bluuebunny/crossref_metadata_2025 huggingface dataset:
The thermal polymerization of orosomucoid
Authors: Spragg S. P. , Halsall H. B. , Flewett T. H. , Barclay Grizel R.
Published: August 2015

1. Orosomucoid was prepared from the urine of a nephrotic patient and polymerized by heating it in a range of salt concentrations at pH4·1. 2. Heating at low ionic strengths produced a ‘chain’ polymer of indefinite length but having the same width as the diameter of the monomer (5·0nm.). Similar treatment in high ionic strengths also produced a spherical (‘ball’) polymer of limited diameter (14·8nm.). 3. The size and shape of both polymers were determined from ultra-centrifuge, gel-filtration and electron-microscope results. The results suggest that eight monomer units condense to form the ball polymer. 4. Heating orosomucoid at pH1·8 hydrolysed the N-acetylneuraminic acid off the molecule; only chains could then be formed, even in high ionic strengths. 5. Both polymers were stable under normal conditions but could be depolymerized in 3m-guanidine hydrochloride. The monomer could be repolymerized on heating: the ‘chain monomer’ only formed chains at all ionic strengths, but the ‘ball monomer’ was indistinguishable from the original monomer in its immunological properties and polymerization reaction.
A comparison of the properties of the phosphofructokinases of the fat body and flight muscle of the adult male desert locust
Authors: Walker P. R. , Bailey E.
Published: August 2015

1. Phosphofructokinase was isolated, and partially purified by ammonium sulphate fractionation, from the fat body and flight muscle of the desert locust. 2. Ammonium sulphate appears to stabilize the enzymes, but does not activate them. 3. Both flight-muscle and fat-body enzymes give sigmoidal hexose monophosphate concentration–activity curves, which are characteristic of regulatory enzymes. 4. At low ATP concentrations both the enzyme activities increase rapidly with increasing ATP concentrations, but above an optimum concentration ATP becomes inhibitory. This optimum concentration is 0·2mm for the fat-body enzyme and 0·1mm for the flight-muscle enzyme. 5. AMP activates both the enzymes; half-maximal activation occurs at 10μm in each case, the effect being independent of substrate concentration. 6. 3′,5′-(cyclic)-AMP (0·5mm) and Pi (1mm) activate the flight-muscle enzyme, but have no effect on the fat-body enzyme. 7. FDP (1mm) inhibits both enzymes, and with the flight-muscle enzyme this inhibition is increased by increasing the ATP concentration. 8. Citrate, phosphoenolpyruvate and α-glycerophosphate have no effect on either enzyme under the assay conditions used. 9. The properties of phosphofructokinases from the locust are compared with those of phosphofructokinases from other sources.
The nature, intergeneric distribution and biosynthesis of isoprenoid quinones and phenols in Gram-negative bacteria
Authors: Whistance G R , Dillon J. F. , Threlfall D R
Published: August 2015

1. Twenty-two aerobically grown Gram-negative bacteria were analysed for demethylmenaquinones, menaquinones, 2-polyprenylphenols, 6-methoxy-2-polyprenylphenols and ubiquinones. 2. All the eight enterobacteria and both the two facultative organisms (Aeromonas punctata and Aeromonas hydrophila) examined contain all the compounds listed above. The principal homologues are octaprenyl; in addition lower (down to tri- or tetra-prenyl for the 2-polyprenylphenols) and sometimes higher homologues are also present. 3. Strict aerobes are of two types, those that contain 2-polyprenylphenols, 6-methoxy-2-polyprenylphenols and ubiquinones, and those that contain ubiquinones only. The principal homologues are generally octa- or nona-prenyl, although one organism (Agrobacterium tumefaciens) has ubiquinone-10 as its principal homologue. As in the enterobacteria, lower homologues of these compounds are also present. 4. In Escherichia coli W, Pseudomonas ovalis Chester and Pseudomonas fluorescens, radioactivity from p-hydroxy[U−14C]benzoic acid is incorporated into 2-polyprenylphenols, 6-methoxy-2-polyprenylphenols, 6-methoxy-3-methyl-2-polyprenyl-1,4-benzoquinones, ubiquinones and a compound tentatively identified as 2-polyprenyl-1,4-benzoquinone. The fact that radioactivity is incorporated into the first three compounds suggests that in these organisms, and indeed in all those Gram-negative bacteria that contain 2-polyprenylphenols and 6-methoxy-2-polyprenylphenols, ubiquinones are formed by a biosynthetic sequence similar to that in Rhodospirillum rubrum. 5. The finding in ‘Vibrio O1’ (Moraxella sp.) and organism PC4 that 2-polyprenylphenols and 6-methoxy-2-polyprenylphenols are chemically and radiochemically undetectable leads to the conclusion that they are not intermediates in the biosynthesis of ubiquinone by these and by other Gram-negative bacteria that do not contain detectable amounts of 2-polyprenylphenols and 6-methoxy-2-polyprenylphenols. However, ‘Vibrio O1’ (organism PC4 was not examined) does contain 6-methoxy-3-methyl-2-polyprenyl-1,4-benzoquinone. 6. In Ps. ovalis Chester, radioactivity from l-[Me−14C]methionine is incorporated into the nuclear C-methyl and O-methyl groups of 6-methoxy-3-methyl-2-polyprenyl-1,4-benzoquinones and ubiquinone-9, and into the O-methyl group of 6-methoxy-2-polyprenylphenols.
The osmotic stability of lysosomes from adult and foetal guinea-pig liver tissue
Authors: Turnbull Julienne M. , Neil M. W.
Published: August 2015

1. Lysosome-rich fractions were obtained from foetal liver tissues as early as 35 days uterine age. Foetal lysosomes showed the same ‘structure-linked latency’ and acid hydrolytic potentiality characteristic of their adult counterparts. 2. The osmotic stability of lysosome-rich fraction from foetal guinea-pig liver tissue was greater than that of the corresponding adult lysosome fractions, p-nitrophenyl-phosphatase being used as marker enzyme. 3. The observation was confirmed by using β-glycerophosphatase and phenolphthalein β-glucuronidase as alternative marker enzymes. p-Nitrophenyl phosphate and β-glycerophosphate appear to act as substrates for the same enzyme. 4. By using p-nitrophenylphosphatase activity measurements it was shown that the osmotic stability of foetal lysosomal fractions decreased with increasing foetal age, but at no time achieved the degree of osmotic instability characteristic of adult lysosomal fractions. 5. The correlation of these findings with the intracellular environment of lysosomes is discussed.
A study of nucleated erythrocytes by density-gradient centrifugation in a zonal rotor
Authors: Mathias A P , Ridge D. , Trezona N. St G.
Published: August 2015

1. Several substances of high molecular weight were examined for their suitability as suspension media in the formation of density gradients for the zonal centrifugation of avian erythrocytes. None proved satisfactory. 2. The behaviour of pigeon erythrocytes in rate-sedimentation experiments in a type A zonal rotor with density gradients of sucrose was examined. The mature cells sediment more rapidly than the younger cells and have a lower RNA/DNA ratio. Maturation is accompanied by a greater loss of RNA from the nucleus than from the cytoplasm. 3. The base composition of the nuclear RNA and of the two species of cytoplasmic ribosomal RNA is reported. 4. The RNA of erythrocytes may be labelled in vivo by injection of inorganic [32P]phosphate. The cells most active in the synthesis of RNA sediment less rapidly than the bulk of the cells. 5. Reticulocyte nuclei sediment more slowly than those from erythrocytes. Reticulocyte nuclei have a mean volume of 35μ3 and are isopycnic with sucrose of density 1·2871 (measured at 20°). Maturation of the nuclei causes them to shrink to a volume of 25μ3 and the density to increase to 1·2944.
Adenosine phosphates and the control of glycolysis and gluconeogenesis in yeast
Authors: Chapman C. , Bartley W
Published: August 2015

1. Changes in dry weight, protein, RNA and DNA were measured in yeast during adaptation to glycolytic metabolism. 2. Only RNA increased significantly during the lag phase, but during the exponential phase all these cellular components increased in parallel. 3. The concentrations of ATP, ADP, AMP and glucose 6-phosphate were measured in respiring yeast and during the transition to glycolytic metabolism. 4. In respiring cells the concentration of AMP was at its highest and that of ATP was at its lowest; this relationship was reversed in glycolysing cells. 5. ADP concentration was similar in respiring and glycolysing cells, but glucose 6-phosphate concentration was much higher in the glycolysing cells. 6. A possible reason for mitochondrial repression is suggested. 7. It is concluded that adenosine phosphates do not control the direction of glycolytic flux in yeast and an alternative control of glycolysis and gluconeogenesis by enzyme activation and inactivation is suggested.
Evidence of a phosphate-transporter system in the inner membrane of isolated mitochondria
Authors: Tyler D. D.
Published: August 2015

1. The organic mercurial sodium mersalyl, formaldehyde, dicyclohexylcarbodiimide and tributyltin each blocked respiratory-chain-linked ATP synthesis in rat liver mitochondria. 2. Mersalyl and formaldehyde also blocked a number of other processes dependent on the entry of inorganic phosphate into mitochondria, including mitochondrial respiration and swelling stimulated by cations and phosphate, the substrate-level phosphorylation reaction of the citric acid cycle, and swelling in ammonium phosphate. 3. Dicyclohexylcarbodi-imide and tributyltin did not inhibit the entry of phosphate into mitochondria. 4. Mersalyl and formaldehyde had a relatively slight effect on succinate oxidation and swelling stimulated by cations when phosphate was replaced by acetate, on succinate oxidation stimulated by uncoupling agents, and on swelling in solutions of ammonium salts other than phosphate or arsenate. 5. Formaldehyde blocked the oxidation of NAD-linked substrates in mitochondria treated with 2,4-dinitrophenol and the ATP-dependent reduction of NAD by succinate catalysed by ox heart submitochondrial particles. Both these effects appear to be due to an inhibition by formaldehyde of the NAD–flavin region of the respiratory chain. 6. Concentrations of dicyclohexylcarbodiimide or tributyltin sufficient to abolish ADP-stimulated respiration blocked the dinitrophenol-stimulated adenosine triphosphatase activity, whereas mersalyl and formaldehyde caused only partial inhibition of ATP hydrolysis. 7. When mitochondria were incubated with dinitrophenol and ATP, less than 10% of the total inorganic phosphate liberated was recovered in the mitochondria and no swelling occurred. In the presence of mersalyl or formaldehyde at least 80% of the total inorganic phosphate liberated was retained in the mitochondria and extensive swelling was observed. This swelling was inhibited by oligomycin but not by antimycin or rotenone. 8. The addition of mersalyl to mitochondria swollen by treatment with valinomycin, K+ and phosphate blocked the contraction induced by dinitrophenol and caused an increase in the phosphate content of the mitochondria, but had no effect on the contraction of mitochondria when phosphate was replaced by acetate. 9. It is concluded that mitochondria contain a phosphate-transporter system, which catalyses the movement of phosphate in either direction across the mitochondrial membrane, and that this system is inactivated by organic mercurials and by formaldehyde. Evidence is presented that the phosphate-transporter system is situated in the inner membrane of rat liver mitochondria and is also present in other types of mammalian mitochondria.
The catabolism of cholesterol in vitro. Formation of 3α,7α,12α-trihydroxy-5β-cholestanoic acid from cholesterol by rat liver
Authors: Mendelsohn D. , Mendelsohn L.
Published: August 2015

1. Both 25-d- and 25-l-3α,7α,12α-trihydroxy-5β-cholestanoic acid were isolated from the gall-bladder bile of Crocodylus niloticus. 2. The catabolism of cholesterol to 25-d- and 25-l-3α,7α,12α-trihydroxy-5β-cholestanoic acid respectively was studied by using a rat liver preparation in vitro. The results show that rat liver can metabolize cholesterol to both forms of 3α,7α,12α-trihydroxy-5β-cholestanoic acid. However, a preference was noted for the formation from [4−14C]cholesterol of 3α,7α,12α-trihydroxy-5β-cholestanoic acid (25-d), which was isolated from the incubations with a specific radioactivity about four times that of 3α,7α,12α-trihydroxy-5β-cholestanoic acid (25-l). 3. The results indicate that 3α,7α,12α-trihydroxy-5β-cholestanoic acid is a normal intermediate in the biosynthesis of bile acids from cholesterol in the rat.
A substrate-induced conformation change in the reaction of alkaline phosphatase from Escherichia coli
Authors: Halford S. E. , Bennett N. G. , Trentham D. R. , Gutfreund H.
Published: August 2015

1. Benzyl phosphonates were prepared and their potentialities as chromophoric reagents for the exploration of the substrate-binding site of Escherichia coli alkaline phosphatase were investigated. 4-Nitrobenzylphosphonate is a competitive inhibitor of the enzyme. 2-Hydroxy-5-nitrobenzylphosphonate changes its spectrum on binding to the enzyme. This spectral change is reversed when the phosphonate is displaced from the enzyme by substrate. 2. The kinetics of the reaction of 2-hydroxy-5-nitrophenylphosphonate were studied by the stopped-flow and the temperature-jump techniques. It was found that the combination of the phosphonate with the enzyme occurred in two successive and reversible steps: enzyme–phosphonate complex-formation followed by rearrangement of the complex. The spectral change is associated with the rearrangement. At pH8 in 1m-sodium chloride at 22° the rate constant is 167sec.−1 for the rearrangement of the initially formed binary complex and is 18sec.−1 for the reverse process. 3. It has previously been proposed that the reactions of phosphatase with its substrates include a distinct step between enzyme–substrate combination and chemical catalysis. The rate constant involved could be predicted but not measured from experiments with substrates. The value for the rate constant measured from the rate of the enzyme–phosphonate rearrangement is in excellent agreement with the predicted value. A model for the reaction mechanism is proposed that includes a conformation change in response to phosphate ester binding before phosphate transfer from substrate to enzyme.
Ion-exchange chromatography of a dinucleotide preparation from controlled alkaline hydrolysis of ribonucleic acids
Authors: Satoh Kimihiko , Inoue Yasuo
Published: August 2015

With the aim of preparing the 16 possible ribodinucleotides derived from the four principal ribonucleotides, we made a kinetic study to determine the optimum conditions for a partial alkaline hydrolysis of RNA. Satisfactory results were obtained when the hydrolysis of RNA (1g.) in 0·2m-sodium hydroxide (100ml.) at 37° was ceased approx. 10min. after the start of the reaction. The relative yields of the monomer, dimer and trimer fractions were approx. 41:30:15 in E260 units, indicating that the reaction conditions were reasonably close to those required from kinetic considerations. The partial alkaline hydrolysate was chromatographed on a column of DEAE-Sephadex A-25 in the presence of 7m-urea. The dinucleotide fraction thus obtained was subjected to a subsequent chromatography on Dowex 1 (X2) under acidic conditions to separate the mixture according to base composition and base sequence. The results were satisfactory, and most of the 32 dinucleotides [i.e. 16 XpYp(3′) plus 16 XpYp(2′)] were fractionated by this single chromatographic procedure. The present method should be useful for further study of oligonucleotides as a time-saving method for the simultaneous preparation of a variety of dinucleotides. Further, examination of the present chromatographic pattern has provided several empirical criteria useful for the identification of oligonucleotides other than dimers appearing in the elution profile of Dowex 1 (X2) column chromatography under similar conditions.
Studies on the advent of ureotelism. The effects of bivalent cations on the capacity of the hepatic arginase of the Mexican axolotl to hydrolyse endogenous arginine
Authors: Palacios R. , Huitrón C. , Soberón G.
Published: August 2015

1. The effects of 19 bivalent cations on the activity, stability and capacity to hydrolyse endogenous arginine of axolotl liver arginase were studied. 2. It was found that Fe2+, Co2+, Ni2+ and Zn2+, as well as Mn2+, stabilize the enzyme and render it able to hydrolyse endogenous arginine. 3. By using different concentrations of Co2+ and Ni2+ it was possible to dissociate the effect of each of the metal ions on the activity and stability of arginase from that on its capacity to hydrolyse endogenous arginine. 4. The effects of Mn2+, Co2+ and Ni2+ on the activity and stability of arginase in the homogenate were also observed with purer preparations of the enzyme. 5. It is suggested that the capacity to hydrolyse endogenous arginine is a consequence of the integration of arginase with the arginine-formation sites.
The inhibition of pyruvate and ls(+)-isocitrate oxidation by succinate oxidation in rat liver mitochondria
Authors: König T. , Nicholls D. G. , Garland P. B.
Published: August 2015

1. The effects of succinate oxidation on pyruvate and also isocitrate oxidation by rat liver mitochondria were studied. 2. Succinate oxidation was without effect on pyruvate and isocitrate oxidation when respiration was maximally activated with ADP. 3. When respiration was partially inhibited by atractylate, succinate oxidation severely inhibited the oxidation of pyruvate and isocitrate. 4. This inhibitory effect of succinate was associated with a two- to three-fold increase in the reduction of mitochondrial NAD+ but no change in the reduction of cytochrome b. 5. It is concluded that, in the partially energy-controlled state, respiration is more severely inhibited at the first phosphorylating site than at the other two. 6. The effects of succinate oxidation are compared with those of palmitoylcarnitine oxidation. It is concluded that a rapid flow of electrons directly into the respiratory chain at the level of cytochrome b is in itself inadequate to inhibit the oxidation of intramitochondrial NADH. 7. The effects of succinate oxidation on pyruvate oxidation were similar in rat heart and liver mitochondria.
Chemical modification of glutamate dehydrogenase by 2,4,6-trinitrobenzenesulphonic acid
Authors: Freedman R. B. , Radda G. K.
Published: August 2015

1. Modification with 2,4,6-trinitrobenzenesulphonic acid was studied for its effect on the structure, activity and response to regulatory effectors of ox liver glutamate dehydrogenase. 2. The modification affected amino groups only, and the relative reactivities of the amino groups of the enzyme are described. 3. A biphasic inactivation of the enzyme was observed and analysis of the course of inactivation and of modification showed that the rapid reaction of one amino group/subunit leads to loss of 80% of the enzymic activity. 4. NADH retarded the inactivation by 2,4,6-trinitrobenzenesulphonic acid, the protection increasing with NADH concentration. This, together with the previous observation, suggests that the rapidly reacting group is essential for the activity of the enzyme. 5. The effects of modification on the optical-rotatory-dispersion and sedimentation behaviour of the enzyme were studied. 6. The enzyme's response to the allosteric effector GTP was rapidly lost on modification, whereas its response to ADP was unaffected. Comparison of the inactivation and desensitization suggests that the reactive amino group is essential for both activity and GTP response, and that only a completely unmodified enzyme oligomer responds fully to GTP. 7. The merits of chemical-modification studies of large enzymes are discussed critically in connexion with the interpretation of these results.
Stereochemistry of phytoene biosynthesis by isolated chloroplasts
Authors: Buggy M. J. , Britton G. , Goodwin T W
Published: August 2015

The incorporation of [2−14C,(5R)-5−3H1]MVA* and [2−14C,5−3H2]MVA into geranylgeraniol and phytoene by a preparation of ‘non-aqueous’ bean leaf chloroplasts has been studied. In the formation of phytoene from two molecules of geranylgeranyl pyrophosphate, the loss of hydrogen is stereospecific, the hydrogen atom lost from C-1 of each molecule of geranylgeranyl pyrophosphate being that which was originally the pro-S hydrogen atom from C-5 of mevalonate. All the pro-R hydrogen atoms from C-5 of mevalonate are retained. These results with a cell-free system confirm and extend the observations made in previous work with tomato slices.
A study of the effects of reaction with formaldehyde on some optical and physical properties of reticulocyte ribosomes
Authors: Cox R A
Published: August 2015

1. The optical rotatory dispersion and ultraviolet-absorption spectrum of ribosomal RNA in situ appear to be unchanged when the ribosome is dissociated into its RNA and protein moieties. 2. Reaction with 0·05% formaldehyde at 20° for 2hr. ‘fixes’ ribosomes so that they remain intact in 1% sodium dodecyl sulphate. 3. The RNA moiety of the ribosome undergoes a conformational change when ribosomes in 8% formaldehyde are heated at 70° for 10min. and cooled to 20°. After this treatment no double-helical character can be detected, but neither the sedimentation coefficient nor the morphology of the ribosome determined by electron microscopy is altered. 4. It is concluded that the RNA moiety of reticulocyte ribosomes is freely accessible to formaldehyde.
Multiple light-induced reactions of cytochromes b and c in Rhodopseudomonas spheroides
Authors: Jones O. T. G.
Published: August 2015

Illumination of chromatophore preparations from Rhodopseudomonas spheroides causes the oxidation of a cytochrome c and a slight oxidation of a cytochrome b with a maximum at 560nm. When illuminated in the presence of antimycin A the oxidation of cytochrome c was more pronounced and cytochrome b560 was reduced; the dark oxidation of cytochrome b560 was biphasic in the presence of succinate, but not in the presence of NADH, a less effective reductant. Split-beam spectroscopy showed that, in addition to the reduction of cytochrome b560, another pigment with maxima at 565 and 537nm. was reduced and was more rapidly oxidized in the dark than cytochrome b560. This pigment, tentatively identified as cytochrome b565, was also detected in spectra at 77°k, after brief illumination at room temperature; the maxima at 77°k were at 562 and 536nm. In the absence of antimycin A, light caused a transient reduction of cytochrome b565 and an oxidation of cytochrome b560. Dark oxidation of b565 was rapid, even in the presence of antimycin A and succinate. Difference spectra, at 77°k, of ascorbate-reduced minus succinate-reduced chromatophores or of anaerobic succinate-reduced minus aerobic succinate-reduced chromatophores suggested that two cytochromes c were present, with maxima at 547 and 549nm. When chromatophores frozen at 77°k were illuminated both these cytochromes c were oxidized, indicating a close association with the photochemical reaction centre. A scheme involving two reaction centres is proposed to explain these results.
Immunoglobin M biosynthesis. Intracellular accumulation of 7s subunits
Authors: Parkhouse R. M. E. , Askonas Brigitte A.
Published: August 2015

Immunoglobulin M biosynthesis was studied with mouse plasma cell tumour MOPC 104E as a model system. Cell suspensions prepared from solid tumours were incubated in vitro with tritiated leucine; the radioactivity incorporated into intracellular and secreted proteins was analysed by polyacrylamide-gel electrophoresis, sucrose-density-gradient centrifugation and precipitation with rabbit antiserum specific for the macroglobulin. The tumour was found to secrete immunoglobulin M and light chains in a 1:2 weight ratio, with lag periods of 20–30min. Within the cells there was a 7s component precipitable with specific antiserum to the macroglobulin that was shown to consist of heavy and light chains. This 7s subunit of the macroglobulin appeared to accumulate in the intracellular environment, so that even after long periods of incubation (3hr.) no more than trace amounts of fully assembled 19s molecules could be detected in cell lysates. Polymerization of the subunits into the pentamer therefore appears to take place shortly before, or simultaneously with, secretion of the molecules.
Heterogeneity of liver acid phosphatases in developing chick embryo
Authors: Wang K.-M.
Published: August 2015

1. The development, localization and heterogeneity of acid phosphatase and a Zn2+-activated acid phosphatase in cellular fractions of developing chick liver were studied. 2. Acid phosphatase is distributed abundantly in the particulate and soluble fractions. The soluble fraction is rich in Zn2+-activated acid phosphatase, which attains its peak activity at about 15 days of incubation. 3. The particulate acid phosphatase activity is inhibited by fluoride but not by sodium l(+)-tartrate or cysteine. On the other hand, the soluble Zn2+-activated acid phosphatase activity is inhibited by sodium l(+)-tartrate and cysteine but not by fluoride. 4. The pH optimum of these two enzymes is similar at about 5·6. 5. The soluble Zn2+-activated acid phosphatase activity appears to be thermally stabilized by the treatment with Triton X-100 or bovine serum albumin.
Yeast cell-wall synthesis
Authors: Sentandreu R. , Northcote D. H.
Published: August 2015

1. A study of wall synthesis has been made by following the incorporation of radioactive glucose and threonine into the cytoplasm and wall of yeast. 2. Both glucose and threonine are incorporated into a mannan glycopeptide. The glucose is also synthesized into a structural glucan of the wall. 3. The mannan glycopeptide contains high-molecular-weight mannan and low-molecular-weight mannose and oligosaccharide units composed of mannose. Both types of carbohydrate are attached to the peptide. The extent of radioactive incorporation into these different carbohydrate constituents of the glycopeptide remained constant during a pulse-chase experiment. No evidence of a sequential synthesis of oligosaccharides and high-molecular-weight mannan was obtained. 4. Cycloheximide inhibits the incorporation of threonine into the wall but only partially inhibits the incorporation of glucose. Thus not all the polysaccharide deposited into the wall is dependent on a simultaneous peptide synthesis and incorporation. 5. Protoplasts grown in an iso-osmotic medium secreted a mannan polymer that was probably a glycopeptide.
Deoxyribonucleic acid–ribonucleic acid hybridization. Annealing and quantitative recovery of intact ribosomal ribonucleic acid molecules from hybrids
Authors: Fry Michael , Artman Michael
Published: August 2015

A simple and efficient method for hybridization and subsequent recovery of non-fragmented ribosomal RNA from the hybrid is described. The procedure involves annealing of immobilized denatured DNA bound on cellulose nitrate membrane filters to complementary RNA in 50% (v/v) formamide–0·33m-potassium chloride–10mm-tris–hydrochloric acid buffer, pH7·4, at 33° for 3hr. Under these conditions no detectable changes in the sedimentation coefficients of the input RNA were detected. The RNA can subsequently be recovered quantitatively from the hybrid in intact form by incubating the filters in formamide or in 85% (v/v) dimethyl sulphoxide. The applicability of the method for the evaluation of the absolute size of ribosomal RNA cistrons in Escherichia coli DNA and for the determination of the size of messenger RNA molecules is discussed.
The effect of magnesium ion deprivation on the synthesis of mucopeptide and its precursors in Bacillus subtilis
Authors: Garrett A J
Published: August 2015

1. Mg2+ or Mn2+ starvation causes suspensions of Bacillus subtilis strain W 23 to accumulate bound amino sugars that are soluble in trichloroacetic acid. 2. The presence of chloramphenicol or puromycin produces higher intracellular concentrations of amino sugars during Mg2+ starvation, but neither compound can stimulate the accumulation when Mg2+ is present. 3. The major component of the amino sugar fraction extracted from cells deprived of Mg2+ is a nucleotide containing uridine, phosphorus, N-acetylmuramic acid, alanine, glutamic acid and α∈-diaminopimelic acid in the molar proportions of 1:2:1:3:1:1. This compound represents at least 80% of the bound N-acetylhexosamine extracted by trichloroacetic acid. 4. Studies of the binding of this nucleotide with vancomycin support the proposal that it is the mucopeptide precursor UDP-N-acetylmuramyl-l-alanyl-d-glutaminyl- α∈-diaminopimelyl-d-alanyl-d-alanine. 5. A method is described for the isolation of this material labelled with [3H]α∈-diaminopimelic acid. 6. When Mg2+ is supplied to cells previously starved of Mg2+, the accumulated pool of amino sugars rapidly decreases. 7. The biosynthesis of mucopeptide is inhibited by 35–50% under conditions of Mg2+ starvation. The presence of EDTA increases this inhibition to 70%. The amount of N-acetylhexosamine that accumulates is balanced exactly by the associated fall in mucopeptide synthesis. 8. ‘Chase’ experiments show that the accumulated N-acetylhexosamine compound is utilized in mucopeptide synthesis.
Calorimetric studies of the haemoglobin–haptoglobin reaction
Authors: Adams Ernest C. , Weiss Margaret R.
Published: August 2015

Haptoglobin binds haemoglobin so firmly that there is practically no dissociation. It would be expected that the heat of the reaction would be relatively large. The development of the microcalorimeter by Benzinger offered the opportunity to measure the heat of reaction. The experiments were carried out in the Beckman 190B Microcalorimeter in two ways: (1) a constant amount of haptoglobin (Kabi; 65mg.) with different amounts of haemoglobin, and (2) a constant amount of haemoglobin (32·5mg.) with different amounts of haptoglobin. The proteins, each in 5ml. of 0·15m-phosphate buffer, pH7·4, were placed in equal-volume calorimeter cells. The heat produced/mg. of haemoglobin was calculated from the slope of the curve for a constant amount of haptoglobin and from the maximum heat for a constant amount of haemoglobin. This heat is about 70kcal./mole at 37°. ΔH varies with temperature, being −70·2 at 37°, −29·7 at 20° and 7·2 at 4°. From the amount of haptoglobin required to attain maximum heat with 32·5mg. of haemoglobin and the amount of haemoglobin required to attain maximum heat with 65mg. of haptoglobin, it appears that at excess of haptoglobin there is competition between the reactions of 2moles of haptoglobin with 1mole of haemoglobin (or 2 αβ-chains) and 1mole of haptoglobin with 1mole of haemoglobin.
Biosynthesis of ethylene. 4-Methylmercapto-2-oxobutyric acid, an intermediate in the formation from methionine
Authors: Mapson L. W. , March J. F. , Wardale D. A.
Published: August 2015

The enzyme responsible for the conversion of methionine into a precursor of ethylene in cauliflower florets is a transaminase. The formation of 4-methyl-mercapto-2-oxobutyric acid by this enzyme has been shown. The oxo acid stimulates the synthesis of ethylene when added to floret tissue, and tracer experiments have shown that 14C is incorporated into ethylene from the labelled oxo acid. The evidence is consistent with the view that the oxo acid is an intermediate in the formation of ethylene from methionine.
Studies on the permeability of rat liver lysosomes to carbohydrates
Authors: Lloyd J B
Published: August 2015

1. The latency of nitrocatechol sulphatase activity was measured in rat liver lysosomes before and after preincubation in 0·25m solutions of 25 different carbohydrates. 2. Preincubation in disaccharides, hexitols, gluconate, glucuronate or lactate gave little or no rise in ‘free’ sulphatase activity, indicating that these compounds do not easily penetrate the lysosomal membrane, but incubation in monosaccharides or the lower glycitols caused a progressive loss of latency. 3. Rates of increase in ‘free’ activity were taken as an indication of rates of solute penetration into lysosomes and were correlated with the structure and molecular weight of each sugar. 4. Additional evidence for non-penetration of maltose was obtained by demonstrating that the latency of lysosomal α-glucosidase is independent of substrate concentration employed. 5. The results are discussed in the light of published data on the latency of lysosomal enzymes.
A new class of chromophoric organomercurials and their reactions with d-glyceraldehyde 3-phosphate dehydrogenase
Authors: McMurray C. H. , Trentham D. R.
Published: August 2015

The syntheses of the following organomercurials are described: 2-chloromercuri-4-nitrophenol, 2-chloromercuri-4,6-dinitrophenol, 4-chloromercuri-2-nitrophenol and 2,6-dichloromercuri-4-nitrophenol. All four organomercurials show large spectral changes in the visible spectrum when thiols displace a more weakly bound ligand such as EDTA from the mercury atom. These spectral changes are primarily associated with pK perturbation of the nitrophenols. The mercurials are therefore chromophoric probes for thiol groups in proteins and other thiols of biological interest. The enzyme d-glyceraldehyde 3-phosphate dehydrogenase from lobster muscle is used as a model system in which the properties of the organomercurials may be illustrated. In particular it is shown how d-glyceraldehyde 3-phosphate dehydrogenase carboxymethylated at the active site may be mercurated at a specific site. This mercurial derivative may be crystallized and shown to be isomorphous with the parent enzyme. The mercurials also act as ‘reporter groups’ by monitoring phosphate or pyrophosphate binding to the enzyme. The mercurials may also be used to estimate cations by an EDTA displacement method.
Enzyme activity and composition of myelin and subcellular fractions in the developing rat brain
Authors: Banik N. L. , Davison A. N.
Published: August 2015

1. Subcellular fractions and myelin were isolated from developing and adult rat brain. 2. Measurements of chemical composition and enzyme activities indicate the presence of a second myelin-like fraction mainly in the brain of developing rats. 3. This membrane fraction has a different lipid composition from myelin, but resembles myelin in its content of phosphohydrolase and aminopeptidase activity. 4. It is suggested that the second myelin-like fraction may be a submicrosomal contaminant or it may be derived from oligodendroglial plasma membrane during myelinogenesis.
The formation of short fibres from native cellulose by components of Trichoderma koningii cellulase
Authors: Halliwell G. , Riaz M.
Published: August 2015

Cellulolytic enzyme components of culture filtrates of Trichoderma koningii were fractionated on ionic and non-ionic forms of Sephadex and on cellulose powder (Whatman) and examined for their ability to hydrolyse soluble carboxymethyl-cellulose, and to saccharify, solubilize and form short fibres from native undegraded cellulose of the type found in cotton. DEAE-Sephadex provided two CM-cellulase components and a C1 component; the C1 component acted weakly and solely on cotton, forming soluble products but not short fibres. The ability to form short fibres was confined almost wholly to one of the CM-cellulase components which completely degraded cotton, minimally to soluble products and extensively to short fibres. The latter action was unaffected by the presence of the other two components. The two CM-cellulase components solubilized cellulose synergistically whereas the short-fibre-forming component and C1 component were inhibitory.
Glucose metabolism in the mucosa of the small intestine. Changes of hexokinase activity during perfusion of the proximal half of rat small intestine
Authors: Mayer R. J. , Shakespeare P. , Hübscher G.
Published: August 2015

1. The effect of perfusion on the activities of hexokinase and lactate dehydrogenase was studied in the proximal half of the small intestine of fed and starved rats. 2. Perfusion of preparations from starved rats with a medium containing glucose caused a significant increase in hexokinase activity of the particle-free supernatant. The increase in activity was observed as early as 5min after the start of perfusion and persisted for up to 66min of perfusion. No increase in hexokinase activity of the particle-free supernatant was observed when a medium containing mannitol was used. As a further control, preparations from fed rats were perfused under the same conditions. With the medium containing glucose, the hexokinase activity of the particle-free supernatant remained unchanged during the first 15min of perfusion and thereafter fell gradually until, after 66min of perfusion, 73% of the original activity was retained. 3. The activity of lactate dehydrogenase in the particle-free supernatant prepared from the proximal half of the untreated small intestine of starved rats was significantly lower than in corresponding preparations from fed animals. However, it did not change significantly on perfusion with media containing either mannitol or glucose. 4. The distribution of hexokinase activity between total particulate fraction and particle-free supernatant was measured in preparations from starved rats after perfusion for 5–10min. In preparations that had not been perfused the ratio of hexokinase activity in total particulate fraction/particle-free supernatant was significantly higher in starved than in fed animals. After perfusion with a medium containing glucose, the total homogenate activity had not changed significantly, whereas the ratio of hexokinase activity in total particulate fraction/particle-free supernatant decreased significantly and approached the value obtained with fed animals. 5. The results agree with the view that the glucose-dependent increase of hexokinase activity in the soluble cell compartment as observed in vivo and in vitro in the intestinal mucosa of starved rats is brought about by a release of hexokinase activity from a particulate subcellular structure(s).
Heat inactivation and Sephadex chromatography of the small-intestine disaccharidases of the chick
Authors: Siddons R. C.
Published: August 2015

1. The maltase, sucrase, isomaltase and palatinase activities of the chick small intestine are localized in particles that sediment when centrifuged at 100000g for 90min. 2. Solubilization of the particle-bound disaccharidases without loss of activity was achieved by digestion with papain. Trypsin was less effective and caused a preferential solubilization of the sucrase, isomaltase and palatinase activities. 3. On Sephadex G-200 columns, the solubilized preparations yielded two disaccharidase peaks. The first peak was eluted close to the void volume of the column and contained all the sucrase, isomaltase and palatinase activities and some of the maltase activity. The remainder of the maltase activity was eluted beyond the total volume of the column. 4. Precipitation with ethanol did not affect the behaviour of the disaccharidases of gel filtration. 5. The maltase activity of the second peak on rechromatography in a buffer containing 0.01m-maltose was eluted close to the void volume. 6. Similar pH optima but different Km values were obtained for the maltase activities of the two peaks. 7. Heat-inactivation studies showed that the first peak contained two disaccharidase enzymes; one hydrolysed sucrose and maltose and the other hydrolysed isomaltose, palatinose and maltose. The second peak contained three disaccharidase enzymes all specific for the hydrolysis of maltose. 8. It is proposed that the intestinal disaccharidases of the chick exist in the form of two complexes: a sucrase–isomaltase complex and a maltase complex.
Glutamate, calcium ion-chelating agents and the sodium and potassium ion contents of tissues from the brain
Authors: Pull I. , McIlwain H. , Ramsay R. L.
Published: August 2015

1. Salts of l-glutamate added to cerebral tissues maintained in glucose–saline–bicarbonate solutions cause the Na+ content of the tissues to increase rapidly and K+ to be lost. Entry of 22Na+ also is accelerated by l-glutamate and this acceleration is inhibited by low concentrations of tetrodotoxin. 2. Tissue Na+ content and its rate of increase after the addition of l-glutamate are affected by the Ca2+ of incubation media. 3. Very rapid and extensive entry of Na+ to the tissue is caused by EDTA, and a moderate entry by citrate and ATP. Calculations of the concentration of free Ca2+ in media after these additions indicate that Na+ entry is sometimes associated with low Ca2+ concentration, but that other substances, especially l-glutamate, act without greatly diminishing Ca2+ concentration. 4. Experiments with 2,4-dinitrophenol and valinomycin are also reported and aspects of the Na+ entry formulated and discussed.
The significance of the incorporation of [14C]leucine into different protein fractions by isolated ox heart mitochondria
Authors: Krymkiewicz Norberto , González-Cadavid Néstor
Published: August 2015

The problem of whether isolated mitochondria are able to synthesize specific proteins was investigated, particular consideration being paid to the possible contribution of micro-organisms to this activity. With ox heart mitochondria it was shown that: (1) The medium used for the incubations inhibits the exponential phase of bacterial growth for at least 8h either in the absence or the presence of fresh mitochondria, but the inhibition disappears after 4h when mitochondria damaged by freezing and thawing are used. (2) The incorporation of [14C]leucine into total proteins is linear up to at least 8h, although part of the radioactivity at the later periods might be due to some incorporation by resting-phase bacteria. (3) A contamination by as little as 800 cells/mg of mitochondrial protein is enough to contribute substantially to the total radioactivity incorporated by the mitochondrial preparations. (4) Purified cytochrome b and cytochrome oxidase are labelled even under conditions of minimal contamination by micro-organisms (less than 60 cells/mg of mitochondrial protein) and the contribution of bacterial proteins to the radioactivity found in cytochromes is negligible, as shown by double-labelling experiments. (5) At 4h the specific radioactivities of cytochrome b and cytochrome oxidase are seven- and 16-fold lower respectively than that of a structural protein-rich fraction, suggesting that the labelling of cytochromes is due to a residual contamination by these proteins.
The stereochemistry of hydrogen elimination during 7,8-double bond formation by Tetrahymena pyriformis
Authors: Wilton D. C. , Akhtar M.
Published: August 2015

It is shown that formation of the 7,8-double bond in the conversion of cholesterol into cholesta-5,7,22-trien-3β-ol involves the removal of the 7β- and 8β-hydrogen atoms.
Thiol and disulphide contents of hen ovalbumin. C-Terminal sequence and location of disulphide bond
Authors: Fothergill L. A. , Fothergill J. E.
Published: August 2015

1. The thiol and disulphide contents of hen ovalbumin were investigated by p-chloromercuribenzoate titration, by determination of cysteic acid content after performic acid oxidation, by measurement of uptake of radioactive iodoacetic acid, and by assay of S-aminoethylcysteine after reaction with ethyleneimine. All results showed that ovalbumin had 6 half-cystine residues. Experiments with and without reducing agents demonstrated that there were 4 thiol groups and 1 disulphide bond. 2. A peptide containing equimolar amounts of S-carboxymethyl-cysteine, serine, valine and proline, but no lysine or arginine, was obtained by radioactive labelling of the cysteine residues with iodo[14C]acetic acid followed by electrophoretic and chromatographic separation of tryptic digests. It was concluded that the C-terminal sequence of ovalbumin is -Cys-Val-Ser-Pro. 3. The location of the disulphide bond was studied by using a double-labelling technique. It was shown that one end of the disulphide was located in this C-terminal peptide.
Studies on ribonucleic acid and homopolyribonucleotide formation in neuronal, glial and liver nuclei
Authors: Kato Takahiko , Kurokawa Masanori
Published: August 2015

1. Various types of nuclear preparations, with different ratios of neuronal to glial nuclei, were isolated from guinea-pig cerebral grey matter and ox cerebral grey matter and white matter. Conditions appropriate for the separate assay of RNA and poly A formation were described. Comparative rates of RNA and poly A formation were studied in cerebral and liver nuclei. 2. RNA polymerase activity per nucleus is higher in neuronal nuclei than in glial nuclei. In liver nuclei, the activity is much lower than in cerebral nuclei. The physical relationship between RNA polymerase and deoxyribonucleoprotein seems to differ in neuronal, glial and liver nuclei. 3. Poly A polymerase activity in liver nuclei is selectively activated by Mn2+ and inhibited by GTP, CTP and UTP. On a DNA basis, the activity in an aggregate enzyme is the same as in intact nuclei. Poly A polymerase activity per nucleus is much higher in liver nuclei than in neuronal nuclei. Glial nuclei show an intermediate activity. 4. It is suggested that, in neuronal nuclei, the synthesis of RNA is more prominent than that of poly A under conditions where both polymers are formed simultaneously. This contrasts with liver nuclei, where more poly A is made than RNA. 5. In neuronal nuclei, the rate of CTP incorporation is much higher than in glial and liver nuclei. This incorporation is most probably due to poly C synthesis.
Acetoacetate metabolism in infant and adult rat brain in vitro
Authors: Itoh T. , Quastel J. H.
Published: August 2015

1. Acetoacetate or dl-β-hydroxybutyrate increases the rate of oxygen consumption to a smaller extent than that brought about by glucose or pyruvate in adult rat brain-cortex slices but to the same extent as that in infant rat brain-cortex slices. 2. The rate of 14CO2 evolution from [1-14C]glucose considerably exceeds that from [6-14C]glucose in respiring infant rat brain-cortex slices, in contrast with adult brain-cortex slices, suggesting that the hexose monophosphate shunt operates at a greater rate in the infant rat brain than in the adult rat brain. 3. The rate of 14CO2 evolution from [3-14C]acetoacetate or dl-β-hydroxy[3-14C]butyrate, in the absence of glucose, is the same in infant rat brain slices as in adult rat brain slices. It exceeds that from [2-14C]glucose in infant rat brain but is less than that from [2-14C]glucose in adult rat brain. 4. Acetoacetate is oxidized in the brain through the operation of the citric acid cycle, as shown by the accelerating effect of glucose on acetoacetate oxidation in adult brain slices, by the inhibitory effects of malonate in both infant and adult brain slices and by its conversion into glutamate and related amino acids in both tissues. 5. Acetoacetate does not affect glucose utilization in adult or infant brain slices. It inhibits the rate of 14CO2 formation from [2-14C]glucose or [U-14C]-glucose the effect not being wholly due to isotopic dilution. 6. Acetoacetate inhibits non-competitively the oxidation of [1-14C]pyruvate, the effect being attributed to competition between acetyl-CoA and CoA for the pyruvate-oxidation system. 7. Acetoacetate increases the rate of aerobic formation of lactate from glucose with both adult and infant rat brain slices. 8. The presence of 0.1mm-2,4-dinitrophenol diminishes but does not abolish the rate of 14CO2 formation from [3-14C]acetoacetate in rat brain slices. This points to the participation of ATP in the process of oxidation of acetoacetate in infant or adult rat brain. 9. The presence of 5mm-d-glutamate inhibits the rate of 14CO2 formation from [3-14C]acetoacetate, in the presence or absence of glucose. 10. Labelled amino acids are formed from [3-14C]acetoacetate in both adult and infant rat brain-cortex slices, but the amounts are smaller than those found with [2-14C]glucose in adult rat brain and greater than those found with [2-14C]glucose in infant rat brain. 11. Acetoacetate is not as effective as glucose as a precursor of acetylcholine in adult rat brain but is as effective as glucose in infant rat brain slices. 12. Acetoacetate or β-hydroxybutyrate is a more potent source of acetyl-CoA than is glucose in infant rat brain slices but is less so in adult rat brain slices.