Showing papers in "Biochemical Journal in 1956"

A study of the conditions and mechanism of the diphenylamine reaction for the colorimetric estimation of deoxyribonucleic acid

Abstract: Of the colour reactions available for the determination and identification of deoxyribonucleic acid (DNA), the reaction with diphenylamine in a mixture of acetic and sulphuric acids at 1000 (Dische, 1930) has been perhaps the most widely used. The present study arose from the observation that a more intense colour was sometimes produced if, instead of being heated at 1000 for 10 min., the reaction mixture was allowed to stand overnight at room temperature. As a result of this observation the procedure has been modified, principally by adding acetaldehyde to the reagents and by allowing the solution to stand for about 17 hr. at 30° instead of heating it at 1000. The modified method is 3-5 times as sensitive as Dische's original procedure, and several substances which interfere in the original method do not do so in the modified procedure. Some observations on the mechanism of the reaction have been made; in particular it was discovered that there is a liberation of inorganic orthophosphate from DNA during the early stages of the reaction. This finding has a bearing on the structure of DNA. The modified method has already been used in an investigation of nucleic acid metabolism during bacteriophage multiplication (Burton, 1955).

The synthesis of porphyrins and bacteriochlorophyll in cell suspensions of Rhodopseudomonas spheroides

Abstract: Free porphyrins are formed by growing cultures of some bacteria and yeasts under conditions in which formation of cytochromes is limited. With Corynebacterium diphtheriae, production of both porphyrin and toxin is dependent upon the concentration of iron salts in the medium; increasing this beyond a critical point decreases both porphyrin and toxin production (Pappenheimer, 1947). Cells grown with iron at concentrations sufficiently high to prevent accumulation of porphyrin contain considerably more cytochrome than those grown under conditions favouring porphyrin formation (Pappenheimer & Hendee, 1947; Rawlinson & Hale, 1949). Bacillus cereus excretes porphyrin (probably coproporphyrin) into the media when grown anaerobically, but only traces of porphyrin occur in aerobic cultures; the concentration of haem compounds in the cells grown anaerobically is considerably smaller than in those from aerobic cultures (Schaeffer, 1952a, b). There have been many observations of porphyrin formation by yeasts (see Lemberg & Legge, 1949). Stich & Eisgruber (1951) found that the amount of coproporphyrin Type I in cells of Saccharomyces anamensis is increased 5to 10-fold by addition of pantothenate to the growth medium, while addition of riboflavin completely suppresses porphyrin formation. Cells grown under conditions which favour porphyrin formation contain little or no haem derivatives, whereas those grown with riboflavin and iron are rich in these substances. Porphyrins may also be precursors of chlorophyll. Induced mutant strains of Chlorella, which are unable to synthesize chlorophyll, form large quantities of protoporphyrin, as well as magnesium protoporphyrin and magnesium vinyl pheophytin a5, when grown in the dark on a glucose--salts medium (Granick, 1948a, b, 1950). Another mutant produces a number of porphyrins containing up to eight carboxyl groups in each molecule, those with two such groups being predominant (Bogorad & Granick, 1953a; Granick, Bogorad & Jaffe, 1953). These observations of porphyrin formation by micro-organisms have been made with growing

A study of the effects of substrate concentration and certain relaxing factors on the magnesium-activated myofibrillar adenosine triphosphatase.

Abstract: Marsh (1952) first showed that whereas the apparent rate of hydrolysis of adenosine triphosphate (ATP) by homogenates of fresh rabbit muscle in an isotonic solution of potassium chloride was comparatively low, after centrifuging and removal of the soluble protein extract a considerable increase in the rate of production of inorganic phosphate by the residue occurred when it was resuspended in fresh potassium chloride solution. On the basis of these findings Marsh concluded that there was present in muscle a factor which inhibited adenosine triphosphatase (ATPase) activity, and he further demonstrated that in the presence of this factor ATP caused an increase in the volume of the cell fragments in the suspension. Bendall (1953 a) extended Marsh's findings and was able to bring about the relaxation of glycerated fibres by the application of ATP in the presence of preparations of the Marsh factor. He also confirmed the inhibitory action of the factor on the ATPase activity of homogenized glycerated fibres. Further investigations by Hasselbach & Weber (1953) on the ATPase activity of these preparations showed that at higher concentrations ATP was inhibitory, and in the presence of the Marsh factor the level at which ATP became inhibitory was considerably reduced. It is clear from the investigations mentioned above and those of Bozler & Prince (1953) that, in general, relaxation in muscle models is associated with a low rate of ATP hydrolysis. Magnesium appears to be of special importance in the contraction-relaxation cycle, yet the precise role of this metal is far from clear. Although magnesium is essential for relaxation (Bendall, 1953a), reagents which form complexes with magnesium, namely ethylenediaminetetraacetate (EDTA) (Bozler, 1954; Watanabe, 1955) and inorganic pyrophosphate (Weber, 1951; Bozler, 1951; Bendall, 1953b) also bring about relaxation of glycerated fibres. Bendall (1953b) has commented on the similarity between the relaxing action of the Marsh factor and that of inorganic pyrophosphate, and it is conceivable that one of the properties possessed in common by these substances is the ability to complex with magnesium. Glycerated fibres consist mainly of myofibrils, but studies on the ATPase activity ofthese fibres are not entirely satisfactory, as preparations may be contaminated with sarcoplasmic granules and nuclei which also possess ATPase activity, and with varying amounts of sarcoplasm. Furthermore, precise analysis ofsuch experiments in terms ofsubstrate and activator concentrations at any point in the fibre is difficult because concentration gradients will be produced by the diffusion of ATP to the myofibrils from the outside of the fibre. To throw more light on the enzymic behaviour of the myofibril a study has been made of the ATPase activity of isolated myofibrils, with particular reference to those conditions that bring about the low level of activity which is characteristic of relaxation. It has been shown that at a low ionic strength the marked inhibition of ATPase activity by ATP is dependent on the magnesium concentration and that this inhibition is not obtained when calcium is the activating metal, nor in the presence of magnesium when inosine triphosphate (ITP) is the substrate. No inhibition could be demonstrated with myokinase, but all the other relaxing factors tested reduced the ATPase activity of isolated myofibrils to a low level. With the exception of findings with EDTA the results are in agreement with the view (Perry, 1955, 1956) that the low rate of ATPase activity associated with relaxation may be due to the binding or removal of magnesium from the system, so that it is no longer available for the myofibrillar ATPase system. A preliminary account of some aspects of this work has already been presented (Perry, 1955).

The mechanism of the reaction of chymotrypsin with p-nitrophenyl acetate.

Abstract: 5. Release of these materials from mycelium treated with fatty acid was greatest at acid pH values, but change of pH had no significant effect upon the release by detergents. 6. Incubation of mycelial pads of these fungi in the presence ofarsenite leads to the accumulation of pyruvate and ax-oxoglutarate together with traces of an unidentified a-keto acid. The yield of ca-keto acids is increased by the presence of glucose in the medium. We wish to thank the Agricultural Research Council for a maintenance grant to one of us (C. C. T.) and for a grant towards the cost of materials. We also gratefully acknowledge a gift of sodium fluoroacetate from Sir Rudolph Peters. REFERENCES

Inhibition of cytochrome systems of heart muscle and certain bacteria by the antagonists of dihydrostreptomycin: 2-alkyl-4-hydroxyquinoline N-oxides.

Abstract: Cormforth, J. W. & James, A. T. (1954). Biochem. J. 58, xlvii. Culvenor, C. C. J. (1953). Rev. Pure appl. Chem., Aust., 3, 83. Gabriel, S. & Gerhard, W. (1921). Ber. dt8ch. chem. Ges. 54, 1071. Hansen, R. P., Shorland, F. B. & Cook, N. J. (1955). Chem. & Ind. p. 92. Hays, E. E., Wells, I. C., Katzman, P. A., Cain, C. K., Jacobs, F. A., Thayer, S. A., Doisy, E. A., Gaby, W. L., Roberts, E. C., Muir, R. D., Carroll, C. J., Jones, L. R. & Wade, N. J. (1945). J. biol. Chem. 139, 725. James, A. T. & Martin, A. J. P. (1956). Biochem. J. 63, 144. Lightbown, J. W. (1950). Nature, Lond., 166, 356. Lightbown, J. W. (1954). J. gen. Microbiol. 11, iv. Lightbown, J. W. & Jackson, F. L. (1956). Biochem. J. 63, 130. McCluskey, K. L. (1922). J. Amer. chem. Soc. 44, 1574. Martin, A. J. P. & James, A. T. (1956). Biochem. J. 63, 138. Pachter, I. J. (1953). J. Amer. chem. Soc. 75, 3026. Stallberg-Stenhagen, S. (1945). Ark. Kemi min. Geol. A, 20, no. 19. Wells, I. C. (1952). J. biol. Chem. 196, 331. Wells, I. C., Elliott, W. H., Thayer, S. A. & Doisy, E. A. (1952). J. biol. Chem. 196, 321.

Some disturbances of erythrocyte metabolism in galactosaemia

Abstract: Galactosaemia is an inborn error of metabolism involving a specific inability to metabolize galactose normally. The affected infants show failure to thrive, enlargement of the liver and spleen, vomiting, jaundice, proteinuria, aminoaciduria, a high blood-galactose level and consequent excretion of large quantities of this sugar in the urine. These disturbances may be avoided or overcome by the early withdrawal of all sources of dietary galactose. Thereafter, the administration of galactose in any form causes this sugar to reappear in blood and urine in significant amounts. If the condition should remain untreated for any length of time death may ensue, whilst the survivors may develop cataract, cirrhosis of the liver and mental retardation. Published reports throw no light on the exact nature of the basic biochemical lesion which is responsible for the syndrome, nor on the possible connexion between the accumulation of galactose in the body and hepatic, renal, cerebral and other disturbances. In an attempt to study these aspects we have sought to use those cells of the patient which are most readily available, the erythrocytes. Since the mammalian erythrocyte is known to metabolize galactose (Katayama, 1926; Feigelson & Conte, 1954; Nossal, 1948) there was ground for the hope that the presence of a biochemical defect in hepatic parenchymal cells might be shared, at any rate to some degree, by the erythrocytes. We were thus led to compare the metabolism of galactosaemic red cells with that of normal cells under the same conditions.

The estimation of arginine.

Abstract: Arginine has been estimated by precipitation as the picrate and flavianate complexes, enzymically by the action of arginase followed by the estimation of the urea formed, microbiologically and colorimetrically (cf. Block & Boiling, 1947). The colorimetric method developed by Sakaguchi (1925a, b) has been most widely used. This method relies upon the formation of a red-coloured complex produced by arginine and other monosubstituted guanido compounds in the presence of a-naphthol and alkaline hypobromite or hypochlorite. Although this method will qualitatively detect small quantities of arginine, it is unreliable quantitatively, as evidenced by the large number of modifications that have been suggested, and which have been reviewed recently by G6mez & Marenzi (1953). A colorimetric method for the estimation of both monoand di-substituted guanidines was introduced by Eggleton, Elsden & Gough (1943). These compounds give a red-coloured complex with diacetyl and a-naphthol in alkaline solution. The authors found that monosubstituted guanidines such as arginine and glycocyamine produced only one-ninth of the colour given by asymmetrically disubstituted guanidines such as creatine. This observation has been confirmed by Ennor & Stocken (1953) and by Roche, Thoai & Hatt (1954). Although this method is less sensitive than the Sakaguchi method, it is simpler and more reliable. This paper describes a modification of the above reaction. When the colour is developed in the presence of n-propanol and an increased concentration of x-naphthol, the sensitivity of the method for monosubstituted guanidines is equal to that previously obtained with disubstituted guanidines. Thus it is possible to estimate arginine in amounts ranging from 10 to 100 ,ug.

Pathways of glucose catabolism in rat liver in alloxan diabetes and hyperthyroidism

Abstract: Small, L. D., Bailey, J. H. & Cavallito, C. J. (1947). J. Amer. chem. Soc. 69, 1710. Stoll, A. & Seebeck, E. (1948). Helv. chim. acta, 31, 189. Stoll, A. & Seebeck, E. (1949a). Helv. chim. acta, 32, 197. Stoll, A. & Seebeck, E. (1949b). Helv. chim. acta, 32, 866. Stoll, A. & Seebeck, E. (1951). Helv. chim. acta, 34, 481. Szymona, M. (1952). Acta Microbiol. Polon., 1, 5. (Quoted from Chem. Abstr. 47, 2412.) Twiss, D. F. (1914). J. chem. Soc. 105, 36. Vinokurow, S. I., Bronz, L. M. & Korsak, S. E. (1947). Bull. Biol. Med. exp. U.R.S.S. 23, 296. (Quoted from Chem. Ab8tr. 42, 6864.) Wills, E. D. (1954). Biochem. J. 57, 109. Wills, E. D. & Wormall, A. (1950). Biochem. J. 47, 158.

Studies on the metabolism of the Protozoa. 7. Comparative carbohydrate metabolism of eleven species of trypanosome.

Abstract: muscle, which is particularly great with DNP. Further experiments on these problems are in progress. SUMMARY 1. The influence of 2:4-dinitrophenol, methylene blue, brilliant cresyl blue, thionine, phenosafranine and Janus Green B on the oxidative phosphoryl-ation of mitochondria isolated from liver, kidney, brain, heart and skeletal muscle of rats has been studied in vitro as well as in vivo. 2. All the substances used produced a high degree of inhibition of oxidative phosphorylation both in vitro and in vivo. In every case, uncoupling of oxidative phosphorylation was accompanied by a change of form of the mitochondria from rod-like to spherical and by swelling. 3. 2:4-Dinitrophenol and brilliant cresyl blue produce activation of adenosinetriphosphatase of liver mitochondria in vitro. 5'-Nucleotidase and both acid and alkaline phosphatases are also stimulated. All the substances used stimulated adenosinetriphosphatase, 5'-nucleotidase and both acid and alkaline phosphatases in vivo. 4. 2:4-Dinitrophenol and brilliant cresyl blue stimulate adenosinetriphosphatase activity of fresh as well as of aged mitochondria. Also soluble adeno-sinetriphosphatase is activated by these substances. 5. Phenosafranine strongly inhibits myokinase of liver mitochondria, both in vitro and in vivo. 6. Prolonged treatment of rats with the substances produces fatty infiltration of the liver. 2:4-Dinitrophenol produces glycogen accumulation instead of fatty infiltration. 7. The significance of the phenomena is discussed. From the numerous papers on the carbohydrate metabolism of trypanosomes which have appeared during the past twenty years, it has become obvious that, with the possible exception of Trypano8oma cruzi (Brand, Tobie, Kissling & Adams, 1949) and the plant trypanosome Strigo-monas oncopelti (Ryley, 1955), cellular motility depends on a supply of extracellular monosac-charide, which is incompletely broken down to a mixture of organic acids; much of this work is admirably summarzed by Brand (1951). Trypano-somes of the brucei-evan8i group [for classification

Purification of penicillin-induced penicillinase of Bacillus cereus NRRL 569: a comparison of its properties with those of a similarly purified penicillinase produced spontaneously by a constitutive mutant strain.

Abstract: We wish to express our thanks to Dr Melvin Cohn for much valuable advice during the preliminary work on methods of purification, and one of us (M.R.P.) is greatly indebted to Dr Jacques Monod of the Institut Pasteur, Paris, for the hospitality of his laboratory in which much of the earlier work on isolation ofpenicillinase was carried out. We are also grateful to Dr T. S. Work for help in the chromatographic estimation of amino acids. The photographs were taken by Mr M. R. Young. The Perkin-Elmer Tiselius apparatus used for electrophoresis analysis was purchased by meauIs of a grant from Eli Lilly and Co.

The excretion of amino acids by the human; a quantitative study with ion-exchange chromatography.

Abstract: Berger, L., Slein, M. W., Colowick, S. P. & Cori, C. F. (1946). J. gen. Phy8iol. 29, 379. Dickens, F. & Glock, G. E. (1951). Biochim. biophy8. Acta, 7, 578. Fiske, C. H. & Subbarow, Y. (1925). J. biol. Chem. 66, 375. Green, D. E., Loomis, W. F. & Auerbach, V. H. (1948). J. biol. Chem. 172, 389. Hochster, R. M. & Quastel, J. H. (1949). Nature, Lond., 164, 865. Hunter, F. E. (1951). Phosphorus Metaboli8m, vol. 1, p. 317. Baltimore: The Johns Hopkins Press. Judah, J. D. & Wiliams-Ashman, H. G. (1951). Biochem. J. 48, 33. Klemperer, H. G. (1955). Biochem. J. 60, 122. Lardy, H. A. & Wellman, H. (1953). J. biol. Chem. 201, 357. Loomis, W. F. & Lipmann, F. (1948). J. biol. Chem. 173, 807. Shacter, B. (1953a). Arch. Biochem. Biophys. 46, 312. Shacter, B. (1953b). Arch. Biochem. Biophy8. 40, 324. Thibault, 0. & Pitt-Rivers, R. (1955). Lancet, 1, 285.