bluuebunny/crossref_metadata_2025 huggingface dataset:
Carbon-13 nuclear-magnetic-resonance spectroscopy of whole cells and of cytochrome C from Neurospora crass grown with (S-Me-13C)methionine
Authors: Eakin R T , Morgan L O , Matwiyoff N A
Published: August 2015

Neurospora crassa cytochrome C biosynthetically labelled with [S-Me-13C]methionine was prepared and analysed by 13C nuclear-magnetic-resonance spectroscopy. The methyl group of methionine is extensively incorporated into an N-trimethyl-lysine-72 residue arise from S-adenosylmethionine transmethylation, and that the methyl carbons of methionine residues are sufficiently close to the haem centre to experience chemical shifts from the ring currents of the tetrapyrrole pi electrons and broadening due to binding of methionine-80 with the haem, as well as interaction of the S-E113C]methyl groups with the paramagnetic iron centre. Although whole cells of the labelled Neurospora produced a 13C resonance at the expected position for the methionyl methyl group most of the methyl label was diverted into N-tetra-alkyl ammonium compounds. After an active state of growth these labelled N-methyl compounds appear, in the main, to be low-molecular-weight derivatives of choline which, if associated with membrane, are in a sufficiently fluid environment to have short rotational correlation times. During a subsequent dormant growth period these compounds become associated to some extent with relatively more immobile phases as a result of membrane binding or an increase in membrane rigidity.
Relation of C-6 glial cells in culture to myelin
Authors: Volpe J J , Fujimoto K , Marasa J C , Agrawal H C
Published: August 2015

C-6 glial cells were shown to contain enriched in their plasma membranes an enzyme, 2′:3′-cyclic nucleotide 3′-phosphohydrolase, characteristic of myelin, and, in addition, proteolipid protein and two basic proteins that are identical in their electrophoretic mobilities with the respective proteins found in myelin. The data indicate that C-6 cells exhibit features of myelin-producing glia as well as astrocytes.
Colchicine inhibition of the heparin-stimulated release of clearing-factor lipase from isolated fat-cells
Authors: Cryer A , McDonald A , Williams E R , Robinson D S
Published: August 2015

When isolated fat-cells are incubated at 25 degrees C in serum-based media containing glucose, insulin and heparin, the rise that occurs in the clearing-factor lipase activity of the incubation medium is inhibited by colchicine. The rise in the fat-cell clearing-factor lipase activity that occurs during similar incubations in the absence of heparin is not affected by colchicine.
Resolution and functional characterization of two mitochondrial iron-sulphur centres of the ‘high-potential iron-sulphur protein’ type
Authors: Ohnishi T , Ingledew W J , Shiraishi S
Published: August 2015

Two distinct iron-sulphur centres of the ‘HiPIP’ (high-potential iron-protein) type are distinguished in both pigeon heart and ox heart mitochondria. These two species, although both are paramagnetic in the oxidized state, exhibit signals which differ in their detailed line shape, field position, and temperature- and power-dependence. They also exhibit different thermodynamic and kinetic behaviour and are located on opposite sides of the mitochondrial coupling membrane. One of these centres corresponds to Centre S-3. The other ‘HiPIP’-type centre is removed readily from the mitochondrial membrane and its physiological function is not known.
Isolation, properties and amino acid sequences of three neurotoxins from the venom of a sea snake, Aipysurus laevis
Authors: Maeda N , Tamiya N
Published: August 2015

Aipysurus laevis venom was chromatographed on CM-cellulose and Bio-Rex 70 columns. Three neurotoxic components, toxins Aipysurus laevis a, b and c, were isolated. The toxins a, b and c corresponded to 22, 33 and 21% respectively of the proteins in the original venom, and accounted for almost all the lethal activity of the venom. The three toxins a, b and c were monodisperse on disc electrophoresis at pH4; toxins a and b moved at the same velocity and c a little faster. They were monodisperse also on sodium dodecyl sulphate-polyacrylamide-disc-gel electrophoresis, giving a molecular weight of 7600. The molecular weight of toxin b estimated by gel filtration was 7000. The amino acid sequence analyses of these toxins revealed that they consisted of 60 amino acid residues and that Aipysurus laevis b was [25-methionine, 28-arginine] Aipysurus laevis a. Aipysurus laevis c was [28-lysine] Aipysurus laevis a, the tryptic peptide sequence relying on homology. The LD50 values of these toxins for 20g mice were 0.076 μg/g body wt. They inhibited the acetylcholine-induced contracture but did not affect the CKl-induced contracture of the isolated muscle.
Factors affecting the activity of citrate synthase of Acetobacter xylinum and its possible regulatory role
Authors: Swissa M , Benziman M
Published: August 2015

The citrate synthase activity of Acetobacter xylinum cells grown on glucose was the same as of cells grown on intermediates of the tricarboxylic acid cycle. The activity of citrate synthase in extracts is compatible with the overall rate of acetate oxidation in vivo. The enzyme was purified 47-fold from sonic extracts and its molecular weight was determined to be 280000 by gel filtration. It has an optimum activity at pH 8.4. Reaction rates with the purified enzyme were hyperbolic functions of both acetyl-CoA and oxaloacetate. The Km for acetyl-CoA is 18 μm and that for oxaloacetate 8.7 μm. The enzyme is inhibited by ATP according to classical kinetic patterns. This inhibition is competitive with respect to acetyl-CoA (Ki = 0.9 mM) and non-competitive with respect to oxaloacetate. It is not affected by changes in pH and ionic strength and is not relieved by an excess of Mg2+ ions. Unlike other Gram-negative bacteria, the A. xylinum enzyme is not inhibited by NADH, but is inhibited by high concentrations of NADPH. The activity of the enzyme varies with energy charge in a manner consistent with its role in energy metabolism. It is suggested that the flux through the tricarboxylic acid cycle in A. xylinum is regulated by modulation of citrate synthase activity in response to the energy state of the cells.
Chemical nature and sequence of alamethicin
Authors: Martin D R , Williams R J P
Published: August 2015

An n.m.r. spectroscopy study of pure alamethicin shows it to be a linear polypeptide of 19 residues. The N-terminus is blocked by an acetyl group, and the eighteenth residue, glutamic acid, is linked by an amide bond on its side chain to phenylalaninol (Fig. 6). The new formula is confirmed by a comparison between pure chemical compounds and the products of partial hydrolysis.
Preparation and activity of guanidinated or acetylated erabutoxins
Authors: Hori H , Tamiya N
Published: August 2015

1. Erabutoxins, a, b and c, neurotoxic proteins of a sea snake Lacticauda semifasciata, were guanidinated with O-methylisourea. The amino groups of all the lysine residues and those at the N-termini of the toxins were modified. The lethal activity of the toxins decreased to 50% (erabutoxins a and b) or 17% (erabutoxin c) of the original value on the modification. The c.d. (circular dichroism) maximum at 227 nm of the modified toxins became lower, whereas the whole profile of the c.d. curve remained unchanged. 2. The amino groups of erabutoxin b were acetylated with acetic anhydride. All the five monoacetyl derivatives were isolated from the reaction products by CM-cellulose and Bio-Rex 70 column chromatography. [1-Nalpha-acetylarginine]-, [15-N6-acetyl-lysine]- and [51-N6-acetyl-lysine]-erabutoxin b retained the toxicity of the native toxin, whereas [27-N6-acetyl-lysine] and [47-N6-acetyl-lysine]-erabutoxin b were 17 and 8% active respectively. The overall profile of c.d. spectrum of erabutoxin b remained unchanged on the monoacetylation.
Acrolein, an irreversible active-site-directed inhibitor of deoxyribose 5-phosphate aldolase?
Authors: Wilton D C
Published: August 2015

The enzyme deoxyribose 5-phosphate aldolase was irreversibly inactivated by the substrate analogue acrolein with a pseudo-first-order rate constant of 0.324 min-1 and a Ki (apparent) of 2.7 × 10(-4) m. No inactivation was observed after prolonged incubation with the epoxide analogues glycidol phosphate and glycidaldehyde. It is suggested that the acrolein is first activated by forming a Schiff base with the enzyme active-site lysine residue and it is the activated inhibitor that reacts with a suitable-active-site nucleophile.
Identification of N-terminal methionine in the precursor of immunoglobulin light chain. Initiation of translation of messenger ribonucleic acid in plants and animals
Authors: Schechter I , Burstein Y
Published: August 2015

The proteins programmed in the wheat-germ cell-free system by the mRNA coding for the MOPC-321 mouse myeloma L (light) chain were labelled with [35S]methionine, [4,5-3H]leucine or [3-3H]serine, and were subjected to amino acid-sequence analyses. Over 95% of the total cell-free product was sequenced as one homogeneous protein, which corresponds to the precursor of the L-chain protein. In the precursor, 20 amino acid residues precede the N-terminus of the mature protein. This extra piece contains one methionine residue at the N-terminus, one serine residue at position 18, and six leucine residues, which are clustered in two triplets at positions 6, 7, 8 and 11, 12, 13. The identification of methionine at the N-terminus of the precursor is in agreement with the evidence showing that unblocked methionine is the initiator residue for protein synthesis in eukaryotes. The absence of methionine at position 20, which precedes the N-terminal residue of the mature protein, suggests that myeloma cells synthesize the precursor. However, within the cell the precursor should be rapidly processed to the mature L chain, since precursor molecules have not yet been found in the intact animal. The abundance (30%) of leucine residues indicates that the extra-piece moiety is quite hydrophobic. The extra piece of the MOPC-321 L-chain precursor synthesized with the aid of the Krebs II ascites cell-free system is of identical size and it has the same leucine sequence [Schechter et al. (1975) Science 188, 160-162]. This indicates that cell-free systems derived from the plant and animal kingdom initiate mRNA translation from the same point. It is shown that the amino acid sequence of minute amounts of a highly labelled protein (0.1 pmol) can be faithfully determined in the presence of a large excess (over 2000 000-fold) of unrelated non-radioactive proteins.
Physical and catalytic properties of α-amylase from Tenebrio molitor L. larvae
Authors: Buonocore V , Poerio E , Silano V , Tomasi M
Published: August 2015

The amylase from Tenebrio molitor L. larvae (yellow mealworm) was characterized according to a number of its molecular and catalytic properties. The insect amylase is a single polypeptide chain with mol.wt. 68000, an isoelectric point of 4.0 and a very low content of sulphur-containing amino acids. The enzyme is a Ca2+-protein and behaves as an α-amylase. Removal of Ca2+ by exhaustive dialysis against water causes the irreversible inactivation of the enzyme. Moreover, the enzyme is activated by the presence in the assay mixture of Cl-, or some other inorganic anions that are less effective than Cl-, and is inhibited by F-. Optimal conditions of pH and temperature for the enzymic activity are 5.8 and 37 degrees C. The insect amylase exhibits an identical kinetic behaviour toward starch, amylose and amylopectin; the enzyme hydrolyses glycogen with a higher affinity constant. Compared with the non-insect α-amylases described in the literature, Tenebrio molitor amylase has a lower affinity for starch.
The prediction of the conformation of membrane proteins from the sequence of amino acids
Authors: Green N M , Flanagan M T
Published: August 2015

The methods of Chou & Fasman [Biochemistry (1974) 13, 211-222, 222-245] and of Lim [J. Mol. Biol. (1974)88, 857-872, 873-894] for predicting secondary structure from amino acid sequence have been applied to five predominantly helical membrane-associated peptides. The predictions from the method of Lim (1974a,b) are consistent with the experimental observations, whereas those from Chou & Fasman (1974a,b), although not inconsistent with α-helix, favour a β-structure for several very hydrophobic regions. The results may be rationalized in terms of the effect of the solvent on the conformation of a polypeptide.
Study on sex-organ development. Oestrogen-receptor translocation in the developing chick Müllerian duct
Authors: Teng C S , Teng C T
Published: August 2015

After oestradiol administration in vivo, 87-95% of the initial concentration of oestradiol receptor in the cytoplasm of the embryonic-chick Müllerian-duct cell was translocated into the nucleus. The process of translocation depends on the amount of oestardiol administered in vivo. At 6 h after oestradiol administration in vivo, about 30% replenishment of the initial content of the cytosol receptor was observed in the cytoplasm. The Müllerian-duct nuclei, after exposure to non-radioactive oestradiol, exhibit saturable exchange with [3H]oestradiol in vitro. The exchange of oestradiol is temperature- and time-dependent. The optimal temperature and time for exchange are 37-41 degrees C and 2h respectively. The [3H]oestradiol-receptor complex extracted from the exchanged nuclei is present in 5-6S form, and its isoelectric point is 6.8. The number of nuclear oestradiol-binding sites of the developing Müllerian duct are 1.66, 2.22, 2.63, and 2.50 pmol/mg of DNA respectively for embryos of 10, 12, 15 and 18 days. The dissociation constants of the nuclear oestradiol receptor of the four observed developmental stages range from 3.0 to 3.1 nM.
A rapid immunological procedure for the isolation of hormonally sensitive rat fat-cell plasma membrane
Authors: Luzio J P , Newby A C , Hales C N
Published: August 2015

1. A rapid method for the isolation of hormonally sensitive rat fat-cell plasma membranes was developed by using immunological techniques. 2. Rabbit anti-(rat erythrocyte) sera were raised and shown to cross-react with isolated rat fat-cells. 3. Isolated rat fat-cells were coated with rabbit anti-(rat erythrocyte) antibodies, homogenized and the homogenate made to react with an immunoadsorbent prepared by covalently coupling donkey anti-(rabbit globulin) antibodies to aminocellulose. Uptake of plasma membrane on to the immunoadsorbent was monitored by assaying the enzymes adenylate cyclase and 5′-nucleotidase and an immunological marker consisting of a 125I-labelled anti-(immunoglobulin G)-anti-cell antibody complex bound to the cells before fractionation. Contamination of the plasma-membrane preparation by other subcellular fractions was also investigated. 4. By using this technique, a method was developed allowing 25-40% recovery of plasma membrane from fat-cell homogenates within 30 min of homogenization. 5. Adenylate cyclase in the isolated plasma-membrane preparation was stimulated by 5 μm-adrenaline.
Amino acid transport in normal and glutathione-deficient sheep erythrocytes
Authors: Young J D , Ellory J C , Tucker E M
Published: August 2015

1. Uptake rates for 23 amino acids were measured for both normal (high-GSH) and GSH-deficient (low-GSH) erythrocytes from Finnish Landrace sheep. 2. Compared with high-GSH cells, low-GSH cells had a markedly diminished permeability to D-alanine, L-alanine, α-amino-n-butyrate, valine, cysteine, serine, threonine, asparagine, lysine and ornithine. Smaller differences were observed for glycine and proline, whereas uptake of the other amino acids was not significantly different in the two cell types.
Regulation of pyruvate dehydrogenase and pyruvate dehydrogenase phosphate phosphatase activity in rat epididymal fat-pads. Effects of starvation, alloxan-diabetes and high-fat diet
Authors: Stansbie D , Denton R M , Bridges B J , Pask H T , Randle P J
Published: August 2015

1. Pyruvate dehydrogenase phosphate phosphatase activity in rat epididymal fat-pads was measured by using pig heart pyruvate dehydrogenase [32P]phosphate. About 80% was found to be extramitochondrial and therefore probably not directly concerned with the regulation of pyruvate dehydrogenase activity. The extramitochondrial activity was sensitive to activation by +, but perhaps less sensitive than the mitochondrial activity.
Tyrosine aminotransferase activity in the rabbit placenta. Evidence for decreased inducibility by insulin and cortisol as a function of gestational age
Authors: Wade R S , Gusseck D J
Published: August 2015

Tyrosine aminotransferase is present at low activity in the rabbit placenta. The activity can be enhanced by injecting the pregnant mother with insulin or cortisol. This response decreases over the last half of gestation (14-28 days gestational age).
The influence of adenine nucleotides and oxidizable substrates on triethyltin-mediated chloride uptake by rat liver mitochondria in potassium chloride media
Authors: Skilleter D N
Published: August 2015

In a 100 mM-KCl medium, pH 6.8, containing ATP increasing concentrations of triethyltin cause an uptake of Cl- into mitochondria with a maximum at 1 muM. This can be inhibited by atractylate or oligomycin, but is virtually unaffected by the presence of rotenone. When the medium contains substrate (pyruvate, β-hydroxybutyrate or succinate), both in the presence and absence of adenine nucleotides, Cl- uptake is greater with a maximum at 1-10 muM-triethyltin. If substrate oxidation is blocked by respiratory-chain inhibitors the Cl- uptake mediated by triethyltin is inhibited except in the media containing ATP, when the characteristics of Cl- uptake similar to that found in the medium containing ATP alone are observed. Under all conditions tested Cl- uptake is decreased by the presence of 2,4-dinitrophenol. It is concluded that energy from either the oxidation of substrate or the hydrolysis of ATP is associated with the generation of sufficient OH- to enable the triethyltin-mediated Cl-/OH- exchange to occur under the metabolic conditions relevant to this action of triethyltin.
L-lactate transport in Ehrlich ascites-tumour cells
Authors: Spencer T L. , Lehninger A L.
Published: August 2015

Ehrlich ascites-tumour cells were investigated with regard to their stability to transport L-lactate by measuring either the distribution of [14C]lactate or concomitant H+ ion movements. The movement of lactate was dependent on the pH difference across the cell membrane and was electroneutral, as evidenced by an observed 1:1 antiport for OH- ions or 1:1 symport with H+ ions. 2. Kinetic experiments showed that lactate transport was saturable, with an apparent Km of approx. 4.68 mM and a Vmax. as high as 680 nmol/min per mg of protein at pH 6.2 and 37°C. 3. Lactate transport exhibited a high temperature dependence (activation energy = 139 kJ/mol). 4. Lactate transport was inhibited competitively by (a) a variety of other substituted monocarboxylic acids (e.g. pyruvate, Ki = 6.3 mM), which were themselves transported, (b) the non-transportable analogues α-cyano-4-hydroxycinnamate (Ki = 0.5 mM), α-cyano-3-hydroxycinnamate (Ki = 2mM) and DL-p-hydroxyphenyl-lactate (Ki = 3.6 mM) and (c) the thiol-group reagent mersalyl (Ki = 125 muM). 5. Transport of simple monocarboxylic acids, including acetate and propionate, was insensitive to these inhibitors; they presumably cross the membrane by means of a different mechanism. 6. Experiments using saturating amounts of mersalyl as an “inhibitor stop” allowed measurements of the initial rates of net influx and of net efflux of [14C]lactate. Influx and efflux of lactate were judged to be symmetrical reactions in that they exhibited similar concentration dependence. 7. It is concluded that lactate transport in Ehrlich ascites-tumour cells is mediated by a carrier capable of transporting a number of other substituted monocarboxylic acids, but not unsubstituted short-chain aliphatic acids.
Dehydrogenation of the phosphonate analogue of glucose 6-phosphate by glucose 6-phosphate dehydrogenase
Authors: Adams P R , Harrison R , Inch T D , Rich P
Published: August 2015

6,7 -Dideoxy-α-D-gluco-heptose 7-phosphonic acid, the isosteric phosphonate analogue of glucose 6-phosphate, was synthesized in six steps from the readily available precursor benzyl 4,6-O-benzylidene-α-D-glucopyranoside. The analogue is a substrate for yeast glucose 6-phosphate dehydrogenase, showing Michaelis-Menten kinetics at pH7.5 and 8.0. At both pH values the Km values of the analogue are 4-5 fold higher and the values approx. 50% lower than those of the natural substrate. The product of enzymic dehydrogenation of the phosphonate analogue at pH8.5 is itself a substrate for gluconate 6-phosphate dehydrogenase.
PH-dependence of the steady-state rate of a two-step enzymic reaction
Authors: Brocklehurst K , Dixon H B F
Published: August 2015

1. The pH-dependence is considered of a reaction between E and S that proceeds through an intermediate ES under “Briggs-Haldane” conditions, i.e. there is a steady state in ES and [S]o greater than [E]T, where [S]o is the initial concentration of S and [E]T is the total concentration of all forms of E. Reactants and intermediates are assumed to interconvert in three protonic states (E equilibrium ES; EH equilibrium EHS; EH2 equilibrium EH2S), but only EHS provides products by an irreversible reaction whose rate constant is kcat. Protonations are assumed to be so fast that they are all at equilibrium. 2. The rate equation for this model is shown to be v = d[P]/dt = (kcat.[E]T[S]o/A)/[(KmBC/DA) + [S]o], where Km is the usual assembly of rate constants around EHS and A-D are functions of the form (1 + [H]/K1 + K2/[H]), in which K1 and K2 are: in A, the molecular ionization constants of ES; in B, the analogous constants of E; in C and D, apparent ionization constants composed of molecular ionization constants (of E or ES) and assemblies of rate constants. 3. As in earlier treatments of this type of reaction which involve either the assumption that the reactants and intermediate are in equilibrium or the assumption of Peller & Alberty [(1959) J. Am. Chem. Soc. 81, 5907-5914] that only EH and EHS interconvert directly, the pH-dependence of kcat. is determined only by A. 4. The pH-dependence of Km is determined in general by B-C/A-D, but when reactants and intermediate are in equilibrium, C identical to D and this expression simplifies to B/A. 5. The pH-dependence of kcat./Km, i.e. of the rate when [S]o less than Km, is not necessarily a simple bell-shaped curve characterized only by the ionization constants of B, but is a complex curve characterized by D/B-C. 6. Various situations are discussed in which the pH-dependence of kcat./Km is determined by assemblies simpler than D/B-C. The special situation in which a kcat./Km-pH profile provides the molecular pKa values of the intermediate ES complex is delineated.
Nitrogenase of Azotobacter chroococcum. Kinetics of the reduction of oxidized iron-protein by sodium dithionite
Authors: Thorneley R N F , Yates M G , Lowe D J
Published: August 2015

The kinetics of the reduction of oxidized Fe-protein of nitrogenase from Azotobacter chroococcum by sodium dithionite were studied by stopped-flow and rapid-freezing e.p.r. (electron-paramagnetic-resonance) spectroscopy. The appearance of the gav. = 1.94 e.p.r. signal (0.24 electron integrated intensity/mol) was associated with a one-electron reduction by SO2- with k greater than 10(8)M-.S-1 at 23 degrees C. A value of k = 1.75s-1 was obtained for the rate of dissociation of S2O42- into 2SO2- at 23 degrees C. Further reductions by SO2- occurred in three slower phases with rate constants in the range 10(4) −10(6)M-1-S-1. These latter phases have no corresponding e.p.r. signal changes and are probably associated with enzymically inactive protein. The high rate of reduction by SO2-of the Fe-protein alone (k greater than 108M1.S-1) relative to the rate of oxidation of the Fe-protein in the catalytically active Fe:Mo-Fe protein complex (k = 2.2 } 102s-1) and the observation that in the steady state the Fe-protein is substantially oxidized means that at normal assay concentrations another reaction must limit the rate of reduction of Fe-protein during turnover.
Neutral proteinases of human spleen. Purification and criteria for homogeneity of elastase and cathepsin G
Authors: Starkey P M. , Barrett A J.
Published: August 2015

1. Human spleen was found to contain proteinases active against azo-casein at neutral and alkaline pH values. 2. The activity was stimulated by high ionic strength and some detergents. 3. Optimal extraction of the proteinases from the tissue was achieved with 1.0M-NaCl containing 0.1% Brij 35 and 0.1% trisodium EDTA. 4. The proteinases were efficiently adsorbed to insoluble material in the absence of salt in the initial stages of purification. 5. Two distinct proteinases were separated by chromatography on DEAE-cellulose, an elastase and a chymotrypsin-like enzyme designated cathepsin G. 6. Both enzymes were highly purified by further column chromatography. 7. The molecular weights of the enzymes were estimated by gel chromatography and sodium dodecyl sulphate-gel electrophoresis. 8. It was shown by isoelectric focusing and gel electrophoresis that both enzymes are cationic proteins that occur in multiple forms.
Separation of rat muscle aminopeptidases
Authors: Parsons M E. , Pennington R J. T.
Published: August 2015

By means of chromatography on DEAE-Sephadex, two arylamidases (hydrolysing L-arginine 2-naphthylamide) and three dipeptidyl peptidases (hydrolysing dipeptide 2-naphthylamides) were distinguished in extracts of rat muscle. However, the arylamidase from the larger peak also hydrolysed the dipeptide 2-naphthylamides. Glycyl-L-arginine amide, an alternative substrate for dipeptidyl peptidase I, was not hydrolysed by arylamidase. L-Leucine amide was hydrolysed by an enzyme, presumed to be leucine aminopeptidase, from a separate peak, but was also hydrolysed by arylamidase. Arylamidase, dipeptidyl peptidase III and most of the leucine aminopeptidase could be extracted from the muscle with a neutral salt solution, but dipeptidyl peptidase I was extracted only in the presence of Triton X-100; dipeptidyl peptidase II showed an intermediate extraction behaviour.
Preparation of the 3-monosulphates of cholic acid, chenodeoxycholic acid and deoxycholic acid
Authors: Haslewood E S. , Haslewood G A. D.
Published: August 2015

1. The 3-sulphates of cholic, chenodeoxycholic and deoxycholic acids were prepared as crystalline disodium salts. 2. The method described shows that it is possible to prepare specific sulphate esters of polyhydroxy bile acids and to remove protecting acyl groups without removing the sulphate. 3. A study of bile acid sulphate solvolysis showed that none of the usual methods give the original bile acid in major yield in a single step. 4. An understanding of the preparation, properties and methods of solvolysis of bile acid sulphates is basic for investigations of cholestasis and liver disease.
The effect of some analogues of disulfiram on the aldehyde dehydrogenases of sheep liver
Authors: Kitson T M.
Published: August 2015

The effect of some close structural analogues of disulfiram on the activity of the cytoplasmic and mitochondrial aldehyde dehydrogenases of sheep liver was studied. Several thiuram disulphides are equally potent inhibitors of the cytoplasmic enzyme, but in all cases the mitochondrial enzyme is much less strongly affected. Tetramethylthiuram monosulphide decreases the activity of the cytoplasmic enzyme in a process apparently involving a covalent interaction.
Reactions involving superoxide and normal and unstable haemoglobins
Authors: Winterbourn C C , McGrath B M , Carrell R W
Published: August 2015

Superoxide ions (O2-) oxidized oxyhaemoglobin to methaemoglobin and reduced methaemoglobin to oxyhaemoglobin. The reactions of superoxide and H2O2 with oxyhaemoglobin or methaemoglobin and their inhibition by superoxide dismutase or catalase were used to detect the formation of superoxide or H2O2 on autoxidation of oxyhaemoglobin. The rate of autoxidation was decreased at about 35% in the presence of both enzymes. The copper-catalysed autoxidation of Hb (haemoglobin) was also shown to involve superoxide production. Superoxide was released on autoxidation of three unstable haemoglobins and isolated alpha and beta chains, at rates faster than with Hb A. Reactions of superoxide with Hb Christchurch and Hb Belfast were identical with those with Hb A, and occurred at the same rate. Hb Koln contrasted with the other haemoglobins in that the thiol groups of residue beta-93 as well as the haem groups reacted with superoxide. Haemichrome formation from methaemoglobin occurred very rapidly with Hb Christchurch and Hb Belfast, as well as the isolated chains, compared with Hb A. The process did not involve superoxide production or utilization. The relative importance of autoxidation and superoxide production compared with haemichrome formation in the haemolytic process associated with these abnormal haemoglobins and thalassaemia is considered.
A low-molecular-weight protein from rat liver that resembles ligandin in its binding properties
Authors: Ketterer B , Tipping E , Hackney J F , Beale D
Published: August 2015

A protein of S20,W 1.6S and mol.wt. 14000, which binds covalently a metabolite of the aminoazodye carcinogen NN-dimethyl-4-amino-3′-methylazobenzene, was isolated from rat liver cytosol from both carcinogen-treated and normal rats. The protein binds non-covalently palmitoyl-CoA, fatty acids, bilirubin, sex steroids and their sulphates, bile acids and salts, bromosulphophthalein, diethylstilboestrol and 20-methylcholanthrene with a wide range of affinities. The protein is isolated as three components with isoelectric points of 5.0, 5.9 and 7.6 by a method involving isoelectric focusing. All three components have closely similar amino acid analyses, tryptic-peptide ‘maps’ and u.v. spectra. Each single component redistributes into all three on further electrophoresis. However, the three forms differ in their binding characteristics, the form of pI 7.6 having much the highest affinity for compounds bound non-covalently. The protein was identified immunologically in rat liver, small intestine, adipose tissue, skeletal muscle, myocardium and testis. The protein was compared with other hepatic binding-protein preparations of similar molecular weight.
Chymotrypsins from the deer (Cervidae) family. Isolation, partial characterization and primary-structure studies of chymotrypsins A and B from both moose (Alces alces) and elk (Cervus elaphus) pancreas
Authors: Lindsay R M , Stevenson K J
Published: August 2015

1. An anionic and a cationic chymotrypsin (EC 3.4.21.1) were isolated from the pancreas glands of the moose (Alces alces) and elk (Cervus elaphus). The A and B chymotrypsins from each species were purified to homogeneity by (NH4)2SO4 fractionation, affinity chromatography on 4-phenylbutylamine-Sepharose and ion-exchange chromatography on DEAE- and CM-cellulose. 2. The molecular weight and pH optimum of each chymotrypsin were similar to those of the corresponding ox A and B chymotrypsins. 3. The substrate specificities of the chymotrypsins were investigated by digestion of glucagon and the oxidized B chain of insulin. The primary specificity of each chymotrypsin for aromatic amino acid residues was further established by determining the Km and kcat for the hydrolysis of a number of synthetic amino acid ester substrates. 4. The amino acid composition and total number of residues of moose and elk chymotrypsin A were similar to those of ox chymotrypsin A. An even greater similarity was observed among the B chymotrypsins of the three species. 5. The A chymotrypsins of moose and elk were fragmented to their constituent ‘A’, ‘B’ and ‘C’ polypeptide chains by succinylation (3-carboxypropionylation), reduction and alkylation of the native enzymes. In each case, the two major chains (‘B’ and ‘C’) were separated and isolated. By comparison of the amino acid compositions of moose, elk and oxy ‘B’ and ‘C’ chains, a greater difference was observed among the three A chymotrypsins than was suggested by the amino acid compositions of the native enzymes alone. 6. Peptides were isolated from the disulphide bridge and active-site regions of the A and B chymotrypsins of moose and elk by diagonal peptide-‘mapping’ techniques. From the amino acid compositions of the isolated peptides (assuming maximum homology) and from a comparison of diagonal peptide ‘maps’, there was established a high degree of primary-structure identity among the mooae, elk and ox chymotrypsins. Tentative sequences were deduced for the peptides isolated by diagonal peptide ‘mapping’. 7. Details of the isolation procedures of the moose and elk chymotrypsins A and B and the amino acid analyses of some peptides obtained by diagonal peptide ‘mapping’ have been deposited as Supplementary Publication SUP 50064 (27 pages) at the British Library Lending Division, Boston Spa, Wetherby, W. Yorkshire LS23 7BQ, U.K., from whom copies can be obtained on the terms indicated in Biochem. J. (1976) 153, 5.
Studies on the proteins from the seeds of Croton tiglium and of Jatropha curcas. Toxic properties and inhibition of protein synthesis in vitro
Authors: Stirpe F , Pession-Brizzi A , Lorenzoni E , Strocchi P , Montanaro L , Sperti S
Published: August 2015

1. Proteins extracted from the seeds of the Euphorbiaceae croton tiglium and Jatropha curcas were separated into three major peaks (I, II, and III) by Sephadex chromatography. 2. The crude protein from both seeds and peaks I and II from Croton and peak I from Jatropha were toxic to mice, to different extents. 3. The crude protein and peak I and peak II from both seeds, inhibited protein synthesis by a reticulocyte lysate; maximum inhibition was exerted by peak II from both seeds. None of these preparations affected protein synthesis in vitro by Ehrlich ascites cells.
Phosphorylation state of cytosolic and mitochondrial adenine nucleotides and of pyruvate dehydrogenase in isolated rat liver cells
Authors: Siess E A , Wieland O H
Published: August 2015

1. Cytosolic and mitochondrial ATP and ADP concentrations of liver cells isolated from normal fed, starved and diabetic rats were determined. 2. The cytosolic ATP/ADP ratio was 6,9 and 10 in normal fed, starved and diabetic rats respectively. 3. The mitochondrial ATP/ADP ratio was 2 in normal and diabetic rats and 1.6 in starved rats. 4. Adenosine increased the cytosolic and lowered the mitochondrial ATP/ADP ratio, whereas atractyloside had the opposite effect. 5. Incubation of the hepatocytes with fructose, glycerol or sorbitol led to a fall in the ATP/ADP ratio in both the cytosolic and the mitochondrial compartment. 6. The interrelationship between the mitochondrial ATP/ADP ratio and the phosphorylation state of pyruvate dehydrogenase in intact cells was studied. 7. In hepatocytes isolated from fed rats an inverse correlation between the mitochondrial ATP/ADP ratio and the active form of pyruvate dehydrogenase (pyruvate dehydrogenase a) was demonstrable on loading with fructose, glycerol or sorbitol. 8. No such correlation was obtained with pyruvate or dihydroxyacetone. For pyruvate, this can be explained by inhibition of pyruvate dehydrogenase kinase. 9. Liver cells isolated from fed animals displayed pyruvate dehydrogenase a activity twice that found in vivo. Physiological values were obtained when the hepatocytes were incubated with albumin-oleate, which also yielded the highest mitochondrial ATP/ADP ratio.
Regulation of carbohydrate metabolism in lymphoid tissue. Nature of the endogenous substrates and their contribution to the respiratory fuel of the sliced rat spleen in vitro
Authors: Suter D , Weidemann M J
Published: August 2015

1. Tissue glycogen contributes, maximally, only 10% of the respiratory fuel of the rat spleen slice in the absence of an added carbon source, and makes no significant contribution when glucose (3mM) is added. 2. The reserves of fatty acid in the form of triglyceride (35.5mumol of fatty acid/g dry wt. of tissue) fall by approx. 25% after incubation of spleen slices with or without added glucose for 2h, and, on this basis, account for 32% of the oxidative fuel. 3. In contrast, the total oxidative contribution of fatty acid reserves to the respiratory fuel, determined on the basis of inhibiton of respiration by 2-bromostearate, is 42-52%. This range includes tissue from both starved and well-fed animals and is not significantly altered by the presence of added glycose (3mM). 4. Large quantities of NH3 (31-35mumol//h per g dry wt. of tissue) are produced by spleen slices incubated in the absence of added substrates, and this value is suppressed by approx. 50% on incubation with glucose (3mM). Adenine nucleotide breakdown can account for only 17% of the total ammonia produced. 5. Individual free amino acid concentrations in spleen were determined, both in vivo and in slices before and after 60 min of incubation. Although the total free amino acid pool size increases by 45% during incubation, owing to protein breakdown, the tissue concentrations of aspartate, glutamate, glutamine and alanine do not increase. It is suggested that these amino acids areoxidized in a net sense to CO2 and water with the liberation of free NH3 via transamination reactions, glutaminase, the purine nucleotide cycle and the tricarboxylic acid cycle. 6. It is concluded that the normal endogenous metabolism of sliced rat spleen (43-52% due to lipids, 30% due to amino acids and 10% due to glycogen) is modified by added glycose only to the extent that glycogen oxidation and 50% of the contribtion made by ino acids are suppressed; endogenous lipid metabolism is unaffected.
The biochemistry of fatty liver and kidney syndrome. Biotin-mediated restoration of hepatic gluconeogenesis in vitro and its relationship to pyruvate carboxylase activity
Authors: Bannister D W
Published: August 2015

Liver slices from chicks affected by the fatty liver and kidney syndrome display an extremely low extent of hepatic gluconeogenesis which is associated with decreased activities of certain rate-limiting gluconeogenic enzymes. Pyruvate carboxylase activity is particularly severely affected, being less than 4% of control values. Incubation of affected slices in a biotin-containing nutrient medium restores both gluconeogenesis and pyruvate carboxylase actiivity (the latter to approx. 35% of the control valve). Activities of the other enzymes studied were not greatly affected by this treatment. Restoration of gluconeogenesis did not occur if biotin was excluded from the nutrient medium, nor was it prevented by protein-synthesis inhibitors. It is concluded that the syndrome involves the lack of available biotin in the liver rather than suppression of apocarboxylase synthesis.
Synthesis of adenosine triphosphate during release of intravesicular and membrane-bound calcium ions from passively loaded sarcoplasmic reticulum
Authors: Vale M G P , Osório V R O E , Castro E , Carvalho A P
Published: August 2015

Sarcoplasmic reticulum isolated from rabbit skeletal muscle and incubated in a medium containing Ca2+ in the absence of ATP retains intravesicular and/or membrane-bound Ca2+. The synthesis of ATP coupled with the release of intravesicular Ca2+ is totally inhibited by the ionophore X-537A. Release of the membrane-bound Ca2+, retained after short periods of incubation (10min) or after release of the intravesicular Ca2+ by ionophore X-537A, still supports some synthesis of ATP. The ratios of Ca2+ released to ATP synthesized are 2.5-3.2, when bound and intravesicular Ca2+ are released simultaneously, and 3.1-4.0, when only bound Ca2+ is released. The results show that the synthesis of ATP by sarcoplasmic reticulum during release of passively accumulated Ca2+ by EGTA [ethanedioxybis(ethylamine)tetra-acetic acid] is accompanied by a loss of membrane-bound Ca2+.
Prevention, by ribosome-bound nascent polyphenylalanine chains, of the functional interaction of t-2 toxin with its receptor site
Authors: Cannon M , Smith K E , Carter C J
Published: August 2015

1. The inhibitory effects of T-2 toxin and trichodermin on poly(U)-directed polyphenylalanine synthesis were studied by using cell-free systems from reticulocytes. Conditions for amino acid incorporation were carefully chosen in an attempt to ensure that the large majority of poly(U) chains bound only one ribosome engaged in protein synthesis and that all such ribosomes carried nascent polyphenylalanine chains containing approximately the same number of residues. 2. Cell-free systems were allowd to synthesize polyphenylalanine, and T-2 toxin and trichodermin were added to the incorporation mixtures at various times. Irrespective of the time of addition, trichodermin (50 μg/ml) inhibited polyphenylalanine synthesis by approx. 70%. In contrast, although T-2 toxin (40 μg/ml), when added at early incubation times, could inhibit polyphenylalanine synthesis with a maximum of 50%, the drug had no effect on the system when added after a critical time-period. 3. It is concluded that although both T-2 toxin and trichodermin can inhibit peptide-bond formation on ribosomes at the level of the peptidyl transferase catalytic centre the presence, on ribosomes, of nascent polyphenylalanine chains above a certain critical chain length excludes T-2 toxin from functional interaction with its receptor site.
Lipid metabolism and mobilization in the guinea pig during pregnancy
Authors: Jones C T
Published: August 2015

The liver of the foetal guinea pig accumulates a large quantity of triacyglycerol late in gestation at the same time that adipose-tissue mass grows at its maximum rate and foetal adipose-tissue lipoprotein lipase activity and sensitivity to lipolytic hormones has substantially declined. The fatty acid for triacyglycerol synthesis is not synthesized in the foetal liver and it is unlikely that it originates from any of the foetal tissues. Before the accumulation of hepatic triacyglycerol the concentration of free fatty acids increases in both the umbilical vein and the maternal inferior vena cava. This occurs at a time when the triacyglycerol lipase activity in maternal adipose tissue is elevated and the rate of lipolysis, but not of fatty acid esterification, is higher than earlier in gestation or than in the non-pregnant state. It is proposed that the increase in lipolysis in maternal adipose tissue, brought about by an increase in circulating lipolytic hormones, mobilizes fatty acid, which passes to the foetus and is partly stored as hepatic triacylglycerol. The foetal liver effectively removes both long-and short-chain fatty acids from umbilical-vein blood. The rate of placental fatty acid transfer is more than adequate to account for the triacylglycerol accumulation.
Nuclear binding of progesterone in hen oviduct. Role of acidic chromatin proteins in high-affinity binding
Authors: Webster R A , Pikler G M , Spelsberg T C
Published: August 2015

The multiple classes of binding sites for the progesterone-receptor complex in hen oviduct muclei were found to be of chromatin origin. The highest-affinity, and presumably most physiologically important class, is localized in oviduct chromatin and contains approx. 6000-10000 sites per nucleus. None of these sites is detected in spleen chromatin. Two new techniques were used for assaying rapidly the binding of steroid-receptor complexes to soluble deoxyribonucleoproteins in vito. The extent of high-affinity binding by the nucleo-acidic protein fraction from spleen chromatin is as great as that by the nucleo-acidic protein from oviduct chromatin. Consequently the tissue-specific nuclear binding of the progesterone receptor is found not to be a consequence of the absence of the nuclear binding sites (acceptors) from chromatin of non-target tissue (spleen), but rather a result of complete masking of these sites. In the target-tissue (oviduct) chromatin, approx. 70% of the high-affinity acceptor sites are also masked. Acidic proteins, and not histones, appear to be responsible for the masking of these acceptor sites. In addition, acidic proteins represent (or at least are an essential part of) these high-affinity sites in the oviduct nucleus. Pure DNA displays a few high-and many low-affinity binding sites. In support of previous work with immature chicks, the acidic protein fraction of the nucleo-acidic results thus support the hypotheis that protein complexed with DNA, and not DNA alone, represent the high-affinity binding sites for the steroid-receptor complexes in nuclear chromatin. The lower-affinity classes of binding sites may represent DNA and/or other nuclear components.
Long-term effects of a high glucose concentration on cyclic nucleotide phosphodiesterase activity in mouse pancreatic islets maintained in tissue culture
Authors: Berne C , Andersson A
Published: August 2015

It has been suggested that the stimulatory effect of glucose on insulin release may be mediated by the adenylate cyclase-cyclic AMP phosphodiesterase system. In this study it was found that exposure of isolated pancreatic islets to an elevated extracellular glucose concentration for 1 week in vitro caused an increase of the cyclic AMP phosphodiesterase activity in the islet cells. These and previous data indicate that there is an increased turnover of cyclic AMP in B-cells exposed for a prolonged time to a high extracellular glucose concentration, which also causes an increased turnover rate of insulin.
Regulation of phosphatidylcholine synthesis in rat liver endoplasmic reticulum
Authors: Sribney M , Knowles C L , Lyman E M
Published: August 2015

The biosynthesis of phosphatidylcholine in rat liver microsomal preparations catalysed by CDP-choline-1,2-diacylglycerol cholinephosphotransferase (EC 2.7.8.2) was inhibited by a combination of ATP and CoA or ATP and pantetheine. ATP alone at high concentrations (20 mM) inhibits phosphatidylcholine formation to the extent of 70%. In the presence of 0.1 mM-CoA, ATP (2 mM) inhibits to the extent of 80% and in the presence of 1 mM-pantetheine to the extent of 90%. ADP and other nucleotide triphosphates in combination with either CoA or pantetheine are only 10-30% as effective in inhibiting phosphatidylcholine synthesis. AMP(CH2)PP [adenosine 5′-(alphabeta-methylene)triphosphate] together with CoA inhibits to the extent of 59% and with pantetheine by 48%. AMP-P(CH2)P [adenosine 5′-(betagamma-methylene)triphosphate] together with either CoA or pantetheine had no significant effect on phosphatidylcholine formation. Other closely related derivatives of pantothenic acid were without effect either alone or in the presence of ATP, as were thiol compounds such as cysteine, homocysteine, cysteamine, dithiothreitol and glutathione. Several mechanisms by which this inhibition might take place were ruled out and it is concluded that ATP together with either CoA or pantetheine interacts reversibly with phosphatidylcholine synthetase to cause temporarily the inhibition of phosphatidylcholine formation.
Glucagon-like and insulin-like hormones of the insect neurosecretory system
Authors: Tager H S , Markese J , Kramer K J , Speirs R D , Childs C N
Published: August 2015

Aqueous extracts of corpus cardiacum-corpus allatum complexes of the adult tobacco hornworm Manduca sexta produced both glycogenolysis and hypoglycaemia when injected into the larval form of the same species. Application of specific radioimmuno assays to similar extracts showed also that these gland complexes contain both glucagon-like and insulin-like peptides. Further, the partially purified immunoreactive peptides had the expected biological activities. The former decreased the glycogen content of the fatbody and the latter the circulating trehalose levels in recipient animals. These results suggest the existence of hormones in these invertebrates having both biological and structural similarities to vertebrate insulin and glucagon.