bluuebunny/crossref_metadata_2025 huggingface dataset:
Biogenesis of mitochondria. A requirement for mitochondrial protein synthesis for the formation of a normal adenine nucleotide transporter in yeast mitochondria
Authors: Haslam J. M. , Perkins Margaret , Linnane Anthony W.
Published: August 2015

1. Parameters of ATP uptake by fully functional Saccharomyces cerevisiae mitochondria, including kinetic constants, binding constants and sensitivity to atractylate, closely resemble those of mammalian mitochondria. Scatchard plots of atractylate-sensitive adenine nucleotide binding indicate two distinct sites of high affinity (binding constant, KD′=1μm), and low affinity (binding constant, KD″=20μm) in the ratio 1:3. Uptake has high Arrhenius activation energies (+35 and +57kJ/mol), above and below a transition temperature of 11°C. Atractylate-insensitive ATP uptake is apparently not saturable and has a low Arrhenius activation energy (6kJ/mol), suggesting a non-specific binding process. 2. Kinetic and binding constants for ATP uptake are not significantly changed in catabolite-repressed or anaerobic mitochondrial structures. 3. Inhibition of the mitochondrial protein-synthesizing system by growth of cells in the presence of erythromycin, or loss of mitochondrial DNA by mutation profoundly alters the adenine nucleotide transporter. ATP uptake becomes completely insensitive to atractylate, and the high-affinity binding site is lost. However, the adenine nucleotide transporter does not appear to be totally eliminated, as a moderate amount of saturable low-affinity ATP binding remains. 4. It is concluded that products of the mitochondrial protein-synthesizing system, probably coded by mitochondrial DNA, are required for the normal function of the adenine nucleotide transporter.
Quantitative characteristics of glutamate transport in rat liver mitochondria
Authors: Bradford Norah M. , McGivan J. D.
Published: August 2015

1. The kinetics of glutamate transport into mitochondria were determined by using Bromocresol Purple to terminate the transport process. 2. Glutamate transport was found to have a Vmax. of 9.1nmol/min per mg of protein at pH6.9 and 20°C; the Km for glutamate was 4mm. 3. The rate of glutamate deamination in intact mitochondria was tenfold slower than in disrupted mitochondria. 4. These results suggest that glutamate deamination may be controlled by the rate of glutamate transport. Possible consequences of these findings are discussed.
Evidence that chick tendon procollagen must be denatured to serve as substrate for proline hydroxylase (Short Communication)
Authors: Murphy Lois , Rosenbloom Joel
Published: August 2015

The ability of chick-embryo proline hydroxylase to hydroxylate [14C]proline-labelled procollagen was investigated between 23° and 37°C. The amount of hydroxy[14C]proline that could be formed increased sharply between 26° and 30°C. This corresponded to the temperature interval in which the [14C]procollagen substrate was thermally denatured, and the results therefore indicate that only denatured molecules can be hydroxylated.
A hydroxyproline-containing glycopeptide released from the walls of sycamore tissue-culture cells by hydrazinolysis
Authors: Heath M. F. , Northcote D. H.
Published: August 2015

Degradation of the sycamore cell wall by hydrazinolysis yielded soluble glycopeptides. The peptide portion of the predominant glycopeptide was shown to be cyclic (4-trans-hydroxy-l-prolyl)-4-trans-hydroxy- l-proline. From this structure and the composition of the major glycopeptide it was deduced that only short oligosaccharides are attached to the hydroxyprolyl residues of the wall glycoprotein. Detailed evidence for the structure of the peptide is given in a supplementary paper that has been deposited as Supplementary Publication SUP 50020, at the National Lending Library for Science and Technology, Boston Spa, Yorks. LS23 7BQ, U.K., from whom copies can be obtained on the terms indicated in Biochem. J. (1973) 131, 5.
Studies by electron paramagnetic resonance on the catalytic mechanism of nitrogenase of Klebsiella pneumoniae
Authors: Smith Barry E. , Lowe David J. , Bray Robert C.
Published: August 2015

The properties and catalytic reactions of the enzyme nitrogenase purified from Klebsiella pneumoniae were studied by electron-paramagnetic-resonance (e.p.r.) spectroscopy at temperatures down to 8°K. The two protein fractions, Kp1 (the iron–molybdenum protein) and Kp2 (the iron protein), were examined alone and in steady-state mixtures and also in pre-steady-state experiments, by using the rapid-freezing method. Kp1 protein in dithionite solution shows a rhombic type of spectrum with g1 4.32, g2 3.63, g3 2.009 at pH6.8 (0°C). Small changes in the spectrum produced by protons (pK=8.7 at 0°C) or by acetylene indicate binding of these oxidizing substrates to this protein fraction. Kp2 protein shows a rhombic spectrum with g1 2.053, g2 1.942, g3 1.865, which integrates to about 0.45 electron/molecule. Binding of ATP, with a dissociation constant of 4×10-4m, changes the spectrum to an axial form with g∥ 2.036, g⊥ 1.929, thus indicating a conformation change of Kp2 protein. The Kp2 protein spectrum disappears reversibly on cautious oxidation. The signals of both proteins are diminished in their steady-state mixtures, obtained in the presence of ATP and dithionite (with an ATP-generating system and Mg2+ ions) and with protons, N2 or acetylene as oxidizing substrate. At the same time as dithionite is consumed in such reactions, the Kp1 protein signal is gradually restored and the Kp2 protein signal diminishes to zero. In rapid-freezing experiments the signals from the two proteins decreased at indistinguishable rates (t½ about 10ms), then they remained constant. Results are interpreted in terms of a scheme in which reducing equivalents pass from dithionite to Kp2 protein, then, in an ATP-dependent reaction to Kp1 protein, this being finally reoxidized by N2 or another oxidizing substrate. In this scheme Kp1 protein cycles between its signal-giving state and a very highly reduced signal-free state.
Oxidation of deuteroferrihaem by hydrogen peroxide
Authors: Jones Peter , Prudhoe Kenneth , Robson Terrence
Published: August 2015

1. The oxidation of deuteroferrihaem by H2O2 to bile pigment and CO was studied both by stopped-flow kinetic spectrophotometry and mass spectrometry, at 25°C, I=0.1m. 2. Spectrophotometric studies imply that, at constant pH, the rate of bile pigment formation is first-order with respect to [H2O2] and also proportional to [deuteroferrihaem monomer]. The effect of pH on the apparent second-order rate constant suggests that acid-ionization of deuteroferrihaem monomer is important in the reaction mechanism. 3. The relative rates of formation of O2 (from catalytic decomposition of H2O2) and CO (from oxidation of ferrihaem) have been measured by mass spectrometry. The results are in excellent agreement with those obtained by combining kinetic data for catalytic decomposition (Jones et al., 1973, preceding paper) with the spectrophotometric results for deuteroferrihaem oxidation.
The structure of a polymer containing galactosamine from walls of Bacillus subtilis 168. (Short Communication)
Authors: Shibaev Vladimir N. , Duckworth Michael , Archibald A. Ronald , Baddiley James
Published: August 2015

Cell walls of Bacillus subtilis 168 contain a phosphorylated polysaccharide composed of N-acetylgalactosamine, glucose and phosphate. Controlled acid hydrolysis gives 3-O-β-d-glucopyranosyl-N-acetylgalactosamine with a phosphomonoester group at the 6-position of glucose. It is likely that the polysaccharide consists of disaccharide units connected by phosphodiester residues joining the 1-position of N-acetylgalactosamine to the 6-position of glucose in the neighbouring unit.
Phosphorylation of myelin basic protein by an adenosine 3′:5′-cyclic monophosphate-dependent protein kinase (Short Communication)
Authors: Carnegie P. R. , Kemp B. E. , Dunkley P. R. , Murray A. W.
Published: August 2015

Myelin basic protein was shown to be a substrate for protein kinase from rabbit muscle. One of the major sites of phosphorylation was the serine residue in the sequence Gly-Arg-Gly-Leu-Ser-Leu. The arginine residue in this sequence is known to be a substrate for a protein methylase.
The steady-state kinetics of isotope exchange for one substrate–one product enzymic reactions
Authors: Darvey Ivan G.
Published: August 2015

Steady-state kinetic equations for isotope exchange are derived for a number of one substrate–one product enzymic mechanisms in which two molecules of substrate or product can be combined with an enzyme molecule at the one time (e.g. allosteric mechanisms). The usual assumption, that the radioactive material is distributed among the substrate and product components according to a first-order law, is not valid. One can recognize whether isotope-exchange kinetics of an enzyme reaction follows first-order behaviour by using various initial concentrations of the labelled substance added to a mixture.
The metabolism of amino acids in the bovine lens. Their oxidation as a source of energy
Authors: Trayhurn P. , Van Heyningen Ruth
Published: August 2015

1. The metabolism by the bovine lens of nine 14C-labelled l-amino acids was studied. These were: alanine, aspartate, glutamate, leucine, lysine, proline, serine, tyrosine and tryptophan. 2. All were taken up by the tissue and incorporated into protein. 3. Aspartate and glutamate, although poorly taken up, were readily metabolized to CO2. Radioactivity from glutamate was also found in glutathione, glutamine, proline and ophthalmic acid. Aspartate was converted into glutamate, glutathione, proline, alanine and lactate. 4. Alanine was largely converted into lactate, which was released into the medium, but incorporation of radioactivity into CO2, glutamate, glutathione, aspartate and lipids also occurred. 5. Radioactivity from leucine was detected in CO2, lipids, glutamate, glutathione, proline and glutamine. 6. Lysine was only slightly broken down by the bovine lens; radioactivity was observed in CO2, glutamate, glutathione, proline and two unidentified compounds. 7. Proline was metabolized to glutamate from which CO2, glutathione and glutamine were formed. Hydroxyproline in the capsule collagen was labelled. 8. Radioactivity from serine was found in CO2, lipids, glutathione, glycine, cystine, ATP, lactate and three unidentified compounds, one of which was probably taurine. 9. Neither tyrosine nor tryptophan were metabolized by the bovine lens. 10. The ability of the lens to metabolize amino acids was also shown by measurement of NH3 production: more NH3 was formed when glucose was absent from the incubation medium. 11. These experiments suggest that oxidation of amino acids is a source of energy for the lens.
The stereochemistry of hydrogen elimination from C-7 during biosynthesis of ecdysones in insects and plants
Authors: Cook I. F. , Lloyd-Jones J. G. , Rees H. H. , Goodwin T. W.
Published: August 2015

1. [7α-3H1]- and [7β-3H1]-Cholesterol were synthesized by a modified method. 2. The stereochemistry of Δ7-bond formation during ecdysone and ecdysterone biosynthesis in the insect, Calliphora erythrocephala and the plants, Taxus baccata and Polypodium vulgare was investigated by using [4-14C,7α-3H1]cholesterol and [4-14C,7β-3H1]cholesterol. 3. In each case, the 7β hydrogen was stereospecifically eliminated. 4. The possible significance of the results is discussed in relation to double-bond formation in other systems and the stage at which the Δ7 bond is introduced during ecdysone biosynthesis.
Calcium ion-dependent p-nitrophenyl phosphate phosphatase activity and calcium ion-dependent adenosine triphosphatase activity from human erythrocyte membranes
Authors: Rega Alcides F. , Richards Donaldo E. , Garrahan Patricio J.
Published: August 2015

In the presence of ATP and of Mg2+, human erythrocyte membranes show a phosphatase activity towards p-nitrophenyl phosphate which is activated by low concentrations of Ca2+. The effect of Ca2+ is strongly enhanced if either K+ or Na+ is also present. Activation of the p-nitrophenyl phosphate phosphatase by Ca2+ reaches a half-maximum at about 8μm-Ca2+ and is apparent only when the ion has access to the inner surface of the cell membrane. Ca2+-dependent phosphatase activity can only be observed if ATP is at the inner surface of the cell membrane, and the presence of ATP seems to be absolutely necessary, since either its removal or its replacement by other nucleoside triphosphates abolishes the activating effect of Ca2+. The properties of the (ATP+Ca2+)-dependent phosphatase are very similar to those of the Ca2+-dependent ATPase (adenosine triphosphatase), also present in erythrocyte membranes, which probably is involved in Ca2+ transport in erythrocytes. The similarities suggest that both activities may be properties of the same molecular system. This view is further supported by the fact that p-nitrophenyl phosphate inhibits to a similar extent Ca2+-dependent ATPase activity and ATP-dependent Ca2+ extrusion from erythrocytes.
Stability of clearing-factor lipase in rat adipose tissue
Authors: Davies P. , Robinson D. S.
Published: August 2015

The stability at 42°C of clearing-factor lipase in adipose tissue, and in intact fat-cells isolated from it, was investigated. That portion of the total activity of the tissue which is associated with the fat-cell is stable under such conditions. This stability is markedly diminished when the fat-cell is disrupted.
Control of 5-aminolaevulinate synthetase activity in Rhodopseudomonas spheroides. The involvement of sulphur metabolism
Authors: Neuberger Albert , Sandy John D. , Tait George H.
Published: August 2015

1. The ‘initial’ 5-aminolaevulinate synthetase activity, that is the activity observed immediately after cell disruption, in extracts prepared from unharvested semianaerobically grown Rhodopseudomonas spheroides, was twice that observed under the same assay conditions in extracts prepared from harvested cells. 2. The effect of oxygenation of a culture on the ‘maximum’ aminolaevulinate synthetase activity, that is the activity observed 1h after disruption of harvested cells, is markedly influenced by the contents of the growth medium. Oxygenation of organisms for 1h in the medium in which they have grown produces an 80–90% decrease in maximum activity, whereas similar treatment of organisms resuspended in fresh medium produces less than a 40% decrease. 3. This protective effect of fresh medium is absolutely dependent on the presence of sulphate. When cells are suspended in sulphate-deficient fresh medium, the maximum activity falls by 65–75% even without oxygenation. A high maximum activity is regenerated when sulphate is resupplied. 4. When organisms are oxygenated in the medium in which they have grown, the cellular contents of GSH+GSSG and cysteine+cystine fall very markedly and homolanthionine is formed. Both the fall in aminolaevulinate synthetase activity and the changes in sulphur metabolism are largely prevented by the addition of compounds which stimulate synthesis of cysteine de novo or inhibit the conversion of cysteine S into homocysteine S. 5. The maximum aminolaevulinate synthetase activity was directly proportional to the GSH+GSSG content of all cell preparations. In glutathione-depleted extracts the ‘low’-activity enzyme could be re-activated in vitro by the addition of GSH, GSSG, cysteine or cystine, whereas in extracts with a high glutathione content the ‘high’-activity enzyme was unaffected by these sulphur compounds. 6. The activation of low-activity enzyme with exogenous sulphur compounds was prevented by excluding air or by adding NADH. Studies with purified enzyme indicate that sulphur compounds do not interact directly with the enzyme, but that their effect is mediated by a number of other endogenous factors.
Inhibition by ricin of protein synthesis in vitro. Ribosomes as the target of the toxin
Authors: Montanaro Lucio , Sperti Simonetta , Stirpe Fiorenzo
Published: August 2015

1. Ricin (a toxic protein from the seeds of Ricinus communis) is a powerful inhibitor of the poly(U)-directed incorporation of phenylalanine into polypeptides catalysed by isolated rat liver ribosomes and elongation factors 1 and 2 (EF 1 and EF 2). The inhibition can be largely overcome by increasing the concentration of ribosomes. 2. The toxin does not affect the binding of phenylalanyl-tRNA to ribosomes catalysed by EF 1, nor does it inhibit the puromycin reaction used as a test for peptide-bond formation catalysed by ribosomes. 3. Ricin inhibits the ribosome-linked GTP hydrolysis catalysed by EF 2. 4. Ribosomes treated with ricin and washed through sucrose gradients containing 0.6m-NH4Cl are functionally inactive in those assay systems that are sensitive to the presence of added toxin. 5. It is suggested that ricin brings about an irreversible modification of ribosomes which impairs their ability to interact with EF 2. Since ricin inhibits at a molar concentration much lower than that of ribosomes it probably acts catalytically. No added cofactor is necessary for the inhibitory action of the toxin.
The effect of thiourea on ureide metabolism in Neurospora crassa
Authors: Nirmala Jasti , Sastry Killampalli Sivarama
Published: August 2015

The wild-type strain of Neurospora crassa Em 5297a can utilize allantoin as a sole nitrogen source. The pathway of allantoin utilization is via its conversion into allantoic acid and urea, followed by the breakdown of urea to ammonia. This is shown by the inability of the urease-less mutant, N. crassa 1229, to grow on allantoin as a sole nitrogen source and by the formation of allantoate and urea by pre-formed mycelia of this mutant. In the wild strain (Em 5297a) thiourea is tenfold more toxic on an allantoin medium than on an inorganic nitrogen medium; allantoin as well as urea counteract thiourea toxicity in the allantoin nitrogen medium. This selective toxicity of thiourea for the mould utilizing allantoin nitrogen does not, however, result in an impairment of allantoin uptake, allantoinase activity or the formation of urea from allantoin. The only process affected by thiourea is the synthesis of urease; urea antagonizes this effect of thiourea in N. crassa.
The metabolism of four sulphonamides in cows
Authors: Nielsen Poul
Published: August 2015

1. The metabolism of sulphanilamide, sulphadimidine (4,6-dimethyl-2-sulphanilamidopyrimidine), sulphamethoxazole (5-methyl-3-sulphanilamidoisoxazole) and sulphadoxine (5,6-dimethoxy-4-sulphanilamidopyrimidine) given by intravenous injection has been examined in cows. 2. The sulphonamides were present mainly as unchanged drugs in blood samples collected 2h after administration. 3. The sulphonamides were excreted in the milk partly as unchanged drugs and partly as conjugated metabolites whereas only small amounts were excreted as the N4-acetyl derivatives. 4. The unchanged drug and the N4-acetyl derivative were the major constituents in urine samples after administration of sulphanilamide, sulphamethoxazole and sulphadoxine. 5. Besides the unchanged drug, the N4-acetyl derivative and the conjugated metabolites, three further metabolites of sulphadimidine were isolated from urine samples and identified. They were 5-hydroxy-4,6-dimethyl-2-sulphanilamidopyrimidine, 4-hydroxymethyl-6-methyl-2-sulphanilamidopyrimidine and sulphaguanidine.
A simplified method for preparing sucrose gradients (Short Communication)
Authors: Stone Alan B.
Published: August 2015

Numerous sucrose gradients can be prepared simultaneously by diffusion in horizontal centrifuge tubes.
Haemoglobulin catabolism: the role of ferrihaems in studies of the degradation pathway (Short Communication)
Authors: Brown N. A. , King R. F. G. J. , Shillcock M. E. , Brown S. B.
Published: August 2015

The importance of ferrihaem aggregation in studies of haemoglobin catabolism is assessed in the light of recent work. Experimental evidence is put forward suggesting that monomeric ferrihaems are degraded much more readily than dimeric species. This may offer an alternative explanation for the apparent ‘apoprotein catalysis’ recently observed.
Separation at neutral pH value of 32P-labelled proteins in a membrane preparation from ox brain. Partial characterization of a component of the sodium-plus-potassium ion-activated adenosine triphosphatase
Authors: Alexander Denis R. , Rodnight Richard
Published: August 2015

1. Ox brain microsomal fractions were labelled with [32P]ATP in the presence of Na+ and the reaction was stopped with sodium dodecyl sulphate. The Na+-dependent bound phosphate was isolated on Sephadex G-25 and by acetone precipitation. The bound phosphate isolated under these neutral conditions was labile to hydroxylamine and gave the same pH profile of hydrolysis as that isolated by precipitation with strong acids. 2. When membrane protein was labelled with [32P]ATP, solubilized with sodium dodecyl sulphate and fractionated on Sepharose 6B, the Na+-dependent label emerged in a peak corresponding to protein of molecular weight 570000–580000. On fractionation of this protein peak on polyacrylamide gels containing detergent and urea, the Na+-dependent label occurred in a single band corresponding to a protein of molecular weight 102000. 3. Fractionation on Sepharose 6B of protein labelled with [32P]ATP in the absence of Na+ revealed three labelled peaks, one of which corresponded in position to the Na+-dependent label. Electrophoresis of this peak material on polyacrylamide gels showed that most of the label occurred in two fast-running bands. Cyclic AMP stimulated the labelling in these two bands, but had no effect on the labelling of the band corresponding in position to the Na+-dependent label. 4. Di-isopropyl [32P]phosphorofluoridate also labelled the band corresponding to the Na+-dependent label on gel electrophoresis. The labelling of this band by the reagent was inhibited by 50–60% by 3mm-ATP, but there was no evidence to suggest that the group labelled is normally phosphorylated by ATP.
A study of the properties of two porphyringlobin species formed in the reaction of protoporphyrin IX with human globin
Authors: Ainsworth Stanley , Treffry Amyra
Published: August 2015

Globin was prepared from the main (A0) component of human haemoglobin and reacted with protoporphyrin IX; the product, when subjected to chromatography on CM-Sephadex, separated into fast- and slow-moving species. These were isolated for examination. The dissociation constant for the tetramer–dimer equilibrium of fast-moving porphyringlobin was determined at 2.8X10−6m; this is to be compared with values of 2.2X10−6m and 8X10−8m determined for oxyhaemoglobin and the slow-moving porphyringlobin respectively. It was also shown that the thiol groups of fast-moving porphyringlobin react with 4,4′dithiodipyridine at an identical rate with those of oxyhaemoglobin; in comparison, the rates of reaction of deoxyhaemoglobin and porphyringlobin are much slower but are again identical with one another. The quenching of porphyringlobin fluorescence by I− ions was also studied. The quenching could not be represented by a simple Stern–Volmer relationship (whereas that of porphyrin–apomyoglobin is), but was represented by a model in which the fluorescence of fast-moving porphyringlobin was more accessible to the quencher than that of the slow-moving component. Similarly, fast-moving porphyringlobin was photodecomposed more rapidly by oxygen than the slow-moving species.
Affinity labelling of proteinases with tryptic specificity by peptides with C-terminal lysine chloromethyl ketone
Authors: Coggins John R. , Kray William , Shaw Elliott
Published: August 2015

Methods are described for the synthesis of peptides terminating in Lys-CH2Cl. The products were examined as affinity labels for several enzymes of trypsin-like specificity which are resistant to Tos-Lys-CH2Cl. In part, the inertness of the latter may be due to the sulphonamide group, since Z-Lys-CH2Cl was more effective. However, a number of tripeptides with C-terminal Lys-CH2Cl were superior in their ability to inactivate subtilisin, thrombin and plasma kallikrein. The possibility of developing enzyme-specific reagents selective for members within the trypsin-like group is demonstrated by Ala-Phe-Lys-CH2Cl, which readily inactivates plasma kallikrein but not thrombin.
The control of ribonucleic acid synthesis in bacteria. Fluctuations in messenger ribonucleic acid synthesis in cultures recovering from amino acid starvation
Authors: Midgley J. E. M. , Smith R. J.
Published: August 2015

Changes in the cell content and rate of synthesis of mRNA were studied in auxotrophs of Escherichia coli recovering from a period of amino acid deprivation. Parallel studies were carried out on bacterial strains inhibited with trimethoprim, when glycine and methionine were added to relieve an amino acid deficiency. In the latter case, protein synthesis was still severely inhibited through a lack of N-formylmethionyl-tRNAfMet for chain initiation, so that fewer ribosomes were attached to mRNA chains. (1) In RCstr strains recovering from amino acid starvation, there was a transient oversynthesis of mRNA, but the amounts returned to normal after about a 15-min period of recovery. RCrel strains did not show this effect; any extra mRNA accumulated during the previous starvation period was rapidly lost, but no oversynthesis occurred during the resumption of growth. (2) In trimethoprim-inhibited cultures supplemented with glycine and methionine, mRNA was produced at the same rate, relative to stable RNA species, as during normal growth. The evidence implied that decreased rates of ribosome attachment had no effect on the functional or chemical lifetime of the mRNA fraction. This suggests that mRNA stability does not depend on the frequency of translation by ribosomes. (3) Changes in the mRNA contents of trimethoprim-inhibited RCstr and RCrel cultures were noted soon after supplementation with glycine and methionine. These closely followed those observed in cultures recovering from simple amino acid withdrawal.
The inhibition of mitochondrial dicarboxylate transport by inorganic phosphate, some phosphate esters and some phosphonate compounds
Authors: Johnson R. N. , Chappell J. B.
Published: August 2015

1. Pi competitively inhibited succinate oxidation by intact uncoupled mitochondria in the presence of sufficient N-ethylmaleimide to block the phosphate carrier, with a Ki of 2.5mm. 2. Of a large number of phosphate esters and phosphonate compounds, phenyl phosphate and phenylphosphonate were found to inhibit competitively uncoupled succinate oxidation by intact but not broken mitochondria. By comparison, benzoate was a relatively weak competitive inhibitor of succinate oxidation by intact mitochondria but a relatively potent inhibitor of succinate dehydrogenase. 3. Phenyl phosphate and phenylphosphonate were non-penetrant, and inhibited Pi-dependent swelling of mitochondria suspended in isosmolar ammonium malate in a manner non-competitive with Pi. The inhibitors did not affect mitochondrial swelling when tested with Pi alone. 4. It is concluded that: (i) phenyl phosphate and phenylphosphonate behaved as non-penetrant analogues of Pi, since their inhibitory properties were in strict contrast with those of benzoate; (ii) phenyl phosphate and phenylphosphonate interacted with the dicarboxylate carrier but not with the phosphate carrier; (iii) Pi was effective as a competitive inhibitor of succinate oxidation because of its being either an alternative substrate for the dicarboxylate carrier or competitive with succinate for the intramitochondrial cations as proposed by Harris & Manger (1968).
Sterol biosynthesis in the free-living nematode Panagrellus redivivus
Authors: Willett James D. , Downey William L.
Published: August 2015

The fate of the known sterol precursor squalene 2,3-oxide was investigated in the free-living nematode Panagrellus redivivus. The nematodes were cultured axenically in the presence of [4-3H]squalene 2,3-oxide. Radioactivity was found in the total lipids of the isolated nematodes. Essentially all of the radioactivity encountered in the total lipids was found in the non-saponifiable fraction. The components present in the non-saponifiable fraction were separated and isolated by t.l.c. Three labelled components were identified by a combination of t.l.c., g.l.c. and mass spectroscopy. It is established that P. redivivus has the capacity for biosynthesis of lanosterol. No labelled C27 sterols could be detected.
The action of α-amanitin in vivo on the synthesis and maturation of mouse liver ribonucleic acids
Authors: Hadjiolov Asen A. , Dabeva Mariana D. , Mackedonski Vladimir V.
Published: August 2015

α-Amanitin acts in vitro and in vivo as a selective inhibitor of nucleoplasmic RNA polymerases. Treatment of mice with low doses of α-amanitin causes the following changes in the synthesis, maturation and nucleocytoplasmic transfer of liver RNA species. 1. The synthesis of the nuclear precursor of mRNA is strongly inhibited and all electrophoretic components are randomly affected. The labelling of cytoplasmic mRNA is blocked. These effects may be correlated with the rapid and lasting inhibition of nucleoplasmic RNA polymerase. 2. The synthesis and maturation of the nuclear precursor of rRNA is inhibited within 30min. (a) The initial effect is a strong (about 80%) inhibition of the early steps of 45S precursor rRNA maturation. (b) The synthesis of 45S precursor rRNA is also inhibited and the effect increases from about 30% at 30min to more than 70% at 150min. (c) The labelling of nuclear and cytoplasmic 28S and 18S rRNA is almost completely blocked. The labelling of nuclear 5S rRNA is inhibited by about 50%, but that of cytoplasmic 5S rRNA is blocked. (d) The action of α-amanitin on the synthesis of precursor rRNA cannot be correlated with the slight gradual decrease of nucleolar RNA polymerase activity (only 10–20% inhibition at 150min). (e) The inhibition of precursor rRNA maturation and synthesis precedes the ultrastructural lesions of the nucleolus detected by standard electron microscopy. 3. The synthesis of nuclear 4.6S precursor of tRNA is not affected by α-amanitin. However, the labelling of nuclear and cytoplasmic tRNA is decreased by about 50%, which indicates an inhibition of precursor tRNA maturation. The results of this study suggest that the synthesis and maturation of the precursor of rRNA and the maturation of the precursor of tRNA are under the control of nucleoplasmic gene products. The regulator molecules may be either RNA or proteins with exceedingly fast turnover.
Reduction and inactivation of superoxide dismutase by hydrogen peroxide
Authors: Bray Robert C. , Cockle Stephen A. , Fielden E. Martin , Roberts Peter B. , Rotilio Giuseppe , Calabrese Lilia
Published: August 2015

Reactions of H2O2 with superoxide dismutase were studied by e.p.r. (electron paramagnetic resonance) spectroscopy and other methods. In agreement with earlier work, the Cu2+ of the enzyme is reduced by H2O2, although the reaction does not go to completion and its kinetics are not simple. With dilute enzyme the time for half-reduction with 9mm-H2O2 is about 150ms. It is suggested that the reaction is a one-electron reduction, involving liberation of O2−. On somewhat more prolonged exposure to H2O2, the enzyme is inactivated. For enzyme in dilute solution and over a limited range of H2O2 concentrations, inactivation is first-order with respect to enzyme and reagent, with k=3.1m−1·s−1 at 20–25°C. Inactivation is accompanied by marked changes in the e.p.r. and visible spectra and appears to be associated with destruction of one histidine residue per subunit. It is suggested that this histidine is close to the metal in the native enzyme and essential for its enzymic activity.
Modified messenger ribonucleic acid release from isolated hepatic nuclei after inhibition of polyadenylate formation
Authors: Schumm Dorothy E. , Webb Thomas E.
Published: August 2015

Treatment of rats with Cordycepin (3′-deoxyadenosine), an inhibitor of nuclear poly(A) synthesis, selectively decreased the energy and cytosol-dependent release of a portion of the mRNA species from the isolated hepatic nuclei in a cell-free system by approximately 40%. An analysis of the differential binding of the transported mRNA containing poly(A) tracts to nitrocellulose filters, and to cellulose or poly(U)–Sepharose columns suggested that the poly(A) tracts in the mRNA transported from the isolated hepatic nuclei of Cordycepin-treated rats, are decreased in size. This size decrease was confirmed through an analysis of the average size of the poly(A) tracts, released from the messengers by ribonuclease activity, on sucrose density gradients.
Macroscopic rate constants involved in the formation and interconversion of the two central enzyme–substrate complexes of the lactate dehydrogenase turnover
Authors: Südi J.
Published: August 2015

The preceding paper (Südi, 1974) reports partial success in describing the conversion of ENADH plus pyruvate into ENAD+ plus lactate in terms of a simple Haldane-type scheme which involves two intermediates (ENADHPyr and ENAD+Lac), where E represents lactate dehydrogenase. This information is completed here by reporting kinetic results obtained by carrying out the same reaction in the opposite direction. The combined results of these two papers confirm the findings of Holbrook & Gutfreund (1973) that the observed spectral changes do take place at the level of resolution of this simple two-intermediate scheme. The following numerical values for the rate (and equilibrium) constants involved in their formation and decomposition are reported: [Formula: see text] It is shown that although the precision of estimation of some of these numerical values is subject to some experimental uncertainty, their derivation from direct experimental observations only involves the principle of microscopic reversibility. This paper describes stopped-flow kinetic observations made with ENAD+ and lactate as the two reactants. It is shown that fluorescence and u.v.-absorption measurements yield the same experimental rate constant for the last reaction step in which ENADH is generated. On the other hand, the generation of ENADHPyr can only be indirectly observed, as a less than stoicheiometric `burst', and by u.v.-absorption measurements only. It is shown that the stoicheiometry of this partial `burst reaction', and a pre-equilibrium factor in the directly observed rate of ENADH-production, yield equivalent information about the reversible oxidation–reduction step. It is further shown that the pre-equilibrium factor that is involved in the generation of ENADH can be determined because k+4=222s−1 is already known (Südi, 1974). Since the fluorescence measurements yield much more precise estimations, and their interpretation is considered by the author to be free of ambiguity, the presented quantitative analysis is based on the fluorescence observations.
Polyphenol–protein interactions
Authors: Haslam Edwin
Published: August 2015

The association of some natural and synthetic polyphenols with β-glucosidase was examined and some observations on the chemical nature of the complex were made.
Pig liver pyruvate carboxylase. Purification, properties and cation specificity
Authors: Warren Graham B. , Tipton Keith F.
Published: August 2015

1. Pyruvate carboxylase was purified to apparent homogeneity from pig liver mitochondria and shown to be free of all kinetically contaminating enzymes. 2. The enzyme has a mol. wt. of 520000 and is composed of four subunits, each with a mol. wt. of 130000. 3. The enzyme can exist as the active tetramer, dimer and monomer, although the tetramer appears to be the form in which the enzyme is normally assayed. 4. For every 520000g of the enzyme there are 4mol of biotin, 3mol of zinc and 1mol of magnesium. No significant concentrations of manganese were detected. 5. Analysis by sodium dodecyl sulphate–polyacrylamide gel electrophoresis indicates three polypeptide chains per monomer unit, each with a mol. wt. of 47000. 6. The amino acid analysis, stoicheiometry of the reaction and the activity of the enzyme as a function of pH are also presented. 7. The enzyme is activated by a variety of univalent cations but not by Tris+ or triethanolamine+. 8. The activity of the enzyme is dependent on the presence of acetyl-CoA; the low rate in the absence of added acetyl-CoA is not due to an enzyme-bound acyl-CoA. The dissociation constant for enzyme-bound acetyl-CoA is a marked function of pH.
Navy (haricot)-bean (Phaseolus vulgaris) lectin. Isolation and characterization of two components from a toxic agglutinating extract
Authors: Andrews A. T.
Published: August 2015

Two protein components were isolated in a highly purified state from a toxic fraction from the navy (haricot) bean (Phaseolus vulgaris). One, a lectin, strongly agglutinated horse erythrocytes and leucocytes, agglutination being readily observable with both types of cell at a lectin concentration of 4μg/ml. The other component (component 1), although possessing some similarity in composition, was thought to be non-agglutinating or, at most, only very weakly so. Component 1 had a mol.wt. of about 143000 and a subunit mol.wt. of about 37000, suggesting a tetrameric structure probably with identical subunits. Alanine was the only N-terminal amino acid identified and the molecule was notable in being devoid of tryptophan and cysteine, low in methionine and high in leucine, glutamic acid and aspartic acid. The lectin was somewhat smaller (mol.wt. about 114000) and apparently also composed of four identical subunits of mol.wt. about 30000. Dansylation showed that arginine occupied the N-terminus of the polypeptide chain. Aspartic acid, serine, threonine and leucine were the predominant amino acids of the lectin, and the sulphur-containing amino acids were entirely absent. Both constituents were glycoproteins and the compositions of the carbohydrate portions (4.9% for component 1 and 8.1% for the lectin) were generally similar, consisting of mannose and glucosamine with smaller amounts of glucose and traces of xylose and arabinose.
Carcinoembryonic antigen-like substances of human urothelial carcinomas. Isolation of components from pathological urine and comparison with colorectal carcinoma antigens
Authors: Nery R. , Barsoum A. L. , Bullman H. , Neville A. M.
Published: August 2015

Urines of several patients with urothelial carcinomas contain inhibitors of the immunoreaction between carcinoembryonic antigen derived from human colorectal carcinomas and monospecific goat antiserum raised against the antigen. These inhibitors range in approximate molecular weights from less than 1000 to several millions, and two have been isolated by a combination of extraction, gel filtration and electrophoretic procedures. These are respectively a macromolecular aggregate, component UCEA-3, which is excluded by Sepharose 4B, and a glycoprotein(s) component, UCEA-1, with mean molecular weight (2×105) similar to that of carcinoembryonic antigen. Comparison of the properties of component UCEA-1 and carcinoembryonic antigen on gel filtration, electrophoresis, immunoelectrophoresis and density gradient ultracentrifugation indicates that these substances of similar molecular size and net charge differ in some immunochemical properties.
The reactivity of the disulphide bonds of wool
Authors: Lindley H. , Cranston R. W.
Published: August 2015

1. Fully reduced and S-carboxymethylated wool samples were prepared in which either the readily reducible cystine bonds or those that could only be reduced with difficulty were specifically labelled with iodo[2-14C]acetate; these two cystine fractions correspond to the (A+B) and (C+D) cystine fractions respectively, of Middlebrook & Phillips (1942). 2. Radioactively labelled peptides were isolated from partial acid hydrolysates of these wool samples. 3. It appears that the (A+B) cystine residues probably owe their increased reactivity to being in a more polar environment. 4. The implication of these results for the problem of characterizing the disulphide bonds of wool is briefly discussed.
Studies on the heparin sulphamidase activity from rat spleen. Intracellular distribution and characterization of the enzyme
Authors: Friedman Yochanan , Arsenis Charalampos
Published: August 2015

A sulphamidase activity present in rat spleen capable of hydrolysing N-[35S]sulphated heparin was studied. This activity was associated with the lysosomal fraction. Studies in vivo showed that the rat is capable of significantly desulphating heparin. Lysosomes in all the major tissues can effectively accumulate heparin. The heparin sulphamidase and arylsulphatase activities from rat spleen were separated by isoelectric focusing. Heparin sulphamidase was a distinct entity from all the arylsulphatase activities.
Histidine residues and the enzyme activity of pig heart supernatant malate dehydrogenase
Authors: Holbrook J. John , Lodola A. , Illsley Nicholas P.
Published: August 2015

1. Supernatant pig heart malate dehydrogenase is completely inhibited by reaction with diethyl pyrocarbonate at pH6.5, when 0.58±0.1 residue of ethoxycarbonylhistidine is formed per NADH-binding site. 2. Oxaloacetate and hydroxymalonate protect the enzyme from inhibition in the absence of coenzyme. 3. Limited ethoxycarbonylation does not alter the binding of NADH to the enzyme but prevents the enzyme–NADH complex from interacting with hydroxymalonate in a ternary complex.
Biosynthesis of glucagon in isolated pancreatic islets of guinea pigs
Authors: Hellerström Claes , Howell Simon L. , Edwards John C. , Andersson Arne , Östenson Claes-Göran
Published: August 2015

1. The biosynthesis of glucagon in guinea-pig A2 cells was investigated by incubation of isolated islets of Langerhans in the presence of [3H]tryptophan for periods of up to 14 days. Proteins were extracted from islets and incubation media and analysed by gel filtration. 2. In addition to very-high-molecular-weight (100000) proteins, the principal tryptophan-containing biosynthetic product after incubation for up to 17h was a protein of minimum mol.wt. 9000, which co-eluted on gel filtration with a peak of glucagon-like immunoreactivity, but was apparently devoid of biological activity in a fat-cell assay. A discrete peak of labelled glucagon was only recovered after incubation for at least 6 days. Losses of glucagon during the extraction and rapid secretion of newly synthesized glucagon into incubation media were excluded as reasons for the lack of recovery of labelled hormone from islets after shorter incubations. 3. The 9000-mol.wt. protein was localized to A2 cells in experiments using B-cell-depleted islets, and to A2-cell granules by subcellular fractionation and electron-microscopic radioautography. Only glucagon was secreted into the incubation medium. 4. Possible relationships between the 9000-mol.wt. protein and glucagon are discussed in the light of postulated mechanisms of glucagon biosynthesis.
The glycoasparagines in urine of a patient with aspartylglycosaminuria
Authors: Pollitt Rodney J. , Pretty Kathleen M.
Published: August 2015

The isolation and analysis of 2-acetamido-N-(4′-l-aspartyl)-2-deoxy-β-d- glucopyranosylamine and 13 higher-molecular-weight derivatives from the urine of a patient with aspartylglycosaminuria is described. Some structural information is presented for nine of these compounds.
The isolation of an easily reversible postsynaptic toxin from the venom of a sea snake, Laticauda semifasciata
Authors: Maeda Nobuyo , Takagi Kenji , Tamiya Nobuo , Chen Yun-Ming , Lee Chen-Yuan
Published: August 2015

A weakly neurotoxic component (Ls-III) was isolated by CM-cellulose column chromatography from the venom of a sea snake Laticauda semifasciata. The content of component LsIII was about 10–20% of the venom as determined by u.v. absorption at 280nm. Component LsIII was homogeneous on rechromatography and disc electrophoresis, and its molecular weight was shown to be 7100 by ultracentrifugation and 7300 by sodium dodecyl sulphate–polyacrylamide-gel disc electrophoresis. The isoelectric point of component LsIII was pH7.2. Component LsIII consisted of 66 amino acid residues including 10 half-cystine residues. The LD50 of component LsIII by intramuscular injection was 1.24μg/g body wt. for mice and 0.45μg/g for baby chicks, which is about eight to ten times less toxic than erabutoxins a, b and c, all of which are contained in the same venom. Experiments with three isolated muscle preparations from different species indicated that component LsIII was a post-synaptically acting toxin, the action of which was easily reversed by washing.
A plasma protein fractionation procedure for use in studies of protein metabolism. Its application to the measurement of synthesis rates of two α1-glycoproteins and other serum proteins in the rat
Authors: Ho J. , Jeejeebhoy K. N. , Painter R. H.
Published: August 2015

A two-step method for the separation of five different plasma proteins on a preparative scale, which is capable of being extended to allow the separation of other plasma proteins, is described. The proteins separated were fibrinogen, two α1-glycoproteins, albumin and transferrin. The α1-glycoproteins were characterized in terms of electrophoretic mobility, ultracentrifugal and immunological characteristics. By using this method, it was shown that a single sample of plasma could be fractionated to yield purified proteins in sufficient quantity to simultaneously measure the synthesis of the two α1-glycoproteins, albumin and transferrin in the rat with McFarlane's technique (McFarlane, 1963; Reeve et al., 1963; McFarlane et al., 1965).
Phospholipid synthesis in rat liver endoplasmic reticulum after the administration of phenobarbitone and 20-methylcholanthrene
Authors: Davison Susan C. , Wills Eric D.
Published: August 2015

1. Phenobarbitone injection did not affect the concentration of phospholipids in the liver endoplasmic reticulum, but it increased the rate of incorporation of [32P]orthophosphate into the phospholipids. 20-Methylcholanthrene caused a transient increase in total phospholipid but a decrease in the turnover rate of the phospholipids. 2. Incorporation of [32P]orthophosphate into phosphatidylcholine, compared with that into phosphatidylethanolamine, was increased by phenobarbitone injection but decreased by 20-methylcholanthrene injection. 3. The activity of S-adenosylmethionine–phosphatidylethanolamine methyltransferase increased 12h after phenobarbitone injection, when incorporation of [32P]orthophosphate into phosphatidylcholine was a maximum, but at other times, and after 20-methylcholanthrene injection, the activity of the enzyme did not correlate with the rate of phosphatidylcholine synthesis. 4. [14C]Glycerol was incorporated more rapidly into phosphatidylcholine than into phosphatidylethanolamine, whereas [32P]orthophosphate and [14C]ethanolamine were incorporated more rapidly into phosphatidylethanolamine than into phosphatidylcholine. 5. Incorporation of [32P]orthophosphate into phosphatidylethanolamine of liver slices incubated in vitro was much more rapid than into phosphatidylcholine, and incorporation into phosphatidylcholine was markedly stimulated by addition of methionine to the medium. Changes in the incorporation of [32P]orthophosphate into phospholipids observed in vivo after injection of phenobarbitone or methylcholanthrene could not be reproduced in slices incubated in vitro. 6. It is concluded that phenobarbitone injection causes an increased rate of turnover of total phospholipids in the endoplasmic reticulum and an increased conversion of phosphatidylethanolamine into phosphatidylcholine, whereas 20-methylcholanthrene injection depresses both the turnover rate of total phospholipids and the formation of phosphatidylcholine.