Showing papers in "Biochemical Journal in 1964"

Estimation of the molecular weights of proteins by Sephadex gel-filtration.

Abstract: Allen, R. J. L. (1940). Biochem. J. 34, 858. Bartlett, P. D., Grimmett, P., Beers, L. & Shelata, S. (1956). J. biol. Chem. 218, 419. Burton, K. (1956). Biochem. J. 62, 315. Caiger, P., Morton, R. K., Jarrett, I. G. & Filsell, 0. H. (1962). Biochem. J. 85, 351. Filsell, 0. H., Jarrett, I. G., Atkinson, M. R., Caiger, P. & Morton, R. K. (1963). Biochem. J. 89, 92. Fishkin, A. F. & Lata, G. F. (1958). Endocrinology, 63, 162. Franz, J. M. & Lata, G. F. (1957). Endocrinology, 60, 602. Gallagher, C. H. (1959). Au8t. J. agric. Res. 10, 854. Gallagher, C. H. & Buttery, S. H. (1959). Biochem. J. 72, 575. Handschumacher, R. E., Mueller, G. C. & Strong, F. M. (1951). J. biol. Chem. 189, 335. Holdsworth, E. S., Neville, E., Nader, C., Jarrett, I. G. & Filsell, 0. H. (1963). Biochim. biophy8. Acta (in the Press). Jarrett, I. G. & Filsell, 0. H. (1958). Aust. J. exp. Biol. med. Sci. 36, 433. Jarrett, I. G. & Filsell, 0. H. (1960). Aust. J. exp. Biol. med. Sci. 38, 347. Jarrett, I. G., Jones, G. B. & Potter, B. J. (1964). Biochem. J. 90, 189. Jarrett, I. G. & Potter, B. J. (1952). Au8t. J. exp. Biol. med. Sci. 30, 207. Kaplan, N. 0. & Lipmann, F. (1948). J. biol. Chem. 174,37. Klein, H. & Lipmann, F. (1953). J. biol. Chem. 203, 101. Krebs, H. A. & Kornberg, H. L. (1957). Ergebn. Physiol. 49, 212. McCandless, E. L. & Dye, J. A. (1950). Amer. J. Physiol. 162, 434. McCarthy, R. D., Shaw, J. C. & Lakshmanan, S. (1958). Proc. Soc. exp. Biol., N. Y., 99, 560. McLean, P. (1958). Biochim. biophys. Acta, 30, 303. Novelli, G. D. (1953). Phy8iol. Rev. 33, 525. Olson, R. E. & Kaplan, N. 0. (1948). J. biol. Chem. 175, 515 Schneider, W. C. (1945). J. biol. Chem. 161, 293. Thompson, M. & Mayer, J. (1962). Amer. J. Physiol. 202, 1005.

A simple method for the preparation of 32P-labelled adenosine triphosphate of high specific activity

Abstract: A method is described for preparing /sup 32/P-labeled ATP of high specific activity by an excbange reaction The method is simple and requires only substrates and enzymes that are available commercially Hydrolysis of the labeled ATP with heavy meromyosin indicates that 98 to 99% of the /sup 32/P is in the 1 -phosphate group (auth)

The separation of synaptic vesicles from nerve-ending particles (`synaptosomes')

Abstract: When brain tissue is homogenized in media iso-osmotic to plasma, the club-like presynaptic nerve endings resist disruption and are snapped or torn off from their attachments to form discrete particles (nerve-ending particles) in which all the main structural features of the nerve ending are preserved. For these particles we propose the name ' synaptosomes' in order to emphasize their relative homogeneity and their resemblance in physical properties to other subcellular organelles. They can be separated as a distinct fraction by differential and density-gradient centrifuging (Gray & Whit-Since acetylcholine is now well established, by all the classical criteria, as a central as well as a peripheral transmitter (for a review see Gaddum, 1961), it seems reasonable to conclude that the particle-bound acetylcholine and choline acetyltransferase of the fraction represent acetylcholine and enzyme localized within synaptosomes derived from chol-inergic neurones. Similarly, the 5-hydroxytrypt-amine and noradrenaline in this fraction are probably due to the presence of synaptosomes derived from neurones containing these amines. This view has been strengthened by the findings of Carlsson, Falck & Hillarp (1962), who have obtained histo-chemical evidence for the neuronal localization of these amines in the brain. The nerve endings and the synaptosomes derived from them have a complex fine structure when examined under the electron microscope with positive staining and thin sectioning (see review by Whittaker & Gray, 1962) or negative staining (Home & Whittaker, 1962). They are seen to consist (Plate 1 d) of thin-walled bags containing cytoplasm packed with synaptic granules or; frequently (though not in this example) one or more mitochondria are also present. The region of the post-synaptic membrane immediately adjacent to the ending is thickened; on homogenization it may remain adherent and accompany the synaptosome through the various steps of the fractionation procedure. Synaptic vesicles appear to be of at least three main kinds: 'hollow', 'dense-cored' and 'compound' (Plate 1 d). They have been proposed as the actual binding sites of transmitters within the nerve endings (De and as the morphological counterpart of the 'quantized' release of acetylcholine detected electrophysiologically (Fatt & Katz, 1952). The dense-cored vesicles are numerous in peripheral adrenergic nerve endings, and have there been proposed as the binding sites of noradrenaline. We have for some time been studying the disruption of synaptosomes with the object of obtaining , as separate fractions, synaptic vesicles, intraneuronal mitochondria, external and post-synaptic membranes and the soluble constituents of the nerve-ending cytoplasm for …

Regulation of insulin secretion studied with pieces of rabbit pancreas incubated in vitro.

Abstract: The purpose of the present study was to develop a simple system in vitro suitable for studies on the control of insulin secretion and based on the rate of release of insulin into the incubation medium from pieces of rabbit pancreas. Rabbit pancreas was chosen for these studies because it is thin (thus permitting ready diffusion of gases and other substances) and because it has a high concentration of insulin relative to that of other species (Marks & Young, 1940). Although the system that has been developed could be used in conjunction with bioassays for insulin, our studies have been greatly facilitated by the immunoassay introduced by Yalow & Berson (1960) and developed in a simplified form in this Laboratory (Hales & Randle, 1963). The latter is more convenient for studies of this sort because of its great sensitivity and specificity and relative simplicity. Earlier studies of the factors affecting insulin secretion have been made both in vivo and in vitro. In studies in vivo the effects of various substances on the concentration of insulin in pancreatic and peripheral venous blood have been investigated with biological and immunological methods of insulin assay (for reviews see Houssay, 1937; Foa, 1956; Randle, 1961; Berson & Yalow, 1962). The most extensive studies in vitro have been carried out with perfused rat pancreas in conjunction with biological or immunological methods for the assay of insulin (Anderson & Long, 1948; Grodsky, Bennett, Batts, McWilliams & Vcella, 1962; Grodsky et at. 1963). The results of these investigations have led to the generally accepted view that an increase in blood glucose concentration provides the major physiological stimulus for insulin secretion. They have also suggested the possibility that growth hormone and glucagon may have a direct influence on insulin secretion (though evidence on this point has been conflicting) and that hypoglycaemic sulphonamide derivatives may provoke release of insulin. Evidence is presented below that the release of insulin by pieces of rabbit pancreas in vitro provides

Distribution of lipids in subcellular particles of guinea-pig brain.

Abstract: It is becoming increasingly apparent that lipids act as essential structural elements in the multienzyme systems associated with the cell organelles. Many of the enzymes of the electron-transport chain in mitochondria can be isolated as discrete lipoproteins (Green, 1959), and the phospholipid contained in these is essential for enzymic activity (Reich & Wainio, 1961). Moreover, lipids play a fundamental role in the microstructure of cell meimbranes and may even participate metabolically in the transport of cations (Hokin & Hokin, 1964). Clearly, therefore, the distribution of individual lipids within the cell becomes of fundamental importance. The technique of differential centrifugation has enabled various subcellular fractions to be prepared and numerous lipid analyses of these fractions have been carried out. With liver tissue, consisting predominantly of hepatic cells, the relative cellular homogeneity allows these analyses to apply to defined morphological entities of the cell such as the nuclei or mitochondria (Spiro & McKibbin, 1956; Macfarlane, Gray & Wheeldon, 1960; Getz & Bartley, 1961). However, in tissues like the brain with its complicated cytological differentiation, the application of the same centrifugation technique produces fractions that are grossly heterogeneous. Lipid analyses of such fractions (Peterson & Schou, 1955; Biran & Bartley, 1961) therefore, although ofgreat value, must be regarded as preliminary. In recent years, the use of density-gradient centrifugation (Hebb & Whittaker, 1958; Whittaker, 1959, 1961; Whittaker, Michaelson & Kirkland, 1963, 1964), coupled with morphological characterization in the electron microscope (Gray & Whittaker, 1960, 1962; Whittaker, 1960; Whittaker etal. 1963, 1964), has permitted the isolation of certain morphologically defined structures in relatively pure form. These include pinched-off nerve endings or 'synaptosomes', and synaptic vesicles which are contained within the synaptosomes and can be released from them by suitable disruptive procedures. The present paper describes detailed analyses of the individual lipids present in such subfractions by using, among other techniques, a recently developed method for the determination of all known phospholipids (Dawson, Hemington & Davenport, 1962). It provides new data on the lipid composition of brain mitochondria, nuclei and microsomes, as well as the subcellular particles peculiar to brain: myelin fragments, synaptosomes and synaptic vesicles.

Reactions of N-ethylmaleimide with peptides and amino acids

Abstract: N-Ethylmaleimide (NEM) has been much used for the chemical modification of proteins, and it is usually assumed that reaction occurs specifically at the thiol groups of cysteinyl residues. However, NEM has been shown to react also with the acamino group of peptides and with the imidazole group of histidine (Smyth, Nagamatsu & Fruton, 1960), and with the a-amnino group of certain amino acids (Smyth et al. 1960; Riggs, 1961). Since the reaction ofNEM with some proteins is much slower than its reaction with the thiol group of cysteine (Gregory, 1955), the possibility should be considered that, when NEM reacts with proteins, groups other than thiol may be involved. The present paper gives the rates of reaction of NEM with the ac-amino group of glycylalanine, with the N-terminal peptide of the a-chain of haemoglobin and with the N-terminal peptide of the ,B-chain of haemoglobin. The results obtained emphasize the necessity for caution in the use of NEM as a specific reagent for thiol groups of proteins. In addition, a detailed investigation is reported on the hydrolysis ofNEM addition products with cysteine, homocysteine, glutathione and reduced ribonuclease. The results obtained with these compounds are used to establish a new procedure for determining the extent of reaction of NEM with the thiol groups of a protein, and for assessing the specificity of the reaction.

The oxidation of citrate, isocitrate and cis-aconitate by isolated mitochondria.

Abstract: There are five oxidative steps involved in the conversion of pyruvate into carbon dioxide and water in the tricarboxylic acid cycle; four of these steps lead to the reduction of nicotinamide nucleotide coenzyme more or less directly. The step catalysed by the succinate dehydrogenase results in the 'energy-linked' reduction of nicotinamide nucleotide coenzyme, under certain conditions (Chance & Hollunger, 1960), but this process is obviously very different from those reductions that occur in the other oxidative events. Two of these latter reactions, involving pyruvate dehydrogenase and oc-oxoglutarate dehydrogenase, require amongst other cofactors lipoate or a derivative (see Krebs & Kornberg, 1957). It appears that the reduced lipoate is re-oxidized by NAD and that this reaction is catalysed by the lipoate dehydrogenase (Hager & Gunsalus, 1953; Cutolo, 1956). The two remaining oxidative steps, utilizing isocitrate dehydrogenase and malate dehydrogenase, are thought to lead to the reduction of nicotinamide nucleotide coenzyme directly. The experimental results given in the present paper lead to the conclusion that the nicotinamide nucleotide coenzyme reduced by the isocitrate dehydrogenase is not available to the respiratory chain and that, despite the presence ofNAD and an active nicotinamide nucleotide transhydrogenase, the reduced nucleotide must react with oxaloacetate, a process catalysed by malate dehydrogenase. The malate is then oxidized by reaction with nicotinamide nucleotide coenzyme, which is, in this case, available to the cytochrome system (Chappell, 1961). The rate at which citrate is oxidized appears to be limited by mitochondrial aconitase activity. This is not the case when C68aconitate serves as substrate. The significance of these findings is discussed in relation to the structural organization of the mitochondrion.

Studies on the nature of polysomes.

Abstract: this fraction. Incorporation into glutamic acid was sufficient to account for the synthesis of at least 85% of the total mycelial ao-amino nitrogen content. The redistribution of label observed after the exhaustion of exogenous nitrogen confirmed previous non-isotopic evidence that a rapid turnover of insoluble nitrogen, involving breakdown to the amino acid level, occurred during nitrogen starvation. Extracellular organic nitrogen released after exhaustion of ammonia arose from the breakdown of insoluble mycelial material. Thanks are due to Mr ID. H. W. Scott for skilled technical assistance in the work describecL in this and the two preceding papers. I am also grateful to Mr R. G. Harrison for carrying out determinations with the mass spectrometer, and to Mr A. F. Henson for help in interpreting the isotopic data. Polysomes are aggregates of ribosomes held together by RNA. These structures have been obtained from rat liver Further, electron-micrographs have been published which indicate that aggregates of ribosomes can be attached to a thin strand which might be units of protein biosynthesis by Wettstein et al. (1963) and Noll et al. (1963). They are called 'ergosomes' by these workers to indicate that these aggregates are the working particles and that the aggregate structure is necessary for protein synthesis in vivo and for amino acid incorporation in vitro. This viewpoint is supported by other workers (Goodman & Rich, 1963) and models of the protein-biosynthetic mechanism have been propounded by them. In this paper we first consider details of conditions necessary for optimum amino acid incorporation into protein with rat-liver polysomes, ribosomes and a total ribonucleoprotein particle preparation. We produce evidence that the strand to which ribo-somes are attached in polysomes isolated from rat liver has some of the properties of messenger RNA. We question some details of the models of the protein synthetic mechanism suggested by Wettstein 19 Bioch. 1964, 92

The microbial oxidation of methanol. 2. The methanol-oxidizing enzyme of Pseudomonas sp. M 27.

Abstract: Kornberg, H. L. & Sadler, J. R. (1960). Nature, Lond., 185, 153. Large, P. J. & Quayle, J. R. (1963). Biochem. J. 87, 386. Leadbetter, E. & Foster, J. W. (1958). Arch. Mikrobiol. 30, 91. MacFadyen, D. A. (1945). J. biol. Chem. 158, 107. Peel, D. & Quayle, J. R. (1961). Biochem. J. 81, 465. Quayle, J. R. (1961). Annu. Rev. Microbiol. 15, 119. Rhodes, M. E. (1958). J. gen. Microbiol. 18, 639. Takamiya, A. (1942). Acta phytochim. 13, 1. Takamiya, A. (1943). Acta phytochim. 13, 193. Takamiya, A. (1953). J. Biochem., Tokyo, 40, 415. Treadwell, F. P. & Hall, W. T. (1935). Analytical Chemistry, vol. 2, p. 369. New York: John Wiley and Sons, Inc. Umbreit, W. W., Burris, R. M. & Stauffer, J. F. (1957). Manometric Techniques. Minneapolis: Burgess Publishing Co. Zatman, L. J. (1946). Biochem. J. 40, lxvii.

Preliminary studies on the isolation and metabolism of an intermediate in aromatic biosynthesis: chorismic acid.

Abstract: Chance, B. & Conrad, H. (1959). J. biol. Chem. 234, 1568. Chance, B. & Hagihara, B. (1961). Proc. 5th int. Congr. Biochem., Mo8cow, Symp. no. 5. Chance, B. & Hollunger, G. (1960). Nature, Lond., 185, 666. Chance, B. & Williams, G. R. (1955). Advanc. Enzymol. 17, 65. Chappell, J. B. (1961 a). In Biological Structure and Function, vol. 2, p. 71. Ed. by Goodwin, T. W. & Lindberg, 0. London: Academic Press (Inc.) Ltd. Chappell, J. B. (1961 b). Fed. Proc. 20, 50. Chappell, J. B. (1962). Biochem. J. 84, 62P. Chappell, J. B. (1963). J. biol. Chem. 238, 410. Chappell, J. B. (1964). Biochem. J. 90, 225. Chappell, J. B., Cohn, M. & Greville, G. D. (1963). In Energy-Linked Functions of Mitochondria, p. 219. Ed. by Chance, B. New York: Academic Press Inc. Chappell, J. B. & Greville, G. D. (1961). Nature, Lond., 190, 502. Chappell, J. B. & Greville, G. D. (1963a). Symp. Biochem. Soc. 23, 39. Chappell, J. B. & Greville, G. D. (1963b). Fed. Proc. 22, 526. Gal, E. M. (1960). Arch. Biochem. Biophy8. 90, 278. Greengard, P., Minnaert, K., Slater, E. C. & Betel, I. (1959). Biochem. J. 73, 637. Krebs, H. A. & Bellamy, D. (1960). Biochem. J. 75, 523. Lardy, H. A., Johnson, D. & McMurray, W. C. (1958). Arch. Biochem. Biophy8. 78, 587. Lardy, H. A. & Wellman, H. (1952). J. biol. Chem. i95, 215. Lehninger, A. L. & Greville, G. D. (1953). Biochim. biophy8. Acta, 12, 188. Lehininger, A. L., Rossi, C. S. & Greenawalt, J. W. (1963a). Biochem. biophy8. Re8. Commun. 6, 444. Lehninger, A. L., Rossi, C. S. & Greenawalt, J. W. (1963 b). Fed. Proc. 22, 526. Pardee, A. B. & Potter, V. R. (1948). J. biol. Chem. 176, 1085. Singer, T. P. & Kearney, E. B. (1956). Arch. Biochem. Biophys. 61, 397. Slater, E. C. & Hiilsmann, W. C. (1959). In Ciba Found. Symp.: Regulation of Cell Metabolism, p. 58. Ed. by Wolstenholme, G. E. W. & O'Connor, G. M. London: J. and A. Churchill Ltd.

Changes in the structure and enzyme activity of Saccharomyces cerevisiae in response to changes in the environment

Abstract: The first report, as a result of work with the electron microscope, of cytoplasmic inclusions in cells of Saccharomyces cerevisiae having the characteristic ultrastructural features of plant and animal mitochondria (Palade, 1953) was published by Agar & Douglas (1957), although the characteristic cristae are barely discernible in their published micrographs. Later electron micrographs of cells fixed in potassium permanganate published by Vitols, North & Linnane (1961) show characteristic mitochondria about 0 5 , in length in aerobically grown S. cerevisiae cells. Linnane, Vitols & Nowland (1962) have shown that, in the yeast Torulopsis utilis, anaerobically grown cells do not contain characteristic mitochondria or cytochromes; the cytoplasm of these cells, however, contains a reticular system of membranes, vacuoles containing electron-dense granules, and concentric membrane systems resembling myelin forms. The aerobically grown cells, however, show characteristic mitochondria, no reticular membrane system, and vacuoles with far fewer and lesselectron-dense granules. Heyman-Blanchet, Zajdela & Chaix (1959) claim to have isolated mitochondria-like structures from anaerobically grown yeast cells, but we have failed so far to detect any mitochondria in anaerobically grown S. cerevisiae cells by electron microscopy. Slonimski (1953, 1955) and Tustanoff & Bartley (1962) have shown that S. cerevisiae cells, when grown anaerobically with glucose as substrate, lose their capacity for oxidation. When this anaerobic yeast is incubated in the presence of oxygen and a low concentration of glucose, respiration develops. Under the conditions of Slonimski (1953) respiration develops gradually over several hours; under the conditions of Tustanoff & Bartley (1962) respiration becomes appreciable and is maximal at about 21 hr. In our experience such anaerobically grown yeast does not develop the ability to oxidize acetate, but yeast grown aerobically on low glucose concentrations eventually becomes able to oxidize acetate. The present paper shows that the growth conditions

Microbial growth on C-1 compounds. 6. Oxidation of methanol, formaldehyde and formate by methanol-grown Pseudomonas AM-1.

Abstract: : The complete oxidation of methanol to carbon dioxide in cell-free extracts of methanol-grown Pseudomonas AM 1 was investigated. By using 3-amino-1,2,4-triazole, a known inhibitor of catalase, the independence of methanol oxidation from catalatic activity was shown. The only enzyme capable of oxidizing methanol that could be demonstrated was a dehydrogenase that can be linked to phenazine methosulphate and required the presence of NH4(+) ions. An aldehyde dehydrogenase that reduced 2,6-dichlorophenol-indophenol or phenazine methosulphate in the presence of formaldehyde was found in cell-free extracts and was purified. A nicotinamide-adenine nucleotidelinked formate dehydrogenase was found in cell-free extracts and purified. The possible significance of these enzymes in the oxidation of C-1 substrates by intact cells is discussed. Cell-free extracts of methanol-grown Protaminobacter ruber, Pseudomonas extroquens and Pseudomonas methanica have been found to possess a methanol dehydrogenases similar to those found in Pseudomonas AM 1. A specific nicotinamide-adenine dinucleotidelinked formaldehyde dehydrogenase was found in high activity in extracts of methanol-grown Pseudomonas methanica, in lower activity in extracts of methanolgrown Pseudomonas extorquens and Protaminobacter ruber.