bluuebunny/crossref_metadata_2025 huggingface dataset:
The intracellular localization of glycollate oxidoreductase in Euglena gracilis
Authors: Lord J. M. , Merrett M. J.
Published: August 2015

1. Lowering of the concentration of carbon dioxide in air available to phototrophically growing Euglena cultures from 5% to the normal value (0.03%) resulted in an increased specific activity of glycollate oxidoreductase. 2. The effects of chloramphenicol and cycloheximide suggested that this increase in activity was due to enzyme synthesis de novo on cytoplasmic ribosomes. 3. The Km for glycollate oxidation by the enzyme in crude cell extracts was 3.0×10−3m. 4. Differential centrifugation established that glycollate oxidoreductase present in phototrophically grown Euglena is a particulate enzyme. The enzyme was partially solubilized by the non-ionic detergent Triton X-100. 5. Sucrose-density-gradient centrifugation achieved the separation of the particulate glycollate oxidoreductase from chloroplasts and mitochondria. 6. Glutamate–glyoxylate aminotransferase was associated with particulate glycollate oxidoreductase.
Allotypically related sequences in the Fd fragment of rabbit immunoglobulin heavy chains
Authors: Mole L. E. , Jackson S. A. , Porter R. R. , Wilkinson J. M.
Published: August 2015

The sequence has been completed of the N-terminal 94 residues of the variable section of the Fd fragment of heavy chains from rabbit immunoglobulin G (IgG) of allotype As1. Most of the sequence of the same section from IgG of allotype Aa3 is also reported. These results, in conjunction with a substantial sequence of the variable region of allotype Aa2 reported elsewhere (Fleischman, 1971), show the presence of 16 positions (including six consecutive positions) in which the residue present correlates with the allotype. No allotype-related sequence variation has been found in the constant section of the Fd fragment. This evidence supports the view that two genes code for the heavy chain and it can be used as evidence in favour of somatic mutation as the origin of the variability in the sequence of the N-terminal section. The evolutionary origin of the ‘a’ locus allotypes of rabbit immunoglobulins remains obscure.
The enzymes forming isopentenyl pyrophosphate from 5-phosphomevalonate (mevalonate 5-phosphate) in the latex of Hevea brasiliensis
Authors: Skilleter D. N. , Kekwick R. G. O.
Published: August 2015

1. Phosphomevalonate kinase and 5-pyrophosphomevalonate decarboxylase have been purified from the freeze-dried latex serum of the commercial rubber tree Hevea brasiliensis. 2. The phosphomevalonate kinase was acid- and heat-labile and required the presence of a thiol to maintain activity. 3. The 5-pyrophosphomevalonate decarboxylase was relatively acid-stable and more heat-stable than the phosphokinase. 4. Maximum activity of the phosphokinase was achieved at pH 7.2 with 0.2mm-5-phosphomevalonate (Km 0.042mm), 2.0mm-ATP (Km 0.19mm) and 8mm-Mg2+at 40°C. The apparent activation energy was 14.8kcal/mol. 5. Maximum activity of 5-pyrophosphomevalonate decarboxylase was achieved at pH5.5–6.5 with 0.1mm-5-pyrophosphomevalonate (Km 0.004mm), 1.5mm-ATP (Km 0.12mm) and 2mm-Mg2+. The apparent activation energy was 13.7kcal/mol. The enzyme was somewhat sensitive to inhibition by its products, isopentenyl pyrophosphate and ADP.
Aminoacyltransferase I-catalysed binding of phenylalanyl-transfer ribonucleic acid to muscle ribosomes from normal and diabetic rats
Authors: Leader D. P. , Wool I. G. , Castles J. J.
Published: August 2015

The aminoacyltransferase I-catalysed binding of phenylalanyl-tRNA (unfractionated Escherichia coli B tRNA acylated with radioactive phenylalanine and 19 non-radioactive amino acids) to skeletal-muscle ribosomes from diabetic rats was less than that to ribosomes from normal rats when the Mg2+ concentration was low (7.5mm); whereas just the reverse was true when the concentration of the cation was higher (15mm). Thus the Mg2+ dependency of aminoacyltransferase I-catalysed binding of phenylalanyl-tRNA to ribosomes from normal and diabetic rats paralleled the effect of Mg2+ concentration on synthesis of polyphenylalanine reported before. During incubation at 7.5mm-Mg2+ phenylalanyl-tRNA was bound only to ribosomes bearing nascent peptidyl-tRNA. There are fewer such ribosomes in a preparation from the muscle of diabetic animals because diabetic animals synthesize less protein in vivo. Thus the difference in polyphenylalanine synthesis in vitro is adequately explained by the difference in enzyme-catalysed binding of phenylalanyl-tRNA to ribosomes, however, the basis of the difference in protein synthesis in vivo is still unknown.
Apiose and mono-O-methyl sugars as minor constituents of the leaves of deciduous trees and various other species
Authors: Bacon J. S. D. , Cheshire M. V.
Published: August 2015

1. Leaves of a number of species were hydrolysed with aqueous sulphuric acid and the resulting mixtures of sugars were fractionated by chromatography on activated charcoal. Paper chromatography of the fractions showed the presence in all the hydrolysates of minor constituents with RF values similar to or greater than those of the common hexoses and pentoses. 2. Two of these were identified as 2-O-methylxylose and 2-O-methylfucose. Estimates of the amounts present in whole leaves, and in fractions prepared from them, showed that they were associated with the hemicelluloses. 3. A third constituent was identified, by the formation of its di-isopropylidene derivative, as apiose. It also was associated chiefly with the hemicellulose fraction; none could be found in aqueous extracts from leaves of Tilia vulgaris, nor in aqueous extracts of Zostera marina, in which apiose is a major constituent of the water-insoluble polysaccharide. 4. A further constituent, after further purification by preparative paper chromatography, was tentatively identified, by gas–liquid chromatography of derivatives, as 3-O-methylgalactose, and was probably accompanied by small amounts of 4-O-methylgalactose. 5. These observations confirm the widespread occurrence of 2-O-methylxylose, 2-O-methylfucose and apiose, but 3-O-methylgalactose was hitherto known only in slippery-elm mucilage, and 4-O-methylgalactose in soil polysaccharides. Some experiments on the digestion of leaf hemicellulose fractions by snail crop-juice suggested that the mono-O-methyl sugars might confer resistance to enzymic degradation.
Calcium binding by human erythrocyte membranes
Authors: Forstner J. , Manery J. F.
Published: August 2015

1. The characteristics of Ca2+ binding to haemoglobin-free human erythrocyte membranes were investigated by using 45Ca and centrifugation partition of ‘ghosts’ from their external incubation medium. Equilibrium of ‘ghosts’ with external Ca2+ required less than 15min. 2. The binding did not vary with temperature in the range 0–37°C. 3. At pH7.4 ‘ghosts’ bound a maximum of 283μmol of Ca2+/g of ‘ghost’ protein, equivalent to 6.85×107 Ca2+ ions per cell. 4. Increasing the ionic strength from 0.01 to 0.46 diminished Ca2+ binding, as did ATP in concentrations ranging from 0 to 15mm in the incubation medium. 5. An increase of the pH from 3.0 to 9.3 caused a marked increase in the amount of Ca2+ bound. 6. Extraction of 45Ca-labelled ‘ghosts’ with chloroform–methanol showed that the distribution of Ca2+ was: 79% protein-bound, 16% lipid-bound, 5% in the aqueous phase, presumably non-bound. Most of the lipid-bound Ca2+ (about 80%) was associated with a phospholipid fraction containing phosphatidylserine, phosphoinositides and phosphatidylethanolamine, giving a molar Ca2+: phosphorus ratio of about 1:2.
Reactions of d-glyceraldehyde 3-phosphate dehydrogenase with chromophoric thiol reagents
Authors: Harrigan P. J. , Trentham D. R.
Published: August 2015

The kinetics of the reaction of d-glyceraldehyde 3-phosphate dehydrogenase with 5,5′-dithiobis-(2-nitrobenzoic acid) show that NAD% dissociates from the enzyme before the reaction. In contrast 2-chloromercuri-4-nitrophenol reacts with the holoenzyme without prior dissociation of NAD%. These studies and observations on the dissociation constant of NAD% to the lobster enzyme show that NAD% must dissociate from sites modified by substrates during the reductive dephosphorylation of 1,3-diphosphoglycerate. All four sites per tetramer of the apoenzyme are acylated by 1,3-diphosphoglycerate. Hydrolysis of the acyl-enzyme occurs at a significant rate even in the absence of NAD%, which may explain previous estimates that only two sites per tetramer can readily be acylated.
Studies on the conversion of pyruvate into fatty acids in white adipose tissue. Effects of insulin, alloxan-diabetes and starvation
Authors: Halperin Mitchell L.
Published: August 2015

The effect of insulin on the conversion of pyruvate into fatty acids in the presence and in the absence of glucose was studied in epididymal adipose tissue of the rat. 1. In adipose tissue from the normal rat, conversion of pyruvate into fatty acids is directly related to its concentration, the maximal rates occurring with 40mm- and the half-maximal rates with approx. 4mm-pyruvate. Insulin treatment did not greatly influence the maximal rates, but the half-maximal rates were at much lower pyruvate concentrations. This effect of insulin could be seen with physiological concentrations of this hormone (50–100μunits/ml). 2. In adipose tissue from acute-alloxan-diabetic and 36h-starved rats the conversion of pyruvate into fatty acids was almost zero until its concentration exceeded 3mm and then increased markedly as the concentration of pyruvate was increased. The lag phase of this S-shaped curve was decreased but not eliminated when insulin was present. This could account for the very low rates of glucose conversion into fatty acids in these metabolic states. Maximum rates of fatty acid synthesis were similar in the presence and in the absence of insulin, but only when 30–40mm-pyruvate was employed. Re-feeding of the starved rats or insulin treatment of the diabetic rats in vivo for several days restored these patterns to normal.
Studies on protein multimers. The association–dissociation behaviour of β-galactosidase in glycerol
Authors: Contaxis C. C. , Reithel F. J.
Published: August 2015

1. The effect of glycerol on the association–dissociation behaviour of β-galactosidases from Escherichia coli is described. Two strains, K12 and ML308, were used as sources of enzyme. The conditions used, involving glycerol at a concentration of 90%, result in dissociation of the active 540000-dalton form to inactive structural subunits of 135000 daltons. 2. A pH-dependent process, assumed to be cyclic in mechanism, allows reassociation to an active form indistinguishable from the initial protein. 3. The apparently identical structural subunits, if produced in the presence of EDTA, were found to give rise to two electrophoretically distinguishable species. 4. Enzymes from both strains of E. coli can be distinguished electrophoretically but exhibit the same behaviour in glycerol. 5. A scheme of the association–dissociation is presented that is consistent with the behaviour observed and that has some predictive value.
Evaluation of the isolated perfused rat hindquarter for the study of muscle metabolism
Authors: Ruderman N. B. , Houghton C. R. S. , Hems R.
Published: August 2015

1. The metabolic integrity of a new isolated rat hindquarter preparation was studied. The hindquarter was perfused with a semi-synthetic medium containing aged human erythrocytes. More than 95% of the oxidative metabolism of the preparation was due to muscle, the remainder being due to bone, adipose tissue and, where present, skin. 2. Consumption of O2, glucose utilization, glycerol release and lactate production were similar in the presence and in the absence of the skin, indicating that the latter contributed little to the overall metabolism of the preparation. 3. After 40min of perfusion, tissue concentrations of creatine phosphate, ATP and ADP were similar to those found in muscle taken directly from intact animals. The muscle also appeared normal under the electron microscope. 4. The hindquarter did not lose K% to the medium during a 30min perfusion. In the presence of insulin it had a net K% uptake. 5. Insulin caused a sixfold increase in glucose uptake, stimulated O2 consumption by nearly 40% and depressed glycerol release to less than half the control value. 6. Bilateral sciatic-nerve stimulation caused severalfold increases in O2 consumption and lactate production. In the absence of insulin nerve stimulation also enhanced glucose uptake; in the presence of insulin it did not further increase the already high rate of glucose uptake. 7. Rates of lactate production and O2 consumption of the rat hindquarter in vivo and the isolated perfused hindquarter were very similar. 8. Ketone bodies were a major oxidative fuel in vivo of the hindquarter of a rat starved for 2 days. If the acetoacetate and 3-hydroxybutyrate removed by the tissue were completely oxidized, they would have accounted for 77% of the O2 consumption. 9. Acetoacetate accounted for 84% of the ketone bodies removed by the hindquarter in vivo even though its arterial concentration was half that of 3-hydroxybutyrate. 10. Similar rates of acetoacetate and 3-hydroxybutyrate utilization were observed in the perfused hindquarter. 11. Acetoacetate utilization by the perfused hindquarter was not diminished by the addition of either oleate or insulin to the perfusate. 12. Oxidation of glucose to CO2 accounted for less than 4% of the O2 consumed by the perfused hindquarter in both the presence and the absence of insulin. 13. The results indicate that the isolated perfused hindquarter is a useful tool for studying muscle metabolism. They also suggest that ketone bodies, if present in sufficient concentration, are the preferred oxidative fuel of resting muscle.
Coupling of glycosaminoglycans to agarose beads (Sepharose 4B)
Authors: Iverius P.-H.
Published: August 2015

1. Heparin, heparan sulphate, chondroitin sulphate and dermatan sulphate were covalently attached to beads of agarose activated by cyanogen bromide. The bond is probably mediated by the amino group of a serine or peptide residue at the reducing end of the polysaccharide chain. 2. The uptake of glycosaminoglycan during the coupling procedure is about 0.9mg/ml of wet gel. However, direct analysis of washed and freeze-dried gels reveals that only about one-third of this amount is firmly attached to the gel. 3. The use of the gels for polysaccharidase analyses is exemplified by a hyaluronidase assay. Further applications, e.g. interaction studies and preparative purposes, are discussed.
The maltase, glucoamylase and transglucosylase activities of acid α-glucosidase from rabbit muscle
Authors: Palmer T. N.
Published: August 2015

1. The maltase and glucoamylase activities of acid α-glucosidase purified from rabbit muscle exhibited marked differences in certain physicochemical properties. These included pH stability, inactivation by thiol-group reagents, inhibition by αα-trehalose, methyl α-d-glucoside, sucrose, turanose, polyols, glucono-δ-lactone and monosaccharides, pH optimum and the kinetics and pH-dependence of cation activation. 2. The results are interpreted in terms of the existence of at least two specific substrate-binding sites or sub-sites. One site is specific for the binding of maltose and probably other oligosaccharides. The second site binds polysaccharides such as glycogen. 3. The sites appear to be in close proximity, since glycogen and maltose are mutually inhibitory substrates and interact directly in transglucosylation reactions. 4. Acid α-glucosidase exhibited intrinsic transglucosylase activity. The enzyme catalysed glucosyl-transfer reactions from [14C]maltose (donor substrate) to polysaccharides (glycogen and pullulan) and to maltose itself (disproportionation). The pH optimum was 5.1, with a shoulder or secondary activity peak at pH5.4. The glucose transferred to glycogen was attached by α-1,4- and α-1,6-linkages. Three major oligosaccharide products of enzyme action on maltose (disproportionation) were detected. 5. The kinetics of enzyme action on [14C]maltose showed that the rate of transglucosylation increased in a sigmoidal fashion as a function of substrate concentration, approximately in parallel with a decrease in the rate of glucose release. 6. The results are interpreted to imply competitive interaction at a specific binding site between maltose and water as glucosyl acceptors. 7. The results are discussed in terms of the possible existence of multiple subgroups of glycogen-storage disease type II.
Interactions of acetate, propionate and butyrate in sheep liver mitochondria
Authors: Smith R. M.
Published: August 2015

1. Interactions in the rates of consumption of acetate, propionate and butyrate in sheep liver mitochondria were examined in the presence and absence of l–malate and α–oxoglutarate. 2. Acetate was not consumed in absence of ancillary substrate but utilization of acetate (7.2nmol/min per mg of protein) occurred in the presence of α–oxoglutarate. This consumption was abolished by propionate or butyrate but the presence of acetate did not affect consumption of propionate or butyrate. 3. Propionate consumption (10.1nmol/min per mg of protein) was unaffected by malate but was stimulated by 63% by butyrate or by 180% by α–oxoglutarate. 4. Butyrate consumption (3.3nmol/min per mg of protein) was stimulated by 117% by malate, by 151% by propionate and by 310% by α–oxoglutarate. 5. In the absence of ancillary substrates the maximum rate of total volatile fatty acid utilization (24.7nmol/min per mg of protein) occurred with a mixture of propionate and butyrate. When both propionate and butyrate were present total consumption was not affected by malate but was stimulated by 24% by α–oxoglutarate. With α–oxoglutarate present, propionate and butyrate each decreased the other's consumption by about 26%, but the total utilization was the greatest observed. 6. The inhibition of acetate consumption by propionate or butyrate is unexplained, but the remaining effects are consistent with an interaction of propionate and butyrate through oxaloacetate together with a general limitation imposed by a need for GTP to rephosphorylate AMP formed during activation of the volatile fatty acids.
Enzymic iodination. A probe for accessible surface proteins of normal and neoplastic lymphocytes
Authors: Marchalonis J J. , Cone R E. , Santer V
Published: August 2015

1. Radioactive iodide was covalently bound to living cells from normal mouse spleen and a variety of lymphoid tumours by a system consisting of lactoperoxidase, hydrogen peroxide and iodide. 2. About 3×105-6×105 molecules of [125I]iodide/cell could be incorporated without affecting cell viability. 3. Electron-micrographic radioautography showed that the radioactive label was associated with the outer surfaces of the cells. 4. Radioiodinated proteins were solubilized in 9m-urea–0.2m-mercaptoethanol and analysed by gel-filtration and disc electrophoresis. 5. Comparison of distinct tumour lines by disc electrophoresis showed qualitative and quantitative differences in protein distribution patterns.
Oximes, nitriles and 2-hydroxynitriles as precursors in the biosynthesis of cyanogenic glucosides
Authors: Tapper B. A. , Butler G. W.
Published: August 2015

The biosynthesis of the cyanogenic glucosides, linamarin and prunasin, was investigated in linen-flax, peach and cherry-laurel shoots. It was shown that related 2-oximino acids, aldoximes, nitriles and 2-hydroxynitriles were generally good precursors of the aglycone moiety. Studies with double-labelled compounds confirmed the retention of the oximino nitrogen atom from 2-oximinoisovaleric acid and isobutyraldoxime in the biosynthesis of linamarin. A general pathway from amino acids to cyanogenic glucosides involving N-hydroxyamino acids, aldoximes, nitriles and 2-hydroxynitriles is proposed.
Testosterone action in the rat ventral prostate. The effects of diethylstilboestrol and cyproterone acetate on the metabolism of [3H]testosterone and the retention of labelled metabolites by rat ventral prostate in vivo and in vitro
Authors: Belham J. E. , Neal G. E.
Published: August 2015

Recent reports have indicated that the prior metabolism of testosterone by the secondary sexual tissues may be necessary for its androgenic effect. The effects of two anti-androgens, diethylstilboestrol and cyproterone acetate (17α-acetoxy-6-chloro-1,2α-methylenepregna-4,6-diene-3,20-dione) used in the chemotherapy of human prostatic carcinoma, have been examined on both the metabolism of testosterone and the retention of its metabolites by the rat ventral prostate gland. Cyproterone acetate was found to inhibit the retention of labelled metabolites of [3H]-testosterone by prostatic nuclei, both in vivo and in vitro. This inhibition appeared to be competitive. In contrast with its effect on nuclear retention of metabolites of testosterone, cyproterone acetate had no significant effect on the metabolism of [3H]testosterone by rat ventral prostate tissue. Diethylstilboestrol similarly had little effect on the metabolism of [3H]testosterone by prostatic tissue, although it did appear partially to inhibit its initial metabolism in all the incubation systems used. Diethylstilboestrol inhibited the nuclear retention of dihydrotestosterone when both [3H]testosterone and diethylstilboestrol were injected intraperitoneally in vivo, but had no effect on dihydrotestosterone retention when both testosterone and diethylstilboestrol were supplied directly to the prostate either in vivo or in vitro. It was concluded that if diethylstilboestrol has an anti-androgenic effect at the level of the target organ as distinct from its effect on androgen production by the testes, then it is probably due to a mechanism differing from that of cyproterone acetate.
Comparison of some metabolic parameters in the perfused and the incubated rat diaphragm muscle with diaphragm muscle in vivo
Authors: Rookledge K. A.
Published: August 2015

1. A method is described for perfusing the rat diaphragm muscle. 2. The following parameters were compared in both perfused and non-perfused incubated preparations: water content, sorbitol space, rate of lactate production, and the concentrations of tissue glucose, pyruvate, lactate, hexose phosphate intermediates, ATP and AMP. No significant differences were found. 3. Significant differences, however, were found on comparison of the tissue kept in vitro with the tissue in vivo. Immediately after removal of the tissue from the animal, the concentrations of the hexose phosphates and ATP were found to be much higher than after incubation or perfusion, and the concentrations of free glucose and of AMP were much lower, possibly indicating that the capacity for oxidative phosphorylation of glucose is impaired in vitro because of hypoxia.
The influence of insulin and of contraction on glucose metabolism in the perfused diaphragm muscle from normal and streptozotocin-treated rats
Authors: Beloff-Chain Anne , Chain E. B. , Rookledge K. A.
Published: August 2015

1. The metabolism of [U-14C]glucose in perfused resting and contracting diaphragm muscle from normal rats and rats made diabetic with streptozotocin was studied in the presence and absence of insulin. 2. The incorporation of [U-14C]-glucose into glycogen and oligosaccharides was stimulated by insulin under all experimental conditions studied. 3. In the normal perfused resting diaphragm muscle the incorporation of radioactivity from [14C]glucose into lactate and CO2 was not affected by insulin. 4. Periodic contractions, induced by electrical stimulation of the perfused diaphragm muscle in the absence of insulin, caused an increased incorporation of 14C into glycogen and hexose phosphate esters, whereas incorporation of 14C into lactate was greatly decreased. Production of 14CO2 in the contracting muscle was not significantly different from that in resting muscle. Addition of insulin to the perfusion liquid caused a further increase in formation of [14C]-glycogen in contracting muscle to values reached in the resting muscle in the presence of insulin. Formation of [14C]lactate was also stimulated by insulin, to values close to those found in the resting muscle in the presence of insulin. 5. In the diabetic resting muscle the rate of glucose metabolism was very low in the absence of insulin. Insulin increased formation of [14C]glycogen to the value found in normal muscle in the absence of insulin. Production of 14CO2 and formation of [14C]hexose phosphate remained unchanged. 6. In the diabetic contracting muscle production of 14CO2 was increased to values approaching those found in normal contracting muscle. Formation of [14C]lactate and [14C]glycogen was also increased by contraction, to normal values. Only traces of [14C]hexose phosphate were detectable. Addition of insulin to the perfusion medium stimulated formation of [14C]glycogen, to values found in normal contracting muscle. Production of [14C]hexose phosphate was stimulated by insulin, to approximately the values found in the normal contracting muscle. Production of 14CO2 and [14C]lactate, however, was not significantly affected by insulin. 7. These results indicate that the defects of glucose metabolism observed in perfused resting diabetic diaphragm muscle can be partially corrected by contraction, and in the presence of insulin the contracting diabetic muscle has a completely normal pattern of glycogen synthesis and lactate production, but CO2 production remains impaired.
Circular-dichroism studies on two β-lactamases from Bacillus cereus
Authors: Dalgleish D. G. , Peacocke A. R.
Published: August 2015

The circular-dichroism (CD) spectra of β-lactamases I and II from Bacillus cereus 569/H are reported, along with that of the β-lactamase II free from carbohydrate. The results show that carbohydrate makes an appreciable contribution to the optical activity of β-lactamase II in the far-ultraviolet, and that removal of carbohydrate greatly affects the optical activity of several aromatic side chains of the protein moiety. Both tyrosyl and tryptophanyl residues are affected, showing that some of these residues must be near to the surface of the protein moiety, close to the site of attachment of the carbohydrate. Although the far-ultraviolet CD spectrum of β-lactamase II resembles that of a protein containing some β-structure, it can be shown that this is a consequence of the optical activity of carbohydrate in this region of the spectrum, and that the protein is likely to contain α-helix rather than β-pleated sheet structure. The overall structures of the protein components of β-lactamases I and II are similar, but not identical, as shown by the dissimilarity of the CD spectra when calculated on a mean residue basis.
Determination of the sequences of protein-derived peptides and peptide mixtures by mass spectrometry
Authors: Morris Howard R. , Williams Dudley H. , Ambler Richard P.
Published: August 2015

Micro-quantities of protein-derived peptides have been converted into N-acetylated permethyl derivatives, and their sequences determined by low-resolution mass spectrometry without prior knowledge of their amino acid compositions or lengths. A new strategy is suggested for the mass spectrometric sequencing of oligopeptides or proteins, involving gel filtration of protein hydrolysates and subsequent sequence analysis of peptide mixtures. Finally, results are given that demonstrate for the first time the use of mass spectrometry for the analysis of a protein-derived peptide mixture, again without prior knowledge of the protein or components within the mixture.
Mescaline-induced changes of brain-cortex ribosomes. Role of spermidine in counteracting the destabilizing effect of mescaline on brain-cortex ribosomes
Authors: Datta R. K. , Antopol W. , Ghosh J. J.
Published: August 2015

1. The effect of spermidine on the mescaline-induced changes of brain-cortex ribosomes was studied by adding spermidine during the treatment of goat brain-cortex slices with mescaline. 2. Mescaline treatment of brain-cortex slices removed a portion of the endogenous spermidine from ribosomes and this removal was significantly prevented when spermidine was present during mescaline treatment. 3. Spermidine present during mescaline treatment of brain-cortex slices counteracted, to some extent, the destabilizing effect of mescaline on ribosomes with respect to heat denaturation. 4. Mescaline treatment of brain-cortex slices made ribosomes more susceptible to breakdown, releasing protein and RNA, and resulting in loss of ribosomal enzymic activities. However, spermidine present during mescaline treatment counteracted moderately the mescaline-induced ribosomal susceptibility to breakdown and ribosomal loss of enzymic activities. 5. Ribosomes of mescaline-treated cortex slices were rapidly degraded by ribonuclease and trypsin. However, if spermidine was present during mescaline treatment of brain-cortex slices the rates of degradation diminished.
A reconstituted cell-free system for the specific transfer of steroid–receptor complexes into nuclear chromatin isolated from rat ventral prostate gland
Authors: Mainwaring W. I. P. , Peterken Brenda M.
Published: August 2015

1. A system has been developed for the specific transfer of [3H]dihydrotestosterone–receptor complexes into prostatic chromatin in vitro. 2. Under optimum conditions the overall transfer of [3H]dihydrotestosterone into purified chromatin in this reconstituted system is entirely consistent with the results obtained in whole tissue both in vivo and in vitro. 3. The transfer of [3H]dihydrotestosterone into chromatin is tissue-specific and maximal into chromatin isolated from androgen-dependent tissues. 4. The tissue specificity is maintained at two levels: first, in the presence of specific cytoplasmic androgen-receptor proteins; secondly, by the nature and composition of the chromatin itself. 5. Evidence is presented that androgenic steroids in vivo may maintain the tissue-specific nature of chromatin in androgen-dependent tissues by the selective induction of nuclear protein synthesis. 6. The relevance of these findings to the mechanism of action of androgenic steroids is discussed.
The energy-linked transhydrogenase reaction in respiratory mutants of Escherichia coli K 12
Authors: Cox G. B. , Newton N. A. , Butlin J. D. , Gibson F.
Published: August 2015

1. Energy-linked and non-energy-linked transhydrogenase activities were assayed in membrane preparations from normal Escherichia coli K 12 and from various mutant strains. 2. The energy-linked transhydrogenase, which uses ATP as energy source, was dependent for activity on the presence of a functional Mg2++Ca2+-stimulated adenosine triphosphatase. 3. Neither of the quinones formed by E. coli, namely ubiquinone-8 and menaquinone-8, was required for normal ATP-dependent energy-linked transhydrogenase activity. 4. The energy-linked transhydrogenase was inhibited by piericidin A at a site unrelated to the sites of inhibition of the electron-transport chain by piericidin A.
Properties of phsophofructokinase from the mucosa of rat jejunum and the relation to the lack of Pasteur effect
Authors: Tejwani G. A. , Ramaiah Abburi
Published: August 2015

1. The properties of phosphofructokinase after its slight purification from the mucosa of rat jejunum were studied. 2. The enzyme is inhibited by almost 100% by an excess of ATP (1.6mm), with 0.2mm-fructose 6-phosphate. AMP, ADP, Pi and NH4+ at 0.2, 0.76, 1.0 and 2mm respectively do not individually prevent the inhibition of phosphofructokinase activity by 1.6mm-ATP with 0.2mm-fructose 6-phosphate to any great extent, but all of them together completely prevent the inhibition of phosphofructokinase by ATP. 3. One of the effects of high concentrations of ATP on the enzyme was to increase enormously the apparent Km value for the other substrate fructose 6-phosphate, and this increase is largely counteracted by the presence of AMP, ADP, Pi and NH4+. At low concentrations of ATP the above effectors individually decrease the concentration of fructose 6-phosphate required for half-maximum velocity and when present together they decrease it further, in a more than additive way. 4. When fructose 6-phosphate is present at a saturating concentration (5mm), 0.3mm-NH4+ increases the maximum velocity of the reaction 3.3-fold; with 0.5mm-fructose 6-phosphate, 4.5mm-NH4+ is required for maximum effect. The other effectors do not change the maximum reaction velocity. 5. The results presented here suggest that NH4+, AMP, ADP and Pi synergistically decrease the inhibition of phosphofructokinase activity at high concentrations of ATP by decreasing the concentration of fructose 6-phosphate required for half-maximum velocity. Such synergism among the effectors and an observed, low ‘energy charge’ [(ATP+½ADP)/(AMP+ADP+ATP)] in conjunction with the possibility of a relatively high NH4+ and fructose 6-phosphate concentration in this tissue, may keep the mucosal phosphofructokinase active and uninhibited by ATP under aerobic conditions, thus explaining the high rate of aerobic glycolysis and the lack of Pasteur effect in this tissue.
Biosynthesis of liver membranes. Incorporation of [3H]leucine into proteins and of [14C]glucosamine into proteins and lipids of liver microsomal and plasma-membrane fractions
Authors: Evans W. H. , Gurd J. W.
Published: August 2015

1. The smooth-and rough-microsomal and the light and heavy plasma-membrane fractions of mouse liver homogenates were prepared and characterized by using biochemical markers. 2. The hexosamine/protein ratio was threefold higher in the plasma membranes than in the smooth-microsomal fraction. Glucosamine was bound only to protein, and galactosamine was attached mainly to lipids. 3. [3H]-Leucine and [14C]glucosamine were injected into animals and the rates of incorporation of radioactivity into the fractions were determined. Both precursors were rapidly incorporated into the microsomal fractions, but plasma membranes showed a slower rate of synthesis which reached a maximum at 2–4h after intravenous administration. 4. The light- and heavy-plasma-membrane fractions showed similar patterns of incorporation, and therefore a precursor–product relationship appears unlikely. 5. Plasma membranes, especially the light subfraction, showed appreciable incorporation of hexosamine into chloroform–methanol-soluble components which were shown to be mainly glycolipids. 6. The results indicate that liver plasma-membrane proteins and glycoproteins are synthesized at similar rates. However, glycolipid synthesis in plasma membranes occurred more rapidly.
Cortisone-evoked decrease of acid-β-galactosidase, β-glucuronidase, N-acetyl-β-glucosaminidase and arylsulphatase in the ileum of suckling rats
Authors: Koldovský O. , Palmieri M.
Published: August 2015

Changes of activity of intestinal acid β-galactosidase, β-glucuronidase, N-acetyl-β-glucosaminidase and arylsulphatase were studied in suckling rats treated with cortisone (5mg/100g body wt. daily, started on day 9 postnatally) and compared with changes in control animals. Specific activities were not changed within the first 72h, but all enzymes decreased similarly 96h after the first injection. Total activities per ileum and animal were not changed within the first 48h, but within 72h a significant decrease was observed. Calculation of the rate of decrease of the hydrolases studied in cortisone-treated animals shows that it proceeds faster than the rate of renewal of enterocytes in this period.
Application of the isotope-dilution principle to the analysis of factors affecting the incorporation of [3H]uridine and [3H]cytidine into cultured lymphocytes. Evaluation of pools in serum and culture media
Authors: Forsdyke D. R.
Published: August 2015

1. Rat lymph-node cells were incubated in serum and medium 199 with [5-3H]uridine or [5-3H]cytidine and acid-precipitable radioactivity was measured. Results were interpreted in terms of an isotope-dilution model. 2. Both serum and medium 199 contained pools that inhibited radioactive labelling in a competitive manner. The serum activity was diffusible and inhibited labelling with [3H]cytidine more than with [3H]uridine; in these respects the activity resembled cytidine (14μm). 3. The pools in serum and plasma were the same size; however, the rate of labelling was greater in plasma, owing to a diffusible factor. 4. Paradoxically, relatively simple media (Earle's salts and Eagle's minimum essential) appeared to have a larger pool than the more complex pyrimidine-containing medium 199; this suggests a contribution to the pool by cells in the simple media. 5. In the absence of pools the average cell was capable of incorporating 2000 radioactive nucleoside molecules/s.
The effects of the stress caused by experimental procedures on alanine aspartate, glutamate and glutamine in rat liver
Authors: Heath D. F. , George D. R. , Rose J. G.
Published: August 2015

Rats were stressed by intravenous injection, tail-warming or moderate restraint for 30s, i.e. by stresses imposed by normal handling during experiment. Liver glutamate concentrations were greatly affected. The results were substantially the same in two varieties of rat (Wistar and Sprague–Dawley), in two laboratories, in experiments carried out by two sets of workers, and after all three stresses. The following detailed results refer to Wistar rats. 1. In starved rats at 20°C and 30°C and in post-absorptive rats at 20°C stress by injection raised liver glutamate concentrations from 1.54, 1.57 and 1.88μmol/g wet wt. 30s after injection to 3.4, 2.7 and 3.6μmol/g wet wt. respectively a few minutes later. In starved rats at 20°C the concentration then fell slowly to 2.3μmol/g wet wt., in starved rats at 30°C it remained steady, and in post-absorptive rats at 20°C it rose slowly to about 4.3μmol/g wet wt. The final values seemed fairly steady and corresponded to an ‘alert’ state. 2. In starved rats at 20°C anaesthesia, with or without injection or cannulation during it, raised glutamate concentrations to the ‘alert’ values, which were maintained for 2–3h. 3. Liver alanine concentration in post-absorptive rats initially fell from 1.5 to 0.8μmol/g, and then stayed fairly constant. 4. Aspartate and glutamine concentrations altered only in starved rats, and proportionately much less than those of glutamate. 5. The necessity for knowing the time-dependence of glutamate concentrations after experimental handling is emphasized. 6. There is no wholly satisfactory explanation of the observations.
Nitrosamine-induced carcinogenesis. The alkylation of N-7 of guanine of nucleic acids of the rat by diethylnitrosamine, N-ethyl-N-nitrosourea and ethyl methanesulphonate
Authors: Swann P. F. , Magee P. N.
Published: August 2015

1. The extent of ethylation of N-7 of guanine in the nucleic acids of rat tissue in vivo by diethylnitrosamine, N-ethyl-N-nitrosourea and ethyl methanesulphonate was measured. 2. All compounds produced measurable amounts of 7-ethyl-guanine. 3. A single dose of diethylnitrosamine or N-ethyl-N-nitrosourea produced tumours of the kidney in the rat. Three doses of ethyl methanesulphonate produced kidney tumours, but a single dose did not. 4. A single dose of diethylnitrosamine produced twice as much ethylation of N-7 of guanine in DNA of kidney as did N-ethyl-N-nitrosourea. A single dose of both compounds induced kidney tumours, although of a different histological type. 5. A single dose of ethyl methanesulphonate produced ten times as much ethylation of N-7 of guanine in kidney DNA as did N-ethyl-N-nitrosourea without producing tumours. 6. The relevance of these findings to the hypothesis that alkylation of a cellular component is the mechanism of induction of tumours by nitroso compounds is discussed.
Glycoprotein of the wall of sycamore tissue-culture cells
Authors: Heath M. F. , Northcote D. H.
Published: August 2015

1. A glycoprotein containing a large amount of hydroxyproline is present in the cell walls of sycamore callus cells. This protein is insoluble and remained in the α-cellulose when a mild separation procedure was used to obtain the polysaccharide fractions of the wall. The glycoprotein contained a high proportion of arabinose and galactose. 2. Soluble glycopeptides were prepared from the α-cellulose fraction when peptide bonds were broken by hydrazinolysis. The soluble material was fractionated by gel filtration and one glycopeptide was further purified by electrophoresis; it had a composition of 10% hydroxyproline, 35% arabinose and 55% galactose, and each hydroxyproline residue carried a glycosyl radical so that the oligosaccharides on the glycopeptide had an average degree of polymerization of 9. 3. The extraction of the glycopeptides was achieved without cleavage of glycosyl bonds, so that the glycoprotein cannot act as a covalent cross-link between the major polysaccharides of the wall. 4. The wall protein approximates in conformation to polyhydroxyproline and therefore it probably has similar physicochemical properties to polyhydroxyproline. This is discussed in relation to the function of the glycoprotein and its effect on the physical and chemical nature of the wall.
Extensive charging of transfer ribonucleic acid by bean leaf extracts in vitro
Authors: Tao K. L. , Hall T. C.
Published: August 2015

1. Phenol was effectively removed from aqueous extracts of RNA by chromatography on Sephadex G-50. 2. Elution of tRNA from Sephadex G-50 columns at pH7.6 was shown to remove 91% of the endogenously bound amino acids. 3. tRNA prepared without recourse to ethanolic precipitation was capable of accepting much greater amounts of amino acids than could redissolved samples of precipitated tRNA. 4. Aminoacyl-tRNA synthetase enzymes were partially purified with calcium phosphate gel. Elution of enzymes from the gel at pH6.5 yielded a fraction having phenylalanine- and alanine-charging activity, but no aspartate-, lysine- or proline-charging activity, whereas elution at pH7.6 gave a fraction having aspartate-, lysine- and proline-charging activity but no phenylalanine- or alanine-charging activity. 5. By using partially synthetase enzymes and tRNA eluted from DEAE-Sephadex A-50 columns, 52% of the theoretical maximum of aminoacyl-tRNA synthesis was obtained in vitro.
Properties of Lubrol-extracted uridine diphosphate glucuronyltransferase
Authors: Howland R. D. , Burkhalter A. , Trevor A. J. , Hegeman S. , Shirachi D. Y.
Published: August 2015

1. A partially purified UDP-glucuronyltransferase was obtained by extracting rat liver microsomal preparations with Lubrol, a non-ionic detergent. 2. The soluble enzyme catalysed conjugation of both o-aminophenol and p-nitrophenol and was extremely stable when compared with untreated microsomal preparations. 3. The characteristics of the conjugation of the two phenols were found to differ with respect to pH optimum, bivalent cation requirement and Michaelis constants, suggesting that more than one enzyme is involved in the conjugation reaction.
Differential sedimentation-velocity and gel-filtration measurements on human apotransferrin and iron–transferrin
Authors: Charlwood P. A.
Published: August 2015

Differential measurements of sedimentation velocity showed that binding of 2 atoms of iron per molecule of human apotransferrin caused an increase in s020,w of about 1.8%. Gel-filtration experiments to compare the elution volumes of apotransferrin and transferrin radioactively labelled with iron showed that binding of the first atom to a molecule produced a decrease in Stokes radius of about 0.7%, and the binding of a second atom an equal decrement. These results confirmed that saturation of human transferrin with iron alters the conformation sufficiently to produce detectable changes in the hydrodynamic properties. They also indicate that the local changes brought about by successive addition of 2 atoms of iron are very similar, if not identical.
Rat liver alcohol dehydrogenase. Purification and properties
Authors: Arslanian M J , Pascoe E , Reinhold J G
Published: August 2015

Alcohol dehydrogenase (EC 1.1.1.1) from the rat liver supernatant fraction has been purified 200-fold and partially characterized. The isolation procedure involved ammonium sulphate fractionation, DEAE-Sephadex chromatography and gel filtration. The purified enzyme behaved as a homogeneous preparation as evaluated by cellulose acetate and polyacrylamide-gel disc electrophoresis. Sulphoethyl-Sephadex chromatography and immunoelectrophoresis with rabbit antiserum indicated the presence of a minor component. Rat liver alcohol dehydrogenase appears to contain 4mol of zinc/mol, has an estimated molecular weight of 65000 and consists of two subunits of similar molecular weight. Heavy-metal ions, thiol-blocking reagents, urea at concentrations below 8m, low pH (5.5) and chelating agents deactivate the enzyme but do not dissociate it into subunits. Deactivated enzyme could not be reactivated. The enzyme is strictly specific for NAD+ and has a broad specificity for alcohols, which are bound at a hydrophobic site. Inhibition occurred with the enzyme equilibrated with Zn2+ at concentrations above 0.1mm.
The proteins of Xenopus ovary ribosomes
Authors: Ford P J
Published: August 2015

1. The preparation of ribosomes and ribosomal subunits from Xenopus ovary is described. 2. The yield of once-washed ribosomes (buoyant density in caesium chloride 1.601g·cm-3; 44% RNA, 56% protein by chemical methods) was 10.1mg/g wet wt. of tissue. 3. Buoyant density in caesium chloride and RNA/protein ratios by chemical methods have been determined for ribosome subunits produced by 1.0mm-EDTA or 0.5m-potassium chloride treatment and also for EDTA subunits extracted with 0.5m-, 1.0m- or 1.5m-potassium chloride, 4. Analysis of ribosomal protein on acrylamide gels at pH4.5 in 6m-urea reveals 24 and 26 bands from small and large EDTA subunits respectively. The actual numbers of proteins are greater than this, as many bands are obviously doublets. 5. Analysis of the proteins in the potassium chloride extract and particle fractions showed that some bands are completely and some partially extracted. Taking partial extraction as an indication of possible doublet bands it was found that there were 12 and 20 such bands in the small and large subunits respectively, making totals of 36 and 46 proteins. 6. From the measured protein contents and assuming weight-average molecular weights for the proteins of large and small subunits close to those observed for eukaryote ribosomal proteins it is possible to compute the total numbers of protein molecules per particle. It appears that too few protein bands have been identified on acrylamide gels to account for all the protein in the large subunit, but probably enough for the small subunit.
The role of Halobacterium cutirubrum deoxyribonucleic acid-dependent ribonucleic acid polymerase subunits in initiation and polymerization
Authors: Louis B. Gregory , Fitt P. S.
Published: August 2015

1. The two subunits α and β of Halobacterium cutirubrum DNA-dependent RNA polymerase are required in equimolar amounts for RNA synthesis to occur in vitro at the maximum rate. 2. In the absence of bivalent cations no interaction occurs between α and β subunits or between the subunits and DNA. 3. Mn2+ causes the subunits to form a 1:1 complex that still does not bind to the template. 4. Mg2+ permits binding of the Mn2+-mediated complex to DNA. 5. The complete enzyme, αβ, is inhibited by rifampicin and only the β subunit relieves the inhibition when added in excess. 6. Rifampicin-insensitive, template-dependent RNA synthesis occurs in the presence of protein α alone provided an oligonucleotide with a 5′-purine terminus is supplied as primer. 7. In the primed reaction with the α protein and an oligonucleotide, the template specificity is independent of the ionic strength, in contrast with the marked effect of salt concentration on the template specificity of the complete enzyme. 8. It is concluded that the β protein controls the specificity of chain initiation and the template specificity of the complete enzyme and also carries the rifampicin-binding site, whereas the catalytic site is on the α subunit.