bluuebunny/crossref_metadata_2025 huggingface dataset:
Islet-cell metabolism during insulin release. Effects of glucose, citrate, octanoate, tolbutamide, glucagon and theophylline
Authors: Montague W , Taylor K W
Published: August 2015

1. Concentrations of glucose 6-phosphate and 6-phosphogluconate were studied in islets of Langerhans isolated from rat pancreas and incubated in the presence of various agents that induce insulin release. 2. In response to rising concentrations of extracellular glucose (2–10mm) there is a linear increase in the intracellular concentration of glucose 6-phosphate, though this is not the case for 6-phosphogluconate, the intracellular concentration of which only increases when the external glucose concentration exceeds 5mm. 3. Tolbutamide, octanoate and citrate, all of which promote insulin secretion from isolated islets, increase the intracellular concentrations of glucose 6-phosphate and 6-phosphogluconate. The results obtained in the presence of octanoate and citrate are compatible with an inhibitory effect of citrate on islet-cell phosphofructokinase. 4. Theophylline and glucagon when incubated with islets in vitro promote insulin release and cause a rise in 6-phosphogluconate concentration and not in that of glucose 6-phosphate. 5. It is suggested that the further metabolism of glucose 6-phosphate through a pathway other than glycolysis is essential for insulin release. One such pathway involves its oxidation to 6-phosphogluconate, which seems to be a necessary accompaniment of insulin secretion due to glucose. The possibility that agents other than glucose promote insulin release by enhancing the oxidation of glucose 6-phosphate through this pathway is discussed.
Transcription of bacteriophage T4 deoxyribonucleic acid in vitro
Authors: Bishop John O. , Robertson Forbes W.
Published: August 2015

1. RNA was synthesized in vitro from a template of bacteriophage T4 DNA, in the presence of Mn2+. A comparison was made of the RNA synthesized by purified RNA polymerase from two sources, Micrococcus lysodeikticus and Escherichia coli; these are referred to as Micrococcus cRNA and E. coli cRNA respectively (where cRNA indicates RNA synthesized in vitro by using purified RNA polymerase and a DNA primer). 2. Both types of RNA were self-complementary as judged by resistance to digestion with ribonuclease after self-annealing, Micrococcus cRNA being more self-complementary (40%) than was E. coli cRNA (30%). The cRNA was found to be much less self-complementary if Mg2+ was present during RNA synthesis instead of Mn2+. 3. Micrococcus cRNA hybridized with a larger part of bacteriophage T4 DNA than did E. coli cRNA. The E. coli cRNA competed with only part (70%) of the Micrococcus cRNA in hybridization-competition experiments. It is concluded that more sequences of bacteriophage T4 DNA are transcribed by Micrococcus polymerase than by E. coli polymerase. 4. The RNA sequences synthesized by Micrococcus RNA polymerase but not by E. coli RNA polymerase are shown by hybridization competition to compete with specifically late bacteriophage T4 messenger RNA sequences. The relevance of this finding to the control of transcription is discussed. 5. In an Appendix, new methods are described for the analysis of hybridization-saturation and -competition experiments. Particular attention is paid to the effects produced if different RNA sequences are present at different relative concentrations. 6. By using cRNA isolated from an enzymically synthesized DNA–RNA hybrid, it is estimated that, of the DNA that is complementary to cRNA, only about half can become hybridized with cRNA under the experimental conditions used.
Comparison of the protein-synthesizing machinery in the skeletal muscle of normal and dystrophic Bar Harbor mice
Authors: Watts D. C. , Reid J. D.
Published: August 2015

1. Although the total weight of leg muscle increased with the age of a normal mouse the DNA and RNA content per leg did not change significantly. 2. The weight of leg muscle from a dystrophic mouse was only about 45% of that from a normal mouse but the DNA and RNA contents were the same and hence similar DNA/RNA ratios were obtained. 3. The total ribosome contents of normal and dystrophic mice were the same on a whole-leg basis, and for both the free ribosomes were about 60% of the total. However, comparison with similar data from liver suggested that some loss of ribosomes occurred during the isolation procedure. 4. The polyribosome patterns obtained by density-gradient centrifugation were the same for normal and dystrophic muscle, and comparable polyribosome fractions of different sizes obtained from such gradients had similar capacities for the incorporation of radioactive amino acids in a standard protein-synthesizing system. 5. By using a standard protein-synthesizing system with normal polyribosomes similar extents of incorporation were found with normal- or dystrophic-muscle pH5 fraction or partially purified transfer RNA preparation. 6. It is concluded that there is no absolute difference between the protein-synthesizing systems of normal and dystrophic mouse muscle and that the observed apparent differences result from concentration differences caused by changes in muscle volume. 7. A possible cause of the failure of dystrophic muscle to resynthesize myofibrils is also suggested.
An analysis of the ribosomal ribonucleic acids of Escherichia coli by hybridization techniques
Authors: Avery R. J. , Midgley J. E. M. , Pigott G. H.
Published: August 2015

From analyses of the hybridization of Escherichia coli rRNA (ribosomal RNA) to homologous denatured DNA, the following conclusions were drawn. (1) When a fixed amount of DNA was hybridized with increasing amounts of RNA, only 0·35±0·02% of E. coli DNA was capable of binding (16s+23s) rRNA. Although preparations of 16s and 23s rRNA were virtually free from cross-contamination, the hybridization curves for purified 16s or 23s rRNA were almost identical with that of the parent specimen containing 1 weight unit of 16s rRNA mixed with 2 weight units of 23s rRNA. The 16s and 23s rRNA also competed effectively for the same specific DNA sites. It appears that these RNA species each possess all hybridizing species typical of the parent (16s+23s) rRNA specimen, though probably in different relative amounts. (2) By using hybridization-efficiency analysis of DNA–RNA hybridization curves (Avery & Midgley, 1969) it was found that (a) 0·45% of the DNA would hybridize total rRNA and (b) when so little RNA was added to unit weight of DNA that the DNA sites were not saturated, only 70–75% of the input RNA would form hybrids. The reasons for the discrepancy between the results obtained by the two alternative analytical approaches were discussed. (3) For either 16s or 23s rRNA, hybridization analysis indicated that two principal weight fractions of rRNA may exist, hybridizing to two distinct groups of DNA sites. However, these groups seem to be incompletely divided between the 16s and 23s fractions. Analysis suggested that (a) 85% of the 16s rRNA was hybridized to about half the DNA that specifically binds rRNA (0·23% of the total DNA). (b) 70% of the 23s rRNA hybridized to a further 0·23% of the DNA and (c) the minor fraction (15%) of 16s rRNA may be competitive with the major fraction (70%) of 23s rRNA. Conversely, the minor fraction (30%) of the 23s rRNA may compete with the major fraction (85%) of 16s rRNA. Models were proposed to explain the apparent lack of segregation of distinct RNA species in the two subfractions of rRNA. (4) If protein synthesis and ribosome maturation were inhibited in cells of an RCrel mutant, E. coli W 1665, by depriving them of an amino acid (methionine) essential for growth, the inhibition had no discernible effect on the relative rates of synthesis of rRNA species. The rRNA that accumulates in RCrel strains of E. coli after amino acid deprivation is apparently identical in its content of RNA species with that of the pre-existing mature RNA in the ribosomes. On the other hand, the messenger RNA is stabilized, and accumulates as about 15% of the RNA formed after withdrawal of the amino acid.
Molecular cohesion in plant cell walls. Methylation analysis of pectic polysaccharides from the cotyledons of white mustard
Authors: Rees D. A. , Wight N. J.
Published: August 2015

Methylation analysis was used to characterize the pectic polysaccharides from mustard cotyledons, a tissue with potential for rapid biological change involving the walls. The methylated sugars were identified by g.l.c. and paper chromatography after conversion of uronic acid derivatives into [3H]hexoses, and confirmed by the formation of crystalline derivatives of most of the main products, which were: 2,3-di-O-methyl-d-[6−3H]galactose, 2-O-methyl-d-[6−3H]galactose, 3,4-di-O-methylrhamnose, 3-O-methylrhamnose, 2,3,5-tri-O-methyl-l-arabinose, 2,3-di-O-methyl-l-arabinose, 2-O-methyl-l-arabinose, 2,3,4-tri-O-methyl-d-xylose and 2,3,4,6-tetra-O-methyl-d-galactose in the molar proportions 1·00:1·14:0·54:0·74:2·86:2·50:2·24:1·88:0·32. The structural units present are similar to those in wellknown polysaccharides from mature tissues, but their proportions are strikingly different. Uninterrupted and unbranched galacturonan segments can therefore contribute little cohesion to these walls, and it is suggested that this correlates with a function of the wall matrix to hydrate and permit readjustment, during germination, of structural elements or wall surfaces or both.
Isolation of a third bovine neurophysin
Authors: Rauch R. , Hollenberg M D , Hope D B
Published: August 2015

1. A third native hormone-binding protein, neurophysin-C, has been isolated from acetone-desiccated bovine pituitary posterior lobes. 2. This protein was detected in lysates of neurosecretory granules isolated from bovine pituitary posterior lobes. 3. The molecular weight appears to be close to 10000. 4. Neurophysin-C is similar in amino acid composition to neurophysin-I and -II; it contains a single residue of tyrosine and of methionine. The N-terminal amino acid in all three neurophysins is alanine. 5. Neurophysin-C accounts for approximately 15% of the total hormone-binding protein present in the pituitary posterior lobes. 6. The new neurophysin forms complexes with oxytocin as well as with [8-arginine]-vasopressin. The complex with vasopressin has been crystallized. 7. Bioassay of the pressor and oxytocic activities of the protein–hormone complexes shows that neurophysin-C binds one molecule of either vasopressin or oxytocin.
The regulation of pea-seed phosphofructokinase by phosphoenolpyruvate
Authors: Kelly G. J. , Turner J. F.
Published: August 2015

1. Pea-seed phosphofructokinase was purified 27-fold by a combination of fractionation with ethanol and ammonium sulphate. Under the conditions of assay, the enzyme was strongly inhibited by phosphoenolpyruvate. This inhibition was reversed by increasing the concentration of fructose 6-phosphate or magnesium chloride, or by lowering the ATP concentration. 2. Citrate, ADP and AMP inhibited phosphofructokinase and increased the sensitivity to phosphoenolpyruvate inhibition. Sulphate and inorganic phosphate stimulated the enzyme activity and decreased the sensitivity to phosphoenolpyruvate. 3. In the presence of inorganic phosphate and low concentrations of ATP, inhibition by phosphoenolpyruvate ceased and phosphoenolpyruvate became stimulatory. 4. The possible significance of these results in the control of plant carbohydrate metabolism is discussed.
Androgen sulphate formation in male and female mice
Authors: Lewis D A
Published: August 2015

1. After the administration of large doses of androsterone, epiandrosterone, dehydroepiandrosterone and testosterone to mice, females excreted more of the dose conjugated with sulphuric acid than did males. 2. Liver slices from female mice conjugated androgens with sulphuric acid to a greater extent than did slices from males. 3. Sulphotransferase preparations from livers of female rats and mice catalysed the formation of dehydroepiandrosterone sulphate at a faster rate than preparations from livers of the male animals. 4. A possible explanation for the observed sex differences is discussed.
Investigations on metabolites of vitamin D in rat bile. Separation and partial identification of a major metabolite
Authors: Bell P. A. , Kodicek E.
Published: August 2015

1. Young rats with cannulated bile ducts were given 0·34mg. of [1α−3H]cholecalciferol or 0·54mg. of [14C]ergocalciferol by intravenous infusion. Of the radioactivity in the dose of [1α−3H]cholecalciferol 31% was recovered in bile within 24hr. 2. The metabolites in bile were separated by gradient-elution column chromatography on silicic acid into five components, all more polar than cholecalciferol or 25-hydroxycholecalciferol. [14C]Ergocalciferol gave a similar pattern of metabolites in bile. 3. The three most polar metabolites were shown to be ionic. The major component has been identified as a glucuronide conjugate, which was not identical with synthetic cholecalciferyl glucuronide.
Temperature and the regulation of enzyme activity in poikilotherms. Fructose diphosphatase from migrating salmon
Authors: Behrisch Hans W.
Published: August 2015

1. The calculated energy charge of the liver cell from migrating salmon is very low (0·464), in keeping with the extended starvation and high rates of muscular and biosynthetic activity of these organisms. 2. Affinity of fructose 1,6-diphosphatase for substrate increases with a decrease in temperature. 3. Arrhenius plots of the saturation kinetics are complex and suggest an interconversion of one or more forms of the enzyme; this interconversion is dependent on the identity of the cofactor. 4. Affinity of salmon fructose 1,6-diphosphatase for its allosteric inhibitor (AMP) is lower than in other fructose 1,6-diphosphatases and this enzyme–AMP interaction is largely insensitive to temperature. The functional significance of diminished AMP-sensitivity is that it allows normal or high fructose 1,6-diphosphatase activity during a low energy charge. 5. These findings suggest mechanisms for the maintenance of high rates of gluconeogenesis in salmon during spawning migration.
Acid-catalysed dehydration of dehydroretinol
Authors: Nayar P. G.
Published: August 2015

1. Treatment of dehydroretinol with hydrogen chloride in light petroleum or toluene-p-sulphonic acid in benzene produces a substance with u.v.-absorption maxima at 284, 296, 310, 350, 368, 388, 408 and 433nm. and antimony trichloride colour maximum at 620nm. This compound is tentatively named compound 433 after its absorption maximum at 433nm. 2. Treatment of dehydroretinol with ethanolic hydrogen chloride produces, besides 3-ethoxyanhydroretinol, some compound 433 also. 3. Compound 433 is present in several freshwater-fish liver oils. 4. The i.r. spectrum of compound 433 shows only a band attributable to a conjugated ethylenic system. 5. The course of changes that lead to the formation of compound 433 is discussed.
Effect of cholesteryl 14-methylhexadecanoate on the activity of some amino acid-transfer ribonucleic acid ligases from mammalian tissues
Authors: Hradec J. , Dušek Z.
Published: August 2015

1. l-Tyrosine–, l-alanine–, l-tryptophan– and l-threonine–tRNA ligases (where tRNA is transfer RNA) were purified from mammalian tissues and the relative contents of cholesteryl 14-methylhexadecanoate were determined in fractions obtained during the isolation. Purified enzymes were extracted with various organic solvents. 2. Cholesteryl 14-methylhexadecanoate contents in purified ligases were up to 210-fold that in the starting material. Different enzymes showed different contents of this cholesteryl ester. 3. Extracted enzymes lost in most cases their ability to catalyse formation of the aminoacylhydroxamate and aminoacyl-tRNA complexes. Enzymes extracted with various solvents showed a variable decreased activity. 4. The original activity could be restored to 70–100% by the addition of cholesteryl 14-methylhexadecanoate. Cholesteryl palmitate, cholesteryl margarate and cholesteryl stearate were inactive in this respect. 5. Incubation mixtures of extracted enzymes with cholesteryl 14-methylhexadecanoate added showed an initial delay in the time-course of both reactions assayed. 6. It is concluded that the effect of cholesteryl 14-methylhexadecanoate on the activity of amino acid–tRNA ligases seems to be specific and that this compound may play some role in the function of these enzymes.
The effect of inhibitors on the formation of phosphoenolpyruvate by rat liver mitochondria
Authors: Bartley W , Dean B.
Published: August 2015

1. Rat liver mitochondria oxidizing malate produce PEP (phosphoenolpyruvate) without the addition of ATP or other nucleotides. 2. The addition of oligomycin in the presence of 2,4-dinitrophenol did not abolish PEP formation and in some instances stimulated its formation. 3. Formation of PEP was inhibited by arsenate. 4. Arsenite decreased PEP formation and caused accumulation of pyruvate. 5. Added GTP and ITP had no effect on PEP formation. 6. PEP formed from malate in the presence of GTP and labelled Pi had a specific radioactivity approximately the same as the Pi with no contribution from the phosphate of the added GTP. 7. There was no parallelism between the effects of inhibitors on PEP formation from malate and their effects on the assayed activity of PEP carboxykinase. 8. In a direct comparison it was shown that the PEP carboxykinase content of mitochondria was insufficient to account for the PEP formation from malate. 9. Consideration of the kinetic characteristics of PEP carboxykinase and mitochondrial content of oxaloacetate and GTP show that this enzyme cannot account for the PEP formed from malate by mitochondria.
Changes in lipid pattern of HeLa cells exposed to immunoglobulin G and complement
Authors: Güttler Flemming , Clausen Jørgen
Published: August 2015

1. Immunoglobulin G was isolated from sera of non-immunized rabbits or rabbits immunized with whole HeLa cell homogenate. The anti-HeLa immunoglobulin G and its Fab fragment precipitated the particulate 400000g-min. fraction of HeLa cell homogenate. 2. Immunoglobulin G from immunized or non-immunized rabbits and fresh or inactivated complement were added to HeLa cell cultures. Changes in the cell count and cellular contents of DNA, RNA, protein, total and individual phospholipids, cholesterol (and esters) and ganglioside were followed. 3. Addition of immunoglobulin G from non-immunized rabbits and guinea-pig serum (complement) caused a transient increase in DNA followed by a permanent increase in RNA, protein, dry weight and number of cells per culture. 4. Addition of anti-HeLa immunoglobulin G and active complement caused an increase in the cellular content of cholesterol, total phospholipids, lysophosphatidylcholine and lysophosphatidylethanolamine greater than the increase of the controls and a decrease in the molar percentages of phosphatidylcholine and phosphatidylethanolamine as compared with the controls. 5. The cholesterol/phospholipid ratio remained constant. 6. The appearance of lysophosphoglycerides was transient, reaching a maximum 3hr. after addition of anti-HeLa immunoglobulin G. 7. The content of lysophosphoglycerides in HeLa cultures exposed to immunoglobulin G from non-immunized rabbits ranged from 50% to 30% of the values obtained from cultures exposed to the anti-HeLa immunoglobulin G and complement. 8. The changes in the lipid pattern of the HeLa cells were associated with the appearance of juxta-nuclear vacuoles in cells, but were apparently not specifically related to the presence of active complement.
The metabolism of d-glucarate by Pseudomonas acidovorans
Authors: Jeffcoat R. , Hassall H. , Dagley S.
Published: August 2015

1. Dehydratases that converted d-glucarate into 4-deoxy-5-oxoglucarate were partially purified from Klebsiella aerogenes and Pseudomonas acidovorans. 2. When d-glucarate was metabolized to 2,5-dioxovalerate it appeared that water and carbon dioxide were removed from 4-deoxy-5-oxoglucarate in one enzymic step: 4,5-dihydroxy-2-oxovalerate was not an intermediate in this reaction. 3. A method for the enzymic determination of d-glucarate is described.
The preparation of iodinated vancomycin and its distribution in bacteria treated with the antibiotic
Authors: Perkins H. R. , Nieto M.
Published: August 2015

Vancomycin was radioactively labelled by iodination with 125I. Iodinated vancomycin was only a little less potent as an antibiotic than vancomycin itself. It was shown, both by chromatography and differential absorption measurements, to combine with acyl-d-alanyl-d-alanine residues. Radioactive vancomycin was used to follow the fate of the antibiotic in bacteria that had been subjected to the least concentration required to inhibit growth. Most of the radioactivity was in the cell walls, although some was found in the membrane fraction. The latter proportion increased during longer incubations with the antibiotic. Pre-formed protoplasts adsorbed very little vancomycin. Mg2+ removed labelled vancomycin from the mucopeptide of Bacillus licheniformis, but had little effect on the antibiotic adsorbed on Micrococcus lysodeikticus, either in vivo or on previously isolated cell walls. Specific peptide was shown to compete with cell walls for vancomycin and it also extracted from cell-wall samples the labelled compound that had been adsorbed on M. lysodeikticus living cells.
The thermodynamics of interaction between Sephadex and penetrating solutes
Authors: Ogston A. G. , Silpananta Panee
Published: August 2015

1. We have measured the partition coefficients of bovine serum albumin with Sephadex grades G-100, G-150 and G-200, and of a dextran ([unk]n 19700) and a polyethylene glycol ([unk]n 8000) with Sephadex G-200. We have also measured the effects of these solutes on the inner volumes of the grades of Sephadex. 2. The results can be described with fair consistency by means of a simple thermodynamic treatment that makes use of the virial coefficients of Sephadex and of the solute, and of a coefficient that expresses their interaction. This coefficient is related to the ‘exclusion volume’ of Sephadex for the solutes. 3. The Sephadex G-200–polyethylene glycol system shows anomalies of behaviour that are ascribed to the occurrence of ‘incompatible’ phase separation within the Sephadex beads.
The disulphide bonds of the heavy chain of rabbit immunoglobulin G
Authors: O'Donnell I. J. , Frangione B. , Porter R. R.
Published: August 2015

Six peptides containing eight half-cystine residues were isolated in good yield, after either oxidation or reduction and carboxymethylation of fragment C-1, which contains the N-terminal half of the heavy chain of rabbit immunoglobulin G. The sequences of five of these peptides had been reported previously (Cebra, Steiner & Porter, 1968b; Wilkinson, 1969) and that of the sixth was established. Other peptides containing half-cystine residues were isolated in much lower yield and are presumed to be derived from minor sequence variants. The cystine-containing peptides from enzymic digests of whole immunoglobulin G and Fc fraction were studied by several techniques and the results obtained enable us to put forward a scheme of the arrangement of the inter- and intra-chain disulphide bonds.
Active accumulation of tetracycline by Escherichia coli
Authors: Franklin T. J. , Higginson B.
Published: August 2015

1. At low concentrations of tetracycline (10μg/ml) net accumulation of the drug by Escherichia coli cells ceased after 7–10min. 2. At higher concentrations of tetracycline (>30μg/ml) the period of net accumulation of the drug was significantly extended. 3. The efflux of tetracycline from E. coli cells transferred from medium containing 10μg of tetracycline/ml to drug-free medium was a rapid temperature-dependent process and was accelerated by 2,4-dinitrophenol. 4. As the concentration of tetracycline in the preloading phase was increased, the rate of subsequent efflux of the drug progressively declined. The efflux of drug from cells preloaded in medium containing 200μg of tetracycline/ml was negligible, although efflux was readily provoked by 2,4-dinitrophenol, by N-ethylmaleimide or by omission of glucose from the medium. 5. The initial rate of uptake of tetracycline by E. coli cells was linearly proportional to the concentration of tetracycline in the medium up to the maximum concentration of drug obtainable under the experimental conditions used (400μg/ml, 0.83mm). 6. Although N-ethylmaleimide strongly inhibited the accumulation of tetracycline by E. coli, no evidence was obtained for the direct involvement of thiol groups in the transport process. It was concluded that N-ethylmaleimide inhibited accumulation by interruption of the energy supply of the cells. 7. Osmotic shock of E. coli cells did not significantly affect the influx of tetracycline, but promoted both efflux of tetracycline and cell lysis in cells treated with a high concentration of tetracycline. 8. A study of the distribution of tetracycline among the subcellular fractions of penicillin-induced spheroplasts preincubated with various concentrations of tetracycline indicated that 60–70% of the accumulated tetracycline was in the high-speed supernatant fraction. Sephadex chromatography showed that the tetracycline of this fraction was present as the free drug. Sephadex chromatography of a detergent extract of the membrane fraction, however, indicated that a significant proportion of the tetracycline radioactivity of this fraction was apparently bound to some macromolecular component. 9. Cellulose phosphate paper chromatography of cold-acid extracts of spheroplasts preloaded with tetracycline indicated that the accumulated drug was chemically unchanged. 10. Membrane preparations isolated from osmotically lysed penicillin-induced spheroplasts showed a temperature-dependent binding of tetracycline that was not energy-dependent and was not inhibited by N-ethylmaleimide. The binding process was stimulated by omitting Mg2+ from the medium, but conversely was profoundly inhibited by EDTA. 11. The relevance of these findings to the probable mechanism of active tetracycline accumulation by E. coli is discussed.
Gluconeogenesis in the kidney cortex. Flow of malate between compartments
Authors: Rognstad R.
Published: August 2015

1. Kidney-cortex slices from starved rats were incubated with l-[U-14C]lactate or l-[U-14C]malate plus unlabelled acetate and the specific radioactivity of the glucose formed was determined. In parallel experiments the specific radioactivity of the glucose formed from [1-14C]acetate plus unlabelled l-lactate and l-malate was determined. 2. By analytical methods the major products formed from the substrates were measured. The glucose formed was purified by paper chromatography for determination of specific radioactivity. 3. The specific radioactivity of the glucose formed from l-[U-14C]lactate agrees with predictions of a model based on interaction of the gluconeogenic and the oxidative pathways. 4. The specific radioactivity of the glucose formed from l-[U-14C]malate agrees with the predicted value if rapid malate exchange between the cytosol and mitochondria is assumed. 5. The rate of malate exchange between compartments was estimated to be rapid and at least several times the rate of glucose formation. 6. The specific radioactivity of the glucose formed from [1-14C]acetate plus unlabelled l-lactate or l-malate agrees with the predictions from the model, again assuming rapid malate exchange between compartments. 7. Malate exchange between compartments together with reversible malate dehydrogenase activity in the mitochondria and cytosol also tends to equilibrate isotopically the NADH pool in these compartments. 3H from compounds such as l-[2-3H]lactate, which form NAD3H in the cytosol, appears in part in water; and 3H from dl-β-hydroxy[3-3H]butyrate, which forms NAD3H in the mitochondria, appears in part in glucose, largely on C-4.
Studies on the chemical modification of the tyrosine residue in bovine neurophysin-II
Authors: Furth Anna J. , Hope D. B.
Published: August 2015

1. Bovine neurophysin-II contains 1mol of tyrosine residue/10000g of protein. This residue could be readily nitrated with tetranitromethane. On hydrolysis and amino acid analysis 1mol of 3-nitrotyrosine was found/10000g of protein. Starchgel electrophoresis at pH8.5 showed that nitration had converted the native protein into a single, more acidic species. The increase in acidity was consistent with the observed fall in pK of the tyrosine hydroxyl from 9.2 in native neurophysin to 7.3 in the nitrated protein. Further, the absence of any intermediate species, even under conditions of minimum substitution, confirmed that the molecular weight of the monomer is 10000. 2. O-Acetylation of the tyrosine residue was carried out with N-acetylimidazole, in conjunction with the reversible blocking of amino groups by citraconylation. The degree of O-acetylation, determined spectroscopically, was 0.9mol of O-acetyltyrosine/10000g of protein. 3. The hormone-binding ability of modified protein was tested by equilibrium dialysis and was found to be unchanged by either nitration or O-acetylation of the tyrosine residue. 4. Interaction of neurophysin-II and [8-arginine]-vasopressin gave rise to a characteristic difference spectrum with a peak at 286.8nm and shoulder at 279.6nm. Part of this hyperchromicity is thought to result from entry of the tyrosine residue at position 2 in the hormone into the hydrophobic environment of the binding site. With nitrated neurophysin-II a second peak appeared at 436nm, showing that the tyrosine of the protein is also perturbed. The very large red shift (84nm) in this region suggests that the 3-nitrotyrosyl residue not only enters a more hydrophobic environment on protein–hormone interaction, but is caused to ionize more fully by the approach of some positively charged group.
Isolation of leucogenenol from bovine and human liver
Authors: Rice F. A. H. , Shaikh Badaruddin
Published: August 2015

A compound has been isolated from both bovine and human liver that on intravenous or intraperitoneal injection into animals induces in 4–12h an increase in the number of peripheral neutrophils, and in 12–24h an increase in the number of peripheral lymphocytes. At 24h after injection there is also a two- to three-fold increase in the relative number of myeloblasts in the bone marrow. The procedure for isolation and the physical and chemical properties identify the compound as leucogenenol, isolated from the metabolic products of Penicillium gilmanii by Rice (1966).
Partial purification and properties of an enzyme from rat liver that catalyses the sulphation of l-tyrosyl derivatives
Authors: Mattock P. , Jones J. G.
Published: August 2015

1. An enzyme that catalyses the transfer of sulphate from adenosine 3′-phosphate 5′[35S]-sulphatophosphate to l-tyrosine methyl ester and tyramine was purified approx. 70-fold from female rat livers. 2. The partially purified preparation is still contaminated with adenosine 3′-phosphate 5′-sulphatophosphate–phenol sulphotransferase (EC 2.8.2.1), but a partial separation of the two enzymes can be achieved by chromatography on columns of Sephadex G-200 and DEAE-Sephadex. 3. The enzyme responsible for the sulphation of l-tyrosine methyl ester and tyramine is activated by dithiothreitol, 2-mercaptoethanol and GSH, the degree of activation being more marked with preparations previously stored at 0 or -10°C. In contrast, the enzymic sulphation of p-nitrophenol is inhibited by all three thiols. Again, there is a quantitative difference in the degree of inhibition of the two enzymes by o-iodosobenzoate, p-chloromercuribenzoate, N-ethylmaleimide and iodoacetate. 4. Mixed-substrate experiments support the hypothesis that the enzyme responsible for the sulphation of l-tyrosine methyl ester and tyramine is separate from that responsible for the sulphation of p-nitrophenol. However, p-nitrophenol is a potent inhibitor of the sulphation of both tyrosyl derivatives whereas these latter compounds have no effect on the sulphation of p-nitrophenol.
An inducible hydrolase from Aspergillus niger, acting on carbon–carbon bonds, for phlorrhizin and other C-acylated phenols
Authors: Minamikawa T. , Jayasankar N. P. , Bohm B. A. , Taylor I. E. P. , Towers G. H. N.
Published: August 2015

1. An inducible enzyme catalysing the hydrolysis of phloretin to form phloroglucinol and phloretic acid has been extracted from the acetone-dried powders of the mycelial felts of an Aspergillus niger strain grown in the presence of phlorrhizin. The enzyme was partially purified by treatment with protamine sulphate, ammonium sulphate fractionation, negative adsorption on tricalcium phosphate gel, and DEAE-cellulose column chromatography. 2. The hydrolytic activity on phloretin appeared to be maximal at about pH9.6. However, the characteristics of the enzyme were studied at pH7.2, because of the lability of the product, phloroglucinol, under alkaline conditions. 3. The apparent Km value at pH7.2 was about 0.3–0.4mm for phloretin and 0.15mm for 3′-methylphloracetophenone. 4. Maximum activity of the enzyme was obtained without the addition of any cofactor or metal ion. The involvement of thiol groups in the reaction was demonstrated by the potent inhibitory action of both heavy-metal ions and p-chloromercuribenzoate. 5. The enzyme showed a rather broad substrate specificity, and some other C-acylated phenols related to phloretin were hydrolysed. It was found that 3′-methylphloracetophenone, phloracetophenone and 2′,4,4′-trihydroxydihydrochalcone were attacked more efficiently than phloretin. We propose the systematic name C-acylphenol acylhydrolase for the enzyme. This enzyme belongs to EC group 3.7.1.
Preparation and characterization of antigen and antibody absorbents covalently coupled to an inorganic carrier
Authors: Weetall Howard H.
Published: August 2015

Immunoadsorbents were prepared by the use of an inorganic carrier. The proteins were covalently coupled to the carrier through a silane coupling agent. An immunoadsorbent prepared with human γ-globulin was found to complex with specific antibodies. These antibodies could be recovered in high yield and purity from the adsorbent. Similarly, insolubilized antibodies to l-asparaginase were used to isolate and purify the enzyme from crude extracts. These insolubilized antibodies are extremely stable with time and may be reused many times.
The amino acid sequence around the active-site cysteine and histidine residues of stem bromelain
Authors: Husain S. S. , Lowe G.
Published: August 2015

Stem bromelain that had been irreversibly inhibited with 1,3-dibromo[2-14C]-acetone was reduced with sodium borohydride and carboxymethylated with iodoacetic acid. After digestion with trypsin and α-chymotrypsin three radioactive peptides were isolated chromatographically. The amino acid sequences around the cross-linked cysteine and histidine residues were determined and showed a high degree of homology with those around the active-site cysteine and histidine residues of papain and ficin.
Histone F1 from rat thymus. Subfractionation and incorporation of [32P]phosphate in vitro
Authors: Buckingham R. H. , Stocken L. A.
Published: August 2015

Four subfractions of histone F1 from rat thymus were obtained by preparative polyacrylamide-gel electrophoresis. These subfractions are closely related in primary structure but show marked differences in radioactivity when derived from rat thymus nuclei labelled in vitro with [32P]phosphate.
Properties and reaction with iodoacetamide of adenosine 5′-triphosphate–creatinine phosphotransferase from human skeletal muscle. Further evidence about the role of the essential thiol group in relation to the mechanism of action
Authors: Kumudavalli I. , Moreland B. H. , Watts D. C.
Published: August 2015

1. The purification of creatine kinase from human and monkey skeletal muscle by horizontal electrophoresis on Sephadex blocks is described. 2. The purified enzymes are shown to have similar chemical and kinetic properties to the rabbit muscle enzyme and a common mechanism is inferred. 3. Iodoacetamide has a similar apparent second-order inhibition constant with the human and rabbit enzymes, but the inhibition does not go to completion with the former. This is even more marked with the monkey enzyme, which has more reactive thiol groups, but inhibition is only about 50%. 4. Single substrates have little effect on the inhibition by iodoacetamide, but with the primate enzymes, in contrast with the rabbit enzyme, high concentrations of ADP–Mg2+ plus creatine convert the essential thiol group from being pH-independent into one with a normal ionization. Low concentrations of ADP–Mg2+ plus creatine first enhance the rate of inactivation, but cause protection as the reaction proceeds. These results are interpreted to indicate an activation of the thiol group on the subunit to which the substrates bind and a co-operatively induced decrease in the activity of the thiol group on the other subunit which lacks substrates. 5. The effects of a substrate equilibrium mixture on the rate of inhibition are essentially those of ADP–Mg2+ plus creatine. 6. Since no substrate combination affords significant protection to the thiol group associated with the catalytic site to which the substrates are bound, it is concluded that any mechanism involving the thiol group in a direct participation in the transition-state complex of the catalytic reaction must be abandoned unless the transition state is only a small part of the time taken for one catalytic cycle.
A variant of rabbit phosphoglucose isomerase
Authors: Welch S. G. , Fitch Linda I. , Parr C. W.
Published: August 2015

An autosomally inherited variant of the rabbit enzyme phosphoglucose isomerase that differs both electrophoretically and kinetically from the usual (wild-type) rabbit enzyme has been investigated. Animals homozygous for this character have an isomerase with considerably decreased activity (in the erythrocytes), an increased Km for fructose 6-phosphate, an increased Ki for 6-phosphogluconate and a decreased stability towards heat and urea. The variant enzyme has been demonstrated in erythrocytes, leucocytes and tissues from the affected rabbits. The kinetic evidence suggests that the mutation present in the variant enzyme affects a histidine residue.
The stereochemistry of hydrogen elimination from C-7 in cholesterol and ergosterol biosynthesis
Authors: Akhtar M. , Rahimtula A. D. , Wilton D. C.
Published: August 2015

The synthesis of [7α-3H]lanosterol is described. It is shown that in the conversion of [7α-3H,26,27-14C2]lanosterol into cholesterol by a rat liver system, it is the 7β-hydrogen atom that is predominantly removed. On the other hand, the conversion of doubly labelled lanosterol into ergosterol by whole yeast cells results in the loss of the 7α-hydrogen atom. These results therefore suggest that the C-7 hydrogen atoms with opposite stereochemistry are labilized by the rat liver and the yeast Δ8–Δ7 steroid isomerases.
The effect of ethylenediaminetetra-acetate on Pseudomonas alcaligenes and the composition of the bacterial cell wall
Authors: Key B. A. , Gray G. W. , Wilkinson S. G.
Published: August 2015

1. EDTA in borate buffer has a marked bactericidal effect on Pseudomonas alcaligenes, which is more sensitive than Pseudomonas aeruginosa. The bactericidal effect is accompanied by solubilization of lipopolysaccharide and release of intracellular solutes. These effects are more pronounced at pH9.2 than 7.1. 2. Cell walls of P. alcaligenes were prepared and from them were obtained the readily extracted lipids and the fractions given by treatment with aqueous phenol. 3. The cell walls and the above components were analysed and results are compared with those for P. aeruginosa. 4. Lipopolysaccharide obtained by treatment of cell walls with aqueous phenol is contaminated with glycosaminopeptide to a variable extent. 5. The lipopolysaccharide contains less neutral sugar but more phosphorus than the lipopolysaccharide of P. aeruginosa; fucosamine is not a component of the lipopolysaccharide of P. alcaligenes.
Intracellular localization of enzymes in white-adipose-tissue fat-cells and permeability properties of fat-cell mitochondria. Transfer of acetyl units and reducing power between mitochondria and cytoplasm
Authors: Martin B. R. , Denton R. M.
Published: August 2015

1. A method is described for extracting separately mitochondrial and extramitochondrial enzymes from fat-cells prepared by collagenase digestion from rat epididymal fat-pads. The following distribution of enzymes has been observed (with the total activities of the enzymes as units/mg of fat-cell DNA at 25°C given in parenthesis). Exclusively mitochondrial enzymes: glutamate dehydrogenase (1.8), NAD–isocitrate dehydrogenase (0.5), citrate synthase (5.2), pyruvate carboxylase (3.0); exclusively extramitochondrial enzymes: glucose 6-phosphate dehydrogenase (5.8), 6-phosphogluconate dehydrogenase (5.2), NADP–malate dehydrogenase (11.0), ATP–citrate lyase (5.1); enzymes present in both mitochondrial and extramitochondrial compartments: NADP–isocitrate dehydrogenase (3.7), NAD–malate dehydrogenase (330), aconitate hydratase (1.1), carnitine acetyltransferase (0.4), acetyl-CoA synthetase (1.0), aspartate aminotransferase (1.7), alanine aminotransferase (6.1). The mean DNA content of eight preparations of fat-cells was 109μg/g dry weight of cells. 2. Mitochondria showing respiratory control ratios of 3–6 with pyruvate, about 3 with succinate and P/O ratios of approaching 3 and 2 respectively have been isolated from fat-cells. From studies of rates of oxygen uptake and of swelling in iso-osmotic solutions of ammonium salts, it is concluded that fat-cell mitochondria are permeable to the monocarboxylic acids, pyruvate and acetate; that in the presence of phosphate they are permeable to malate and succinate and to a lesser extent oxaloacetate but not fumarate; and that in the presence of both malate and phosphate they are permeable to citrate, isocitrate and 2-oxoglutarate. In addition, isolated fat-cell mitochondria have been found to oxidize acetyl l-carnitine and, slowly, l-glycerol 3-phosphate. 3. It is concluded that the major means of transport of acetyl units into the cytoplasm for fatty acid synthesis is as citrate. Extensive transport as glutamate, 2-oxoglutarate and isocitrate, as acetate and as acetyl l-carnitine appears to be ruled out by the low activities of mitochondrial aconitate hydratase, mitochondrial acetyl-CoA hydrolyase and carnitine acetyltransferase respectively. Pathways whereby oxaloacetate generated in the cytoplasm during fatty acid synthesis by ATP–citrate lyase may be returned to mitochondria for further citrate synthesis are discussed. 4. It is also concluded that fat-cells contain pathways that will allow the excess of reducing power formed in the cytoplasm when adipose tissue is incubated in glucose and insulin to be transferred to mitochondria as l-glycerol 3-phosphate or malate. When adipose tissue is incubated in pyruvate alone, reducing power for fatty acid, l-glycerol 3-phosphate and lactate formation may be transferred to the cytoplasm as citrate and malate.
The use of equilibrium-density-gradient methods for the preparation and characterization of blood-group-specific glycoproteins
Authors: Creeth J. M. , Denborough M. A.
Published: August 2015

1. The method of sedimentation equilibrium in a gradient of caesium chloride has been applied to the preparation of blood-group-specific glycoproteins from human ovarian-cyst fluids: it is shown that virtually complete separation from contaminating protein is easily accomplished in a single step. 2. The glycoproteins isolated in this way have been characterized by analytical density-gradient experiments in both caesium chloride and caesium sulphate and values of the buoyant density, selective solvation and apparent molecular weight have been obtained. 3. In some cases, materials prepared from the same cysts by solvent extraction methods have also been characterized in these terms. 4. The selective solvation values are about 0.1 and 0.5g of water/g of glycoprotein in caesium chloride and caesium sulphate respectively. 5. The apparent molecular-weight values are much lower than the weight-average molecular weights, and it is shown that the origin of the discrepancy is heterogeneity in density of the glycoproteins. 6. Some sources of error in the interpretation of density-gradient schlieren patterns are examined.
Aspects of the stereochemistry of torularhodin biosynthesis
Authors: Tefft R. E. , Goodwin T. W. , Simpson K. L.
Published: August 2015

1. The incorporation of [2-14C]acetate, [2-14C]mevalonic acid and [2-14C,2-3H2]-mevalonic acid into torulene and torularhodin by Rhodotorula rubra and Rhodotorula glutinis was studied. 2. A recovery of 14.3% of the label was obtained on decarboxylation of the torularhodin biosynthesized from [2-14C]mevalonic acid. 3. An analysis of the 3H/14C ratio in torularhodin gave a value of 9.44:8. 4. These results, obtained by different experimental techniques, show that the reactions in the conversion of the dimethyl group of isopentenyl pyrophosphate into the 16′,17′-position of torularhodin must be free from randomization. A mechanism for the isomerization of isopentenyl pyrophosphate to dimethylallyl pyrophosphate is suggested.
The composition of the cyst wall of the beet cyst-nematode Heterodera schachtii
Authors: Clarke A. J.
Published: August 2015

1. Cyst walls of the beet cyst-nematode (Heterodera schachtii Schmidt) were obtained by sieving a suspension of crushed cysts; about 15mg of dried cyst walls was obtained from 1000 cysts. 2. The cyst walls contained 68% protein calculated from nitrogen content. Glutamic acid, glycine, proline and hydroxyproline made up about 54% by weight of the amino acids obtained on acid hydrolysis. 3. Minor constituents of the cyst wall were hexosamine (3.3%), lipid (6%), carbohydrate (2%) and phenols (2%). The hexosamine was identified as galactosamine. 4. The cyst walls contained inorganic material (ash 17%), most of which was extractable with EDTA, but not with water. Major inorganic components were calcium and phosphorus (1.7% and 1.5% respectively, by weight). Carbon dioxide (about 1% by weight) was liberated from the cyst walls on acidification. 5. The cyst walls of H. schachtii and the potato cyst-nematode (Heterodera rostochiensis) contained different amounts of the same amino acids. They also differed in their inorganic content and in the nature of the hexosamine present.
Sedimentation-equilibrium studies on the heterogeneity of two β-lactamases
Authors: Lloyd P. H. , Peacocke A. R.
Published: August 2015

Solutions of crystalline β-lactamase I and β-lactamase II, prepared by Kuwabara (1970), were examined in the ultracentrifuge and their sedimentation coefficients, diffusion coefficients, molecular weights and heterogeneity determined. Each sample was shown to consist of a major component comprising at least 97% of the material and a minor component of much higher molecular weight. The molecular weights of the major components were 27800 for β-lactamase I and 35600 for β-lactamase II. Emphasis is placed on a straightforward practical way of analysing the sedimentation-equilibrium results on mixtures of two macromolecular components rather than on a strict theoretical solution. Appendices describe the theory of systems at both chemical and sedimentation equilibrium and the procedure for calculating the combined distribution of two components.
The isolation and characterization of lysosomal particles from myxamoebae of the cellular slime mould Dictyostelium discoideum
Authors: Wiener Edith , Ashworth J. M.
Published: August 2015

1. The myxamoebae of the cellular slime mould Dictyostelium discoideum possess several typically lysosomal enzyme activities. 2. These enzymes are present in the cell in association and in a lysosome-like particle. 3. The lysosomes of myxamoebae grown axenically have a different enzymic composition and a different density from those grown on bacteria. 4. During cell differentiation the specific activities of the lysosomal enzymes change. 5. It is suggested that both during growth and differentiation the amounts of lysosomal enzymes present in the cell are regulated.