bluuebunny/crossref_metadata_2025 huggingface dataset:
Nuclear binding of oestradiol-17β and induction of protein synthesis in the rat uterus during postnatal development
Authors: Sömjen Dalia , Sömjen G. , King R. J. B. , Kaye A. M. , Lindner H. R.
Published: August 2015

1. The uterine response to a single injection of oestradiol-17β during postnatal development of the rat was studied with respect to (i) nuclear binding of oestradiol-17β; (ii) induction of the synthesis of a specific cytoplasmic protein (‘induced protein’ of Gorski); (iii) rate of incorporation of 3H-labelled amino acids into total protein and into nuclear acid-soluble and acid-insoluble protein; and (iv) rate of [3H]thymidine incorporation into DNA. 2. Specific nuclear binding of oestradiol-17β could be demonstrated even at birth. Administration of oestradiol-17β in vivo caused a significant increase in the number of nuclear binding sites in rats aged 10 days or older. 3. A rapid method is described for the detection of the ‘induced protein’, based on cellulose acetate electrophoresis. Induction of this protein could be demonstrated at the age of 10, 15 and 20 days, but not in 5-day-old rats. 4. In 20-day-old rats the rate of 3H-labelled amino acid incorporation into protein increased by 3h after oestradiol administration. Incorporation into the different protein fractions reached peak values asynchronously: at 3–4h for acid-insoluble nuclear protein, at 6h for total protein and at about 12h for acid-soluble protein. 5. Treatment with oestradiol failed to stimulate amino acid incorporation into protein in 5- or 10-day-old rats; at the age of 15 to 30 days the hormone caused a significant increase in incorporation into total protein and into both types of nuclear protein. 6. Since the capacity for nuclear binding of oestradiol and for synthesis of the induced protein is demonstrable in the rat uterus before it acquires the ability to respond to the hormone with enhanced general protein synthesis and DNA synthesis, it appears that nuclear binding and the synthesis of the induced protein may be necessary but not sufficient conditions for the trophic action of oestradiol.
Synthesis and degradation of mitochondrial components in hypertrophied rat heart
Authors: Albin Richard , Dowell Russell T. , Zak Radovan , Rabinowitz Murray
Published: August 2015

The accumulation of inner mitochondrial components of rat heart was studied 1 and 3 days after constriction of the ascending aorta of rats. By 1 day after aortic constriction, the activities of three mitochondrial respiratory enzymes/mg of cardiac homogenate protein were increased; after 3 days, specific activities had levelled off or decreased. Selective accumulation of inner mitochondrial membrane components 24h after aortic constriction was further indicated by increased left ventricular cytochrome c concentration (nmol/mg of protein). By 3 days after surgery, cytochrome c concentration was significantly diminished. Low-temperature spectroscopy of isolated mitochondria showed that the ratios of cytochromes c, b and a+a3 remained unchanged after aortic constriction, suggesting that cytochrome c was a good indicator of the response of the other mitochondrial inner-membrane cytochromes as well. The effect of cardiac hypertrophy on the turnover of cytochrome c was also examined. Cytochrome c was labelled in its haem group with δ-amino[2,3-3H2]laevulinate 3 days before aortic constriction. By 1 day after surgery the total ventricular radioactivity in cytochrome c of aortic banded animals was significantly higher than in sham-operated controls, indicating a decreased degradation rate in the former during the first postoperative day. δ-Aminolaevulinate was shown to be a particularly suitable precursor for such turnover studies, since it results in rapid pulse-labelling of cytochrome c (peak activity in 90min), is rapidly removed from the precursor pool (t½=30min) and is not reutilized.
Isoenzyme patterns in parenchymal and non-parenchymal cells isolated from regenerating and regenerated rat liver
Authors: Bonney Robert J. , Walker P. Roy , Potter Van R.
Published: August 2015

Parenchymal and non-parenchymal cells were isolated from adult rat liver that had been fully regenerated after a 70% partial hepatectomy. The characteristics of the parenchymal cell preparations from regenerated rat liver indicated that they were a homogeneous population and comparable with parenchymal cells isolated from intact liver. The parenchymal cells from regenerated adult rat liver contain glucokinase, hexokinase, pyruvate kinase type I and aldolase B. The non-parenchymal cells contain hexokinase, pyruvate kinase type III and aldolase B. When cells were isolated at different times of the day from rats on controlled feeding schedules, variation of tyrosine aminotransferase activity and liver glycogen content were observed in the parenchymal cells in keeping with the reported diurnal oscillations found in whole liver extracts. When parenchymal cells were isolated from rats 48 and 72h after partial hepatectomy, different isoenzyme patterns were observed. These cells appeared to synthesize pyruvate kinase type III, a function that was assigned previously to non-parenchymal cells or to foetal rat liver hepatocytes.
Thymidine kinase and deoxyribonucleic acid metabolism in growing and regenerating livers from rats on controlled feeding schedules
Authors: Hopkins Harold A. , Campbell Harold A. , Barbiroli Bruno , Potter Van R.
Published: August 2015

Rats accustomed to eating during the first 8h of a daily 12h dark period re-established about 80% of intact liver weight, protein and DNA within 4 days following partial hepatectomy; further increases were not observed. Liver thymidine kinase activity and thymidine incorporation into liver DNA exhibited marked daily oscillations during liver regeneration. Maximum values were observed near the end of the dark period both in intact growing rats and in rats partially hepatectomized 2h before the end of the dark period. The time of day of surgery affected thymidine kinase activity and thymidine incorporation into DNA at specific times following partial hepatectomy. This seriously affects the interpretation of reports of experiments where the time of day of killing has been held constant and time of surgery varied. Highly significant correlation coefficients were observed for thymidine incorporation before killing versus thymidine kinase activity at time of killing and for thymidine versus orotic acid incorporation into DNA of livers from rats partially hepatectomized 2h before the end of the dark period and killed at 12h intervals. Thymidylate phosphatase activity returned to the normal amount at a rate similar to that for liver protein. Thymidylate phosphatase did not affect the validity of the thymidine kinase assay. The relationship of [14C]orotic acid to [3H]thymidine incorporation into liver DNA varied with the time of day, with the age of the rat and during the regeneration of the liver.
The interaction of the calcium-binding protein (troponin C) with bivalent cations and the inhibitory protein (troponin I)
Authors: Head J. F. , Perry S. V.
Published: August 2015

1. The molecular weight of the calcium-binding protein of rabbit white skeletal muscle was estimated to be 18500 by sedimentation equilibrium and electrophoresis in sodium dodecyl sulphate. 2. Addition of 2 Ca2+ ions per molecule produced reversible changes in the u.v.-absorption spectrum that are interpreted as arising from conformational changes in the structure of the protein. 3. Cd2+ was almost as effective as Ca2+ in producing the spectral changes. Other bivalent metal ions, particularly Mg2+, were less effective. 4. Binding of Ca2+ by the calcium-binding protein produced an increase in mobility to the anode on electrophoresis in 6m-urea at pH8.6. The Ca2+-saturated form of the protein was more retarded on gel filtration than the Ca2+-free form. 5. In the presence of Ca2+ the calcium-binding protein formed an equimolar complex with the inhibitory protein. This complex was stable in 8m-urea and in the pH range 7.0–8.6. 6. An isotope-dilution method for the measurement of the content of calcium-binding protein in whole muscle is described. In rabbit psoas muscle the ratio of actin monomers to molecules of calcium-binding protein was approx. 7:1. Similar values were obtained for red skeletal and cardiac muscle. 7. Evidence is presented indicating that in the rabbit the inhibitory protein of the troponin complex of red skeletal and cardiac muscles is different from the inhibitory protein of white skeletal muscle.
Studies on partially reduced mammalian cytochrome oxidase. Reactions with carbon monoxide and oxygen
Authors: Greenwood Colin , Wilson Michael T. , Brunori Maurizio
Published: August 2015

A number of methods were used to prepare a species of mammalian cytochrome oxidase (EC 1.9.3.1, ferrocytochrome c–oxygen oxidoreductase) in which only cytochrome a3 is reduced and in combination with CO. The kinetics of CO binding by cytochrome a32+ in this species is significantly different from that exhibited by cytochrome a32+ in the fully reduced enzyme. The second-order rate constant for combination was 5X104m−1·s−1 and the ‘off’ constant was 3X10−2s−1. The kinetic difference spectra cytochrome a32+–cytochrome a32+–CO reveal further differences between the mixed-valence and the fully reduced enzyme. The reaction between cytochrome a32+ and oxygen in the mixed-valence species was followed in flow–flash experiments and reveals a fast, oxygen-dependent (8X107m−1·s−1 at low oxygen) rate followed by a slow process, whose rate is independent of oxygen but whose amplitude is dependent on [O2]. The fast oxygen-dependent reaction yields as the first product the so-called ‘oxygenated’ enzyme. We conclude from these experiments that the ligand-binding behaviour of cytochrome a3 depends on the redox state of its partners, a fact which represents clear evidence for site–site interaction in this enzyme. The fact that oxygen reacts rapidly with this enzyme species in which only one component, namely cytochrome a3, is reduced represents clear and unequivocal evidence that this is indeed the O2-binding site in cytochrome oxidase and may indicate that reduction of oxygen can proceed via single electron steps.
The reaction of alkylating agents with bacteriophage R17. Biological effects of phosphotriester formation
Authors: Shooter Kenneth V. , Howse Ruth , Merrifield R. Kenneth
Published: August 2015

The extent of biological inactivation and of the degradation of the RNA after reaction of bacteriophage R17 with ethyl methanesulphonate, isopropyl methanesulphonate and N-ethyl-N-nitrosourea was studied. Formation of breaks in the RNA chain probably results from hydrolysis of phosphotriesters formed in the alkylation reactions. Near neutral pH the ethyl and isopropyl phosphotriesters are sufficiently stable for the kinetics of the hydrolysis reaction to be followed. Results indicate that the rate of hydrolysis increases rapidly as the pH is raised. The evidence shows that a phosphotriester group does not itself constitute a lethal lesion. The extent of phosphotriester formation by the different agents is discussed in terms of reaction mechanism.
Preparation and properties of a phosphoenzyme from the phosphoglucomutase of Micrococcus lysodeikticus
Authors: Clarke John B. , Birch Michael , Britton Hubert G.
Published: August 2015

1. Phosphoglucomutase from Micrococcus lysodeikticus was incubated with 14C- and 32P-labelled glucose 1,6-diphosphate and separated from the cofactor on a Sephadex column. 32P-labelled phosphate (0.7mol/mol of enzyme) was associated with the enzyme, but no 14C label was. 2. The 32P-labelled enzyme exchanged its label with the substrates. When the labelled enzyme was incubated in Tris buffer, pH8.3, at 30°C the proportion of exchangeable label slowly fell indicating a half-life of the phosphoenzyme of about 50h. 3. When HClO4 was added to the labelled phosphoenzyme all of the label was precipitated with the protein and none was released as Pi. On alkaline hydrolysis Pi was released at a rate comparable with the rate of hydrolysis of the phosphoenzyme from rabbit muscle. 4. We conclude that the phosphoenzyme from Micrococcus lysodeikticus yields a relatively stable, catalytically active phosphoenzyme when treated with cofactor, and that there is no evidence for the formation of an enzyme–glucose 1,6-diphosphate complex. The properties of the phosphoenzyme, which resemble those of rabbit muscle phosphoglucomutase, suggest that the phosphate may be bound to serine.
The purification and specificity of a neutral endopeptidase from rabbit kidney brush border
Authors: Kerr M. A. , Kenny A. J.
Published: August 2015

1. A neutral peptidase, previously shown to be located in the brush border of the proximal tubule, and assayed by its ability to hydrolyse [125I]iodoinsulin B chain was purified from rabbit kidney. 2. The starting material for the purification was a microsomal pellet prepared from a homogenate of cortical tissue. The membrane-bound enzymes were solubilized by treatment with toluene and trypsin. About half the neutral peptidase activity was released by this treatment in a form that no longer sedimented with the microsomal pellet and which penetrated polyacrylamide gels when subjected to disc electrophoresis. Other treatments with detergents or proteolytic enzymes either inactivated the peptidase or failed to convert it into a genuinely soluble form. 3. Chromatography with successive columns of Sephadex G-200, DEAE-cellulose and hydroxyl-apatite yielded an enzyme that was free of other brush-border peptidase activities and which was homogeneous on disc electrophoresis and ultracentrifugation. 4. The purified enzyme attacked [125I]iodoglucagon at a rate comparable with that for [125I]iodoinsulin B chain. It did not appear to attack proteins (insulin, albumin and casein) that had been similarly iodinated. 5. Unlabelled insulin B chain and unlabelled glucagon were substantially hydrolysed by the endopeptidase, whereas insulin and albumin released only trivial amounts of ninhydrin-reacting material. The resistance of insulin to attack by endopeptidase, even after prolonged incubation, was confirmed by biological and immunoassay. 6. The specificity of the peptidase was determined by analysis of the products after incubating unlabelled insulin B chain, and some oligopeptide substrates, including pentagastrin, with the enzyme. All of the bonds readily cleaved were those involving the α-amino group of hydrophobic residues, i.e. x-Leu-, x-Val-, x-Tyr-, x-Phe- and x-Met-, provided that the residues were not C-terminal. 7. The enzyme showed only endopeptidase activity. Substrates suitable for aminopeptidases, carboxypeptidases or esterases were not attacked.
The molecular weight and properties of a neutral metallo-endopeptidase from rabbit kidney brush border
Authors: Kerr M. A. , Kenny A. J.
Published: August 2015

1. Some properties of a brush-border neutral endopeptidase purified from rabbit kidney were investigated. The peptidase was assayed by its ability to hydrolyse [125I]iodoinsulin B chain. 2. The enzyme was found to be homogeneous when studied in the analytical ultracentrifuge and stained as a single glycoprotein band after electrophoresis in polyacrylamide gels. 3. The molecular weight was estimated by gel filtration in columns of Sephadex G-200, by polyacrylamide-gel electrophoresis in the presence of 2-mercapto-ethanol and sodium dodecyl sulphate and by sedimentation equilibrium in the ultra-centrifuge. The estimates fell within the range 87000–96000. The mean from two sedimentation equilibrium experiments was 93000, though this estimate may be slightly inflated because of the carbohydrate component of the enzyme. No evidence of dissociation into smaller subunits was obtained in the presence of thiol, sodium dodecyl sulphate or guanidine hydrochloride. 4. The endopeptidase was maximally active at pH6.0, although in phosphate buffer, which was strongly inhibitory, an optimum above pH8 was observed. 5. The enzyme was not affected by di-isopropyl phosphofluoridate nor by several thiol reagents. It was, however, strongly inhibited by many thiols and by EDTA and other chelating agents. 6. Although activity of the EDTA-treated enzyme could be partially restored by various bivalent metal ions, the optimum concentration for its reactivation by Zn2+ was lower than that for other ions. This metal was detected in the enzyme preparation by atomic absorption spectrophotometry in an amount equivalent to approximately one atom/mol. 7. The enzyme is the only endopeptidase shown to be located in the kidney brush border and is the first mammalian example of a neutral Zn2+- activated endopeptidase to be characterized.
Chromatography of transforming deoxyribonucleic acid on methylated albumin and by gel filtration
Authors: Barker G. R. , Hodges P.
Published: August 2015

1. Native DNA from two strains of Bacillus subtilis was chromatographed by stepwise elution from MAK (methylated albumin on kieselguhr). 2. Transforming activity was confined to two out of the three main fractions, activity being distributed between the two peaks differently for DNA from the different strains. 3. Fractionation of DNA from both strains on 2% agarose gel gave two components. Approx. 75% of the material was eluted within the void volume of the column. Approx. 25% of the material consisted of degradation products of lower molecular weight. 4. Chromatography on MAK of the material of high molecular weight eluted from agarose gel gave a number of peaks differing in molecular weight, indicating that degradation of the DNA takes place during chromatography on MAK. 5. The distribution of transforming activity among the fractions from MAK suggests that degradation occurs preferentially in certain regions of the DNA.
Differential anion effects on thermal stability of collagen in the dispersed and aggregated states
Authors: Russell A. E.
Published: August 2015

The effects of KCNS and KI on thermal transition temperatures of calf skin collagen molecules in dilute acid solution and precipitated collagen fibrils from the same source were compared as a function of salt concentration and pH. The two salts produced qualitatively similar effects on each collagen form, but the response shown by single collagen molecules in dilute solution differed from that observed for molecular aggregates present in native-type fibrils.
Regulation of glucose and ketone-body metabolism in brain of anaesthetized rats
Authors: Ruderman Neil B. , Ross Peter S. , Berger Michael , Goodman Michael N.
Published: August 2015

1. The effects of starvation and diabetes on brain fuel metabolism were examined by measuring arteriovenous differences for glucose, lactate, acetoacetate and 3-hydroxybutyrate across the brains of anaesthetized fed, starved and diabetic rats. 2. In fed animals glucose represented the sole oxidative fuel of the brain. 3. After 48h of starvation, ketone-body concentrations were about 2mm and ketone-body uptake accounted for 25% of the calculated O2 consumption: the arteriovenous difference for glucose was not diminished, but lactate release was increased, suggesting inhibition of pyruvate oxidation. 4. In severe diabetic ketosis, induced by either streptozotocin or phlorrhizin (total blood ketone bodies >7mm), the uptake of ketone bodies was further increased and accounted for 45% of the brain 's oxidative metabolism, and the arteriovenous difference for glucose was decreased by one-third. The arteriovenous difference for lactate was increased significantly in the phlorrhizin-treated rats. 5. Infusion of 3-hydroxybutyrate into starved rats caused marked increases in the arteriovenous differences for lactate and both ketone bodies. 6. To study the mechanisms of these changes, steady-state concentrations of intermediates and co-factors of the glycolytic pathway were determined in freeze-blown brain. 7. Starved rats had increased concentrations of acetyl-CoA. 8. Rats with diabetic ketosis had increased concentrations of fructose 6-phosphate and decreased concentrations of fructose 1,6-diphosphate, indicating an inhibition of phosphofructokinase. 9. The concentrations of acetyl-CoA, glycogen and citrate, a potent inhibitor of phosphofructokinase, were increased in the streptozotocin-treated rats. 10. The data suggest that cerebral glucose uptake is decreased in diabetic ketoacidosis owing to inhibition of phosphofructokinase as a result of the increase in brain citrate. 11. The inhibition of brain pyruvate oxidation in starvation and diabetes can be related to the accelerated rate of ketone-body metabolism; however, we found no correlation between the decrease in glucose uptake in the diabetic state and the arteriovenous difference for ketone bodies. 12. The data also suggest that the rates of acetoacetate and 3-hydroxybutyrate utilization by brain are governed by their concentrations in plasma. 13. The finding of very low concentrations of acetoacetate and 3-hydroxybutyrate in brain compared with plasma suggests that diffusion across the blood –brain barrier may be the rate-limiting step in their metabolism.
Induction of primary alkylsulphatases and metabolism of sodium hexan-1-yl sulphate by Pseudomonas C12B
Authors: Fitzgerald John W. , Dodgson Kenneth S. , Payne William J.
Published: August 2015

Sodium hexan-1-yl sulphate and certain related alkyl sulphate esters have been shown to serve as inducers of the formation of primary alkylsulphatases (designated as P1 and P2) in Pseudomonas C12B. When the organism is grown on sodium hexan-1-yl [35S]sulphate as the sole source of sulphur or as the sole source of carbon and sulphur only the P2 alkylsulphatase is formed and inorganic 35SO42- is liberated into the media. Cell extracts contain this anion as the major 35S-labelled metabolite although two unidentified labelled metabolites as well as choline O-[35S]sulphate occur in trace quantities in some extracts. Dialysed cell extracts are capable of liberating inorganic 35SO42- from sodium hexan-1-yl [35S]sulphate without the need to include cofactors known to be required for the bacterial degradation of n-alkanes. The collective results suggest that sodium hexan-1-yl sulphate can act as an inducer of P1 alkylsulphatase formation without the need for prior metabolic modification of the carbon moiety of the ester.
Specific inhibition of pyruvate transport in rat liver mitochondria and human erythrocytes by α-cyano-4-hydroxycinnamate (Short Communication)
Authors: Halestrap Andrew P. , Denton Richard M.
Published: August 2015

α-Cyano-4-hydroxycinnamate greatly inhibits the transport of pyruvate but not that of acetate or butyrate in liver mitochondria and erythrocytes. In the latter, lactate uptake is also inhibited. It is concluded that a specific carrier is involved in membrane transport of pyruvate and that the plasma-membrane carrier may also be involved in lactate transport.
Guinea-pig liver tryptophan pyrrolase. Absence of detectable apoenzyme activity and of hormonal induction by cortisol and possible regulation by tryptophan
Authors: Badawy Abdulla A.-B. , Evans Myrddin
Published: August 2015

1. When assayed in fresh homogenates, guinea-pig liver tryptophan pyrrolase exists only as holoenzyme. It does not respond to agents that activate or inhibit the rat liver enzyme in vitro. Only by aging (for 30min at 5°C) does the guinea-pig enzyme develop a requirement for ascorbate. 2. The guinea-pig liver enzyme is activated by the administration of tryptophan but not cortisol, salicylate, ethanol or 5-aminolaevulinate. 3. The tryptophan enhancement of the guinea-pig liver pyrrolase activity is prevented by 0, 34 and 86% by pretreatment with actinomycin D, cycloheximide or allopurinol respectively. 4. The guinea-pig liver tryptophan pyrrolase is more sensitive to tryptophan administration than is the rat enzyme. On the other hand, the concentrations of tryptophan in sera and livers of guinea pigs are 45–52% less than those in rats. 5. It is suggested that tryptophan may regulate the activity of guinea-pig liver tryptophan pyrrolase by mobilizing a latent form of the enzyme whose primary function is the detoxication of its substrate.
Simulation of the pentose cycle in lactating rat mammary gland
Authors: Haut Michael J. , London Jack W. , Garfinkel David
Published: August 2015

A computer model representing the pentose cycle, the tricarboxylic acid cycle and glycolysis in slices of lactating rat mammary glands has been constructed. This model is based primarily on the studies, with radioactive chemicals, of Abraham & Chaikoff (1959) [although some of the discrepant data of Katz & Wals (1972) could be accommodated by changing one enzyme activity]. Data obtained by using [1-14C]-, [6-14C]- and [3,4-14C]-glucose were simulated, as well as data obtained by using unlabelled glucose (for which some new experimental data are presented). Much past work on the pentose cycle has been mainly concerned with the division of glucose flow between the pentose cycle and glycolysis, and has relied on the assumption that the system is in steady state (both labelled and unlabelled). This assumption may not apply to lactating rat mammary glands, since the model shows that the percentage flow through the shunt progressively decreased for the first 2h of a 3h experiment, and we were unable to construct a completely steady-state model. The model allows examination of many quantitative features of the system, especially the amount of material passing through key enzymes, some of which appear to be regulated by NADP+ concentrations as proposed by McLean (1960). Supplementary information for this paper has been deposited as Supplementary Publication SUP 50023 at the British Museum (Lending Division) (formerly 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.
Protein synthesis in the cotyledons of Pisum sativum L. Protein factors involved in the binding of phenylalanyl-transfer ribonucleic acid to ribosomes
Authors: Wells Gary N. , Beevers Leonard
Published: August 2015

1. Proteinaceous factors contained in a 0.5m-KCl extract of ribosomes from pea cotyledons form a ternary complex at 0°C with [14C]phenylalanyl-tRNA and poly(U). The complex is measured by its quantitative retention on Millipore filters. 2. Complex-assembly is optimal at 5mm-Mg2+ and is independent of GTP and ribosomes. 3. The addition of ribosomes is required to stabilize the complex at 34°C. The complex binds to a puromycin-sensitive site on the ribosome. 4. Soluble factors from the 250000g supernatant of pea cotyledon form a Millipore-retainable complex dependent on GTP and ribosomes. 5. Complex-formation by soluble factors has a Mg2+ optimum of 10–12mm and forms a puromycin-insensitive complex with ribosomes. 6. The function of the ribosomal protein factors and the supernatant fraction in initiation of protein synthesis is discussed.
Purification and specificity of a human microsomal epoxide hydratase
Authors: Oesch Franz
Published: August 2015

Epoxide hydratase was solubilized from human liver microsomal fractions and purified to an extent where the specific activity was 40-fold greater than that of the liver homogenate. Combination of homogenate and purified preparation showed that the increase in activity was not due to the removal of an inhibitor. Monosubstituted oxiranes with a lipophilic substituent larger than an ethyl group (isopropyl, t-butyl, n-hexyl, phenyl) readily interacted as substrates or inhibitors with this purified human epoxide hydratase, whereas those with a small substituent (methyl, ethyl, vinyl) were inactive, probably reflecting greater affinity of the former epoxides owing to lipophilic binding sites near the active site of the enzyme. In a series of oxiranes having a lipophilic substituent of sufficient size (styrene oxides), monosubstituted as well as 1,1- and cis-1,2-disubstituted oxiranes readily served as substrates or inhibitors of the enzyme, but not the trans-1,2-disubstituted, tri- or tetra-substituted oxiranes. trans-Substitution at the oxirane ring apparently prevents access of the oxirane ring to the active site by steric hindrance. Epoxide hydratase was also solubilized from microsomal fractions of rat and guinea-pig liver and purified by the same procedure. Structural requirements for effective interaction of substrates, inhibitors and activators were qualitatively identical for epoxide hydratase from the three sources. However, several quantitative differences were observed. Thus human hepatic epoxide hydratase seems to be very similar to, although not identical with, the enzyme from guinea pig or rat. Studies with epoxide hydratase from the latter two species therefore appear to be significant with respect to man. In addition, knowledge of structural requirements for epoxides to serve as substrates for human epoxide hydratase may prove useful for drug design. Compounds which need aromatic or olefinic moieties for their desired effect would not be expected to lead to accumulation of epoxides if their structure was such as to allow for a metabolically produced epoxide to be rapidly consumed by epoxide hydratase.
The amine oxidases of human placenta and pregnancy plasma
Authors: Bardsley William G. , Crabbe M. James C. , Scott Ian V.
Published: August 2015

1. The purification of monoamine oxidase and diamine oxidase from normal human term placental tissue is described. 2. The properties of these enzymes are reported and compared with the properties of unpurified human pregnancy plasma. 3. This comparison shows that the amine oxidase of pregnancy plasma has properties corresponding to purified placental diamine oxidase, suggesting a placental origin for the plasma enzyme system. 4. Detailed kinetic study of the purified placental diamine oxidase suggests that it has a Ping Pong sequence, a mechanism of action and rate-limiting step similar to the diamine oxidase of pig kidney. 5. It is suggested that the enzyme system is important in protecting the foeto-placental unit from excesses of biogenic amines.
Iron and aconitase activity
Authors: Gawron Oscar , Waheed Abdul , Glaid Andrew J. , Jaklitsch Anna
Published: August 2015

Aconitase activated with Fe2+, cysteine and ascorbate incorporates 1 g-atom of Fe2+/mol. Loss of this Fe2+ by transfer to ferrozine, a Fe2+ chelator, results in loss of activity. Ascorbate increases the rate of transfer of the essential Fe2+ whereas citrate retards the rate of transfer. Transfer of Fe2+ from inactive aconitase, 2 g-atoms of Fe/mol, can be accomplished in the presence of urea and ascorbate. The correlation of activity with the presence of an added g-atom of Fe2+/mol leads to the conclusion that active aconitase has only one active site per mol.
The direct linear plot. A new graphical procedure for estimating enzyme kinetic parameters
Authors: Eisenthal Robert , Cornish-Bowden Athel
Published: August 2015

A new plot is described for analysing the results of kinetic experiments in which the Michaelis–Menten equation is obeyed. Observations are plotted as lines in parameter space, instead of points in observation space. With appropriate modifications the plot is applicable to most problems of interest to the enzyme kineticist. It has the following advantages over traditional methods of plotting kinetic results: it is very simple to construct, because it is composed entirely of straight lines and requires no calculation or mathematical tables; the kinetic constants are read off the plot directly, again without calculation; it may be used during the course of an experiment to judge the success of the experiment, and to modify the experimental design; it provides clear and accurate information about the quality of the observations, and identifies aberrant observations; it provides a clear indication of the precision of the kinetic constants; constructed with care, it provides unbiased estimates of the kinetic constants, the same as those provided by a computer program; it may be used to simulate results for illustrative purposes very rapidly and simply.
Equilibrium relations between the oxidation—reduction reactions and the adenosine triphosphate synthesis in suspensions of isolated liver cells
Authors: Wilson David F. , Stubbs Marion , Veech Richard L. , Erecińska Maria , Krebs Hans A.
Published: August 2015

1. The redox state of cytochrome c, cytochrome a and the mitochondrial NAD couple, and the phosphorylation state of the adenine nucleotides, were measured in suspensions of isolated rat liver cells. 2. The ΔG for the transfer of two electrons from the mitochondrial NAD to the cytochrome c couple is calculated to be 104kJ (24.8kcal). 3. The ΔG associated with the synthesis of ATP at the measured phosphorylation state is calculated to be 95kJ (22.7kcal)/2mol of ATP. 4. The near equality of ΔG of the electron-transport process and ΔG required for ATP synthesis indicates near-equilibrium between the mitochondrial respiratory chain and the extramitochondrial phosphorylation state. 5. The existence of near-equilibrium in the coupled reactions implies that the respiratory activity depends on the ratio [ATP]/[ADP][Pi] and not on the concentrations of the individual reactants. 6. If the overall system of oxidative phosphorylation is at near-equilibrium, all intermediary reactions must also be at equilibrium. Hence if the intramitochondrial and extramitochondrial phosphorylation states are indeed different, it follows that any differences in the activities of ATP, ADP and Pi must be coupled to ion gradients and/or potentials across the inner mitochondrial membrane in such a way that translocation occurs without loss of free energy. 7. The metabolic state of the mitochondria in the cell can be defined by the turnover number of the cytochromes, the cytoplasmic phosphorylation state, and the oxidation–reduction potential of the NAD couple, rather than by the availability of ADP, substrate and O2.
Microbial oxidation of amines. Partial purification of a trimethylamine mono-oxygenase from Pseudomonas aminovorans and its role in growth on trimethylamine
Authors: Boulton Christopher A. , Crabbe M. James C. , Large Peter J.
Published: August 2015

1. A mono-oxygenase, which oxidizes trimethylamine and other tertiary amines bearing methyl or ethyl groups, was partially purified sixfold from Pseudomonas aminovorans grown on trimethylamine as sole carbon source. 2. The preferred electron donor was NADPH. The enzyme had a pH optimum of 8.0–9.4 for trimethylamine oxidation, and 8.8–9.2 for dimethylamine oxidation. 3. The oxidation product of trimethylamine was shown to be trimethylamine N-oxide. Other tertiary amines were probably also converted into N-oxides. 4. The enzyme also oxidized secondary amines. 5. The oxidation of trimethylamine was only slightly inhibited by CO and not at all by KCN or proadifen hydrochloride (SKF 525-A), but was inhibited by trimethylsulphonium chloride, tetramethylammonium chloride, 2,4-dichloro-6-phenylphenoxyethylamine (Lilly 53325) and its NN-diethyl derivative (Lilly 18947). 6. The oxidation of dimethylamine showed a similar response to inhibitors and a parallel loss in activity on heating at 35°C. 7. The activities of the trimethylamine mono-oxygenase, trimethylamine N-oxide demethylase and the secondary-amine mono-oxygenase increased severalfold during adaptation of succinate-grown bacteria to growth on trimethylamine, and the trimethylamine mono-oxygenase was the first enzyme to show an increase in activity. It is concluded that all three enzymes are involved in growth on trimethylamine by this organism.
Microbial metabolism of the pyridine ring. Metabolism of 2- and 3-hydroxypyridines by the maleamate pathway in Achromobacter sp
Authors: Cain Ronald B. , Houghton Charles , Wright Keith A.
Published: August 2015

1. Washed suspensions of two Achromobacter species (G2 and 2L), capable of growth upon 2- and 3-hydroxypyridine respectively as sources of C and N, rapidly oxidized their growth substrate pyridine-2,5-diol (2,5-dihydroxypyridine) and the putative ring-cleavage product maleamate without a lag. Suspensions derived from fumarate plus (NH4)2SO4 cultures were unable to do so. 2. Extracts of both bacteria oxidized pyridine-2,5-diol with the stoicheiometry of an oxygenase forming 1mol of NH3/mol of substrate. 3. Heat-treated extracts, however, formed maleamate and formate with little free NH3. 4. The conversion of maleamate into maleate plus NH3 by extracts of strain 2L, fractionated with (NH4)2SO4, and the metabolism of maleamate and maleate to fumarate by extracts of both strains demonstrated the existence of the enzymes catalysing each reaction of the maleamate pathway in these bacteria. 5. The pyridine-2,5-diol dioxygenase (mol.wt. approx. 340000) in extracts of these Achromobacter species required Fe2+ (1.7μm) to restore full activity after dialysis or treatment with chelating agents; the enzyme from strain 2L also had a specific requirement for l-cysteine (6.7mm), which could not be replaced by GSH or dithiothreitol. 6. The oxygenase was strongly inhibited in a competitive manner by the isomeric pyridine-2,3- and -3,4-diols.
High-affinity binding of oestradiol-17β by cytosols from testis interstitial tissue, pituitary, adrenal, liver and accessory sex glands of the male rat
Authors: Van Beurden-Lamers Wilma M. O. , Brinkmann Albert O. , Mulder Eppo , Van Der Molen Henk J.
Published: August 2015

The specificity of the binding of oestradiol-17β by cytoplasmic fractions of several tissues of the male rat was investigated. 1. Agar-gel electrophoresis, Sephadex chromatography, adsorption by dextran-coated charcoal and sucrose-gradient centrifugation were used to estimate the binding capacity and specificity. The four different methods all gave similar results for the capacity of the specific oestradiol-17β-binding macromolecules in the testis. 2. The presence of a specific saturable binding protein with a sedimentation coefficient of 8S was demonstrated in liver, adrenal, pituitary, prostate, epididymis and testis interstitial tissue. The highest concentration of oestradiol-17β-binding macromolecules was found in testis interstitial tissue (0.12pmol/mg of protein) and in the pituitary (0.075pmol/mg of protein). 3. The oestradiol-17β receptor in the testis cytosol showed the characteristics of a protein with respect to Pronase treatment and temperature sensitivity. In competition experiments with different steroids the receptor showed a high affinity for oestradiol-17β, a moderate affinity for diethylstilboestrol and oestradiol-17α and a low affinity for oestrone, oestriol, testosterone and 5α-dihydrotestosterone (17β-hydroxy-5α-androstan-3-one). 4. The wide distribution of oestradiol-17β receptors in the male rat is in apparent contradiction to the current concept of the specificity of steroid-hormone action. Further research is required to investigate a possible physiological meaning of the presence of specific receptors in the different tissues.
Involvement of binding lipoproteins in the absorption and transport of α-tocopherol in the rat
Authors: Rajaram O. V. , Fatterpaker P. , Sreenivasan A.
Published: August 2015

1. Specific lipoproteins binding α-tocopherol but not its known metabolites have been isolated and identified from cytosol of rat intestinal mucosa and from serum. 2. A timestudy of the appearance of the orally administered α-[3H]tocopherol with these lipoproteins indicates that very-low-density lipoprotein of serum acts as a carrier of the vitamin. 3. The involvement of the mucosal lipoprotein in the absorption of the vitamin from the intestine has been inferred from observations on the amounts of α-tocopherol in serum of orotic acid-fed rats where release of lipoproteins from the liver to serum is completely inhibited. A considerable decrease in the association of α-tocopherol with serum very-low-density lipoprotein under this condition is interpreted to mean that serum lipoproteins are limiting factors for the transport of the vitamin across the intestine and that this is possibly effected by exchange of α-tocopherol between serum very-low-density lipoprotein and mucosal lipoprotein.
Compartmentation of free amino acids for protein synthesis in rat liver
Authors: Airhart Judith , Vidrich Alda , Khairallah Edward A.
Published: August 2015

The concept that a general intracellular pool serves as the sole precursor of amino acids for protein biosynthesis has been vigorously debated in recent years. To help resolve this controversy, we followed the distribution of intraperitoneally administered [3H]valine in the tRNA and the extracellular and intracellular compartments of rat liver. The specific radioactivity of the valine released from isolated tRNA was 2–3 times higher than that of intracellular valine, suggesting that the intracellular pool cannot be the sole precursor of amino acids used for charging tRNA. In addition, the specific radioactivity of the tRNA was only half that of the extracellular valine. Therefore it is unlikely that the valyl-tRNA is charged exclusively with amino acids derived from the extracellular pool. A model is proposed which stipulates that both extracellular and intracellular amino acids contribute to a restricted compartment that funnels amino acids towards protein biosynthesis.
Kinetic properties of cerebral pyruvate kinase
Authors: Nicholas Peter C. , Bachelard Herman S.
Published: August 2015

Partly purified guinea-pig brain pyruvate kinase is not activated by fructose 1,6-diphosphate and gives hyperbolic substrate-saturation curves with phosphoenolpyruvate. It is therefore different from the L-type pyruvate kinase of mammalian liver. Inhibition by MgATP2- was competitive for MgADP- but not for phosphoenolpyruvate, and the enzyme is therefore different from the M-type pyruvate kinase, which is said to be competitively inhibited by MgATP2- with respect to both substrates. The Ki(MgATP2-) value of approx. 8mm for the brain enzyme is higher than the values (about 2mm) reported for the muscle enzyme. Stimulation of enzymic activity was observed at low (1–2mm) concentrations of MgATP2-. Substrate kinetic constants were Km (MgADP-)=0.47mm, Km (phosphoenolpyruvate)=0.08mm. Free Mg2+ at very high concentrations (over 10mm) was inhibitory (Ki=20–32mm). Neither ADP3- nor 5′-AMP2- inhibited the activity. The brain enzyme was concluded to be different from both the M-type and the L-type of other mammalian organs such as muscle and liver.
The nature of the multiple forms of cytoplasmic aspartate aminotransferase from pig and sheep heart
Authors: John Robert , Jones Richard
Published: August 2015

Starch-gel electrophoresis of sheep heart aspartate aminotransferase was carried out over the range pH7.0–8.5. The enzyme separates into three subforms in the same way as the pig heart enzyme. As the pH was increased the distance migrated by each subform increased by the same amount, so that they remained the same distance apart. Titration of the enzyme over the appropriate pH range was used to calculate the difference in charge between the subforms and it was concluded that they differ by one charged group per dimer from their nearest neighbour on the electrophoretogram over the whole pH range studied. It was also shown that the pig-heart α and β subforms differ by almost one charged group per dimer in the range pH5.5–5.7 and that the spacing between the subforms on starch-gel electrophoresis at pH8.0 is the same as that for the sheep enzyme. Since the charge difference between the subforms is maintained over such a wide range of pH, it is concluded that they probably differ from each other in covalent structure, because of the improbability that conformational differences can give rise to such behaviour. The relationship between the subforms and inactive binding of the coenzyme is also examined.
Structural studies of α-melanocyte-stimulating hormone and a novel β-melanocyte-stimulating hormone from the neurointermediate lobe of the pituitary of the dogfish Squalus acanthias
Authors: Bennett Hugh P. J. , Lowry Philip J. , McMartin Colin , Scott Alexander P.
Published: August 2015

A melanocyte-stimulating hormone (MSH) has been isolated from extracts of the neurointermediate lobe of the pituitary of the dogfish Squalus acanthias by gel-filtration and ion-exchange chromatography. It had approximately 1% of the potency of mammalian α-MSH on bioassays in vitro on frog skin and dogfish skin. Sequence analysis revealed it to be a hexadecapeptide with the following primary structure: Asp-Gly-Asp-Asp-Tyr-Lys-Phe-Gly-His-Phe-Arg-Trp-Ser-Val-Pro-Leu. It appears to be related to the β-MSH species of mammalian species but has only the sequence -His-Phe-Arg-Trp- in common with the heptapeptide core -Met-Glu-His-Phe-Arg-Trp-Gly- which is characteristic not only of the MSH peptides but also of the adrenocorticotrophins and lipotrophins studied so far. An α-MSH was also isolated, 50% of which was amidated at the C-terminus group. Sequence data from this study taken in conjunction with those from a previous study (Lowry & Chadwick, 1970b) revealed it to be a tridecapeptide which is identical with the N-terminal sequence of dogfish adrenocorticotrophin.
Structures for polyinosinic acid and polyguanylic acid
Authors: Arnott Struther , Chandrasekaran Rengaswami , Marttila Constance M.
Published: August 2015

X-ray-diffraction analysis of oriented, partially crystalline fibres of polyinosinic acid has resulted in a new molecular model. This model consists of four identical polynucleotide chains related to one another by a fourfold rotation axis. The coaxial helices are righthanded (screw symmetry 232) and have an axial translation per residue h=0.341nm and a rotation per residue t=31.3°. Incorporated in the model are standard bond lengths, bond angles and C-2-endo furanose rings. The nucleotide conformation angles, determined by linked-atom least-squares methods, are orthodox and the fit with the X-ray intensities is good. Each hypoxanthine base is linked to two others by hydrogen bonds involving O-6 and N-1. Further stability may arise from intrachain hydrogen bonds between each ribose hydroxyl group and the phosphate oxygen O-3. If guanine were to be substituted for hypoxanthine in an isogeometrical molecular structure, additional hydrogen bonds could be made between every N-2 and N-7.
Hexose phosphate synthase in trimethylamine-grown bacterium 2B2, a facultative methylotroph
Authors: Cox Roger B. , Zatman L. J.
Published: August 2015

Hexose phosphate synthase and hexulose phosphate isomerase activities were found in trimethylamine-grown bacterium 2B2, a facultative methylotroph. The activities were separated by column chromatography of cell extracts on DEAE-cellulose. Hexulose phosphate isomerase activity was measured spectrophotometrically by using the product of the hexose phosphate synthase reaction as substrate.
The binding of oxidized and reduced nicotinamide–adenine dinucleotides to bovine liver uridine diphosphate glucose dehydrogenase
Authors: Gainey Paul A. , Phelps Charles F.
Published: August 2015

The binding of NAD+and NADH to bovine liver UDP-glucose dehydrogenase was studied by using gel-filtration and fluorescence-titration methods. The enzyme bound 0.5mol of NAD+and 2 mol of NADH/mol of subunit at saturating concentrations of both substrate and product. The dissociation constant for NADH was 4.3μm. The binding of NAD+to the enzyme resulted in a small quench of protein fluorescence whereas the binding of NADH resulted in a much larger (60–70%) quench of protein fluorescence. The binding of NADH to the enzyme was pH-dependent. At pH8.1 a biphasic profile was obtained on titrating the enzyme with NADH, whereas at pH8.8 the titration profile was hyperbolic. UDP-xylose, and to a lesser extent UDP-glucuronic acid, lowered the apparent affinity of the enzyme for NADH.
A kinetic analysis of enzyme systems involving four substrates
Authors: Elliott Keith R. F. , Tipton Keith F.
Published: August 2015

A treatment of kinetic data for enzyme mechanisms involving four substrates is described. The initial-rate equations and product-inhibition patterns for such mechanisms are presented. The treatment is extended to include analysis of enzyme mechanisms involving three substrates in which two molecules of one substrate are used.