Showing papers in "Biochemical Journal in 1961"

Biochemistry of dystrophic muscle. Mitochondrial succinate–tetrazolium reductase and adenosine triphosphatase

Abstract: Little is known about biochemical abnormalities which accompany or are responsible for the morphological changes which occur in the primary myopathies. A number of such diseases have been characterized in humans (Walton & Nattrass, 1954). The discovery (Michelson, Russell & Harman, 1955) of a muscular dystrophy in a strain of mice, inherited by an autosomal recessive gene, has facilitated the search for biochemical alterations in myopathy. Although this condition may not be identical with any of the types of human muscular dystrophy, its investigation may help to throw light on the biochemistry ofhuman muscular dystrophies. It seems probable that many, at least, of the secondary biochemical changes may be common to various types of muscle disease. A few workers (e.g. Weinstock, Epstein & Milhorat, 1958; Hazzard & Leonard, 1959; White, 1959) have reported altered concentrations of certain muscle enzymes in the mouse myopathy. In view of reported morphological abnormalities in mitochondria of dystrophic mouse muscle (Dr G. W. Pearce, unpublished work; Ross, Pappas & Har-

Analytical separations by high-voltage paper electrophoresis. Amino acids in protein hydrolysates.

Abstract: The determination of the amino acid composition of peptides and proteins is usually carried out on acid hydrolysates by ion-exchange chromatography, by following the methods developed by Moore & Stein and their collaborators (Moore, Spackman & Stein, 1958). This method is precise but time-consuming, requiring up to 600 individual determinations for a single protein analysis. Automatic scanning of the column effluent (Spackman, Stein & Moore, 1958) obviates this objection, but increases complication and cost. An alternative group of methods for amino acid analysis are based on paper-chromatographic separation of the individual amino acids, followed by quantitative determination by means of some modification of the ninhydrin reaction. A considerable literature (reviewed by Lederer & Lederer, 1957; Hanes, 1961) describes the many variants of this method, but most are subject to considerable errors due mainly to the limitations of the ninhydrin reaction, and to losses on the paper during the chromatographic separation and the subsequent manipulations. An exhaustive study of these sources of error has been made by Hanes and his associates (Connell, Dixon & Hanes, 1955; Hanes, 1961; Wade, Matheson & Hanes, 1961; Hanes, Harris, Moscarello & Tigane, 1961; Matheson, Tigane & Hanes, 1961; Tigane, Wade, Tze-Fei Wong & Hanes, 1961), who have developed two new paper-chromatographic systems for the separation of 16 of the 18 common amino acids, and also a modified ninhydrin reagent. These improvements constitute a considerable advance over previous methods, but at the cost of a relatively complex technique. Recoveries of individual amino acids were within ± 10 %. No applications to protein hydrolysates were reported. Levy (1954) has described a method of amino acid analysis based on the paper-chromatographic separation of the dinitrophenyl derivatives, followed by spectrophotometric determination at 360 mp. This method appears to be markedly inferior to ion-exchange chromatography (see Hedbom, 1961, for a direct comparison). Whitehead (1958) has described an isotope-dilution method based on acetylation of the amino acids with [14C]and [3H]-acetic anhydride, and a combined chromatographic separation of the acetyl compounds. This method does not appear to have been widely used, although it is applicable to microgram amounts of protein. Recent advances in the technique of high-voltage paper electrophoresis (reviewed comprehensively by Michl, 1958) have provided an analytical method of extremely high resolving power for small molecules. The present paper describes systems for the complete separation of all the amino acids commonly occurring in protein hydrolysates. Combined with the improved cadmium-ninhydrin method ofHeilmann, Barollier & Watzke (1957) this provides a new method of amino acid analysis, which reduces the estimations required to one for each amino acid present in the hydrolysate, while the precision is comparable with that obtained with the ion-exchange method; 250 ,ug. of protein suffices for a complete amino acid. analysis. Rowe, Ferber & Fischer (1958) have described an analogous method, which was, however, applicable to only 10 of the 18 commonly occurring amino acids. Preliminary accounts of the present work have already been published (Atfield & Morris, 1960a, b).

The partition of solutes between buffer solutions and solutions containing hyaluronic acid.

Abstract: Holden, H. F. (1934). Auat. J. exp. Biol. med. Sci. 12, 55. Holden, H. F. (1935). Aust. J. exp. Biol. med. Sci. 13, 103. Kellaway, C. H. & Williams, F. C. (1933). Au8t. J. exp. Biol. med. Sci. 11, 75, 81. Lovelock, J. E. (1955). Biochem. J. 60, 692. McCosker, D. E. & Daniel, L. J. (1959). Arch. Biochem. Biophys. 79, 1. Neumann, W. P. & Habermann, E. (1952). Naturwi88enschaften, 39, 286. Neumann, W. P. & Habermann, E. (1954). Arch. exp. Path. Pharmak. 222, 367. Neumann, W. P. & Habermann, E. (1955). Biochem. Z. 327, 170. North, E. A. & Doery, H. M. (1958). Nature, Lond., 182, 1374. Rous, P. & Turner, J. R. (1916). J. exp. Med. 23, 219. Saunders, L. (1957). J. Pharm., Lond., 9, 834. Trethewie, E. R. (1939). Aust. J. exp. Biol. med. Sci. 17, 145.

The biosynthesis of trehalose in the locust fat body.

Abstract: Schi8tocerca gregaria, the haemolymph of which may contain up to 2 % of trehalose (Howden & Kilby, 1956). Treherne (1958a, b) demonstrated that the introduction of radioactive glucose into the alimentary canal or directly into the haemolymph of S. gregaria in vsvo resulted in the appearance of radioactive trehalose in the haemolymph within a short time, but the site and mode of conversion of glucose into trehalose were not investigated. We have been able to show that the fat body is the most active tissue of the locust in this respect. The insect fat body is a conspicuous organ which extends throughout the abdominal and thoracic cavities, and consists of a loose meshwork of anastomosing lobes formed of sheets of single or double layers of yellow cells. It occupies the space between the gut and the body wall and is everywhere in contact with the blood, so a ready interchange of metabolites between fat-body cells and the blood would be expected. One function of the fat body which has long been recognized is that of a storage organ, since the cells become loaded with globules of fat, protein and glycogen during the development of the insect. Recently it has been shown that fat-body tissue is active in carrying out a number of different metabolic reactions (Kilby & Neville, 1957; Bellamy, 1958; Zebe & McShan, 1959), and the organ may possibly be considered as an equivalent in some respects of the mammalian liver as a site of intermediary metabolism. For these reasons, it was thought that it might be involved in trehalose biosynthesis. Fat body forms a very convenient tissue for biochemical investigation as it is readily dissected from the insect and can be obtained almost free from other tissues. It was found that the fat body ofS. gregaria would convert radioactive glucose into trehalose in vitro, and the subsequent preparation of active cell-free extracts from it facilitated the study of the pathway of trehalose biosynthesis. Their yeast preparation also contained a specific phosphatase for trehalose phosphate. In the present paper we describe the identification of similar enzymes in the fat body of S. gregaria, and also of other enzymes for the regeneration of uridine diphosphate glucose and the formation of glucose 6-phosphate. Parts of this work have been briefly reported elsewhere (Candy & Kilby, 1959, 1960).

Regulation of glucose uptake by muscle. 5. Effects of anoxia, insulin, adrenaline and prolonged starving on concentrations of hexose phosphates in isolated rat diaphragm and perfused isolated rat heart.

Abstract: Membranetransport ofglucose andintracellular phosphorylation ofthesugar are bothaccelerated by anoxiain theperfused isolated ratheart (Morgan, Randle& Regen,1959). Inisolated rat diaphragm, membranetransport ofglucose isaccelerated byanoxia, buttheeffect ofanoxiaon glucose phosphorylation inthistissue isnotknown (Randle & Smith,1958a, b).Crane& Sols(1953) haveshownthatmusclehexokinase isinhibited by glucose 6-phosphate andthisobservation suggested thatanoxiamightaccelerate glucose phosphorylation inmusclebylowering theintracellular concentration ofglucose 6-phosphate andthereby increasing theactivity ofhexokinase. Theresults of experiments recorded hereappear toshowthat anoxia can lowertheintracellular concentration of glucose 6-phosphate inheartanddiaphragm, and further measurements havebeenmadeoftheconcentrations offructose 6-phosphate and fructose 1:6-diphosphate inan attempt todefine theenzymic steporsteps whichareaffected byanoxiaandwhich bringaboutthischangeintheconcentration of glucose 6-phosphate. The possibility thatmembrane transport of monosaccharides inmusclemightberegulated by theintracellular concentration ofglucose 6-phosphatehasalsobeeninvestigated bystudying the effects ofagents whichaltertheintracellular concentration ofglucose 6-phosphate, on themembrane transport ofD-xylose andD-3-0-methylglucose in

Protein synthesis in mitochondria. Requirements for the incorporation of radioactive amino acids into mitochondrial protein

Abstract: of inactivated (heated at 80° for 30 min.) intestinal mucosa of hens, and the optimum pH for the enzyme of mature fowl was not altered by inactivated mucosa of chicks. 3. A mixture of the enzyme preparations of chicks and mature fowl exhibited a pH-activity curve and quantitative reaction velocities which would be expected from a combination of two enzymes possessing different pH optima. 4. In rabbits the shift in pH optima for intestinal phosphatase was to lower values with increasing age, the major change taking place in the first 2 months of life. 5. There was no change in pH optima of the phosphatase of intestinal mucosa of rats as their age increased from 6 weeks to 1 year. The pH optima for the phosphatase of a preparation of the whole duodenum of 1-day-old rats were higher than those of the mucosal enzyme of older rats. 6. The pH optima for the phosphatases of bone, liver and kidney of fowl and rabbits appeared to be constant regardless of their age. The authors wish to express their indebtedness to Miss G. Ritcey for invaluable technical assistance.

Carbohydrates in protein. 3. The preparation and some of the properties of a glycopeptide from hen's-egg albumin

Abstract: material. The studywas initiated partlyinordertoinvestigate thenature oftheprotein-carbohydrate linkage, butthisgoal couldnot be achievedby themethodsthen available. Someprogress hasbeenmade inthis problemby more recentstudies and Johansen, Marshall & Neuberger (1960) havegiventhemost probable values forthemannose,glucosamine and acetyl contents ofthewholeprotein andofaglycopeptide isolated fromit.Theprobability thatthese aretheonlysugarspresentwasindicated. Itis thepurposeofthispapertodescribe thepreparationand some oftheproperties ofthisglycopeptide, andtoconsider thenatureofthechemical bondlinking thecarbohydrate to theprotein. A briefdescription ofthisworkwas reported earlier (Johansen, Marshall & Neuberger, 1958). Cunningham, Nuenke& Nuenke (1957)and Jevons(1958) havealsogivenshortaccountsof their findings onthesamesubject.

Sudden freezing as a technique for the study of rapid reactions.

Abstract: Anderson, L. & Plaut, W. G. E. (1949). In Respiratory Enzymes, p. 71. Ed. by Lardy, H. A. Minneapolis, U.S.A.: Burgess Publishing Co. Avis, P. G., Bergel, F. & Bray, R. C. (1955). J. chem. Soc. p. 1100. Avis, P. G., Bergel, F., Bray, R. C., James, D. W. F. & Shooter, K. V. (1956a). J. chem. Soc. p. 1212. Avis, P. G., Bergel, F. & Bray, R. C. (1956b). J. chem. Soc. p. 1219. Beinert, H. & Sands, R. H. (1959). Biochem. biophys. Re8. Commun. 1, 171. Beinert, H. & Sands, R. H. (1960). Biochem. biophys. Res. Commun. 3, 41. Bergel, F. & Bray, R. C. (1958). Symp. biochem. Soc. 15,64. Bleaney, B. & Ingram, D. J. E. (1952). Proc. phys. Soc., Lond., A 65, 953. Bowers, K. D. & Owen, J. (1955). Rep. Progr. Phys. 18, 304. Bray, R. C. (1959). Biochem. J. 73, 690. Bray, R. C. (1961). Biochem. J. 81, 196. Bray, R. C., Malmstr6m, B. G. & Vgnng&rd, T. (1959). Biochem. J. 73, 193. Gibson, J. F., Ingram, D. J. E. & Schonland, D. (1958). Disc. Faraday Soc. 26, 72. Gutfreund, H. & Sturtevant, J. M. (1959). Biochem. J. 73, 1. Haddow, A., de Lamirande, G., Bergel, F., Bray, R. C. & Gilbert, D. A. (1958). Nature, Lond., 182, 1144. Hollocher, T. C. & Commoner, B. (1960). Proc. nat. Acad. Sci., Wash., 40, 416. Larson, M. L. (1960). J. Amer. chem. Soc. 82, 1223. Malmstr6m, B. G. & Vainngard, T. (1960). J. molec. Biol. 2, 118. Michaelis, L. & Schubert, M. P. (1938). Chem. Rev. 22, 437. Morell, D. B. (1952). Biochem. J. 51, 657. Perrin, D. D. (1958). J. Amer. chem. Soc. 80, 3540. Sacconi, L. & Cini, R. (1954). J. Amer. chem. Soc. 76, 4239. Sands, R. H. & Beinert, H. (1960). Biochem. biophys. Bes. Commun. 3, 47. Selwood, P. W. (1956). Magnetochemistry. New York: Interscience Publishers Inc. Theorell, H. & Ehrenberg, A. (1951). Ark. Fys. 3, 299. Totter, J. R. & Comar, C. C. (1956). In Inorganic Nitrogen Metabolism, p. 513. Ed. by McElroy, W. D. & Glass, B. Baltimore: The Johns Hopkins Press. VanngArd, T., Bray, R. C., Malmstr6m, B. G. & Pettersson, R. (1961). In Symp. Free Radicals in Biological Systems, p. 209. New York: Academic Press Inc. Weissman, S. I. & Cohn, M. (1957). J. chem. Phy8. 27, 1440. Whitby, L. G. (1953). Biochem. J. 54, 437.

A colorimetric method for the estimation of monoamine oxidase.

Abstract: Michael, S. E. (1958). Ab8tr. Comm. 4thint,. Congr. Biochem., Vienna, no. 2-106, p. 28. Pedersen, K. 0. (1958). J. phys. Chem. 62, 1282. Popjaik, G. & McCarthy, E. F. (1946). Biochem. J. 40, 789. Ram, J. S. & Maurer, P. H. (1958). Arch. Biochem. Biophys. 76, 28. Reichmann, M. E. & Charlwood, P. A. (1954). Canad. J. Chem. 32, 1092. Schwert, G. W. (1957). J. Amer. chem. Soc. 79, 139. Smith, D. B., Wood, G. C. & Charlwood, P. A. (1956). Canad. J. Chem. 34, 364. Svedberg, T. & Pedersen, K. 0. (1940). The Ultracentrifuge. Oxford University Press. Warren, R. L. & Charlwood, P. A. (1953). Nature, Lond., 171, 353. Wieme, R. J. (1959). Clin. chim. Acta, 4, 317. Williams, J. W., van Holde, K. E., Baldwin, R. L. & Fujita, H. (1958). Chem. Rev. 58, 715.