bluuebunny/crossref_metadata_2025 huggingface dataset:
The metabolism of S-methyl-l-cysteine
Authors: Sklan Naomi M. , Barnsley E. A.
Published: August 2015

1. Methylsulphinylacetic acid, 2-hydroxy-3-methylsulphinylpropionic acid and methylmercapturic acid sulphoxide (N-acetyl-S-methyl-l-cysteine S-oxide) were isolated as their dicyclohexylammonium salts from the urine of rats after they had been dosed with S-methyl-l-cysteine. 2. A fourth sulphoxide was isolated but not identified. 3. The excretion of sulphate in the urine of rats dosed with S-methyl-l-cysteine was measured. 4. The metabolism of S-methyl-l-cysteine by the hamster and guinea pig was examined chromatographically. 5. The preparation of the following compounds is reported: (−)-dicyclohexylammonium methyl-mercapturate sulphoxide; the dicyclohexylammonium salts of the optically inactive forms of 2-hydroxy-3-methylthiopropionic acid, 2-hydroxy-3-methyl-sulphinylpropionic acid and methylsulphinylacetic acid.
Ultramicro determination of plasma testosterone by electron-capture detection of testosterone diheptafluorobutyrate
Authors: Exley D.
Published: August 2015

1. A new highly sensitive and accurate ultramicro method for the estimation of testosterone in human peripheral plasma is described. The method uses paper-and thin-layer-chromatographic separation of plasma testosterone, which is determined as testosterone diheptafluorobutyrate by electron-capture detection after gas–liquid chromatography. 2. The average difference between duplicates is ±2% (range 1–5%) with as little as 2·5ml. of human male peripheral plasma. With 10ml. of plasma the method is sensitive enough for the accurate determination of testosterone in human female plasma. The high order of accuracy is achieved by the use of a radioactive label and an internal standard for gas chromatography, and by obtaining several gas chromatograms from the same plasma sample. 3. As little as 40μμg. of peripheral plasma testosterone can be detected. The method is 20 times as sensitive as electron-capture techniques with the monochloroacetate derivative. 4. The method is simpler and quicker than double-isotope-derivative methods, and slightly more sensitive. The advantages of the method, which is specific for testosterone, are its high sensitivity and accuracy, which are achieved with relative convenience.
Investigations on the oligosaccharide units of an A myeloma globulin
Authors: Dawson G. , Clamp J. R.
Published: August 2015

The carbohydrate content of an A myeloma globulin was investigated. The carbohydrate content was found to be unchanged when the protein was isolated from the patient over a period of 18 months. The various polymeric forms of the protein contained similar proportions of carbohydrate. The A myeloma globulin contained approx. 2 residues of 6-deoxy-l-galactose (l-fucose), 14–15 of d-mannose, 12–13 of d-galactose, 12–13 of 2-acetamido-2-deoxy-d-glucose (N-acetyl-d-glucosamine), 6 of 2-acetamido-2-deoxy-d-galactose (N-acetyl-d-galactosamine) and 5 of N-acetylneuraminic acid (sialic acid), and these were distributed between six oligosaccharide units all of which were present on the heavy polypeptide chains. The oligosaccharide units showed two kinds of heterogeneity, which have been termed central and peripheral. Central heterogeneity was shown by the presence of three completely different core units, which had the following compositions: (1) 3 residues of d-galactose and 3 of 2-acetamido-2-deoxy-d-galactose, joined to protein by an O-glycosidic linkage between acetamidohexose and serine; (2) 3 residues of d-mannose, 2 of d-galactose and 3 of 2-acetamido-2-deoxy-d-glucose, joined to protein by an N-glycosidic linkage between acetamidohexose and aspartic acid; (3) 4 residues of d-mannose and 3 of 2-acetamido-2-deoxy-d-glucose with a linkage similar to that in (2). The core oligosaccharide units showed peripheral heterogeneity in the attachment of 6-deoxy-l-galactose, 2-acetamido-2-deoxy-d-glucose and N-acetylneuraminic acid. Tentative structures are proposed for these various types of oligosaccharide unit. Glycopeptides were isolated in which the sialic acid content exceeded that of d-galactose. Explanations are given for the electrophoretic mobility and staining characteristics of the various glycopeptides.
Studies on the enzymic N-acylation of amino sugars in the sheep colonic mucosa
Authors: Allen A. , Kent P W
Published: August 2015

1. d-[2−14C]Glucose, [2−14C]acetate, hydroxy[3−14C]pyruvate, [3−14C]pyruvate and [U−14C]glycine were incorporated by surviving scrapings of sheep colonic mucosal tissue into glycoprotein. 2. d-[2−14C]Glucose, [2−14C]acetate, incorporated hydroxy-[3−14C]pyruvate and [3−14C]pyruvate resulted in labelling of each of the monosaccharide residues of the glycoprotein, namely N-glycollylneuraminic acid, N-acetylneuraminic acid, galactose, fucose, glucosamine and galactosamine. [U−14C]Glycine was incorporated as glycyl and seryl residues of the glycoprotein. 3. Despite N-glycollylneuraminic acid being quantitatively the predominant sialic acid (N-glycollylneuraminic acid and N-acetylneuraminic acid were 8·5 and 5·2% by weight of the glycoprotein respectively) the corresponding ratio of the radio-active labelling from d-[2−14C]glucose in N-glycollylneuraminic acid to that in N-acetylneuraminic acid was 1·00:7·27 (expressed as percentages of the total radioactivity in the glycoprotein). Neutral sugar, hexosamine and N-acetylneuraminic acid residues of the mucoprotein were each labelled to a similar extent. 4. Similarly, the ratio of the radioactivity in N-glycollylneuraminic acid to that in N-acetylneuraminic acid in the mucoprotein from tissue incubations with [2−14C]-acetate was 1·0:4·0. 5. Both [2−14C]acetate and [2−14C]glucose with whole tissue led to labelling of the N-glycollyl substituent and of the main nonose skeleton of the N-glycollylneuraminic acid. In whole-tissue incubations, [3−14C]pyruvate was also a precursor of radioactive N-glycollylneuraminic acid. 6. Hydroxy[3−14C]-pyruvate and [U−14C]glycine caused labelling of the carbohydrate and peptide residues of the glycoprotein, but did not give rise to labelling in the N-glycollylneuraminic acid residues. 7. With a wide variety of possible N-glycollyl precursors (fructose 6-phosphate, hydroxypyruvate, glycollate and chemically synthesized glycollyl-CoA) biosynthesis of N-glycollylglucosamine was not observed in cell-free preparations.
Influence of temperature acclimatization on the ionic activation of goldfish intestinal adenosine triphosphatase
Authors: Smith M W , Colombo V. E. , Munn E A
Published: August 2015

1. An adenosine triphosphatase membrane system, dependent on Mg2+ and activated further by Na++K+, was prepared from goldfish anterior intestine by differential centrifugation of homogenized intestinal scrapings. 2. The affinity of this preparation for Na+ in the presence of K++Mg2+, for K+ in the presence of Na++Mg2+ and for Mg2+ alone, measured at 37°, did not depend on the previous environmental temperature of the fish. When Na++K+ were added to preparations from 8°-acclimatized fish the affinity for Mg2+ increased; this was not seen with preparations from 30°-acclimatized fish. 3. Part of the Mg2+-activated adenosine triphosphatase was inhibited by Na+ and the amount of inhibition appeared to increase at high acclimatization temperatures. 4. This Na+-inhibited adenosine triphosphatase was separated from the (Na++K+)-activated enzyme by centrifugation on sucrose density gradients. 5. Preparations from 8°-acclimatized fish contained less Mg2+-activated and more (Na++K+)-activated adenosine triphosphatase than did similar fractions from 30°-acclimatized fish. 6. Acclimatization to different environmental temperatures might involve one form of adenosine triphosphatase changing to another. The origin of various membranes seen in microsomal fractions must, however, be established before this hypothesis can be tested further.
Choice between autotrophy and heterotrophy in Pseudomonas oxalaticus. Utilization of oxalate by cells after adaptation from growth on formate to growth on oxalate
Authors: Blackmore Maureen A. , Quayle J. R. , Walker I O
Published: August 2015

1. The labelling patterns of phosphoglycerate obtained from formate-grown or oxalate-grown Pseudomonas oxalaticus after exposure for 15sec. to [14C]formate or [14C]oxalate respectively were determined. 2. The phosphoglycerate obtained from the formate-grown cells contained 78% of the radioactivity in the carboxyl group. This is in accord with that predicted for operation of the ribulose diphosphate cycle of carbon dioxide fixation. 3. The labelling pattern of the phosphoglycerate obtained from the oxalate-grown cells approached uniformity, as predicted for the heterotrophic pathway of oxalate assimilation. The departure from complete uniformity may have been due to concurrent 14CO2 fixation into C4 dicarboxylic acids. 4. The labelling pattern of phosphoglycerate obtained from cells that had just started to grow on oxalate after adaptation from formate was determined after incubation of the cells for 15sec. with [14C]oxalate. This pattern approached uniformity. 5. The pathway of incorporation of 14CO2 into cells that had just started to grow on oxalate after adaptation from formate, in the presence of either formate or oxalate as energy source, was studied by chromatographic and radio-autographic analysis. 6. It is concluded from the isotopic data that a mixed heterotrophic–autotrophic metabolism, with the former mode predominating, operates in the initial stages of growth on oxalate after adaptation from growth on formate.
Characterization and biosynthesis of cytochrome b5 in rat liver microsomes
Authors: Sargent J. R. , Vadlamudi B. P.
Published: August 2015

1. Cytochrome b5 is released from rat liver microsomes by both proteolytic enzymes and by treatments that disrupt phospholipids. Cytochrome P-420 is only released to a marked extent by treatments that disrupt phospholipids. 2. Cytochrome b5 was isolated in a pure state from both the rough and smooth fractions of rat liver microsomes after treatment with trypsin, and was shown to contain two cytochrome components with identical spectral properties. 3. Amino acid analyses of the two components are presented, together with peptide ‘fingerprint’ patterns of tryptic digests of the two components. 4. Studies based on the direct isolation of cytochrome b5 after administration of a single dose of radioactive amino acid to rats demonstrate that the cytochrome is synthesized initially in the rough fraction of microsomes and only subsequently appears in the smooth fraction. 5. Isolated rat liver microsomes are capable of incorporating radioactive amino acids into cytochrome b5 under standard conditions. 6. Under these conditions the amino acid is incorporated into peptide linkage in the cytochrome.
The effect of reaction with formaldehyde on the sedimentation rates of ribonucleic acids
Authors: Fenwick M. L.
Published: August 2015

It has been reported that the RNA of several bacteriophages and that of the larger ribosomal sub-units of mammalian cells sediment faster in the presence of 0·1m-sodium chloride than is expected from their estimated molecular weights. The effect of blocking the hydrogen-bonding amino groups of these and other types of RNA was studied. The RNA of phage R17 no longer sedimented anomalously fast after treatment with formaldehyde. In contrast, the larger ribosomal RNA of HeLa cells appeared more aberrant than before, sedimenting faster than tobacco-mosaic-virus RNA (mol.wt. 2×106) in the presence of formaldehyde. The rapidly labelled nuclear 45s RNA of HeLa cells still sedimented faster than the larger ribosomal RNA after reaction with formaldehyde, showing no evidence of disaggregation. It is suggested that both the large ribosomal RNA and the 45s RNA of HeLa cells may have a non-linear structure.
Gluconeogenesis in developing rat kidney cortex
Authors: Zorzoli A. , Turkenkopf I. J. , Mueller V. L.
Published: August 2015

1. Gluconeogenesis in developing rat kidney cortex was studied by assaying the activities of two enzymes, glucose 6-phosphatase and phosphoenolpyruvate carboxykinase, and by measuring glucose formation in tissue slices. 2. Glucose 6-phosphatase and phosphoenolpyruvate carboxykinase are present in late foetal (21–22-day-old) tissue and increase rapidly postnatally. Maximum activity of phosphoenolpyruvate carboxykinase occurs at 7 days of age, followed by a decline to the adult level. Glucose 6-phosphatase activity rises during the first 2 postnatal weeks and then declines. 3. Late foetuses synthesize glucose from both pyruvate and l-glutamate. The rate increases during the first 2 weeks to above adult levels. Synthesis is always higher from pyruvate than from glutamate. 4. The effect of 24hr. starvation was studied in perinatal animals. The results indicate that the ability to increase the rate of glucose synthesis as a result of starvation is not present at birth, but develops some time after the second postnatal day.
Lactate dehydrogenase isoenzymes of sperm cells and testes
Authors: Clausen Jørgen
Published: August 2015

1. The kinetic and metabolic properties of lactate dehydrogenase isoenzyme LDHx from human sperm cells and rat testes were studied. 2. LDHx shows a sensitivity to inhibition by stilboestrol diphosphate, urea and guanidinium chloride different from that of the LDH-H4 and LDH-M4 isoenzymes. 3. About 10 and 20% of the total lactate dehydrogenase activity of testes and sperm cells respectively were associated with particulate fractions. In sperm cells 11% was localized in the middle piece and 18·8% in the head fraction. LDHx was found in all particulate fractions of sperm cells. The middle piece contained 41·0% of total LDHx activity and showed high succinate dehydrogenase activity. 5. The pH-dependence of lactate/pyruvate and NAD+/NADH concentration ratios were estimated. Lactate dehydrogenase in sperm cells has maximal activity with NADH as coenzyme at pH7·5 and with NADPH as coenzyme at pH6·0. At pH6·0 a 10% greater oxidation of NADPH than of NADH was found. At acid pH lactate hydrogenase may function as an enzyme bringing about transhydrogenation from NADPH to NAD+. 6. In agreement with the stoicheiometry of the lactate de- hydrogenase reaction, the lactate/pyruvate concentration ratio decreased with increasing pH. 7. The lactate/pyruvate and NAD+/NADH concentration ratios were estimated with glucose, fructose and sorbitol as substrates and as a function of time after addition of these substrates. During a 20min. period after the addition of the substrates, changes in lactate/pyruvate and NAD+/NADH concentration ratios were noticed. Increasing concentration of the substrates mentioned gave rise to asymptotic increases in lactate and pyruvate. 8. Sorbitol did not act as a substrate for LDHx. 9. The findings described are consistent with the idea that LDHx is different from other known lactate dehydrogenase isoenzymes, but that it has a metabolic function similar to that of the isoenzymes of other tissues.
A product-inhibition study of the mechanism of mitochondrial octanoyl-coenzyme A synthetase
Authors: Graham A. B. , Park M. V.
Published: August 2015

By a study of the product-inhibition kinetics of the octanoyl-CoA synthetase from ox liver mitochondria, evidence was obtained consistent with the hypothesis that the enzyme reacts by a Bi Uni Uni Bi Ping Pong type of mechanism in which the order of addition and evolution of substrates and products is CoA, octanoate, octanoyl-CoA, ATP, PPi and AMP. There is also evidence that more than one molecule of CoA can add to the enzyme and that it may act as an allosteric activator.
Temperature and the regulation of enzyme activity in poikilotherms. Properties of rainbow-trout fructose diphosphatase
Authors: Behrisch H. W. , Hochachka P. W.
Published: August 2015

1. The properties of fructose diphosphatase from the liver of rainbow trout (Salmo gairdnerii) were examined over the physiological temperature range of the organism. 2. Saturation curves for substrate (fructose 1,6-diphosphate) and a cofactor (Mg2+) are sigmoidal, and Hill plots of the results suggest a minimum of two interacting fructose 1,6-diphosphate sites and two interacting Mg2+ sites per molecule of enzyme. 3. Mn2+-saturation curves are hyperbolic, and the Ka for Mn2+, which inhibits the enzyme at high concentrations, is 50–100-fold lower than the Ka for Mg2+. 4. Fructose diphosphatase is inhibited by low concentrations of AMP; this inhibition appears to be decreased and reversed by increasing the concentrations of Mg2+ and Mn2+. Higher concentrations of AMP are required to inhibit the trout fructose diphosphatase in the presence of Mn2+. 5. The affinities of fructose diphosphatase for fructose diphosphate and Mn2+ appear to be temperature-independent, whereas the affinities for Mg2+ and AMP are highly temperature-dependent. 6. The pH optimum of the enzyme depends on the concentrations of Mg2+ and Mn2+. In addition, pH determines the Ka for Mg2+; at high pH, Ka for Mg2+ is lowered. 7. The enzyme is inhibited by Ca2+ and Zn2+, and the inhibition is competitive with respect to both cations. 8. The possible roles of these ions and AMP in the modulation of fructose diphosphatase and gluconeogenic activity are discussed in relation to temperature adaptation.
Lysosomal enzyme changes in growing and regressing mammary tumours
Authors: Shamberger Raymond J.
Published: August 2015

Rat mammary tumours induced by 7,12-dimethylbenz[a]anthracene can undergo repeated growth and regression during successive pregnancies. In a 10-day period after birth about half of the tumours regressed 50% or more. The concentrations of the lysosomal enzymes increased in regressing mammary tumours to the following multiples of the initial values: β-glucuronidase, 7·7; β-galactosidase, 3·9; cathepsin, 2·9; acid ribonuclease, 2·1; arylsulphatase A, 1·5; acid phosphatase, 1·4. In contrast, several non-lysosomal enzymes failed to increase. Activities in the post-partum uterus increased to the following multiples of the initial values: β-glucuronidase, 5·8; cathepsin, 5·5; acid ribonuclease, 4·3; β-galactosidase, 2·2; acid phosphatase, 1·8. Arylsulphatase A in the post-partum uterus decreased significantly, suggesting a non-lysosomal distribution or a special function related to pregnancy. No other significant changes were observed in the lysosomal or non-lysosomal enzymes in the hormone-independent liver or hormone-dependent normal mammary gland. The ratio of free to bound arylsulphatase A and acid ribonuclease decreased slightly 1–3 days after birth because of problems in homogenizing the tumours. At days 4–8, however, there was a dramatic increase in the ratio of the free to bound activities. The results can be explained in terms of the lysosomal theory of intracellular digestion.
Metabolism of parathyroid hormone. Degradation of 125I-labelled hormone by kidney enzyme
Authors: Martin T. J. , Melick R. A. , de Luise M.
Published: August 2015

A study was made of the enzymic degradation of 125I-labelled parathyroid hormone by rat kidney microsomes. Incubation with microsomes resulted in rapid destruction of the labelled hormone. The microsomal factor was not separable by dialysis, and the reaction was favoured by pH values in the physiological range. Velocity of the reaction varied directly as the substrate concentration, and additional crude parathyroid hormone (trichloroacetic acid-precipitated, 3·68mg./ml.) inhibited destruction of labelled hormone. There was much less inhibition with added trichloroacetic acid-precipitated calcitonin (3·92mg./ml.) and virtually none with added pig insulin (3·80mg./ml.). Gel filtration of control medium on P6 (Bio-Gel) yielded one radioactive peak at the void volume. After incubation with microsomes three further peaks were obtained on gel filtration. Only the void-volume peak contained intact 125I-labelled parathyroid hormone, indicating that the microsomal enzyme degraded labelled hormone to a number of smaller fragments.
Study of the methylation and lack of deamination of deoxyribonucleic acid by N-methyl-N′-nitro-N-nitrosoguanidine
Authors: Craddock Valda M.
Published: August 2015

1. DNA labelled with 14C in the purine residues was prepared by treating newborn rats with [14C]formate and killing them for preparation of nucleic acids at 11–17 months. This DNA was incubated with N-methyl-N′-nitro-N-nitrosoguanidine, and then analysed for products of methylation and deamination reactions. 2. Evidence was found for the formation of 7-methylguanine and a smaller amount of 3-methyladenine, and, after preliminary denaturation of the DNA, 1-methyladenine was detected. The presence of cysteine increased the extent of methylation. No evidence was found for the formation of xanthine or hypoxanthine, even at pH5·5.
Importance of the osmolarity of the incubation medium on amino acid incorporation into protein by isolated rat liver mitochondria
Authors: Haldar D. , Freeman K. B.
Published: August 2015

1. Incorporation of [14C]leucine into protein by isolated rat liver mitochondria was examined by using incubation media similar to those used by Sandell, Löw & Decken (1967) (medium A) and Roodyn, Reis & Work (1961) (medium B). The incorporation process was found to be almost completely inhibited in medium A. 2. By decreasing the amount of sucrose and omitting tris–hydrochloric acid from medium A, incorporation proceeded at a rate higher than that found in medium B. It was found that the inhibitory action of medium A was due to its high osmolarity. 3. Oxidative phosphorylation and RNA synthesis by the isolated mitochondria proceeded at the same rate in media essentially the same as media A and B. 4. There was a partial inhibitory action of medium A on leucine uptake by the mitochondria and also on the formation of leucyl-transfer-RNA. The major block of inhibition by the hyperosmolarity of medium A seemed to be located at a later step of protein synthesis involving mitochondrial ribosomes. 5. Protein synthesis by Escherichia coli B was only slightly inhibited, if at all, in hyperosmotic media in which protein synthesis by isolated mitochondria was completely stopped.
The metabolism of arylazoisoxazolones by rats and dogs
Authors: Daniel J. W.
Published: August 2015

1. When rats were given a single oral dose of the lipid-soluble fungicide 4-(2-chlorophenylhydrazono)-3-methyl[4−14C]isoxazol-5-one ([14C]drazoxolon), about 75% of the label was excreted in the urine and 13% in the faeces in 96hr. An additional 7% of the radioactivity was recovered as 14CO2 in 48hr. 2. About 8% of the label was excreted by rats in the bile in 0–24hr. and an additional 6% was excreted by the same route in 24–48hr. 3. When dogs were given a single oral dose of [14C]drazoxolon about 35% of the label was excreted in the urine and a similar amount was excreted in the faeces in 96hr. 4. The major metabolites in the urine of the rat and the dog were identified as 2-(2-chloro-4-hydroxyphenylhydrazono)acetoacetic acid (dog, 14%), the corresponding ether glucosiduronic acid (dog, 12%; rat, 13%) and ester sulphate (rat, 65%). 5. When rats were given a single oral dose of 3-methyl-4-([U−14C]phenylhydrazono)isoxazol-5-one about 75% of the label was excreted in the urine and 15% in the faeces in 96hr. The major metabolite in the urine was identified as the ester sulphate conjugate of 2-(4-hydroxyphenylhydrazono)-acetoacetic acid. 6. Reduction of the azo link was of minor quantitative significance. 7. These results are discussed in their relation to species differences in the toxicity of these compounds.
The localization of proteolytic activity in rat liver mitochondria and its relation to mitochondrial swelling and aging
Authors: Alberti K. G. M. M. , Bartley W.
Published: August 2015

1. On storage of rat liver mitochondria at 0°, water content, total amino acid content and leakage of protein all rose steadily over a 72hr. period. The initial ratio of intramitochondrial to extramitochondrial amino acid concentration lay between 18 and 24. Initially this rose, but it then fell to 1·9 at the end of storage. The concentration gradient between internal and external amino acids was relatively constant throughout the period. These processes were accentuated at 22° and 40°, the concentration gradient reaching 70μmoles/ml., water content rising to 8·3mg./mg. dry wt. and protein leakage reaching 42% of total mitochondrial protein. ‘Swelling agents’ produced no correlated changes in amino acid production and swelling. 2. Added glutamate was not concentrated within the pellet of whole or disrupted mitochondria. Endogenous amino acids were distributed evenly between the pellet and the supernatant of disrupted mitochondria. It is concluded that amino acids are produced within mitochondria and that adsorption and uptake from the medium do not contribute significantly to amino acids in the pellet. 3. β-Glycerophosphate, a lysosome protectant, increased amino acid production by rat liver mitochondria. Treatment with Triton X-100 and disruption by freezing and thawing showed that 56% of proteolytic activity was ‘free’ in whole mitochondria, whereas only 11% of acid phosphatase activity, a lysosomal enzyme, was ‘free’. 4. ‘Light’ mitochondria contained 30% more neutral proteolytic activity but 300% more acid phosphatase activity than ‘heavy’ mitochondria. 5. Electron micrographs of mitochondrial preparations showed less than one particle in 500 that could be identified as a lysosome. Treatment with Triton X-100 disrupted the structure of roughly 50% of the mitochondria; the rest appeared to retain their membrane, cristae and ground substance. Freezing and thawing caused gross swelling and loss of ground substance and rupture of external membranes. 6. Of the recovered proteolytic activity, 81% at pH7·4 and 70% at pH5·8 were found in the high-speed supernatant of broken mitochondria. A further fivefold increase in specific activity was found in the first protein fraction obtained by Sephadex G-50 gel filtration. 7. Between 60 and 80% of proteolytic activity was found in the 40–60%-saturated ammonium sulphate precipitate. Almost all of the soluble-fraction proteolytic activity could be recovered in a pH5·0 supernatant. 8. The results give no support to the view that mitochondrial neutral proteolytic activity reflects lysosomal content. 9. The possible role of intramitochondrial amino acid production and the proteolysis of internal barriers in passive swelling of mitochondria is discussed.
Isolation of mycobactins from various mycobacteria. The properties of mycobactins S and H
Authors: White A. J. , Snow G. A.
Published: August 2015

Mycobactin S has been isolated from Mycobacterium smegmatis and from Mycobacterium sp. Olitzky & Gershon, strain 2, and mycobactin H from M. thermoresistible; all three organisms were grown on synthetic media of low iron content. These two mycobactins are mixtures of compounds having the same nucleus but differing in their fatty side chains. The nucleus of mycobactin S has a chemical structure identical with that of mycobactin T but differs in the optical configuration at the β-carbon atom of the hydroxy acid fragment; the configuration in mycobactin S is S whereas that in mycobactin T is R (the previous assignment of this configuration was incorrect). The cobactin fragment of mycobactin H is identical with that of mycobactin S, but the mycobactic acid moiety differs in having methyl groups at position 6 in the benzene ring and at position 5 in the oxazoline ring. The configurations of all the asymmetric centres have been established for both mycobactins. Improved and simplified methods for the extraction and purification of mycobactins are described.
The C4-dicarboxylic acid pathway of photosynthesis. Identification of intermediates and products and quantitative evidence for the route of carbon flow
Authors: Johnson Hilary S. , Hatch M. D.
Published: August 2015

1. When leaves with the C4-dicarboxylic acid pathway of photosynthesis are exposed to 14CO2 the major labelled compounds formed, in order of labelling, are dicarboxylic acids, 3-phosphoglycerate, bexose phosphates and sucrose. During the present studies several quantitatively minor intermediates were identified and their labelling behaviour is described. 2. The pattern of labelling of dihydroxyacetone phosphate, fructose 1,6-diphosphate and ribulose di- and mono-phosphates during radiotracer pulse–chase experiments was consistent with their operation as intermediates in the pathway of carbon dioxide fixation. 3. Serine, glycine, alanine and glutamate had labelling patterns typical of products secondary to the main flow of carbon. 4. The mechanism of the transfer of label from C-4 of dicarboxylic acids to C-1 of 3-phosphoglycerate was also examined. Evidence consistent with pyruvate being derived from C-1, C-2 and C-3 of oxaloacetate, and for a relationship between ribulose 1,5-diphosphate and the acceptor for the C-4 carboxyl group, was obtained. 5. Evidence is provided that, under steady-state conditions, essentially all the label incorporated from 14CO2 into C-1 of 3 phosphoglycerate enters via C-4 of the dicarboxylic acids. These and other studies indicated that the route via dicarboxylic acids is essentially the sole route for entry of carbon into 3-phosphoglycerate.
The chemical composition of the lipopolysaccharide of Pseudomonas aeruginosa
Authors: Fensom A. H. , Gray G. W.
Published: August 2015

1. Lipopolysaccharide was isolated from both cell walls and acetone-dried whole cells of Pseudomonas aeruginosa (N.C.T.C. 1999). 2. Closely similar products are obtained, although that from whole cells cannot be completely freed from small amounts (2–7%) of residual nucleic acids. 3. The lipid moiety (23–33%) has a similar amino sugar backbone to that of lipids of enterobacterial lipopolysaccharides, but contains different hydroxy acids (2- and 3-hydroxydodecanoic acid and 3-hydroxydecanoic acid). 3-Hydroxytetradecanoic acid is absent, and 3-hydroxydodecanoic acid is the main N-acylating acid. No clear evidence permitting a distinction between the possibilities that phosphodiester or glycosidic linkages exist between the glucosamine residues was obtained. 4. Identifiable sugars (glucose, rhamnose, 3-deoxy-2-octulonic acid and heptose) account for less than 20% of the lipopolysaccharide, and alanine, galactosamine and fucosamine are apparently components of the polysaccharide moiety. 5. The polysaccharide moiety is unusual in that it is not readily obtained from the lipopolysaccharide by treatment with dilute acetic acid, which does, however, solubilize much of the phosphorus of the lipopolysaccharide. 6. The ‘polysaccharide’ fraction (approx. 21%) obtained by treatment with dilute acetic acid contains only a small proportion of the total polysaccharide components, and in one case only 45% of the fraction was accountable for in terms of identifiable components. 7. Evidence suggests that unidentified nitrogenous components are concentrated in the residual material after removal of both the lipid and the ‘polysaccharide’ fraction from the lipopolysaccharide.
The excretion of lactate dehydrogenase in human urine after ingestion of aspirin
Authors: Leathwood P. D. , Plummer D. T.
Published: August 2015

1. Cells present in normal human urine contain 5–10% of the total lactate dehydrogenase excreted. The enzyme released from these cells by ultrasonication contained a distribution of isoenzymes similar to that found in the bulk of the urine and it is suggested that these cells are the main source of urinary lactate dehydrogenase. 2. Cells were thoroughly washed before examination so it is unlikely that the enzyme found in urinary sediment was simply adsorbed. In addition, full recoveries of added lactate dehydrogenase isoenzymes LDH1 and LDH5 showed that adsorption did not occur. 3. Most of the cells in normal urine are of the non-squamous epithelial type and their excretion is greatly increased after the ingestion by the subject of 3g. of aspirin. The possible origin of these non-squamous cells from the kidney is discussed. 4. Starch-block electrophoresis and relative activity measurements of lactate dehydrogenase excreted after the subject had taken aspirin show that the enzymes present in urine and cells are very similar, confirming the conclusion reached above (point 1). They have slightly more M subunits than the normal, shown particularly as an increase in isoenzyme LDH2. The isoenzyme pattern is like that of the kidney medulla and the possible reasons for this are discussed in terms of the concentration of salicylic acid in various parts of the kidney. 5. The results confirm the previous suggestion that the kidney is the main source of urinary lactate dehydrogenase.
Substituent effect on lysozyme-catalysed hydrolysis of some β-aryl di-N-acetylchitobiosides
Authors: Tsai C. S. , Tang J. Y. , Subbarao S. C.
Published: August 2015

Measurements are reported on the kinetics of the lysozyme-catalysed hydrolysis of several β-aryl di-N-acetylchitobiosides, some of which have been synthesized for the first time. The catalytic rate constants (kcat.) at 45° yield a curved Hammett plot (concave up) and the plot of ΔH‡ versus ΔS‡ has a sharp break. Substrates with electron-withdrawing groups exhibit a kinetic deuterium isotope effect (kHcat./kDcat.), whereas those with electron-donating groups show no such isotope effect. The results suggest the operation of different mechanisms for the two types of substrates.
The catabolism of plasma glycoproteins in normal and injured rats
Authors: Robinson G B
Published: August 2015

The catabolism of 14C-labelled plasma glycoprotein in rats was studied after injecting homologous plasma protein labelled in the N-acetylglucosamine and sialic acid moieties. In normal animals the catabolism was approximately described by a four-compartment model. The fractional rate of catabolism of the plasma-protein amino sugar was found to be 0·0305hr.−1, corresponding to the degradation of 2·75μmoles/hr. The 14C label was eliminated from the animals largely as carbon dioxide with a small proportion appearing in the urine. Freely circulating amino sugars or glycopeptides did not appear in the plasma as a result of the catabolic processes, and there was no evidence that the protein-bound amino sugars were reutilized in biosynthetic processes. A study of the distribution of 14C label in the carcasses of animals 24hr. after injection provided evidence that the gastrointestinal tract accounted for 25–38% of the total catabolic pool; the lungs, kidneys, spleen and liver also appeared to contribute to catabolism. Studies were conducted with rats that had been treated with turpentine to induce an inflammatory reaction; the results could not be analysed kinetically, since the metabolism of plasma proteins in these animals did not appear to be in a steady state. The injected plasma protein disappeared from the intravascular pool more quickly than in normal animals, but there were no significant differences in the rates of excretion of the 14C label.
The interaction of aflatoxin B1 with polynucleotides and its effect on ribonucleic acid polymerase
Authors: King A. M. Q. , Nicholson B. H.
Published: August 2015

1. The interaction of aflatoxin B1 with different polynucleotides was studied spectrophotometrically. Equations were derived that enable the degree of binding to be determined without first determining the extinction coefficient of the bound form. 2. The interaction with calf thymus DNA obeys first-order relationships with an association constant of 0·40mm−1, but there is some evidence for a secondary binding process from results obtained at 390nm. 3. The spectral shifts decreased in the order polyadenylic acid+polyuridylic acid>DNA>polyadenylic acid>polyadenylic acid+polyinosinic acid. Polycytidylic acid, polyuridylic acid, polyinosinic acid (both single- and triple-stranded), AMP, CMP, GMP and UMP did not interact with aflatoxin. It was concluded that there is a requirement for the amino group of adenine (or possibly guanine) for binding of aflatoxin to polynucleotides to occur. 4. Binding is reversed by increasing ionic strength, and by Mn2+ and Mg2+ in the concentration range studied (0–5mm). The effect of the Mn2+ or Mg2+ was far greater than would be expected on the basis of their ionic strength. With both the bivalent cations and sodium chloride the reversal is greatest with double-stranded polynucleotides. 5. Inhibition in vitro of the DNA-dependent RNA polymerase of Escherichia coli by aflatoxin B1 was detected only in the absence of Mg2+ and at concentrations of Mn2+ below the optimum for RNA synthesis in vitro. 6. The degree of inhibition (maximally 30%) was dependent on the concentration of Mn2+ and decreased during incubation.
Complex-formation between reduced xanthine oxidase and purine substrates demonstrated by electron paramagnetic resonance
Authors: Pick Frances M. , Bray R C
Published: August 2015

The origin of the Rapid molybdenum electron-paramagnetic-resonance signals, which are obtained on reducing xanthine oxidase with purine or with xanthine, and whose parameters were measured by Bray & Vänngård (1969), was studied. It is concluded that these signals represent complexes of reduced enzyme with substrate molecules. Xanthine forms one complex at high concentrations and a different one at low concentrations. Purine forms a complex indistinguishable from the low-concentration xanthine complex. There are indications that some other substrates also form complexes, but uric acid, a reaction product, does not appear to do so. The possible significance of the complexes in the catalytic cycle of the enzyme is discussed and it is suggested that they represent substrate molecules bound at the reduced active site, waiting their turn to react there, when the enzyme has been reoxidized. Support for this role for the complexes was deduced from experiments in which frozen samples of enzyme–xanthine mixtures, prepared by the rapid-freezing method, were warmed until the signals began to change. Under these conditions an increase in amplitude of the Very Rapid signal took place. Data bearing on the origin of the Slow molybdenum signal are also discussed. This signal disappears only slowly in the presence of oxygen, and its appearance rate is unaffected by change in the concentration of dithionite. It is concluded that, like other signals from the enzyme, it is due to Mov but that a slow change of ligand takes place before it is seen. The Slow species, like the Rapid, seems capable of forming complexes with purines.
A possible structure of the rabbit reticulocyte ribosome. An exercise in model building
Authors: Cox R A , Bonanou Sophia A.
Published: August 2015

It is suggested that the location of each of the diverse ribosomal proteins in the ribosome, or in the ribosomal subparticles in their native or derived forms, is determined by the nucleotide sequence of the 16s and 23–30s RNA moieties, i.e. that the 16s and 23–30s RNA species provide a unique binding site for each species of ribosomal protein. The ways of assembling such a thread into a ribosome-like structure appear limited if the ribosome is largely stabilized by protein–protein interactions. The nucleoprotein thread was built into a structure, having the general features of a hollow cylinder, that is consistent with the known dimensions and properties of the rabbit reticulocyte ribosome. It appears possible to test the model by experiment.
The incorporation of a hydrogen atom at C-15 of cholesterol biosynthesized from squalene
Authors: Akhtar M , Rahimtula A. D. , Wilton D. C.
Published: August 2015

Cholesterol is biosynthesized from squalene in the presence of tritiated water. Chemical degradation reveals that a considerable percentage of the total radioactivity is present at C-15. This result confirms the previous observations on the involvement of a C-15 hydrogen atom in cholesterol biosynthesis.
Factors affecting the lactate dehydrogenase isoenzyme pattern of cultured kidney-cortex cells
Authors: Güttler Flemming , Clausen Jørgen
Published: August 2015

1. The lactate dehydrogenase isoenzyme pattern of cultured calf kidney-cortex cells was correlated to growth phase, changes in oxygen supply, mean generation time and changes in nutritional supply. 2. During culture of free cells and intact explants the lactate dehydrogenase isoenzyme pattern changed towards a dominance of isoenzymes containing the M subunit. 3. Of the shift in monomer proportion, 58% occurred during the lag phase and 42% during the initial part of the exponential growth phase. During the stationary phase the shift in monomer proportion reversed slightly. It was possible to relate the observed shift in monomer proportion to the glycolytic rate. 4. Factors that depressed glycolysis decreased the shift in monomer proportion. Oxygen was found to limit the decrease in the H subunit/M subunit ratio caused by anaerobic culture in vitro. 5. The results obtained support the view that the altered lactate dehydrogenase isoenzyme pattern of urine in renal ischaemia may be explained by anaerobic changes in the lactate dehydrogenase isoenzyme pattern of cortical tubule cells.
Inhibition of pyrophosphatase activity of mouse duodenal alkaline phosphatase by magnesium ions
Authors: Nayudu P. R. V. , Miles P. L.
Published: August 2015

Duodenal alkaline phosphatase of juvenile (11-day-old) mice, like other non-specific alkaline phosphatases, has the ability to hydrolyse PPi. When a constant Mg2+/PPi concentration ratio is maintained, plots of velocity as a function of PPi concentration are consistent with Michaelis–Menten kinetics. Mg2+ activates pyrophosphate hydrolysis and maximal activity is obtained at a constant Mg2+/PPi concentration ratio of 0·66. At higher ratios there is strong inhibition. At constant concentrations of Mg2+ and increasing concentrations of PPi, the velocity–substrate (PPi) concentration plots show sigmoidal dependence. By assuming that the true substrate is MgP2O72− complex, and using complexity constants, the concentrations of free Mg2+, Mg2P2O7 and MgP2O72− were calculated in assay mixtures ranging in PPi concentration from 0·1 to 2·5mm and in total Mg2+ concentration from 0·6 to 2·6mm. From these data, the concentrations of added Mg2+ and PPi in the assay mixtures were selected so that the velocity could be measured (1) at three fixed concentrations of free Mg2+ ions with varied concentrations of MgP2O72− and (2) at four fixed concentrations of Mg2P2O7 with varied concentrations of MgP2O72−. Lineweaver–Burk and Hill plots from these data showed that the inhibition is caused by free Mg2+ ions, of a mixed type and consistent with Michaelis–Menten kinetics. The sigmoidal dependence observed between velocity and PPi concentration at constant concentration of total Mg2+ is therefore not due to allosteric inhibition. It is due to a combined effect of (1) inhibition by free Mg2+ ions, (2) depletion of the true substrate, MgP2O72−, owing to the formation of Mg2P2O7 and (3) the manner in which the concentrations of these three molecular or ionic species change when PPi concentration is increased maintaining the total Mg2+ concentration constant.
Activation of liver succinate dehydrogenase in rats exposed to hypobaric conditions
Authors: Aithal H. N. , Ramasarma T.
Published: August 2015

1. On brief exposure of rats to hypobaric conditions, the activity of hepatic mitochondrial succinate dehydrogenase was raised from the basal state to a ‘partially activated state’. This was further raised to ‘fully activated state’ by preincubation of mitochondria with succinate, as was the activity in mitochondria from normal rats. 2. On washing mitochondria with the homogenizing sucrose medium the activity excess obtained on preincubation with succinate was lost in mitochondria from both normal and treated rats. 3. The enzyme in the ‘partially activated state’ from animals exposed to hypobaric conditions was stable to the washing procedure but was labilized and reverted to a low basal state of activity on freezing and thawing of the isolated mitochondria. 4. The results suggest that activation of succinate dehydrogenase under hypobaric conditions represents a conformational change leading to a stable, partially activated, form of the enzyme system: this is the first evidence of physiological modulation of this rate-limiting step in the control of the rate of oxidation of succinate.
The mass-spectrometric identification of hypoxanthine and xanthine (‘oxypurines’) in skeletal muscle from two patients with congenital xanthine oxidase deficiency (xanthinuria)
Authors: Parker R. , Snedden W. , Watts R. W. E.
Published: August 2015

1. The presence of hypoxanthine and xanthine in the skeletal muscle of two patients with congenital xanthine oxidase deficiency (xanthinuria) was demonstrated by high-resolution mass spectrometry. 2. Evidence was obtained for the presence of a trace of hypoxanthine only in normal muscle. 3. Dry pulverized tissue was introduced directly into the mass spectrometer and preliminary chemical processing of the tissue was therefore unnecessary. 4. The criteria for the mass-spectrometric identification of hypoxanthine and xanthine in the tissue and the significance of the observations are discussed.
Purine metabolism in germinating wheat embryos
Authors: Price C. E. , Murray A. W.
Published: August 2015

1. Both the acid-soluble fraction and the nucleic acid fraction of wheat embryos were extensively labelled after incubation for 6hr. in the presence of [8−14C]adenine. Subsequent incubation in the absence of labelled adenine resulted in no loss of radioactivity to the medium during a 48hr. period. Radioautography indicated that during this period there was a continuous increase in the radioactivity present in the acid-insoluble fractions of the root and leaf tissues relative to that present in the coleorhiza and coleoptile. 2. During incubation at 25° there was a 26-fold increase in the activity of 3′-nucleotidase between 4hr. and 24hr.; the activities of enzymes hydrolysing AMP and IMP increased to a smaller extent. The activities of adenine phosphoribosyltransferase and hypoxanthine phosphoribosyltransferase increased three- to five-fold during incubation at 25° for 24hr. 3. Adenosine kinase, inosine phosphorylase and 5-phosphoribosyl pyrophosphate synthetase activities were high in extracts from dry embryos and did not increase during 48hr. at 25°. 4. The increase in 3′-nucleotidase activity was prevented by cycloheximide, cryptopleurine or incubation at 4°, but not by actinomycin D; these treatments did not depress the activity of the other enzymes measured. 5. The results are discussed in relation to RNA translocation within the wheat embryo during germination.
Metal complexes of mycobactin P and of desferrisideramines
Authors: Snow G. A.
Published: August 2015

Crystalline gallium mycobactin P and chromic mycobactin P have been prepared. The chromic compound, unlike other metallic complexes of mycobactin P, does not detectably exchange its metal with ferric iron; it competitively antagonizes the growth-promoting action of mycobactin P towards Mycobacterium johnei. Mycobactin P, desferrioxamine B and desferrichrysin form coloured 1:1 complexes with ammonium vanadate. The vanadyl complexes of the water-soluble desferrisideramines are formed in aqueous solution. Two distinct forms occur at pH7 and pH3; these are slowly interconvertible when the pH is changed. The complexes show other changes at lower pH values; unlike other metallic desferrisideramine complexes, the vanadyl compounds do not dissociate even in the strongest acids, but dissociate above pH9. Their properties have been studied spectrophotometrically, by electrophoresis and by electrometric titration. The affinity of mycobactin for ferric iron is greater than that of desferrioxamine B under two different conditions of measurement.