Showing papers in "Biochemical Journal in 1941"

Nicotinamide, cozymase and tissue metabolism.

Abstract: THE importance of cozymase as a fundamental constituent of certain respiratory processes in the living cell is now well established. It is known that cozymase is necessary for the activity in animal tissues of specific dehydrogenases oxidizing alcohol, lactate, malate, triosephosphate, etc. Its mode of action is reasonably clear. After combination with a dehydrogenase, it is reduced by a substrate (for example, lactate) with the formation of reduced cozymase and the oxidized product of the substrate (for example, pyruvate). The reduced cozymase is oxidized to its original form by other enzyme systems in the cell, transfer of hydrogen from the substrate to the final oxidizing agent being thus accomplished. Cozymase acts as a catalyst in the transfer of hydrogen in the cell, and in the intact tissue a dynamic equilibrium between reduced and oxidized cozymase is presumably always maintained.

Studies on diffusing factors: The hyaluronidase activity of testicular extracts, bacterial culture filtrates and other agents that increase tissue permeability.

Abstract: AQUEOUS extracts of mammalian testicle contain a factor that dramatically increases the permeability of the tissues to injected fluids [McClean, 1930; 1931; Hoffman & Duran-Reynals, 1931]. This can be demonstrated by the rapid disappearance of the bleb following intracutaneous injection of these extracts and by the rapid spread through a large area of skin of any suitable coloured indicator that may be injected together with them. This factor is associated with the germinal epithelium and can be extracted from spermatozoa [McClean, 1931]. Factors with similar diffusing properties have been obtained from the most diverse sources, for example, from ifitrates and extracts of staphylococcus and streptococcus [Duran.Reynals, 1933], from organisms of the gas-gangrene group and virulent pneumococci [McClean, 1936], from extracts of malignant tissues [Duran-Reynals & Stewart, 1931; Boyland & McClean, 1935], from snake and spider venoms [Duran-Reynals, 1939] and'from leeches [Claude, 1937]. Various methods for the partial purification of testicular extracts and of baclFrial culture ifitrates of the gas-gangrene group have been described [Morgan & McClean, 1932; Favilli, 1936; Claude & Duran-Reynals, 1937; McClean, 1936; Madinaveitia, 1938; 1939, 1, 2; Mannozzi-Torini, 1939], but no naturally occurring diffusing factor has been isolated in the pure state. Nobody had succeeded in elucidating the mechanism whereby this diffusion effect is produced until Chain & Duthie [1939] reported that purified preparations of testicular diffusing factor exhibit a remarkable mucolytic activity characterized by a rapid fall in the viscosity of mucoprotein preparations and the liberation therefrom of reducing substances. This observation suggested that the mucolytic and diffusing -activities might be due to the same factor and that the spread in the tissues might be due to the action of the enzyme on the mucin-like interfibrillar substance in the collagen of the dermis. It was obviously important to confirm the observations of Chain & Duthie, to determine whether diffusing factors from other sources exhibit a similar mucolytic activity and to make a comparative study of these enzymes. Madinaveitia & Quibell [1940] have already described a viscosimetric method whereby testicular mucinase may be accurately estimated and they have investigated many of the characters of the reaction. During the progress of our own observations Meyer et al. [1940, 1] reported the hydrolysis of hyaluronic acid by bacterial enzymes; it had previously been shown by Meyer & Palmer [1936] that the mucoproteins of vitreous humour and umbilical cord contain a, mucopolysaccharide which they had designated by the term hyaluronic acid. More recently

Separation of the higher monoamino-acids by counter-current liquid-liquid extraction: the amino-acid composition of wool.

Abstract: THE possibility of employing counter-current liquid'liquid-extraction for the separation of amino-acids in the form of acyl derivatives from protein 'hydrolysates was briefly discussed by Synge [1939, 1-4], who described preliminary experiments on the separation of some of the naturally occurring 'monoaminoacids'. In the present paper we review the literature on counter-current liquidliquid extraction, discuss the mathematical and physical basis of the separation of acylamino-acide by liquid-liquid extraction, and describe the construction, operation and testing of a multi-plate chloroform-water counter-current extraction train. We then describe the use of this machine in a control analysis of a known mixture of amino-acids for such components as methionine, valine, proline, the leucines and phenylalanine, together with the application of the new technique to an analysis of wool for these amino-acids. In conclusion we compare the results with those obtained by other methods, and summarize existing knowledge of the amino-acid composition of wool.

A colorimetric method for the standardization of heparin preparations.

Abstract: IT is a fact well known to histologists that certain basic dyes undergo a change of tint when taken up by particular tissue elements, especially cartilage, mucus and the granules of the so-called 'mast cells'. The chemistry of this phenomenon, named 'metachromasia' by Ehrlich, has been studied in some detail by Lison [1935], This investigator concluded that the change of colour in the dye is due to the formation of the tautomeric imino-base, accompanied by an increased degree of molecular aggregation; and that all the substances which show the metachromatic staining contain organically combined sulphuric acid. Any sulphuric acid ester, in fact, provided that its molecular weight is not too low, will produce the characteristic colour change when added to an aqueous solution of a metachromatic dye. Now most of the substances known to be active in delaying the clotting of blood have large molecules containing esterified sulphuric acid; and, conversely, most substances having this structure appear to be anticoagulants [cf. Huggett & Rowe, 1933; Bergstrom, 1936; Chargaff et al. 1936]. Thus anticoagulant activity, and the capacity for giving the metachromatic reaction, are related to the same type of chemical structure. The effectiveness of any substance in producing the colour change appears, indeed, as will be shown, to be roughly proportional to its anticoagulant potency. Heparin co7ntains esterified sulphuric acid [Jorpes, 1935], and gives the metachromatic reaction with toluidine blue [Jorpes et al. 1937]. The reactionhas ,been used with success as a histochemical test for heparin [Jorpes et al. 1937; Wilander, 1939]. It has not, however, been applied-hitherto for the standardization of heparin preparations; and the purpose of this paperdis to specify conditions under which such an application is possible. The dye which has been chosen is toluidine blue.

Esters of phosphoric acid: Phosphoryl hydroxyamino-acids

Abstract: PHOSPHOTYROSINE was made by Levene & Schormuller [1933] by the action of POCl3 in CC14 on formyltyrosine in presence of MgO. From the Mg salt of formylphosphotyrosine the formyl group was removed by hydrolysis with HCI and phosphotyrosine was obtained through the Ba and Pb salts. Levene & Schormiiller [1934, 1] tried three methods of preparing phosphodl-serine of which only direct phosphorylation with H3PO4 and P205 was successful. The Ba salt was isolated. Later Levene & $chormiiller [1934,2] prepared larger quantities and separated the stereoisomers by fractional crystallization of the brucine salts. They also isolated-the Ba salt of phospho-l-hydroxyproline after direct phosphorylation. Phosphohydroxyaspartic acid and phosphohydroxyglutamic acid could not be made. Neither phosphoserine nor phosphohydroxyproline were prepared and very little information was given of the properties of the compounds. Further information was desirable, especially concerning the hydrolysis of these esters by acid, alkali and phosphatase. to compare with caseinogen, caseo-phosphopeptone and other esters of phosphoric acid. EXPERIMENTAL

A study of the partial acid hydrolysis of some proteins, with special reference to the mode of linkage of the basic amino-acids.

Abstract: where R, R', R\" are the side-chains of the different amino-acids, of which only 20 odd molecular species have been isolated so far from the whole range of living organisms. It seems clear, therefore, that the distinctive properties of proteins reside in the arrangement rather than the nature of the amino-acid residues. Bergmann & Niemann [1936; 1937; 1938] have put forward a theory of protein structure based on the idea that amino-acid residues are repeated at regular intervals along the peptide chain, and, further, that the 'frequency' of such recurrency must be factorizable entirely by 2 and/or 3, as must the total number of amino-acid residues in the molecule, and therefore the total number of residues of each amino-acid species in the molecule. This hypothesis imposes considerable limitations on the astronomical possibilities of protein isomerism permitted by the Hofmeister-Fischer hypothesis. The main positive chemical evidence presented in its favour by Bergmann and others has been the results of amino-acid analysis of complete hydrolysates of proteins. It is clear that the only direct evidence in favour of a hypothesis concerning the order of amino-acid residues in a peptide chain can come from the isolation and identification of fragments of this chain containing at least two amino-acid residues. Since n molecular species of simple amino-acids can only give rise to n2 molecular species of dipeptides, an investigation of the dipeptides resulting from the partial hydrolysis of proteins should be possible technically, and, if we can be sure that the dipeptides studied do not arise by rearrangement, but are true fragments of the original peptide chain of the protein, the characterization of these compounds should yield information as to the structure of the original protein molecule. A considerable body of data of this sort has been accumulated during the past 40 years. In the course of an examination of this literature undertaken by one of us (R. L. M. S.), special attention was paid to possible rearrangements occurring in the course of preparing and isolating the products of partial hydrolysis. No evidence could be found in the literature suggesting that partial

The excretion of vitamin A in urine.

Abstract: SINCE vitamin A is insoluble in water one would not expect to find it in urine. As far as normal human urine is concerned this expectation appears to be correct. An early claim by Cooper [1924] to have detected vitamin A in human urine by biological means was criticized by Rowntree [1930], who could not confirm the presence of the vitamin in urine from children even when the diet contained milk, eggs, carrots and cod-liver oil. Kaufmann & Drigalski [1933] obtained negative results from human urine by colorimetric methods. In the urine of rats Davies & Moore [1934] found no vitamin A by the colorimetric method even when toxic overdoses of the vitamin were given. Przezdziecka [1935] claimed to have detected vitamin A in urine by a colorimetric method, which was, however, applied in so unusual a manner as to leave doubt as to the significance of the observation. In urine from pathological subjects, however, Boiler & Brunner [1936] obtained positive results by the SbCl3 method in 10 out of 42 cases examined. Half of the patients excreting the vitamin had cancer. Later, Boiler et al. [1937] published results on 321 cases. Vitamin A was found in the urine in icterus with closure of the biliary duct, chronic nephritis, nephrosis, lobar pneumonia before crisis and cirrhosis of the liver. Large oral doses of vitamin A had little effect on the amounts excreted in the urine. Excretion, was reduced by pyramidone. The presence of vitamin A in pathological urine was confirmed by Schneider & Weigand [1937, 1, 2]. Excretion was observed in cancer, tuberculosis and chronic infections. The excretion was not due to specific renal damage, nor was it caused by increased ingestion of the vitamin. It was claimed that excretion only took place in hypovitaminosis C, and that it could be checked by giving ascorbic acid. Lindqvist [1937] found that in pneumonia the excretion of vitamin A varied from 228 to 3060 I.u. xdaily before crisis, failing to zero after crisis. Urinary excretion was associated with a lowered vitamin A content of the blood, which returned to normal after crisis. Gaehtgens [1937] reported the presence of vitamin A in urine from 8 out of 31 cases ofnormal pregnancy. After large doses of vitamin A had been given it was found in the urine in 19 cases. In chronic nephritis Hedberg & Lindqvist [1938] observed constant or irregular excretion of vitamin A in 23 out of 25 cases. In contrast with the finding in pneumonia the vitamin A content of the blood was often high. In livers taken at autopsy the reserves were frequently low, but bore no relation to

Cytochrome system in Bacterium coli commune.

Abstract: THE aerobic respiration of Bact. coli, like that of Bacillus subtilis, baker's yeast and other aerobic cells, is strongly and reversibly inhibited by KCN, H2S, NaN3 and CO, but, unlike that of B. subtilis or cells of baker's yeast, the inhibition with CO is not light-sensitive. The absorption spectrum of cytochrome in B. coli differs markedly from that of B. subtilis or yeast cells. Thus,,the band a is absent from B. coli, and is replaced by a hardly perceptible shading a, at 590 m,u and a feeble band a2 at 628 mp, which is similar to the band a2 of Azotobacter. This band in organisms exposed to CO moves to 634 m,u, on oxidation it is shifted to 645 mp, and in presence of KCN and 02 it disappears completelyl [Keilin, 1934; Fugita & Kodama, 1934]. The band a2 belongs therefore to an autoxidizable haemochromogen compound which reacts with KCN and with CO. According to Lemberg & Wyndham [1937] this compound can be considered as a bile pigment haemochromogen with -an open tetrapyrrolic chain kept together in a ring form by the central iron atom. Lemberg ascribes to a2 the structure of a biliviolin-haemochromogen which is obtained by dehydrogenation of verdohaemochromogen. The component a2 appears therefore to belong to a bile pigment derivative of haematin a, and, as in all the derivatives with an open tetrapyrrolic chain [Holden & Lemberg, 1939], its absorption spectrum is devoid of the Soret or y-band. The properties of a2., namely its reactions with 02, C0 and KCN, suggest that in B. coli, as in Azotobacter [Negelein & Garischer, 1934], this component may act as cytochrome oxidase. On the other hand, the failure to find any parallelism between the concentration of a2 and the respiratory activity of cells containing this component leaves open the possibility of the existence in B. coli of yet another autoxidizable haematin compound acting as cytochrome oxidase and capable of oxidizing the component b,. The bands b and ; of a typical cytochrome are replaced in B. coli by one band which lies at about 560 nip and which represents the oc-band of a non-autoxidizable haemochromogen compound bl. This band is asymmetric and shows on its short-wave side a reinforcement which, according to Yamagutchi [1937], may represent the band c masked by an extension of the band bl. On freezing and cooling the suspension of B. coli to the temperature of liquid air, the short-wave side of the band b, becomes greatly intensified and appears at 551 m, united by a shading to a much fainter band at 559 m,. The oc-band of cytochrome c which, under similar conditions, is also markedly intensified is, however, shifted from ita usual position at 649-5 to 547 m,. This clearly shows, even if we assume that the asymmetric band b, of B. coli is composed of two