bluuebunny/crossref_metadata_2025 huggingface dataset:
A simple fluorimetric microassay for adenine compounds in platelets and plasma and its application to studies on the platelet release reaction
Authors: Gordon John L. , Drummond Alan H.
Published: August 2015

1. The formation of a stable fluorescent product between chloroacetaldehyde and adenine or its derivatives provides the basis of a rapid simple assay for total adenine compounds in blood platelets and in plasma. The assay will measure down to 200pmol of adenine nucleotides. An evaluation of the method established the optimum conditions for the production of maximum fluorescence. 2. Values obtained for total adenine compounds in platelets were 12.9nmol/108 cells in man and 7.8nmol/108 cells in rat. These closely agree with previous values for total platelet adenine nucleotides found by using a firefly luciferase assay, or a recycled NAD-linked photometric assay. This supports the concept that the chloroacetaldehyde reaction measures total adenine nucleotides in platelets. 3. Platelet aggregation induced by collagen was studied photometrically in 0.1ml volumes of citrated platelet-rich plasma, and total adenine nucleotides were assayed in platelets and plasma before and after aggregation. During aggregation 58% of adenine nucleotides were released from human platelets, and 36% from rat platelets. 4. The chloroacetaldehyde assay is no substitute for more sophisticated procedures, but is a simple sensitive means of monitoring the release of adenine nucleotides from blood platelets and is particularly valuable when small plasma samples must be used.
Rat brain microsomal gangliosides. Accessibility to a neuraminidase preparation and the possible existence of different pools in relation to their biosynthesis
Authors: Maccioni H. J. F. , Arce A. , Landa C. , Caputto R.
Published: August 2015

1. Treatment of rat brain microsomal membranes with a neuraminidase preparation from Clostridium perfringens resulted in an almost complete conversion of polysialogangliosides into monosialogangliosides. 2. Neuraminidase treatment of the membranes did not increase the incorporation of N-[3H]acetylneuraminic acid from CMP-N-[3H]acetylneuraminic acid into the gangliosidic fraction, indicating that a monosialoganglioside is an acceptor of N-acetylneuraminic acid in these membranes only if, in addition to having the right chemical structure, it is in a proper position, probably in relation to the endogenous sialyltransferases. 3. These experiments also indicated that no independent turnover of the neuraminidase-labile N-acetylneuraminyl groups of gangliosides occurred in vitro. 4. N-[3H]Acetylneuraminic acid from endogenous polysialogangliosides labelled in vitro was released by neuraminidase at a slower rate than N-acetylneuraminic acid from unlabelled gangliosides of the same membranes. From this it was concluded that recently synthesized polysialogangliosides (completed in vitro) are in the membranes in a position less accessible to neuraminidase than are those synthesized earlier which were present in the membranes at the start of the labelling experiment.
Fractionation of iodinated particles and mitochondria from thyroid by zonal centrifugation and a study of their heterogeneity
Authors: Miquelis Raymond , Simon Claude
Published: August 2015

1. The subcellular particles of horse and rat thyroids were fractionated in a B XIV zonal rotor on a non-linear gradient of Ficoll after labelling with radioactive iodine in vitro (horse) or in vivo (rat). In the horse, the resulting fractions were analysed for radioactive iodine, protein and enzymes representative of certain subcellular particles. In the rat, iodine turnover and thyrotrophin stimulation were studied. 2. The population of iodinated particles could be subdivided into three main classes, characterized by differences in β-galactosidase and acid phosphatase content and position in the gradient. The presence of a fourth class of particles is suggested. 3. It is concluded that iodinated particles isolated from the thyroid are essentially secondary lysosomes. Their heterogeneity is established with respect to their position in the gradient, their content of acid hydrolases and their iodine turnover. 4. The iodine pools of these secondary lysosomes are increased by thyrotrophin without any change in their number. 5. Their functional significance is discussed. 6. The distribution of mitochondria as judged by succinate dehydrogenase was also studied. The succinate dehydrogenase was spread throughout the gradient with a maximum of activity (40%) in the upper layer of the gradient. Separation of mitochondria from lysosomes by this method was not successful.
The effects of acetylcolletotrichin on the mitochondrial respiratory chain
Authors: Foucher Bernard , Chappell J. B. , McGivan J. D.
Published: August 2015

1. Acetylcolletotrichin is a phytotoxic compound that has been isolated from the culture medium of the fungus Colletotrichum capsici (Grove et al., 1966). 2. With isolated liver and kidney mitochondria acetylcolletotrichin markedly inhibited the oxidation of succinate and those substrates with NAD-linked dehydrogenases, but did not inhibit the oxidation of ascorbate in the presence of tetramethyl-p-phenylenediamine. In this respect its action was similar to that of antimycin A. 3. Acetylcolletotrichin differed from antimycin in that, even at high concentrations which produced a maximal inhibitory effect, its action was partially reversed by uncoupling agents. Also acetylcolletotrichin had no detectable effect on the oxidative activity of blowfly flight-muscle mitochondria and was not very effective with heart mitochondria. 4. Acetylcolletotrichin inhibited the oxidative activity of liver mitochondria more markedly when respiration was stimulated by ADP together with phosphate and was less effective when respiration was stimulated by uncoupling agents. 5. There was an unusual interaction between the succinate oxidation system and the oxidation of glutamate together with malate. Thus, glutamate together with malate, even in the presence of rotenone, markedly decreased the effectiveness of acetylcolletotrichin in inhibiting succinate oxidation. 6. These effects were paralleled in the observed redox changes of cytochrome c. 7. The unusual behaviour of the cytochromes b in the presence of acetylcolletotrichin is described, and it is suggested tentatively that this inhibitor acts between cytochromes b with absorption maxima at 30°C of approximately 560 and 565nm.
The inhibition of human placental diamine oxidase by substrate analogues
Authors: Crabbe M. James C. , Bardsley William G.
Published: August 2015

1. The oxidation of p-dimethylaminomethylbenzylamine by purified placental diamine oxidase was followed by measuring the change in E250 caused by the production of p-dimethylaminomethylbenzaldehyde. 2. The inhibition of this reaction by substrate analogues such as isothiouronium, guanidinium, dimethylsulphonium and trimethylammonium compounds was extensively studied. 3. The type and degree of inhibition by mono- and bis-onium compounds is described, and a theory is developed to explain the type of inhibition produced.
Haem ligands of the ferricytochrome c of Ustilago sphaerogena
Authors: Vinogradov Serge N. , Bitar Kamal G. , Lowenkron Steven
Published: August 2015

The mammalian-type cytochrome c of the basidiomycete Ustilago sphaerogena contains in a single polypeptide chain of 107 residues, two histidine residues located at positions 18 and 33, and one methionine residue situated at position 80 (Bitar et al., 1972). The reaction of Ustilago ferricytochrome c with bromoacetate at neutral pH resulted in the modification of histidine-33, but not of histidine-18 or of the invariant methionine residue. The activities of Ustilago cytochrome c with mitochondrial cytochrome c oxidase and with NADH–cytochrome c reductase were unaltered by the modification. The equilibrium constants for the formation of low-spin complexes of the ferrihaem octapeptide of horse cytochrome c (residues 14–21, including the haem bound covalently to cysteines 14 and 17) with imidazole, N2-acetylhistidine and monocarboxymethyl derivatives of N2-acetylhistidine were determined spectrophotometrically. Alkylation of the imidazole side-chain group of N2-acetylhistidine resulted in a marked decrease in its ability to form low-spin ferrihaem complexes. These results indicate that in Ustilago ferricytochrome c in solution histidine-33 is not involved in the central co-ordination complex. Since side-chain groups of residues other than histidine and methionine do not appear to be involved in the central complexes of other mammalian-type cytochromes c (Hettinger & Harbury, 1964, 1965; Myer & Harbury, 1965) it is likely that in Ustilago ferricytochrome c in solution at neutral pH, the side-chain groups of histidine-18 and methionine-80 are involved in the central co-ordination complex. The latter is stable over the pH range 2.6–8.4.
Studies of the polysaccharide fraction from the lipopolysaccharide of Pseudomonas alcaligenes
Authors: Lomax James A. , Gray George W. , Wilkinson Stephen G.
Published: August 2015

Studies of the lipopolysaccharide of Pseudomonas alcaligenes strain BR 1/2 were extended to the polysaccharide moiety. The crude polysaccharide, obtained by mild acid hydrolysis of the lipopolysaccharide, was fractionated by gel filtration. The major fraction was the phosphorylated polysaccharide, for which the approximate proportions of residues were; glucose (2), rhamnose (0.7), heptose (2–3), galactosamine (1), alanine (1), 3-deoxy-2-octulonic acid (1), phosphorus (5–6). The heptose was l-glycero-d-manno-heptose. The minor fractions from gel filtration contained free 3-deoxy-2-octulonic acid, Pi and PPi. The purified polysaccharide was studied by periodate oxidation, methylation analysis, partial hydrolysis, and dephosphorylation. All the rhamnose and part of the glucose and heptose occur as non-reducing terminal residues. Other glucose residues are 3-substituted, and most heptose residues are esterified with condensed phosphate residues, possibly in the C-4 position. Free heptose and a heptosylglucose were isolated from a partial hydrolysate of the polysaccharide. The location of galactosamine in the polysaccharide was not established, but either the C-3 or C-4 position appears to be substituted and a linkage to alanine was indicated. In its composition, the polysaccharide from Ps. alcaligenes resembles core polysaccharides from other pseudomonads: no possible side-chain polysaccharide was detected.
The identification of intermediates in the reaction of pig heart lactate dehydrogenase with its substrates
Authors: Whitaker J. R. , Yates D. W. , Bennett N. G. , Holbrook J. J. , Gutfreund H.
Published: August 2015

Pig heart lactate dehydrogenase was studied in the direction of pyruvate and NADH formation by recording rapid changes in extinction, proton concentration, nucleotide fluorescence and protein fluorescence. Experiments measuring extinction changes show that there is a very rapid formation of NADH within the first millisecond and that the amplitude of this phase (phase 1) increases threefold over the pH range 6–8. A second transient rate (phase 2) can also be distinguished (whose rate is pH-dependent), followed by a steady-state rate (phase 3) of NADH production. The sum of the amplitudes of the first two phases corresponds to 1mol of NADH produced/mol of active sites of lactate dehydrogenase. Experiments that measured the liberation of protons by using Phenol Red as an indicator show that no proton release occurs during the initial very rapid formation of NADH (phase 1), but protons are released during subsequent phases of NADH production. Fluorescence experiments help to characterize these phases, and show that the very rapid phase 1 corresponds to the establishment of an equilibrium between ENADLactate ⇌ H+ENADHPyruvate. This equilibrium can be altered by changing lactate concentration or pH, and the H+ENADHPyruvate species formed has very low nucleotide fluorescence and quenched protein fluorescence. Phase 2 corresponds to the dissociation of pyruvate and a proton from the complex with a rate constant of 1150s-1. The observed rate constant is slower than this and is proportional to the position of the preceding equilibrium. The ENADH formed has high nucleotide fluorescence and quenched protein fluorescence. The reaction, which is rate-limiting during steady-state turnover, must then follow this step and be involved with dissociation of NADH from the enzyme or some conformational change immediately preceding dissociation. Several inhibitory complexes have also been studied including ENAD+Oxamate and ENADHOxamate' and the abortive ternary complex ENADHLactate. The rate of NADH dissociation from the enzyme was measured and found to be the same whether measured by ligand displacement or by relaxation experiments. These results are discussed in relation to the overall mechanism of lactate dehydrogenase turnover and the independence of the four binding sites in the active tetramer.
The inhibition of mitochondrial calcium transport by lanthanides and Ruthenium Red
Authors: Reed Ken C. , Bygrave Fyfe L.
Published: August 2015

An EGTA (ethanedioxybis(ethylamine)tetra-acetic acid)-quench technique was developed for measuring initial rates of 45Ca2+ transport by rat liver mitochondria. This method was used in conjunction with studies of Ca2+-stimulated respiration to examine the mechanisms of inhibition of Ca2+ transport by the lanthanides and Ruthenium Red. Ruthenium Red inhibits Ca2+ transport non-competitively with Ki 3×10-8m; there are 0.08nmol of carrier-specific binding sites/mg of protein. The inhibition by La3+ is competitive (Ki=2×10-8m); the concentration of lanthanide-sensitive sites is less than 0.001nmol/mg of protein. A further difference between their modes of action is that lanthanide inhibition diminishes with time whereas that by Ruthenium Red does not. Binding studies showed that both classes of inhibitor bind to a relatively large number of external sites (probably identical with the ‘low-affinity’ Ca2+-binding sites). La3+ competes with Ruthenium Red for most of these sites, but a small fraction of the bound Ruthenium Red (less than 2nmol/mg of protein) is not displaced by La3+. The results are discussed briefly in relation to possible models for a Ca2+ carrier.
Leucyl-transfer ribonucleic acid synthetase in senescing tobacco leaves
Authors: Nathan Ilana , Richmond Amos
Published: August 2015

1. The activity of leucyl-tRNA synthetase obtained from tobacco leaves declined by 50% over a period of 4 days senescence induced by detachment. In addition tRNALeu from senescing leaves was charged to a lesser extent than tRNALeu extracted from mature leaves immediately after detachment. 2. tRNALeu was charged with a synthetase preparation from either mature or senescent leaves and chromatographed on an RPC 3 column. The elution profile showed that the marked decline in specific activity of leucyl-tRNA synthetase in senescent leaves was not associated with a loss of acylation of any isoacceptor of tRNALeu.
Hepatic drug hydroxylation and lipid peroxidation in riboflavin-deficient rats
Authors: Patel J. M. , Galdhar N. R. , Pawar S. S.
Published: August 2015

The effect of riboflavin deficiency and phenobarbital pretreatment on drug hydroxylation and lipid peroxidation was investigated. A significant decrease in aniline and acetanilide hydroxylation as well as NADPH-linked and ascorbate-induced lipid peroxidation was observed during 4- and 7-week riboflavin deficiency in both adult male and adult female rats. The drug-hydroxylation and lipid-peroxidation activities were further lowered with the increase in riboflavin deficiency. The phenobarbital pretreatment induced aniline and acetanilide hydroxylase activity even in riboflavin-deficient animals. Drug hydroxylation inhibits lipid peroxidation in both deficient and normal rats. The administration of riboflavin was followed by a significant increase in drug hydroxylation and lipid peroxidation.
Effect of nitrate on the synthesis and decay of nitrate reductase of Neurospora
Authors: Sorger George J. , Debanne Maria Teresa , Davies Jacqueline
Published: August 2015

1. A method was developed to examine the turnover of nitrate reductase by the use of tungstate. 2. Evidence is presented which suggests that the disappearance of nitrate reductase activity from Neurospora mycelia exposed to non-inducing conditions is due to the disappearance of the enzyme protein(s) from the mycelia, and not merely due to the disappearance of its (their) catalytic power. 3. The presence of NO3− in the culture medium slows down the rate of degradation of nitrate reductase in Neurospora in vivo.
The role of ribonuclease II in the maturation of precursor 16S ribosomal ribonucleic acid in Escherichia coli
Authors: Dean John R. , Sykes John
Published: August 2015

The suggested involvement of ribonuclease II in the maturation of rRNA has been examined directly by determining the activity of the enzyme and the amount of p16S rRNA in cell-free extracts from Escherichia coli A19 and its temperature-sensitive derivative N464 grown under experimental conditions designed to vary the amounts of enzyme and precursor independently. In strain A19 the enzyme showed maximum activity in circumstances where the amount of p16S rRNA was normal (e.g. exponential-phase cells) or raised eight times (e.g. during inhibition of growth by methionine starvation of the relaxed auxotroph or by chloramphenicol or puromycin treatment). In strain N464 at the non-permissive temperature the ribonuclease II activity may be decreased by 50% without effect upon the amount of p16S rRNA, whereas in methionine starvation of this strain the enzyme activity is at a maximum and the p16S rRNA is eight times that in exponential-phase cells. These observations are discussed in relation to the previously implied role of ribonuclease II in the maturation of rRNA within ribosome precursors.
Isolation and characterization of a hydroxyproline-containing protein from soluble extracts of the leaves of sandal (Santalum album L.)
Authors: Mani Uliyar V. , Radhakrishnan Amurtur N.
Published: August 2015

1. A hydroxyproline-containing protein was isolated from the soluble fraction of sandal leaves (Santalum album L.) and the purified protein was homogeneous by disc electrophoresis. 2. It is a glycoprotein containing 16% carbohydrate, the components of which were mainly arabinose, with only small amounts (about 5%) of galactose. The principal amino acids were glutamic acid, aspartic acid, glycine, alanine, arginine, lysine, proline and hydroxyproline, which together comprised 60% of the total. The number of acidic amino acids exceeds the number of basic amino acids. By Sephadex gel filtration, the approximate molecular weight was found to be about 63000. The ratio of residues of hydroxyproline to those of arabinose was 1:2. 3. The native protein is resistant to the action of several proteolytic enzymes. After partial hydrolysis with 0.1m-HCl, the protein became susceptible to attack by Pronase but remained resistant to collagenase.
Kinetics of alkaline phosphatase from pig kidney. Mechanism of activation by magnesium ions
Authors: Ahlers Jan
Published: August 2015

The mechanism of activation of alkaline phosphatase (EC 3.1.3.1) from pig kidney by Mg2+ ions was investigated with the aid of kinetic measurements. Mg2+ ions are essential for enzyme activity. The following model (Scheme 1 of the text) for the reaction of enzyme, substrate and Mg2+ ions was derived: [Formula: see text] The binding of the substrate to the enzyme is independent of the binding of the activator, and vice versa. Mg2+ must therefore play a part in the substrate decomposition. It is not possible to determine whether the Mg2+ ions are involved directly in the catalytic process, or whether they act as regulatory effectors. Because of the strong affinity existing between the alkaline phosphatase and Mg2+, it is necessary to adjust the metal-ion concentration with the aid of a metal buffer. In the Appendix the necessary equations are derived for calculating the concentration of free metal ions in a system with several different metal ions. A FORTRAN IV program for solving these equations and for graphic presentation of the results has been deposited as Supplementary Publication SUP 50030 at the British Library (Lending Division) (formerly the National Lending Library for Science and Technology), Boston Spa, Yorks. LS 23 7 BQ, U.K., from whom copies may be obtained on the terms indicated in Biochem. J. (1973), 131, 5.
Electron-spin-resonance studies of the structure and formation of bacterial diazodiphenoquinone pigments (Short Communication)
Authors: Ashworth Paul
Published: August 2015

The formation of diazodiphenoquinone pigments during bacterial oxidations of pyridinediols can be accounted for in terms of a free-radical mechanism similar to that operating for oxidations in vitro involving azasemiquinone radicals. Electron-spin-resonance spectra are of great value in pigment structure determinations.
Peroxidatic oxidation of tyrosine to melanin in supernatant of crude mouse melanoma homogenates
Authors: Patel Ravindra P. , Okun Milton R. , Edelstein Leon M. , Cariglia Nancy
Published: August 2015

Peroxide-dependent enzymic oxidation of tyrosine to dopachrome and melanin was demonstrated in cell-free melanoma homogenates. Histochemical methods for distinguishing peroxidase activity from aerobic dopa (3,4-dihydroxyphenylalanine) oxidase activity are not reliable with cell-free preparations. Therefore the presence of peroxidase activity in such preparations precludes assay of cresolase activity of mammalian ‘tyrosinase’.
Alterations in cholesterol and fatty acid biosynthesis in rat liver homogenates by aryloxy acids
Authors: Olson Robert J. , Trumble Thomas E. , Gamble Wilbert
Published: August 2015

2,4-Dichlorophenoxyacetic acid and 2,4,5-trichlorophenoxyacetic acid inhibited the incorporation of [2-14C]mevalonate into cholesterol and non-saponifiable lipids. Both compounds inhibited the conversion of [1-14C]isopentenyl pyrophosphate into cholesterol and the synthesis of cholesterol and fatty acids from [2-14C]acetate. There was no inhibition of the conversion of [1-14C]mevalonate into CO2. At low concentrations (0.5mm) of the compounds there was a stimulation of acetate incorporation into fatty acids.
The effects of 5α-dihydrotestosterone on the kinetics of cell proliferation in rat prostate
Authors: Lesser Barry , Bruchovsky Nicholas
Published: August 2015

The regenerating rat prostate was used as an experimental model to determine the effects of 5α-dihydrotestosterone on certain parameters of cell proliferation, including the duration of the phases of the cell cycle and the size of the cellular growth fraction. Rats castrated 7 days previously were treated with daily subcutaneous injections of 5α-dihydrotestosterone for 14 days; 48h after the beginning of therapy, cells in the process of DNA synthesis were labelled with a single injection of radioactive thymidine and the progress of these cells through the division cycle was observed. Cell-cycle analysis was performed by fractionating prostatic nuclei according to their position in the cell cycle by using the technique of velocity sedimentation under unit gravity. The results indicate that during regeneration the cell population undergoes 1.8 doublings with a doubling time of 40h, and that the process involves almost four rounds of cell division with a cell-generation time of 20h. The growth fraction at any time is about 0.5, and about half the daughter cells produced do not re-enter the proliferative cycle. All cells present at the start of regeneration eventually undergo at least one division during the course of regeneration, although any given cell can divide from one to four times.
Inhibition of protein synthesis by ricin: experiments with rat liver mitochondria and nuclei and with ribosomes from Escherichia coli
Authors: Greco Margherita , Montanaro Lucio , Novello Francesco , Saccone Cecilia , Sperti Simonetta , Stirpe Fiorenzo
Published: August 2015

1. Ricin, a toxic protein from the seeds of Ricinus communis which inhibits poly(U)-directed polyphenylalanine synthesis by rat liver ribosomes (Montanaro et al., 1973), does not affect protein synthesis by isolated rat liver mitochondria. 2. The toxin is ineffective also on poly(U)-directed polyphenylalanine synthesis in reconstituted systems with ribosomes isolated from rat liver mitochondria or from Escherichia coli. 3. Ricin inhibits protein synthesis by isolated rat liver nuclei, but at concentrations much higher than those affecting rat liver ribosomes.
Crystallization of macromolecular heparin (Short Communication)
Authors: Atkins Edward D. T. , Nieduszynski Ian A. , Horner Alan A.
Published: August 2015

X-ray fibre-diffraction photographs were obtained from oriented films of the sodium salt of macromolecular heparin (molecular weight approx. 106) prepared from rat skin. Two distinct molecular chain conformations corresponding to two different crystal lattices were observed as a function of relative humidity. The first conformation, obtained at 78% relative humidity, has a layer-line spacing of 1.73nm, which can be interpreted as an approximate twofold helix. On increasing the relative humidity to 84% a second phase with a layer-line repeat of 1.65nm is obtained with the reflexions indexing on a triclinic unit cell similar to that obtained previously (Nieduszynski & Atkins, 1973) for pig mucosal heparin.
An inhibitor of phospholipase D in saliva
Authors: Dawson Rex M. C. , Hemington Norma
Published: August 2015

1. Bovine, dog and human saliva contain substances which inhibit the soluble phospholipase D present in grass leaf or celery stalk. 2. The inhibitor in bovine saliva is of high molecular weight and exhibits considerable stability to heat, acids and alkalis. 3. The inhibitor has been purified free from salivary mucoprotein. 4. It is suggested that the inhibitor could protect the upper alimentary tract of a herbage-eating animal from the necrotic action of phospholipase D.
Electron-paramagnetic-resonance studies on cobalt(II) carbonic anhydrase–sulphonamide complexes
Authors: Cockle Stephen A. , Lindskog Sven , Grell Ernst
Published: August 2015

Sulphonamide adducts of three Co(II) carbonic anhydrases were investigated by e.p.r. (electron paramagnetic resonance) at helium temperatures. The highly anisotropic 9 GHz spectra exhibited only three distinct features, with g values between 6.3 and 1.5. Such spectra arise from an electronic state with effective spin S′=½, indicating that the high-spin (S=3/2) ground level is split into two spin doublets differing in energy by an amount large compared with the microwave quantum, but small in relation to thermal energies at ambient temperature. This situation would occur in a tetrahedral system suffering a large rhombic distortion. Calculations based on this model accounted for apparent discrepancies in integrated spectral intensities, and yielded magnetic moments in good agreement with independent measurements, especially in the case of certain small Co(II) complexes resembling the enzyme adducts in their e.p.r. signals. Precise sets of g values, reflecting a particular co-ordination geometry, were found to be representative of each enzyme variant and the type of sulphonamide inhibitor, whether benzocyclic or heterocyclic. A series of substituted benzene sulphonamides bound to the same enzyme gave rise to closely similar spectra despite a wide range of pKi values. Thus benzocyclic and heterocyclic sulphonamides were evidently held in the active-site cleft in characteristic orientations irrespective of side chains that might considerably influence the total binding strength. Visible absorption spectra of various sulphonamide adducts at room temperature showed a similar pattern of inhibitor dependence to the e.p.r. spectra, suggesting a correspondence between the co-ordination structures in liquid and frozen solution. E.p.r. spectra of the sulphonamide complexes were remarkable not only for their range of g values, but also for their variations in line-width and spin-lattice relaxation behaviour. Addition of glycerol to the medium produced marked enhancement in resolution, owing to the creation of a more homogeneous frozen matrix. The non-uniform spin relaxation was probably a consequence of the large anisotropy in effective g tensor.
Properties of pig heart aconitase
Authors: Gawron Oscar , Kennedy Mary C. , Rauner Richard A.
Published: August 2015

Comparison of pig heart aconitase (Kennedy et al., 1972) with yeast (Candida lipolytica) aconitase (Suzuki et al., 1973) reveals similarities in molecular weight and iron content but not in sulphide content. Comparison with the Mildvan & Villafranca (1971) pig heart aconitase preparation reveals differences in iron ligands, specific activity and other properties; these differences possibly arise from protein association as pig heart protein associates under a variety of conditions. The electron spin resonance spectrum, g 4.25, and the low molar relaxivity, 473m−1·s−1, of water H+ suggest the presence of high-spin Fe(III) unco-ordinated to water in the enzyme. The iron chromophore on acid titration at 320nm gives a curve with an inflexion at pH4.2. Ten of 16 expected thiol equivalents are titrated with p-hydroxymercuribenzoate suggesting the presence of cystine as well as cysteine residues. Inhibition of the activation of inactive (activatable) enzyme is sigmoidally related to the molar ratio, p-hydroxymercuribenzoate/enzyme with 10–11mol of mercurial compound causing complete inhibition. Active enzyme, free from activating reagents, requires high molar ratios of mercurial compound for rapid inhibition. In terms of p-hydroxymercuribenzoate the enzyme then lacks an essential thiol group.
The effect of systematic error on the accuracy of Michaelis constants and maximum velocities estimated by using the integrated Michaelis–Menten equation (Short Communication)
Authors: Newman Peter F. J. , Atkins Gordon L. , Nimmo Ian A.
Published: August 2015

Systematic errors in initial substrate concentration (s0), product concentration and reaction time give much larger errors in the Michaelis–Menten parameters unless s0 is treated as an unknown parameter. These errors are difficult to detect because the fitted curve deviates little from the data. The effect of non-enzymic reaction is also examined.
Changes in respiratory activities during the cell-cycle of the fission yeast Schizosaccharomyces pombe 972h- growing in the presence of glycerol
Authors: Poole Robert K. , Lloyd David
Published: August 2015

1. The specific activities of cytochrome c oxidase, catalase, succinate dehydrogenase, succinate–cytochrome c oxidoreductase, NADH–cytochrome c oxidoreductase, and NADPH–cytochrome c oxidoreductase in mid-exponential-phase batch cultures of glycerol-grown Schizosaccharomyces pombe indicated that the organisms were catabolite-de-repressed. 2. In cultures growing synchronously in the presence of glycerol as sole carbon source, the respiration rate showed two abrupt increases at about 0.45 and 0.95 of the cell-cycle and remained constant in the periods between successive rises. 3. Catalase, succinate dehydrogenase, NADH–cytochrome c oxidoreductase and acid p-nitrophenyl-phosphatase all showed peak patterns of expression in synchronous cultures. 4. Cytochrome c oxidase and cytochromes a+a3 both showed step patterns of expression with two rises per cell-cycle. 5. Cytochromes c548, b554 and b560 all followed similar time-courses in step patterns of expression, but these were distinct from, and more complex than, that of cytochromes a+a3. 6. These results are compared with those previously obtained with glucose-grown cultures, and the part played by catabolite repression in the expression of respiratory activities in the cell-cycle is assessed.
Inhibitory effect of carbon monoxide on the N-and ring-hydroxylation of 2-acetamidofluorene by hamster hepatic microsomal preparations (Short Communication)
Authors: Lotlikar P D , Zaleski K
Published: August 2015

The effect of CO on N-, 3-, 5- and 7-hydroxylation of 2-acetamidofluorene by liver microsomal fractions from control and 3-methylcholanthrene-pretreated hamsters was studied. All hydroxylations were inhibited by CO, but the degree of inhibition was different for each hydroxylation. The ratios of CO to O2 needed for 50% inhibition of the N-, 3-, 5- and 7-hydroxylations by control preparations were 8.0:1, 8.2:1, 4.2:1 and 7.1:1 respectively and by preparations from treated animals were 4.2:1, 8.9:1, 2.3:1 and 3.2:1 respectively. These results are discussed in terms of the possible presence of more than one type of cytochrome P-450 involved in hydroxylations of 2-acetamidofluorene by liver microsomal fractions from both control and pretreated hamsters.
The carbon assimilation pathways of Methylococcus capsulatus, Pseudomonas methanica and Methylosinus trichosporium (OB3B) during growth on methane
Authors: Strøm T , Ferenci T , Quayle J R
Published: August 2015

D-arabino-3-Hexulose 6-phosphate was prepared by condensation of formaldehyde with ribulose 5-phosphate in the presence of 3-hexulose phosphate synthase from methane-grown Methylococcus capsulatus. The 3-hexulose phosphate was unstable in solutions of pH greater than 3, giving a mixture of products in which, after dephosphorylation, allulose and fructose were detected. A complete conversion of d-ribulose 5-phosphate and formaldehyde into d-fructose 6-phosphate was demonstrated in the presence of 3-hexulose phosphate synthase and phospho-3-hexuloisomerase (prepared from methane-grown M. capsulatus). d-Allulose 6-phosphate was prepared from d-allose by way of d-allose 6-phosphate. No evidence was found for its metabolism by extracts of M. capsulatus, thus eliminating it as an intermediate in the carbon assimilation process of this organism. A survey was made of the enzymes involved in the regeneration of pentose phosphate during C1 assimilation via a modified pentose phosphate cycle. On the basis of the presence of the necessary enzymes, two alternative routes for cleavage of fructose 6-phosphate are suggested, one route involves fructose diphosphate aldolase and the other 6-phospho-2-keto-3-deoxygluconate aldolase. A detailed formulation of the complete ribulose monophosphate cycle of formaldehyde fixation is presented. The energy requirements for carbon assimilation by this cycle are compared with those for the serine pathway and the ribulose diphosphate cycle of carbon dioxide fixation. A cyclic scheme for oxidation of formaldehyde via 6-phosphogluconate is suggested.
Regulation of hepatic l-serine dehydratase and l-serine-pyruvate aminotransferase in the developing neonatal rat
Authors: Snell K , Walker D G
Published: August 2015

1. The activities of l-serine dehydratase and l-serine–pyruvate aminotransferase were determined in rat liver during foetal and neonatal development. 2. l-Serine–pyruvate aminotransferase activity begins to develop in late-foetal liver, increases rapidly at birth to a peak during suckling and then decreases at weaning to the adult value. 3. l-Serine dehydratase activity is very low prenatally, but increases rapidly after birth to a transient peak. After a second transient peak around the time weaning begins, activity gradually rises to the adult value. Both of these peaks have similar isoenzyme compositions. 4. In foetal liver both l-serine dehydratase and l-serine–pyruvate aminotransferase activities are increased after injection in utero of glucagon or dibutyryl cyclic AMP. Cycloheximide or actinomycin D inhibited the prenatal induction of both enzymes and actinomycin D blocked the natural increase of l-serine dehydratase immediately after birth. Glucose or insulin administration also blocked the perinatal increase of l-serine dehydratase. 5. After the first perinatal peak of l-serine dehydratase, activity is increased by cortisol and this is inhibited by actinomycin D. After the second postnatal peak, activity is increased by amino acids or cortisol and this is insensitive to actinomycin D inhibition. Glucose administration blocks the cortisol-stimulated increase in l-serine dehydratase and also partially lowers the second postnatal peak of activity. 6. The developmental patterns of the enzymes are discussed in relation to the pathways of gluconeogenesis from l-serine. The regulation of enzyme activity by hormonal and dietary factors is discussed with reference to the changes in stimuli that occur during neonatal development and to their possible mechanisms of action.
Radio-enzymic assay of oxo steroids by their reduction with the tritiated form of reduced nicotinamide-adenine dinucleotide
Authors: Norymberski J K , Stoddart N , Wolstenholme J B
Published: August 2015

A method of analysis is proposed based on the enzyme-catalysed transfer of tritium from [3H]NADH to suitable substrates. Its practicability is demonstrated on the examples of oestrone and progesterone with the respective use of the 3 beta, 17 beta- and 3 alpha, 20 beta-hydroxysteroid oxidoreductase. Specificity is tested by application to the analysis of the plasma of pregnant women and measurement of the 3H/14C ratios on purification of the enzymic reduction products.
Active-sitve titration of pig plasma benzylamine oxidase with phenylhydrazine
Authors: Buffoni F , Ignesti G
Published: August 2015

Pig plasma benzylamine oxidase is a protein containing cupric copper and pyridoxal phosphate. The pyridoxal phosphate is stably linked to the enzyme. Discrepancies in the numbers of active sites per molecule of enzyme are reported in the literature. This paper shows that the fully active pure enzyme contains 3 mol of pyridoxal phosphate per mol, whereas enzymes with a lower specific activity are shown by titration with phenylhydrazine to have a lower pyrdoxal phosphate content.
Separation of a proteoglycan fraction from Kurloff cells stimulating protein synthesis in macrophages
Authors: Dean M F , Muir H , Shires T K
Published: August 2015

Proteoglycan from Kurloff cells, when present in the medium in low concentrations, increased the incorporation of (3-H)leucine into trichloracetic acid-precipitable material by macrophages from peritoneal exudates, in addition to inhibiting their migration from capillary tubes, as observed previously. After treatment with 0.5 M-NaOH, followed by dialysis or ultrafiltration, material with the distinctive u.v. and i.r. spectra of the whole proteoglycan appeared in the diffusate, and biological activity was lost from the proteoglycan which remained in the dialysis residue. The diffusible material absorbed near 260 nm and had i.r. bands at 805 cm-minus-1 and 1260 cm-minus-1, but did not display the i.r. bands characteristic of chondrotin 4-sulphate. It contained little sulphate, no hexosamine and less than 1% of the uronic acid present in the whole proteoglycan, and there were only trace amounts of amino acids, xylose and galactose. However, significant amounts of ribose and organic phosphate were present, each representing about 1% of the whole proteoglycan. After proteolysis and chondroitanase digestion of the proteoglycan, a fraction with absorbance at 260 mn was eluted from Dowex 1 with water which stimulated the incorporation of (3H)leucine by macrophages and inhibited their migration from capillary tubes.
Purification and heterogeneity of inorganic pyrophosphatase of pig scapula cartilage
Authors: Felix R , Fleisch H
Published: August 2015

Inorganic pyrophosphatase (pyrophosphate phosphohydrolase; EC 3.6.1.1) was purified from pig scapula cartilage by fractionation with N-cetylpyridinium chloride and (NH4)2SO4, followed by ion-exchange and gel-filtration chromatography. Enzyme preparations of high purity were obtained, with specific activities (100-400 units/mg) higher than those previously described for mammalian pyrophosphatases. The enzyme activity could be separated into several subfractions on ion-exchange columns.
Subcellular localization and properties of mouse adrenal C19-steroid 5β-reductase
Authors: Collins W , Cameron E H
Published: August 2015

The localization and some characteristics of mouse adrenal C19-steroid 5 β-reductase were determined by the incubation of subcellular fractions of mouse adrenal tissue with [7 α-3H]androst-4-ene-3,17-dione. This enzyme was present only in the soluble fraction and was NADPH-dependent, although a small activity in the presence of NADH was also detected. The soluble fraction also contained 3α-, 3β- and a small amount of 17 β-hydroxy steroid dehydrogenase. These and other steroid-metabolizing enzymes present in the remaining subcelluar fractions are also described briefly. To measure 5 β-androstane-3,17-dione production by the mouse adrenal soluble fraction, all 5 β products first had to be oxidized to 5 β-androstane-3,17-dione, and the recovery of radio-activity between the substrate androst-4-ene-3,17-dione and product 5 β-androstane-3,17-dione of 96.1 +/-3.2% validated this technique. C19-steroid 5 β-reductase has a pH optimum of 6.5 and at low substrate concentrations the Km and Vmax. for 5 β reduction of [7 α-3H]androst-4-ene-ene-3,17-dione was 2.22 times 10(-6) ±0.48 times 10(-6) M and 450+/- 53 pmol/min per mg of protein respectively. At high substrate concentration, inhibition of the reaction occurred, which was shown to be due to increasing product concentration.
Ribonucleic acid synthesis in isolated rat liver nuclei under conditions of ribonucleic acid processing and transport
Authors: McNamara D J , Racevskis J , Schumm D E , Webb T E
Published: August 2015

A cell-free system is described which permits a significant and prolonged synthesis of RNA in isolated rat liver nuclei, under conditions previously demonstrated to support normal nuclear processing and transport of both rRNA and mRNA. The system contains cytosol but not (NH4)2SO4 or other non-physiological components. Evidence is presented for cytosol factors which stimulate ribosomal, and to a lesser degree, non-ribosomal RNA synthesis.
Separation of a series of chromophores and fluorophores present in elastin
Authors: Thornhill D P
Published: August 2015

Purified elastin was hydrolysed with HCl and manipulated under conditions that minimized oxidation. Gel-permeation chromatography on polyacrylamide gel and ion-exchange chromatography on dextran cation-exchanger each resulted in the separation of a series of yellow fluorescent fractions. These hitherto unreported ampholytes have fluorescence spectra that approximate to that of the intact protein, and account for its characteristic optical properties. Since the coloured fluorophores are confined to enzyme-resistant regions of the protein molecule they appear to have important structural implications.
Chemical and metabolic properties of adenosine diphosphate ribose derivatives of nuclear proteins.
Authors: Smith J A , Stocken L A
Published: August 2015

1. ADP-ribose is found in rat liver nuclei covalently bound to histone F1, to a non-histone protein, and to a small peptide. 2. A single unit of ADP-ribose, covalently bound to phosphoserine, was isolated from an enzymic hydrolysate of histone F1. ADP-ribose-bearing peptides were isolated from a tryptic digest of the histone. 3. It is proposed that the 1′-hydroxyl group of ADP-ribose is linked to the phosphate group of phosphoserine in histone F1. 4. The incorporation of 32P into ADP-ribose on histone F1 a parallels the DNA content through the cell cycle. An increased incorporation of the nucleotide into the other derivatives is observed during S phase. 5. It is suggested that the ADP-ribose derivative of histone F1 has a role in maintaining the G0 state and that one or both of the other derivatives is concerned with control of DNA synthesis.
The aerobic metabolism of cyclohexanecarboxylic acid by Acinetobacter anitratum
Authors: Rho E M , Evans W C
Published: August 2015

1. The aerobic metabolism of cyclohexanecarboxylic acid by a bacterium isolated from garden soil (Acinetobacter anitratum) was investigated. 2. Evidence for the formation of cyclohex-1-ene-1-carboxylate, 2-hydroxycyclohexanecarboxylate and pimelate when either cell suspensions or cell-free extracts were incubated in the presence of cyclohexanecarboxylic acid is presented. 3. Crude cell-free extracts required ATP, CoA, FAD and Mg2+ as cofactors for the production of pimelate from cyclohexanecarboxylic acid, suggesting the existence of an activating reaction with formation of CoA esters, in this system.
Stimulation of glycosaminoglycan biosynthesis by amyloid fibrils
Authors: Palmoski M J , Brandt K D
Published: August 2015

Since amyloid-laden organs have an increased glycosaminoglycan content, the effect of amyloid fibrils on glycosaminoglycan metabolism by normal fibroblasts was examined. In comparison with controls, synthesis of glycosaminoglycans, primarily hyaluronic acid, was increased by an average of 48 and 93% respectively when 0.1 and 1.0 mg of amyloid fibrils/ml was added to the cultures.
Iron removal from transferrin by a cell-free amphibian system
Authors: James G T , Frieden E
Published: August 2015

1. Transferrin-bound iron was released by a cell-free solution prepared from Rana catesbeiana immature erythrocytes, showing that cellular integrity is not necessarily a requirement for this process as previously thought. 2. The biological molecule(s) involved in the removal of iron from transferrin is relatively small.
Essentiality of ubiquinone for choline oxidation in rat liver mitochondria
Authors: Barrett M C , Dawson A P
Published: August 2015

Rat liver mitochondria treated extensively with n-pentane are incapable of oxidizing choline. Choline oxidation is more sensitive than is succinate oxidation to serial n-pentane extraction of mitochondria. The ability to oxidize choline is restored by the addition of ubiquinone-2 or ubiquinone-10 to the oxidase assay medium.
Structure and biosynthesis of the ribosomal ribonucleic acids from the oncogenic bacterium Agrobacterium tumefaciens
Authors: Grienenberger J M , Simon D
Published: August 2015

The rRNA of the oncogenic bacterium Agrobacterium tumefaciens was extracted by several methods and analysed by polyacrylamide-gel electrophoresis. The large rRNA of this bacterium is degraded in vivo during the maturation of the ribosome. The influence of Mg2+ and denaturation on degradation of 23S RNA was studied. In pulse and chase experiments, we identified two precursors of the rRNA with mol.wts. of 1.04 × 106 and 0.70 × 106. From studies of the structure of the large rRNA, we propose that it could have arisen from a gene duplication. This structure is discussed in relation to a recent hypothesis involving such gene duplication as a means of origin of 23S rRNA.
Effects of adenine nucleotides on rice-root adenosine triphosphate sulphurylase activity in vitro
Authors: Onajobi F D
Published: August 2015

The presence of ATP sulphurylase activity in the 30000g supernatant fraction of rice-root homogenate has been demonstrated. Studies of the effects of adenosine and its nucleotides on the enzymic activity showed that AMP activated but that ADP and adenosine inhibited the enzyme.
Stopped-flow fluorescence studies on saccharide binding to lysozyme
Authors: Halford S E
Published: August 2015

The binding of the β-1-4-linked trimer of N-acetyl-D-glucosamine to hen egg-white lysozyme was studied by rapid-reaction-kinetic methods with tryptophyl fluorescence observation of the transients. It was found that discrete segments of the fluorescence-difference spectrum from this reaction were perturbed at different time-points during the binding process. The results were interpretated as the formation of the initial complex, the fast phase of the reaction, perturbing the environment of tryptophan-62 and a subsequent and slower rearrangement of the initial complex perturbing the environment of tryptophan-108. At pH 4.4, the release of protons from aspartate-101 occurred during the rearrangement step of the binding reaction. A model for the reaction is presented (E, enzyme; L, ligand): (see article) The association of this ligand with lysozyme may be visualized in three-dimensional terms as initial complex-formation across the top of the active-site cleft followed by a diving motion of the ligand into the cleft.