D J , Pawlik R T , Bray R C
Published: August 2015

E.p.r- (electron-paramagnetic-resonance) spectroscopy was used to compare chemical environment and reactivity of molybdenum, flavin and iron-sulphur centres in the enzyme xanthine dehydrogenase from Veillonella alcalescens (Micrococcus lactilyticus) with those of the corresponding centres in milk xanthine oxidase. The dehydrogenase is frequently contaminated with small but variable amounts of a species resistant to oxidation and giving a new molybdenum (V) e.p.r. signal, “Resting I”. There is also a “desulpho” form of the enzyme giving a Slow Mo(V) signal, indistinguishable from that of the milk enzyme. Molybdenum of the active enzyme behaves in a manner analogous to that of the milk enzyme, giving a Rapid Mo(V) signal on partial reduction with substrates or dithionite. Detailed comparison shows that molybdenum in each enzyme must have the same ligand atoms arranged in the same manner. As with the milk enzyme, complex-formation between reduced dehydrogenase and purine substrate molecules, presumably interacting at the normal substrate-binding site, modifies the Rapid signal, confirming that such substrates interact near molybdenum. The dehydrogenase-flavin semiquinone signal is identical with that of the oxidase but, in contrast, there is only one iron-sulphur signal. The latter gives an e.p.r. spectrum similar to that of aldehyde oxidase.
Some properties of an alcohol dehydrogenase partially purified from baker's yeast grown without added zinc
Authors: Dickenson C J , Dickinson F M
Published: August 2015

Alcohol dehydrogenase was partially purified from yeast (Saccharomyces cerevisiae) grown in the presence of 20 μM-MnSO4 without added Zn2+ and from yeast grown in the presence of 1.8 μM-MnSO4. The enzyme from yeast grown with added Zn2+ has the same properties as the crystalline enzyme from commercial supplies of baker's yeast. The enzyme from yeast grown without added An2+ has quite different properties. It has a mol.wt. in the region of 72000 and an S 20 w of 5.8S. The values can be compared with a mol.wt. of 141000 and an S 20 w of 7.6S for the crystalline enzyme. ADP-ribose, a common impurity in commercial samples of NAD+, is a potent competitive inhibitor of the new enzyme (K1 = 0.5 μM), but is not so for the crystalline enzyme. The observed maximum rate of ethanol oxidation at pH 7.05 and 25 degrees C was decreased 12-fold by the presence of 0.06 mol of inhibitor/mol of NAD+ when using the enzyme from Zn2+-deficient yeast, but with crystalline enzyme the maximum rate was essentially unchanged by this concentration of inhibitor. The kinetic characteristics for the two enzymes with ethanol, butan-1-ol, acetaldehyde and butyraldehyde as substrates are markedly different. These kinetic differences are discussed in relation to the mechanism of catalysis for the enzyme from Zn2+-deficient yeast.
Bile acids of snakes of the subfamily Viperinae and the biosynthesis of C-23-hydroxylated bile acids in liver homogenate fractions from the adder, Vipera berus (Linn.)
Authors: Ikawa S , Tammar A R
Published: August 2015

1. Analysis of bile salts of four snakes of the subfamily Viperinae showed that their bile acids consisted mainly of C-23-hydroxylated bile acids. 2. Incubations of 14C-labelled sodium cholate (3 α, 7 α, 12 α-trihydroxy-5 β-cholan-24-oate) and deoxycholate (3 α, 12 α-dihydroxy-5 β-cholan-24-oate) with whole and fractionated adder liver homogenates were carried out in the presence of molecular oxygen and NADPH or an NADPH-generating system. The formation of C-23-hydroxylated bile acids, namely bitocholic acid (3 α, 12 α, 23xi-trihydroxy-5 β-cholan-24-oic acid) and 3 α, 7 α, 12 α, 23 ξ-tetrahydroxy-cholanic acid (3 α, 7 α, 12 α, 23 ξ-tetrahydroxy-5 β-cholan-24-oic acid), was observed mainly in the microsomal fraction and partly in the mitochondrial fraction. 3. Biosynthetic pathways of C-23-hydroxylated bile acids are discussed.
A study of the supposed hydroxylation of tyrosine catalysed by peroxidase
Authors: Smith P I , Swan G A
Published: August 2015

The claim that peroxidase (rather than tyrosinase) is the enzyme responsible for the conversion of tyrosine into dopa (3,4-dihydroxyphenylalanine) in melanogenesis was investigated. The spectral changes that occurred during the action of horseradish peroxidase in the presence of H2O2 on dopa, tyrosine and mixtures of dopa with tyrosine or other phenolic compounds were studied. The effect of ascorbic acid or dihydroxyfumaric acid on some of these changes was also investigated. No evidence was found that tyrosine was hydroxylated by peroxidase in the presence of H2O2 and dopa as cofactor, although tyrosine or other phenolic compounds increased the rate of oxidation of dopa to dopachrome (indoline-5,6-quinone-2-carboxylic acid). Peroxidase was, however, effective in oxidizing tyrosine to dopa in the presence of dihydroxyfumaric acid and oxygen.
Purification and some properties of acetyl-coenzyme A carboxylase from rabbit mammary gland
Authors: Manning R , Dils R , Mayer R J
Published: August 2015

1. Acetyl-Coa carboxylase from lactating-rabbit mammary gland was purified to homogeneity by the criterion of polyacrylamide-gel electrophoresis in the presence of sodium dodecyl sulphate. 2. Use of phosphate buffer throughout the purification gave low recovery of enzyme. Consequently, Tris buffers were used in the extraction and in selected stages of the purification procedure. 3. The purified enzyme had a specific activity of 5.15 +/- 0.3 μmol of bicarbonate incorporated/min per mg of protein (mean +/- S.E.M. of five preparations). This represents a purification of 257 +/- 16-fold and a yield of 4.3 +/- 0.13%. 4. The kinetic parameters of the purified enzyme were similar to those reported for the enzyme from other tissue sources. 5. The enzyme was assayed by a spectrophotometric assay and by a [14C]bicarbonate-fixation assay. Short incubation were used in the radio-chemical assay to avoid substantial loss of [14C]bicarbonate.
Isolation of the liver N-aspartyl-β-glucosaminidase in aspartylglucosaminuria
Authors: Savolainen H
Published: August 2015

The isolation of liver N-aspartyl-beta-glucosaminidase in human aspartylglucosaminuria, where this enzyme activity is diminished, yields an enzyme molecule with the same molecular weight and pH optimum as the normal enzyme. Its activity is 10% of that of the control preparation. Combination of both enzymes results in the summation of both activities, and the pathological enzyme does not inhibit the control preparation. It is concluded that no change into a totally different isoenzyme has occurred in aspartylglucosaminuria.
Synthesis and turnover of plasma-membrane proteins and glycoproteins in a neuroblastoma cell line
Authors: Mathews R A , Johnson T C , Hudson J E
Published: August 2015

A kinetic analysis of the appearance of 14C-labelled proteins in the surface membranes isolated from exponentially growing neuroblastoma cells (N2a) showed that the total membrane proteins reached a steady-state specific radioactivity in 18-20 h. However, examination of individual protein bands resolved by sodium dodecyl sulphate-urea-polyacrylamide-gel electrophoresis illustrated that differences in the kinetics of specific surface-membrane proteins could be detected. Although most of the protein bands reached a steady-state specific radioactivity at a time similar to that for total membrane proteins, at least two bands (mol. wt. 180000 and 130000) attained the steady-state within 8-10 h. It was shown by the use of dual-labelling techniques that these two protein bands turned over in the surface membranes of neuroblastoma N2a cells at least 180 and 150% faster than the total membrane protein. These two proteins were glycosylated and located on the outer surface of the cells, since they were labelled with radioactive carbohydrates and readily removed by treatment of the intact neuroblastoma cell with proteinases.
An unusual precursor of 50S ribosomes in a mutant of Escherichia coli
Authors: Markey F , Wild D G
Published: August 2015

Escherichia coli strain 15-28 is a mutant with a defect in ribosome synthesis that leads to the accumulation of large amounts of ribonucleoprotein (“47S”) particles during exponential growth. These particles are precursors to 50S ribosomes, but are distinct from precursors detected by pulse-labelling of the parent strain and also from ribosome precursors that accumulate during inhibition of growth by CoC12. Either ribosome assembly in the mutant differs from that in the wild-type strain, or 47S particles represent a hitherto unstudied stage in the synthesis of 50S ribosomes.
Nature of the facilitated messenger ribonucleic acid transport from isolated nuclei
Authors: Yannarell A , Schumm D E. , Webb T E
Published: August 2015

Cytoplasmic macromolecules were previously identified which regulate both qualitatively and quantitatively the release of messenger-like RNA from isolated nuclei. These macromolecules are now shown to be denatured at 45-50 degrees C and their synthesis is sensitive to pactamycin or cycloheximide. The putative regulatory proteins are essentially quantitatively precipitated with high specificity from the cytosol by streptomycin at a concentration 10-fold higher than that used to precipitate RNA. The nuclear concentration-dependence of RNA transport from successive samples of nuclei strongly suggests that the regulatory factors are recycled. Quantitative changes in the sequences transported at various dilutions of the cytosol suggest that not all the different classes of the putative regulatory macromolecules are present in an effective concentration at any one dilution.
Poly(adenosine diphosphate ribose) polymerase activity and nicotinamide adenine dinucleotide in differentiating cardiac muscle
Authors: Claycomb W C.
Published: August 2015

Poly(ADP-ribose) polymerase activity in nuclei isolated from differentiating cardiac muscle of the rat has been characterized and its activity measured during development. Optimum enzyme activity is observed at pH 8.5. Poly(ADP-ribose) polymerase is inhibited by ATP, thymidine, nicotinamide, theophylline, 3-isobutyl-1-methylxanthine and caffeine and stimulated by actinomycin D. The activity measured under optimal assay conditions increases during differentiation of cardiac muscle and is inversely related to the rate of DNA synthesis and to the activities of DNA polymerase α and thymidine kinase. When DNA synthesis and the activity of DNA polymerase α are inhibited in cardiac muscle of the 1-day-old neonatal rat by dibutyryl cyclic AMP or isoproterenol, the specific activity of poly(ADP-ribose) polymerase measured in isolated nuclei is increased. The concentration of NAD+ in cardiac muscle increases during postnatal development. In the adult compared with the 1-day-old neonatal rat the concentration of NAD+ relative to fresh tissue weight, DNA or protein increased 1.7-fold, 5.2-fold or 1.4-fold respectively. The concentration of NAD+ in cardiac muscle of the 1-day-old neonatal rat can be increased by approx. 20% by dibutyryl cyclic AMP. These data suggest that NAD+ and poly(ADP-ribose) polymerase may be involved with the repression of DNA synthesis and cell proliferation in differentiating cardiac muscle.
A deoxyribonucleic acid-replication intermediate in the growing mosquito
Authors: Kao P C , Beyer C F. , Lang C A.
Published: August 2015

In previous experiments on growth and aging in the yellow-fever mosquito, Aedes aegypti, a low mol. wt. (500000) DNA species was found in the supernatant fraction after ultracentrifugation of homogenates of rapidly-growing larvae. This DNA species, “sDNA”, constituted 30-40% of total DNA in 2-4-day-old larvae, but was less than 5% in older larvae, pupae and adults. We have now isolated and characterized sDNA and initiated experiments to determine its metabolic role. Isolated sDNA has the same physical and chemical characteristics as bulk DNA, “pDNA”, and differs only in size. In CsCl isopycnic centrifugation the buoyant densities of sDNA and pDNA were 1.700 and 1.697 g/cm3 respectively. The “melting” temperature of both DNA species was 84 degrees C. Base compositions calculated from these data and other methods were 38.9 mol% of guanine-plus-cytosine for sDNA, and 38.5 for pDNA. Also, the size of newly-synthesized DNA was investigated by pulse-labelling and pulse-chase experiments. In neutral sucrose gradients the labelled DNA component after a 2h pulse had a sedimentation coefficient of about 8S, but after a 4h pulse sedimented in a broad band from 10-19S. In alkaline sucrose gradients a single peak around 7S was observed for pulse times up to 4h. After a 6h pulse and a 1 day “chase”, labelled DNA species had sedimentation coefficients ranging from 10-15S in alkaline sucrose, and after a 2-day chase the values were 17-31S, similar to those of pDNA under alkaline conditions. These results suggest that sDNA represents an intermediate form in the replication of DNA in mosquito larvae.
The biosynthesis of deoxyribonucleic acid during the life-span of the mosquito
Authors: Mills B J , Lang C A.
Published: August 2015

Previous results from this laboratory [Kao et al, (1976) Biochem. J. 154, 471-480] demonstrated that a low-molecular-weight DNA species, “sDNA”, present in high concentrations during rapid larval growth of the yellow-fever mosquito, Aedes aegypti, may be an intermediate in DNA replication. We have now determined the life-span profiles of synthetic rates and the steady-state contents of sDNA and total DNA by radiometric and biochemical procedures and by using mosquitoes grown and maintained under defined and controlled conditions, including axenic culture and synchronization of stages. 2. Our results showed exponential increases in DNA contents and synthetic rates which paralleled the changes in weight during larval growth. Of special importance was the finding that in rapidly-growing larvae and newly-emerged adult mosquitoes high contents and rates of synthesis of sDNA were correlated with increases in total DNA/organism, which suggests that sDNA content may be an index of DNA replication. In senescent mosquitos a twofold increase in the synthetic rate of bulk DNA (“pDNA”) without an increase in sDNA may reflect repair synthesis. 3. The significance of this investigation is that it may be the first quantitative and systematic description of the synthetic rates and contents of both DNA-replication intermediates and total DNA in an axenic, eukaryotic organism rather than a specific cell type. Equally important is that these findings describe DNA changes during growth and also during aging through senescence in an age-characterized organism.
Development and properties of fructose 1,6-bisphosphatase in the endosperm of castor-bean seedlings
Authors: Youle R J. , Huang A H. C.
Published: August 2015

1. The activity of fructose 1,6-bisphosphatase (EC 3.1.3.11) in the fatty endosperm of castor bean (Ricinus communis) increases 25-fold during germination and then declines. The developmental pattern follows that of catalase, a marker enzyme for gluconeogenesis in this tissue. 2. The enzyme at its peak of development was partially purified, and its properties were studied. It has an optimal activity at neutral pH (7.0-8.0). The apparent Km value for fructose 1,6-bisphosphate is 3.8 × 10-5 M. The activity is inhibited by AMP allosterically with an apparent Ki value of 2.2 × 10-4 M. The enzyme hydrolyses fructose 1,6-bisphosphate and not ribulose 1,5-bisphosphate or sedoehptulose 1,7-bisphosphate. 3. Treatment of the partially purified enzyme with acid leads to an 80% decrease in activity. The remaining activity is insensitive to AMP and has optimal activity at pH 6.7 and a high apparent Km value (2.5 × 10-4 M) for fructose 1.6-bisphosphate. Enzyme extracted from the tissue with water instead of buffer has a similar modification. The effect of acid explains the discrepancies between this report and previous ones on the properties of the enzyme in this tissue. 4. The storage tissues of various fatty seedlings all contain a ‘neutral’ fructose 1,6-bisphosphatase. The activities of the enzyme from some of the tissues are inhibited by AMP. 5. The properties of the enzyme in fatty seedlings and in green leaves are discussed in comparison with that in animal tissues.
The regulation of transport of glucose, gluconate and 2-oxogluconate and of glucose catabolism in Pseudomonas aeruginosa
Authors: Whiting P H. , Midgley M , Dawes E A.
Published: August 2015

1. The induction by glucose and gluconate of the transport systems and catabolic enzymes for glucose, gluconate and 2-oxogluconate was studied with Pseudomonas aeruginosa PAO1 growing in a chemostat under conditions of nitrogen limitation with citrate as the major carbon source. 2. In the presence of a residual concentration of 30mM-citrate an inflowing glucose concentration of 6-8 mM was required to induce the glucose-transport system and associated catabolic enzymes. When the glucose concentration was raised to 20mM the glucose-transport system was repressed, but the transport system for gluconate, and at higher glucose concentrations, that for 2-oxogluconate, were induced. No repression of the glucose-catabolizing enzymes occurred at the higher inflowing glucose concentrations. 3. In the presence of 30mM-citrate no marked threshold concentration was required for the induction of the gluconate-transport system by added gluconate. 4. In the presence of 30mM-citrate and various concentrations of added glucose and gluconate, the activity of the glucose-transport system accorded with the proposal that a major factor concerned in the repression of this system was the concentration of gluconate, produced extracellularly by glucose dehydrogenase. 5. This proposal was supported by chemostat experiments with mutants defective in glucose dehydrogenase. Such mutants showed no repression of the glucose-transport system by high inflowing concentrations, but with a mutant apparently defective only in glucose dehydrogenase, the addition of gluconate caused repression of the glucose-transport system. 6. Studies with the mutants showed that both glucose and gluconate can induce the enzymes of the Entner-Doudoroff system, whereas for the induction of the gluconate-transport system glucose must be converted into gluconate.
Energy coupling to active transport in anaerobically grown mutants of Escherichia Coli K12
Authors: Gutowski S J. , Rosenberg H
Published: August 2015

1. Anaerobic uptake of proline requires either the presence of a coupled Mg2+-stimulated adenosine triphosphatase or anaerobic electron transport. 2. Anaerobic uptake of glutamine does not require anaerobic electron transport even in the absence of a coupled Mg+2-stimulated adenosine triphosphatase. 3. These results support previous suggestions [Berger (1973) Proc. Natl. Acad. Sci. U.S.A. 70, 1514––1518; Berger & Heppel (1974) J. Biol. Chem. 249, 7747-7755; Kobayashi, Kin & Anraku (1974) J. Biochem. (Tokyo) 76, 251-261] that two distinct mechanisms of energy coupling to active transport exist in Escherichia coli in that energization of anaerobic proline uptake requires the ‘high-energy membrane state’, whereas the energization of anaerobic glutamine uptake does not.
Distribution of membranes, especially of plasma-membrane fragments, during zonal centrifugations of homogenates from glucose-repressed Saccharomyces Cerevisiae
Authors: Nurminen T , Taskinen L , Suomalainen H
Published: August 2015

1. The distributions of several enzymes and other marker components were examined after zonal centrifugations of whole homogenates from glucose-repressed Saccharomyces cerevisiae on sucrose and iso-osmotic Ficoll, and the composition and morphology of the fractions were investigated. 2. After high-speed zonal centrifugation most of the protein, acid and alkaline phosphatases, alkaline pyrophosphatase, adenosine monophosphatase, β-fructofuranosidase, α-mannosidase, NADPH-cytochrome c oxidoreductase and an appreciable amount of phospholipid and sterol were non-sedimentable, i.e. were at densities below 1.09 (g/cm3). Most of the RNA was at p=1.06-1.08 in Ficoll and at p=1.09-1.11 in sucrose. 3. The bulk of the Mg2+-dependent adenosine triphosphatase (Mg-ATPase) was coincident with the main peak of phospholipid and sterol, at median density 1.10, which was also rich in smooth-membrane vesicles. In Ficoll, a minor peak of phospholipid and sterol at p-1.12-1.15 contained a smaller part of the oligomycin-insensitive Mg-ATPase and heavy membrane fragments. In sucrose, several minor peaks of Mg-ATPase were in the mitochondrial density range, and a peak of oligomycin-insensitive Mg-ATPase coincident with a minor peak of phospholipid and sterol at around p-1.25 contained heavy membrane fragments of high carbohydrate content, especially mannose. 4. Further purification of the oligomycin-insensitive Mg-ATPase containing membrane preparations was performed on Urografin gradients. 5. It is argued that the oligomycin-insensitive Mg-ATPase containing membranes are fragments of the plasma membrane, but have different densities because they contain different amounts of glycoprotein particles.
The binding of calcium to a salivary phosphoprotein, protein A, common to human parotid and submandibular secretions
Authors: Bennick A
Published: August 2015

The binding of Ca2+ to a previously described phosphoprotein from human parotid saliva, protein A [Bennick (1975) Biochem J. 145, 557-567] was studied by means of equilibrium dialysis. In 5 mM-Tris/HC1 buffer, pH7.5, protein A bound 664nmol of Ca/mg of protein. Km was determined to be 181 muM and the binding of Ca2+ to the protein was non-co-operative. The binding of Ca2+ apparently occurs to side-chain carboxyl groups in the protein, but protein phosphate is of minor if any importance in calcium binding. Hydrolysis of protein A by trypsin and collagenase or heating of the protein at 60 degrees or 100 degrees C did not affect Ca2+ binding. The Ca2+ binding decreases with increased concentration of the dialysis buffer and on the addition of SrCl2, or MgCl2 or MnCl2 to the dialysis buffer. Protein A does not aggregate in the presence of Ca2+, since the s20,w was identical when determined in the presence (1.30S) and absence (1.35S) of CaCl2. By use of a specific antiserum to protein A it was found that protein C [Bennick & Connell (1971) Biochem. J. 123, 455-464] and perhaps minor related components cross-reacted with protein A. No other salivary proteins showed immunological similarity. Proteins A and C were also present in submandibular saliva. The possible functions of protein A are discussed.
Spin-label study of the mobility of enzyme-bound lipoic acid in the pyruvate dehydrogenase multienzyme complex of Escherichia coli
Authors: Ambrose M C. , Perham R N.
Published: August 2015

The lipoic acid residues covalently bound to the transacetylase component of the pyruvate dehydrogenase multienzyme complex of Escherichia coli were selectively modified by reaction with 4-maleimido-2,2,6,6-tetramethylpiperidino-oxyl. The electron-spin-resonance spectrum of the spin-labelled enzyme indicates that the bound nitroxide groups have high mobilities relative to the protein molecule. This physicochemical evidence is consistent with the view that the dithiolane ring of a lipoyl residue is capable of rapid migration between the active sites of the component enzymes in the catalytic mechanism.
Preferential inhibition by poly(adenylic acid) of minus-strand synthesis by bacteriophage Qβ ribonucleic acid replicase
Authors: Kondo M
Published: August 2015

Among the homopolymers examined, poly(A) was found to inhibit preferentially the synthesis of the minus strand by bacteriophage Qβ RNA replicase in the presence of host factor. A specific interaction of poly(A) with the host factor is suggested to be a principal cause for the observed preferential inhibition by poly(A) of the host-factor-requiring Qβ RNA replicase reaction.
A method for the purification of milligram quantities of stable human phosphatidylcholine-cholesterol acyltransferase
Authors: Varma K G , Soloff L A
Published: August 2015

A method for processing 3 litres of human plasma for the purification of phosphatidylcholine-cholesterol acyltransferase is described. The method involves (NH4)2SO4 fractionation, citric acid treatment, and DEAE-cellulose and hydroxyapatite chromatography. At this stage the enzyme preparation is purified approx. 8000-fold. This preparation appears to be free of lipoproteins as determined by immunoelectrophoresis against anti-human serum and is minimally contaminated with albumin (less than 30 mug/mg of enzyme protein) as determined by immunodiffusion. The activity of the enzyme was stable for 4 days, but most of its activity was lost after 20 days On electrophoresis on 5% polyacrylamide gel, a fast-moving band with enzyme activity and a slow-moving band with no enzyme activity was observed. A faint band of albumin was also present. Extracts of enzymically active bands cut from ten gels and then pooled and extracted with 0.15 M-NaC1/4mM-sodium phosphate, pH 7.0, showed a single band on re-electrophoresis on 5% polyacrylamide gel.
The nature of the 5'-linked 5' nucleotide sequence at the 5' end of rabbit globin messenger ribonucleic acid
Authors: Hunt J A , Oakes G N
Published: August 2015

Poly(A)-containing messenger RNA isolated from rabbit reticulocytes as estimated by periodate oxidation and condensation with [3H]isoniazid has two oxidizable end groups per molecule of mol. wt. 220000. When the mRNA is subjected to stepwise degradation by beta-elimination, only one oxidizable end-group is found. This indicates that one of the 2′,3′ hydroxyl end-groups is linked through the normal 3′-5′ phosphodiester bond, but that the other is linked in such a way that after stepwise degradation no new 2′,3 hydroxyl group is revealed. This structure could be a 5′-linked 5′-phospho di- or tri-ester. On digestion with ribonuclease the isoniazid-labelled RNA produced oligonucleotide hydrazones consistent with a poly(A) sequence at the 3′ end plus fragments that are not found after stepwise degradation. These fragments have a charge of -6 and -8 from pancreatic ribonuclease or -7 from ribonuclease T1 digestion. These charges are changed to -3.4 and -4.1 after pancreatic ribonuclease, ribonuclease T2 and alkaline phosphatase digestion. methyl-3H-labelled-poly(A)-containing RNA isolated from late erythroid cells contain a methyl-labelled fragment resistant to endonuclease and phosphodiesterase II digestion. After digestion with phosphodiesterase I this fragment produces methyl-3 H-labelled nucleotides with the electrophoretic mobility of pm7G and pAm. It is concluded that globin mRNA has the 5′ sequences m7G(5′)ppp′AmpYpGp ... and m7G(5′)pppAmpApGpYp.
Dihydrofolate reductase and thymidylate synthetase activities in the liver of rabbit foetuses during the haemapoietic period
Authors: Bourbon J R
Published: August 2015

Dihydrofolate reductase activity did not change with variations of mitotic rate in foetal rabbit haemopoietic tissue, whereas the activity of thymidylate synthetase declined with decrease in cell divisions. Both the activities were normal in the brachydactylia strain, in which a failure in folate metabolism was previously assumed.