bluuebunny/crossref_metadata_2025 huggingface dataset:
Subcellular distribution of Δ5-3 β-hydroxy steroid dehydrogenase in the granulosa cells of the domestic fowl (Gallus domesticus)
Authors: Armstrong D G
Published: August 2015

1. The distribution of 3 beta-hydroxy steroid dehydrogenase was examined in the subcellular fractions of granulosa cells collected from the ovary of the domestic fowl. 2. 3 beta-hydroxy steroid dehydrogenase activity was observed in the mitochondrial (4000g for 20min) and microsomal (105 000g for 120min) fractions. 3. Approximately three times more 3 beta-hydroxy steroid dehydrogenase activity was associated with the cytochrome oxidase activity (a mitochondrial marker enzyme) in anteovulatory-follicle granulosa cells than with that of the postovulatory follicle. 4. Comparison of the latent properties of mitochondrial 3 beta-hydroxy steroid dehydrogenase with those of cytochrome oxidase and isocitrate dehydrogenase indicated that 3 beta-hydroxy steroid dehydrogenase is located extramitochondrially. 5. This apparent distribution of 3 beta-hydroxy steroid dehydrogenase is explained on the basis that the mitochondrial activity is either an artefact caused by a redistribution in the subcellular location of the enzyme, occurring during homogenization, or by the existence of a functionally heterogeneous endoplasmic reticulum that yields particles of widely differing sedimentation properties.
The role of disulphide bonds in human intestinal mucin
Authors: Forstner Janet F. , Jabbal Inderjit , Qureshi Rauf , Kells David I. C. , Forstner Gordon G.
Published: August 2015

Goblet-cell mucin (mucin 1) was isolated and purified from human small-intestinal scrapings. After application of mucin 1 to DEAE-Bio-Gel (A) columns, most of the glycoprotein (76–94% of hexoses) was eluted in the first peak (designated mucin 2). Minor amounts of acidic glycoproteins were eluted with 0.2m- and 0.4m-NaCl in later peaks. Analyses of mucin 1 and mucin 2 revealed mucin 2 to be a monodisperse highly glycosylated glycoprotein containing 6.3% by wt. of protein, N-acetylgalactosamine, N-acetylglucosamine, galactose and fucose. Mucin 1 was similar in composition, but was polydisperse and contained more protein (12.3% by wt.) as well as N-acetylneuraminic acid. Analytical CsCl-gradient ultracentrifugation showed both mucin 1 and mucin 2 to have a major component with an average buoyant density of 1.47000g/ml. Mucin 1 also contained a slightly less-dense minor glycoprotein component. After exhaustive reduction and alkylation mucin 1 retained its major component, but partly dissociated into two lighter glycoprotein components. Mucin 2, in contrast, did not change its density distribution after reduction. Band ultracentrifugation in 2H2O-containing iso-osmotic buffers showed that mucin 1 contained a major fast-sedimenting component (so=37±2S), and a minor amount of a slower-sedimenting component. After reduction there was an increased quantity of the latter component, for which an so value of 14.5S was calculated. In contrast, mucin 2 was unaltered by reduction (so=33±2S). These findings indicate that the major component of goblet-cell mucin (mucin 2) does not dissociate after S–S-bond reduction, and thus does not apparently rely for its polymeric structure on the association of subunits through covalent disulphide bonds. However, the effects of reduction on mucin 1 suggest that in the native mucin intramolecular disulphide bonds in the minor glycoproteins may stabilize their structure, permitting secondary non-covalent interactions to develop with the major dense mucin (mucin 2) protein.
Inhibition of degradation of insulin by ophthalamic acid and by a bovine pancreatic proteinase inhibitor
Authors: Offord R E , Philippe J , Davis J G , Halban P A , Berger M
Published: August 2015

We have previously observed that, on subcutaneous administration, a significant proportion of insulin is degraded at the site of injection. The present paper reports that the degradative activity of slices of rat adipose tissue can be inhibited in vitro by ophthalmic acid, a natural analogue of glutathione, and by bovine pancreatic proteinase inhibitor, whereas it is increased by the addition of reduced glutathione.
Tri-iodothyronine-induced increase in rat liver nuclear thyroid-hormone receptors associated with increased mitochondrial α-glycerophosphate dehydrogenase activity
Authors: Hamada S , Nakamura H , Nanno M , Imura H
Published: August 2015

The effect of tri-iodothyronine injection on the nuclear tri-iodothyronine receptor (putative thyroid-hormone receptor) was examined in rat liver. Nuclear receptors were extracted from isolated nuclei with 0.4 M-KCl, and their association constants (Ka) and maximal binding capacities (Cmax.) were determined by Scatchard analyses with and without correction for the endogenous hormone. The amount of endogenous tri-iodothyronine bound to non-histone protein was estimated on the basis of the specific radio-activity of [125I]tri-iodothyronine injected 2 h before the rats were killed. It was demonstrated that Cmax. of the nuclear receptors was 2.5-fold higher in severely hyperthyroid than in hypothyroid rats. However, irrespective of the thyroid status, the Ka of the receptors remained unchanged when corrected for endogenous tri-iodothyronine bound to non-histone protein. The validity of the correction was supported by experiments in vitro in which nuclear receptors were preincubated with unlabelled tri-iodothyronine. The increase in Cmax. of nuclear receptors was directly related to mitochondrial alpha-glycerophosphate dehydrogenase activity. These results suggest a hormonal modulation of the nuclear receptors which is associated with hormonal action.
Protein phosphorylation in human peripheral blood lymphocytes. Phosphorylation of endogenous plasma membrane and cytoplasmic proteins
Authors: Chaplin David D. , Wedner H. James , Parker Charles W.
Published: August 2015

Phosphorylation of endogenous proteins in subcellular fractions of human peripheral-blood lymphocytes was studied by one- and two-dimensional polyacrylamide-gel electrophoresis. Studies using extensively purified subcellular fractions indicated that the endogenous phosphorylating activity in the particulate fractions was derived primarily from the plasma membrane. Electrophoresis of 32P-labelled subcellular fractions in two dimensions [O'Farrell (1975) J. Biol. Chem.250, 4007–4021] provided much greater resolution of the endogenous phosphoproteins than electrophoresis in one dimension, facilitating their excision from gels for quantification of 32P content. More than 100 cytoplasmic and 20 plasma-membrane phosphorylated species were observed. Phosphorylation of more than 10 cytoplasmic proteins was absolutely dependent on cyclic AMP. In the plasma membrane, cyclic AMP-dependent phosphoproteins were observed with mol.wts. of 42000, 42000, 80000 and 90000 and pI values of 6.1, 6.3, 6.25 and 6.5 respectively. Phosphorylation of endogenous cytoplasmic and plasma-membrane proteins was rapid with t½=5–12s at 25°C. Between 40 and 70% of the 32P was recovered as phosphoserine and phosphothreonine when acid hydrolysates of isolated plasma-membrane phosphoproteins were analysed by high-voltage paper electrophoresis. The presence of cyclic AMP-dependent protein kinase and endogenous phosphate-acceptor proteins in the plasma membranes of lymphocytes provides a mechanism by which these cells might respond to plasma-membrane pools of cyclic AMP generated in response to stimulation by mitogens or physiological modulators of lymphocyte function.
The maximum activities of enzymes that are involved in substrate cycles in liver and muscle of obese mice
Authors: Newsholme E A , Brand K , Lang J , Stanley J C , Williams T
Published: August 2015

The maximum activities of some dehydrogenases and enzymes involved in substrate cycles are increased in both liver and muscle of obese mice. This suggests that endocrine control rather than maximum capacity of the activities of enzymes constituting such cycles is impaired in obese mice.
Lipid environment of gastric potassium ion-stimulated adenosine triphosphatase
Authors: Sen P C , Ray T K
Published: August 2015

The K+-stimulated ATPase associated with the purified gastric microsomal fraction can be completely inactivated by treatment with 15% (v/v) ethanol for 60s at 37 degrees C, but not at 25 degrees C. Sequential exposure of the microsomal fraction to 15% ethanol at 25 degrees C and 37 degrees C caused release of 2.5% and 2.9% of the total membrane phospholipids respectively. Restoration of the enzyme activity was achieved by sonication with phosphatidylcholine in the presence of Mg2+, K+ and ATP, which were essential for the reconstitution. Our data suggest that the phospholipids extracted by 15% ethanol at 37 degrees C are derived primarily from the immediate lipid environment of the enzyme, and ATP, together with the metal ions, helps the partially delipidated enzyme to retain the appropriate configuration for the subsequent reconstitution.
Polyamines and cellular adenosine 3′ :5′-cyclic monophosphate
Authors: Clô C , Caldarera C M , Tantini B , Benalal D , Bachrach U
Published: August 2015

The effect of polyamines on the cellular concentrations of cyclic AMP was studied. It was shown that 1 microM-spermine caused a decrease in cyclic AMP in chick-embryo heart cells, chick-embryo fibroblasts, neuroblastoma, glioma and neuroblastoma-glioma hybrid cells, grown in culture. A similar decrease was observed when polyamines were added to cells in the presence of a phosphodiesterase inhibitor or after stimulating the cells with various hormones. Noradrenaline was used in cultures of heart cells, prostaglandin E1 and adenosine for neuroblastoma and neuroblastoma-glioma hybrids, whereas isoproterenol was used for the stimulation of glioma cells. Polyamines at higher concentrations were either without effect or caused a slight increase in cyclic AMP. Spermidine (10 microM) also caused a decrease in cellular cyclic AMP, as did 0.1 microM-putrescine. It is suggested that the effect of polyamines on cellular cyclic AMP may be explained by the effect of these polycations on the activity of cellular phosphodiesterase.
Corticotropin regulation of the synthesis of a specific rat adrenal cytosolic protein. Effects of hypophysectomy and actinomycin D
Authors: Dazord Alice , Gallet Dominique , Cohen Helene , Saez Jose M.
Published: August 2015

The mechanism of corticotropin stimulation of the synthesis of a specific rat adrenal cytosolic protein was investigated. This protein (protein E) has a mol.wt. of approx. 30000. It is detected by polyacrylamide-gel electrophoresis of cytosol prepared from adrenal slices from rats treated with corticotropin in vivo and control rats, the slices being incubated with [3H]- and [14C]-leucine respectively. In rats 1–15 days after hypophysectomy, corticotropin, like dibutyryl cyclic AMP, induces an increase in protein E similar to that induced in control rats, even though both compounds no longer stimulate total protein synthesis. Corticotropin stimulation of protein E synthesis is mediated by cyclic AMP but not by corticosterone, since aminoglutethimide, a steroidogenic inhibitor, does not affect corticotropin stimulation, and dexamethasone alone has no effect. Actinomycin D, when injected in vivo 1h before or after corticotropin injection, prevents the effect of corticotropin on protein E synthesis, which is interpreted as evidence that mRNA synthesis is necessary for the stimulation of protein E synthesis. When injected more than 2h after corticotropin, actinomycin D does not prevent corticotropin stimulation of protein E synthesis, but completely blocks corticotropin stimulation of total protein synthesis. This is interpreted as meaning that, after stimulation of mRNA coding for protein E, corticotropin has no effect on the synthesis of protein E. On the other hand, corticotropin stimulation of protein E synthesis persists after hypophysectomy even though it no longer stimulates total protein synthesis. These data suggest that the factor(s) involved in the synthesis of protein E are more stable than those involved in total protein synthesis.
Biosynthesis and degradation of prolactin in the rat anterior pituitary gland. Time course of incorporation of label in vitro and evidence for rapid degradation
Authors: Shenai R , Wallis M
Published: August 2015

Biosynthesis of prolactin was studied in anterior pituitary glands from female rats, incubated in vitro. In this system [3H]leucine was incorporated into pituitary proteins, including somatotropin (growth hormone) and prolactin. The rate of uptake of label into prolactin (and to a lesser extent into total protein) slowed considerably during the first 2 h of incubation, although the rate of uptake into somatotropin was constant for 8 h. The most probable explanation for this apparent decrease in the rate of prolactin synthesis is degradation of prolactin in the gland. Degradation of this hormone was also demonstrated by incubating prelabelled pituitaries in unlabelled medium and following the content of labelled prolactin, and by studying the hormonal content of pituitary glands (by radioimmunoassay) before and after incubation. Degradation of prolactin appears to be much more rapid than that of somatotropin, and may represent a physiological mechanism whereby over-accumulation of prolactin is prevented when secretion of the hormone has been rapidly switched off.
A study of the glycerol phosphate acyltransferase and dihydroxyacetone phosphate acyltransferase activities in rat liver mitochondrial and microsomal fractions. Relative distribution in parenchymal and non-parenchymal cells, effects of N-ethylmaleimide, palmitoyl-coenzyme A concentration, starvation, adrenalectomy and anti-insulin serum treatment
Authors: Bates E J , Saggerson E D
Published: August 2015

1. GPAT (glycerol phosphate acyltransferase) and DHAPAT (dihydroxyacetone phosphate acyltransferase) activities were measured both in subcellular fractions prepared from fed rat liver and in whole homogenates prepared from freeze-stopped pieces of liver. 2. GPAT activity in mitochondria differed from the microsomal activity in that it was insensitive to N-ethylmaleimide, had a higher affinity towards the palmitoyl-CoA substrate and showed a different response to changes in hormonal and dietary status. 3. Starvation (48 h) significantly decreased mitochondrial GPAT activity. The ratio of mitochondrial to microsomal activities was also significantly decreased. The microsomal activity was unaffected by starvation, except after adrenalectomy, when it was significantly decreased. Mitochondrial GPAT activity was decreased by adrenalectomy in both fed and starved animals. 4. Acute administration of anti-insulin serum significantly decreased mitochondrial GPAT activity after 60 min without affecting the microsomal activity. 5. A new assay is described for DHAPAT. The subcellular distribution of this enzyme differed from that of GPAT. The highest specific activity of DHAPAT was found in a 23 000 gav. pellet obtained by centrifugation of a post-mitochondrial supernatant. This fraction also contained the highest specific activity of the peroxisomal marker uricase. DHAPAT activity in mitochondrial fractions or in the 23 000 gav. pellet was stimulated by N-ethylmaleimide, whereas that in microsomal fractions was slightly inhibited by this reagent. The GPAT and DHAPAT activities in mitochondrial fractions had a considerably higher affinity for the palmitoyl-CoA substrate. 6. Total liver DHAPAT activity was significantly decreased by starvation (48 h), but was unaffected by administration of anti-insulin serum. 7. The specific activities of GPAT and DHAPAT were lower in non-parenchymal cells compared with parenchymal cells, but the GPAT/DHAPAT ratio was 5–6-fold higher in the parenchymal cells.
Biosynthesis of ethylene from methionine. Isolation of the putative intermediate 4-methylthio-2-oxobutanoate from culture fluids of bacteria and fungi
Authors: Billington D C , Golding B T , Primrose S B
Published: August 2015

Methods are described for identifying the 2,4-dinitrophenylhydrazones of 4-methylthio-2-oxobutanoate by means of t.l.c., n.m.r. and mass spectroscopy. By using these methods 4-methylthio-2-oxobutanoate, a putative intermediate in the biosynthesis of ethylene from methionine, has been identified in culture fluids of Aeromonas hydrophila B12E and a coryneform bacterium D7F grown in the presence of methionine. Relative to 4-methylthio-2-oxobutanoate, the yield of 3-(methylthio)propanal (methional) from the same cultures was less than 1%. Because 4-[2H]methylthio-2-oxobutanoate was obtained from cultures grown on [Me-2H]methionine, the 4-methylthio-2-oxobutanoate must be derived from methionine. By means of t.l.c. alone, 4-methylthio-2-oxobutanoate was identified in the culture fluids of a range of bacteria, the yeast Saccharomyces cerevisiae and the fungus Penicillium digitatum. A photochemical assay developed for 4-methylthio-2-oxobutanoate shows it to be a product of the metabolism of methionine by Escherichia, Pseudomonas, Bacillus, Acinetobacter, Aeromonas, Rhizobium and Corynebacterium species.
Purification and properties of Klebsiella aerogenes
d-arabitol dehydrogenase
Authors: Neuberger M S , Patterson R A , Hartley B S
Published: August 2015

An Escherichia coli K12 strain was constructed that synthesized elevated quantities of Klebsiella aerogenes D-arabitol dehydrogenase; the enzyme accounted for about 5% of the soluble protein in this strain. Some 280 mg of enzyme was purified from 180 g of cell paste. The purified enzyme was active as a monomer of 46,000 mol.wt. The amino acid composition and kinetic constants of the enzyme for D-arabitol and D-mannitol are reported. The apparent Km for D-mannitol was more than 3-fold that for D-arabitol, whereas the maximum velocities with both substrates were indistinguishable. The enzyme purified from the E. coli K12 construct was indistinguishable by the criteria of molecular weight, electrophoretic mobility in native polyacrylamide gel and D-mannitol/D-arabitol activity ratio from D-arabitol dehydrogenase synthesized in wild-type K. aerogenes. Purified D-arabitol dehydrogenase showed no immunological cross-reaction with K. aerogenes ribitol dehydrogenase. During electrophoresis in native polyacrylamide gels, oxidation by persulphate catalysed the formation of inactive polymeric forms of the enzyme. Dithiothreitol and pre-electrophoresis protected against this polymerization.
Preparation and properties of bilirubin photoisomers
Authors: Stoll M S , Zenone E A , Ostrow J D , Zarembo J E
Published: August 2015

Polar photoisomers of bilirubin were formed by irradiation of bilirubin in chloroform solution in the absence of O2. Two pairs of compounds were isolated with molecular weights identical with bilirubin. One pair reverted to bilirubin in polar media and gave chemical reactions similar to bilirubin; the other pair were not reconverted into bilirubin by chemical means and gave reactions distinct from those of bilirubin. However, both groups were reconverted into bilirubin by irradiation in chloroform solution in the absence of O2. The probable role of these photoisomers in the catabolism of bilirubin during phototherapy of neonatal jaundice is discussed.
Comparison of radioactive peptides obtained from specifically labelled human renal and placental alkaline phosphatases
Authors: Whitaker K B , Moss D W
Published: August 2015

Peptides with different chromatographic and electrophoretic properties were obtained from human placental and renal alkaline phosphatases by tryptic digestion of the enzymes labelled with radioactive orthophosphate at their active centres. These results provide structural evidence for the distinct genetic origins of the two isoenzymes that had previously been inferred from their different properties and from the observed phenotypic variation of placental phosphatase.
Purification and properties of plaice metallothionein, a cadmium-binding protein from the liver of the plaice (Pleuronectes platessa)
Authors: Overnell J , Coombs T L
Published: August 2015

A low-molecular-weight protein induced in the liver of the plaice (Pleuronectes platessa) by exposure to cadmium was purified and characterized. It is closely similar to mammalian metallothioneins in all of its properties in that it is a single-chain cadmium-binding protein of approx. 7000 mol.wt. with a high cysteine content (31 mol%) and no aromatic amino acid residues. The thiol groups of the cysteine residues complex with the cadmium in a SH/Cd molar ratio of 3:1 and produce a characteristic absorption maximum at 250 nm. Unlike the mammalian metallothioneins, however, metal analyses reveal only traces of zinc and copper in addition to cadmium. The presence of carbohydrate previously assumed from a positive reaction with periodic acid/Schiff reagent has now been disproved, and the positive reaction attributed to interaction with the thiol groups in the protein.
Specificity for guanine nucleotide activation and stabilization of rabbit cardiac adenylate cyclase
Authors: Snyder Floyd F. , Carter Robert J.
Published: August 2015

These studies examined the structural specificity for guanine nucleotide-facilitated hormonal activation and guanine nucleotide stabilization of cardiac adenylate cyclase. 1. The phosphonate analogues of GTP, p[CH2]ppG (guanosine 5′-[βγ-methylene]-triphosphate) and pp[CH2]pG (guanosine 5′-[αβ-methylene]triphosphate), were the most effective activators of adenylate cyclase. Other nucleotides producing significant activation (P<0.01) were, in decreasing order of activation: ITP, GDP, GMP, GTP, XTP, CTP, p[NH]ppG (guanosine 5′-[βγ-imido]triphosphate), dGTP and 2′-O-methyl-GTP. Guanosine, cyclic GMP, UTP and ppppG (guanosine tetraphosphate) had no effect, and 7-methyl-GTP caused a decrease in the activity. 2. Preincubation of membranes at 37°C for 15min before assay at 24°C produced an 80% decrease in adenylate cyclase activity, and preincubation with p[CH2]ppG and pp[CH2]pG protected and resulted in a net increase in activity. Other nucleotides that completely or partially preserved activity in decreasing order of effectiveness were p[NH]ppG, GDP, GTP, dGTP, ITP, ppppG, 2′-O-methyl-GTP, GMP, CTP and XTP. Several compounds had no effect, including guanosine, cyclic GMP and UTP, whereas preincubation with 7-methyl-GTP produced a further decrease (P<0.05) in activity. 3. The concentration-dependence for activation and stabilization by the naturally occurring guanine nucleotides was examined in the absence of a regenerating system and revealed GMP to have no stabilizing effect and to be less potent than either GDP or GTP in activating adenylate cyclase. 4. A significant correlation (r=0.90) was found between the properties of activation and stabilization for the compounds examined. These findings are consistent with there being a single nucleotide site through which both the activation and stabilization of adenylate cyclase are mediated.
Limited proteolysis of complement components C2 and factor B. Structural analogy and limited sequence homology
Authors: Kerr M A
Published: August 2015

A method is described for the simultaneous purification of milligram quantities of complement components C2 and Factor B. Both products are homogeneous by the criteria of polyacrylamide-gel electrophoresis and N-terminal sequence analysis. Component C2 is cleaved by serine proteinase C1s at an X-Lys bond to give fragment C2a (approx. mol.wt. 74000) and fragment C2b (approx. mol.wt. 34000). The two fragments can be separated by gel filtration without the need for reducing or denaturing agents. Fragment C2b represents the N-terminal end of the molecule. Similar results were seen on cleavage of Factor B by Factor D in the presence of component C3. Again two non-covalently linked fragments are formed. The smaller, fragment Ba (approx. mol.wt. 36,000),) has threonine as the N-terminal residue, as does Factor B; the larger, fragment Bb (approx. mol. wt. 58000), has lysine as the N-terminal residue. A similar cleavage pattern is obtained on limited proteolysis of Factor B by trypsin, suggesting an Arg-Lys-or Lys-Lys bond at the point of cleavage. Although component C2 and Factor B show no apparent N-terminal sequence homology, a limited degree of sequence homology is seen around the sites of proteolytic cleavage.
Relationship between activity of hepatic 3-hydroxy-3-methylglutaryl-coenzyme A reductase and secretion of very-low-density-lipoprotein cholesterol by the isolated perfused liver and in the intact rat
Authors: Goh E H , Heimberg M
Published: August 2015

The hepatic output of triacylglycerol and cholesterol from very-low-density lipoprotein (VLD lipoprotein), and the activity of 3-hydroxy-3-methylglutaryl-coenzyme A reductase were compared in the isolated perfused rat-liver preparation and in the intact rat. The output of triacylglycerol and cholesterol from VLD lipoprotein by the perfused liver was stimulated by oleate concomitant with stimulation of hepatic microsomal hydroxymethylglutaryl-coenzyme A reductase activity. In the intact animal treated with Triton WR-1339, the magnitude of secretion of triacylglycerol and cholesterol from VLD lipoprotein coincided with the diurnal rhythm of hepatic hydroxymethylglutaryl-coenzyme A reductase activity, which was maximal at 24:00 h and minimal at 12:00 h. These observations suggest that the stimulation of the reductase and of the secretion of cholesterol from VLD lipoprotein by non-esterified fatty acids, as observed with the isolated perfused rat liver preparation in vitro, may also be an important physiological mechanism in vivo. Hepatic cholesterogenesis may be stimulated under conditions conductive to the secretion of the VLD lipoprotein, the primary transport form for triacylglycerol in the postabsorptive state.
Metabolism of palmitate in perfused rat liver. Effect of ethanol in livers from rats fed on a high-fat diet with or without ethanol
Authors: Kondrup Jens , Lundquist Frank , Damgaard Stig E.
Published: August 2015

1. Rats were treated for 4 weeks with liquid diets that contained, on the basis of energy content, 35% fat, 18% protein and 47% carbohydrate (high-fat diet) or 35% fat, 18% protein, 11% carbohydrate and 36% ethanol (high-fat/ethanol diet). 2. The livers were perfused with 1mm-[1-14C]palmitate and with 0, 10mm- or 80mm-ethanol. The oxidation and esterification of palmitate was measured. Two subcellular pools of triacylglycerol were separated; one contained triacylglycerol from cytoplasmic lipid droplets and the other contained triacylglycerol from the endoplasmic reticulum and Golgi apparatus. 3. In the presence of ethanol, liver from rats fed on the high-fat diet esterified about 70% of the [1-14C]palmitate taken up compared with 90% in liver from rats fed chow (containing 11% fat on the basis of energy content). Compared with chow diet the high-fat diet did not potentiate the effect of ethanol on storage of [1-14C]palmitate in hepatic triacylglycerol. The relation between the fat content of the diet and the degree of fatty liver induced by by ethanol [Lieber & DeCarli (1970) Am. J. Clin. Nutr.23, 474–478] is discussed. 4. The ethanol-containing diet increased the hepatic content of triacylglycerol 4-fold and the increase was exclusively found in the fraction suggested to contain lipid from cytoplasmic lipid droplets. The ethanol-induced fatty liver, perfused with ethanol, esterified and oxidized palmitate at rates that were quite similar to the rates found in high-fat control livers perfused without ethanol. This suggests that the fatty liver had adapted to the presence of ethanol with respect to palmitate metabolism. 5. O2 and ethanol uptake by the livers were not affected by the ethanol-containing diet.
Characterization of subcellular components in synchronized hepatoma cells as a function of the cell cycle
Authors: Quintart Joël , Bartholeyns Jacques , Baudhuin Pierre
Published: August 2015

The specific activity and subcellular distribution of marker enzymes for the main subcellular components were analysed in homogenates of synchronized hepatoma cells (Morris 7288c), obtained by selective detachment at mitosis combined with a metaphase block with Colcemid. Markers for lysosomes, mitochondrial outer membrane, plasma membrane and cytosol are synthesized throughout the cycle at the same rate as the bulk of cellular protein. Larger variations are observed for a Golgi marker; after a decrease around mitosis, the specific activity of galactosyltransferase increases steadily from middle G1-phase on, and at the end of G2-phase it is nearly twice that observed at the beginning of G1-phase. Our results show that synthesis of cytochrome oxidase may occur preferentially in G2-phase. Large modifications of the density distribution of lysosomes are observed during the cell cycle; the median equilibrium density of lysosomal markers decreases in G1-phase, and some increase in soluble activity occurs at the same time. Reverse changes occur progressively during S- and G2-phases. At mitosis, Golgi galactosyltransferase shows a more dispersed distribution, and modifications in the density distribution of endoplasmic-reticulum NADPH–cytochrome c reductase are observed. The latter can be most easily explained by a detachment of ribosomes from endoplasmic-reticulum membranes. No significant modifications occur in mitochondrial and plasma-membrane markers.
Influence of neonatal hypothyroidism on the development of ketone-body-metabolizing enzymes in rat brain
Authors: Patel M S
Published: August 2015

Neonatal hypothyroidism markedly retarded the postnatal development of three ketone-body-metabolizing enzymes in rat brain during the first 4 weeks after birth. In contrast with normal animals, the brains of hypothyroid rats did not show decreases in the activities of these enzymes during the immediate postweaning period. The activities of ketone-body-oxidizing enzymes were markedly diminished in both non-synaptic and synaptic mitochondria isolated from 4-week-old hypothyroid rats compared with age-matched normal animals.
Transient complexes. A structural model for the activation of adenylate cyclase by hormone receptors (guanine nucleotides/irradiation inactivation)
Authors: Martin B. Richard , Stein Janet M. , Kennedy Edwina L. , Doberska Christine A. , Metcalfe James C.
Published: August 2015

1. The irradiation-inactivation procedure was used to study changes in the state of association of the protein components of adenylate cyclase in intact rat liver plasma membranes by measurement of alterations in the target size determined from the catalytic activity of the enzyme. 2. A decrease in target size at 30°C in response to p[NH]ppG (guanosine 5′-[βγ-imido]triphosphate) or GTP was demonstrated, which we take to reflect the dissociation of a regulatory subunit. The effect of GTP is potentiated by glucagon. This effect is not observed at 0°C. 3. An increase in target size was observed in response to glucagon in the absence of guanine nucleotides, which we take to reflect the association of glucagon receptor with adenylate cyclase. 4. We propose a model for the activation of adenylate cyclase by glucagon in which the binding of the hormone to its receptor causes an initial association of the receptor with the catalytic unit of the enzyme and a regulatory subunit to form a ternary complex. The subsequent activation of the adenylate cyclase results from the dissociation of the ternary complex to leave a free catalytic unit in the activated state. This dissociation requires the binding of a guanine nucleotide to the regulatory subunit. 5. The effects of variation of temperature on the activation of adenylate cyclase by glucagon and guanine nucleotides were examined and are discussed in relation to the irradiation-activation data. 6. The effectiveness of hormones, guanine nucleotides and combinations of hormone and guanine nucleotides as activators of adenylate cyclase in both rat liver and rat fat-cell plasma membranes was studied and the results are discussed in relation to the model proposed, which is also considered in relation to the observations published by other workers.
Insulin action on adipocytes. Evidence that the anti-lipolytic and lipogenic effects of insulin are mediated by the same receptor
Authors: Thomas S H L , Wisher M H , Brandenburg D , Sönksen P H
Published: August 2015

1. The dose-response relationships of insulin stimulation of lipogenesis and inhibition of lipolysis were studied simultaneously by using rat adipocytes to determine whether these different effects of insulin are mediated through the same or different sets of receptors. 2. The sensitivity (defined as the concentration of insulin required to produce a half-maximal effect) of the stimulated lipogenic response to insulin was not significantly different from the sensitivity of the anti-lipolytic response to insulin. The addition of different adrenaline and glucose concentrations did not alter the half-maximal concentration of insulin required to inhibit lipolysis. 3. The specificities of the lipogenic and antilipolytic responses were studied by using insulin analogues. The sensitivities of the lipogenic and anti-lipolytic responses were the same for five chemically modified insulins and hagfish insulin, which have potencies compared with bovine insulin of between 3 and 90%. 4. Starving rats for 48h significantly increased the sensitivities of both the antilipolytic and lipogenic responses to insulin, but the changes in the sensitivities of both lipogenesis and anti-lipolysis returned to that of fed rats. 5. We conclude that insulin stimulates lipogenesis and inhibits lipolysis over the same concentration range. These observations provide powerful evidence that the different effects of insulin are mediated through the same set of receptors.
Stoichiometry of carbon dioxide release and oxygen uptake during glycine oxidation in mitochondria isolated from spinach (Spinacia oleracea) leaves
Authors: Arron G P , Spalding M H , Edwards G E
Published: August 2015

Mitochondria isolated from spinach (Spinacia oleracea) leaves oxidized glycine with a stoichiometry of CO2 evolution to O2 uptake of 2 : 1. In the absence of added substrate, the mitochondria exhibited an extremely low endogenous rate of O2 uptake.
Studies of acidosis in the ischaemic heart by phosphorus nuclear magnetic resonance
Authors: Garlick P B , Radda G K , Seeley P J
Published: August 2015

1. Phosphorus-nuclear-magnetic-resonance measurements were made on perfused rat hearts at 37 degrees C. 2. With the improved sensitivity obtained by using a wide-bore 4.3 T superconducting magnet, spectra could be recorded in 1 min. 3. The concentrations of ATP, phosphocreatine and Pi and, from the position of the Pi resonance, the intracellular pH (pHi) were measured under a variety of conditions. 4. In a normal perfused heart pHi = 7.05 +/- 0.02 (mean +/- S.E.M. for seven hearts). 5. During global ischaemia pHi drops to 6.2 +/- 0.06 (mean +/- S.E.M.) in 13 min in a pseudoexponential decay with a rate constant of 0.25 min-1. 6. The relation between glycogen content and acidosis in ischaemia is studied in glycogen-depleted hearts. 7. Perfusion of hearts with a buffer containing 100 mM-Hepes before ischaemia gives a significant protective effect on the ischaemic myocardium. Intracellular pH and ATP and phosphocreatine concentrations decline more slowly under these conditions and metabolic recovery is observed on reperfusion after 30min of ischaemia at 37 degrees C. 8. The relation between acidosis and the export of protons is discussed and the significance of glycogenolysis in ischaemic acid production is evaluated.
The isolation and characterization of a sequence-specific endonuclease from Anabaena subcylindrica
Authors: Hughes S G , Bruce T , Murray K
Published: August 2015

An endonuclease, AsuI, was isolated from extracts of Anabaena subcylindrica on the basis of gel-electrophoretic analysis of digests of bacteriophage-lambda DNA with the paritally purified extracts. The enzyme requires Mg2+, but no other cofactors. Endonuclease AsuI recognizes the interrupted tetranucleotide sequence: (Formula: see tex), and breaks the phosphodiester bonds indicated by the arrows to leave single-stranded trinucleotide projections at the 5'-termini of the DNA fragments.
Analysis of bilirubin and bilirubin mono- and di-conjugates. Determination of their relative amounts in biological samples
Authors: Blanckaert N
Published: August 2015

1. A novel method for determination of the relative amounts of unconjugated bilirubin and sugar mono- and di-conjugates of bilirubin in biological samples, including serum, is described and illustrated by its application to the analysis of bilinoids in rat bile. 2. The method is based on specific conversion of the carbohydrate conjugates of bilirubin into the corresponding mono- or di-methyl esters by base-catalysed transesterification in methanol. Under the selected reaction conditions, unconjugated biliru-in remains intact and no dipyrrole exchange in the bilinoids is detectable; transesterification of bilirubin mono- or di-glucuronide is virtually complete (approx. 99%), and sponification is negligible (less than 1%); recovery of the pigments is approx. 95%. 3. The reaction products bilirubin and its methyl esters are separated by t.l.c. and determined spectrophotometrically; the two isomeric bilirubin-IX alpha monomethyl esters are separated and therefore can be determined individually. 4. Reference bilirubin mono- and di-methyl esters have been synthesized and characterized, and the two isomers of bilirubin-IX alpha monomethyl ester and bilirubin dimethyl ester were obtained individually, in crystalline form. 5. With this new method, virtually all bilinoids (over 99%) in normal rat bile have been found to be conjugated, with diconjugates (71%) predominating. A significantly increased proportion of monoconjugates is present in bile collected from heterozygous Gunn rats or from normal rats that were refused with large amounts of bilirubin.
Characterization of the low-temperature intermediates of the reaction of fully reduced soluble cytochrome oxidase with oxygen by electron-paramagnetic-resonance and optical spectroscopy
Authors: Clore G M , Andréasson L E , Karlsson B , Aasa R , Malmström B G
Published: August 2015

The reaction of fully reduced soluble bovine heart cytochrome oxidase with O2 at 173K was investigated by low-temperature optical and e.p.r. spectroscopy, and the kinetics of the reaction were analysed by non-linear optimization techniques. The only e.p.r. signals seen during the course of the reaction are those attributable to low-spin cytochrome a3+ and CuA2+. Quantitative analysis of e.p.r. signals shows that, at the end point of the reaction at 173K, nearly 100% of CuA is in the cupric state but only about 40% of cytochrome a is in the ferric low-spin state. The optical spectra recorded at this stage of the reaction show incomplete oxidation of haem and the absence of a 655 nm absorption band. The only reaction scheme that accounts for both the e.p.r. and optical data is a four-intermediate mechanism involving a branching pathway. The reaction is initiated when fully reduced cytochrome oxidase reacts with O2 to form intermediate I. This is then converted into either intermediate IIA or intermediate IIB. Of these, intermediate IIB is a stable end product at 173 K, but intermediate IIA is converted into intermediate III, which is the stable state at 173 K in this branch of the mechanism. The kinetic analysis of the e.p.r. data allows the unambiguous assignments of the valence states of cytochrome a and CuA in the intermediates. Intermediate I contains cytochrome a2+ and CuA+, intermediate IIA contains low-spin cytochroma a3+ and CuA+, intermediate IIB contains cytochrome a2+ and CuA2+, and intermediate III contains low-spin cytochrome a3+ and CuA2+. The electronic state of the O2-binding CuBa3 couple during the reoxidation of cytochrome oxidase is discussed in terms of an integrated structure containing CuB, cytochrome a3 and O2.
Inhibition of β-lactamase of Bacillus licheniformis 749/C by compound PS-5, a new β-lactam antibiotic
Authors: Fukagawa Y , Takei T , Ishikura T
Published: August 2015

By use of a new computer-assisted u.v.-spectrophotometric assay method, the kinetic parameters of the reaction catalysed by Bacillus licheniformis 749/C beta-lactamase were re-examined and the mode of inhibition of the enzyme by compound PS-5, a novel beta-lactam antibiotic, was studied with benzylpenicillin as substrate. (1) The fundamental assay conditions for the determination of Km and V were examined in detail with benzylpenicillin as substrate. In 0.1 M-sodium/potassium phosphate buffer, pH 6.8, at 30 degrees C, initial substrate concentrations of benzylpenicillin above 0.7 mM were very likely to lead to substrate inhibition. The Km value of the enzyme for benzylpenicillin at initial concentrations from 1.96 to 0.07 mM was calculated to be 97-108 microM. (2) The Km values of the enzyme for 6-aminopenicillanic acid, ampicillin and cephaloridine were found to be 25, 154-161 and 144-161 microM respectively. (3) Compound PS-5 was virtually unattacked by Bacillus licheniformis 749/C beta-lactamase. (4) The activity of the enzyme was diminished by compound PS-5, to extents depending on the duration of incubation and the concentration of the inhibitor. The rate of inactivation of the enzyme by compound PS-5 followed first-order kinetics. (5) In an Appendix, a new computer-assisted u.v.-spectrophotometric enzyme assay method, in which a single reaction progress curve of time-absorbance was analysed by the integrated Michaelis-Menten equation, was devised for the accurate and precise determination of the kinetic constants of beta-lactamase. For conversion of absorbance readings into molar substrate concentrations, the initial or final absorbance reading that was independent of the reaction time was used as the basis of calculation. In calculation of Km and V three systematic methods of data combination were employed for finer analysis of the reaction progress curve. A list of the computer program named YF6TAIM is obtainable from the author on request or as Supplementary Publication SUP 50100 (12 pages) from the British Library Lending Division, Boston Spa, Wetherby, West Yorkshire LS23 7BQ, U.K., on the terms indicated in Biochem. J. (1978) 169, 5.
The glycoprotein of measles virus. External radioactive labelling of its carbohydrate and partial characterization of the glycopeptide
Authors: Anttonen O , Jokinen M , Salmi A , Vainionpää R , Gahmberg C G
Published: August 2015

Measles virus was propagated in VERO cells and purified from the culture supernatants by two successive tartrate-density-gradient centrifugations. Surface carbohydrates were labelled both in vitro and in vivo with 3H after treatment with galactose oxidase/NaB3H4 or with [3H]glucosamine. The major labelled glycoprotein in measles virions had a mol.wt. of 79 000. After labelling with periodate/NaB3H4, which would result in specific labelling of sialic acid residues, the 79 000-mol.wt. glycoprotein was very weakly labelled. This suggests that there is no or a very low amount of sialic acid in the virions. Further analysis of the glycoprotein showed that galactose is the terminal carbohydrate unit in the oligosaccharide, and the molecular weight of the glycopeptide obtained after Pronase digestion is about 3000. The oligosaccharide is attached to the polypeptide through an alkali-stable bond, indicating a N-glycosidic asparagine linkage.
Active-site amino acid residues in γ-glutamyltransferase and the nature of the γ-glutamyl-enzyme bond
Authors: Elce John S.
Published: August 2015

Active-site residues in rat kidney γ-glutamyltransferase (EC 2.3.2.2) were investigated by means of chemical modification. 1. In the presence of maleate, the activity was inhibited by phenylmethanesulphonyl fluoride, and the inhibition was not reversed by β-mercaptoethanol, suggesting that a serine residue is close to the active site, but is shielded except in the presence of maleate. 2. Treatment of the enzyme with N-acetylimidazole modified an amino group, exposed a previously inaccessible cysteine residue and inhibited hydrolysis of the γ-glutamyl-enzyme intermediate, but not its formation. 3. After reaction of the enzyme successively with N-acetylimidazole and with non-radioactive iodoacetamide/serine/borate, two active-site residues reacted with iodo[14C]acetamide. One of these possessed a carboxy group, which formed a [14C]glycollamide ester, and the other was cysteine, shown by isolation of S-[14C]carboxymethylcysteine after acid hydrolysis. When N-acetylimidazole treatment was omitted, only the carboxy group reacted with iodo[14C]acetamide. 4. Isolation of the γ-[14C]glutamyl-enzyme intermediate was made easier by prior treatment of the enzyme with N-acetylimidazole. The γ-glutamyl-enzyme bond was stable to performic acid, and to hydroxylamine/urea at pH10, but was hydrolysed slowly at pH12, indicating attachment of the γ-[14C]glutamyl group in amide linkage to an amino group on the enzyme. Proteolysis of the γ-[14C]glutamyl-enzyme after performic acid oxidation gave rise to a small acidic radioactive peptide that was resistant to further proteolysis and was not identical with γ-glutamyl-ε-lysine. 5. A scheme for the catalytic mechanism is proposed.
Isolation and preliminary characterization of epithelial-specific messenger ribonucleic acids and their products during embryonic tooth development
Authors: Zeichner-David M , Weliky B G , Slavkin H C
Published: August 2015

Experiments were designed to identify and characterize tissue-specific proteins involved in the process of tooth organogenesis. Epithelial and mesenchymal proteins were extracted from intact molar organs or mechanically separated tissues obtained from 25-day New Zealand White rabbit embryos. Labelling experiments with [35S]methionine followed by radioautography or gel electrophoresis and fluorography showed the presence of label only in epithelial proteins. Most of these proteins range from 43 000 mol.wt. and higher, except for one band of approx. 16 000 mol.wt. A mRNA fraction of 16-26S was isolated by ultracentrifugation on sucrose gradients. When translated in a reticulocyte-lysate cell-free system, the mRNA obtained from intact molar organs resulted in the synthesis of three proteins, of mol.wts. 65 000, 58 000 and 43 000. A similar mRNA fraction obtained from dental-pulp mesenchyme gave only the 43 000-mol.wt. protein, indicating that the 65 000- and 58 000-mol.wt. proteins are derived from epithelial cells.
Differential effect of L-thyroxine on phospholipid biosynthesis in mitochondria and microsomal fraction
Authors: Pasquini J M , Faryna de Raveglia I , Capitman N , Soto E F
Published: August 2015

1. The action of L-thyroxine on the incorporation of radioactive choline or CDP-choline into phosphatidylcholine in vitro was explored in liver and brain microsomal fraction and mitochondria obtained from young adult rats. 2. In liver mitochondria isolated from animals treated with L-thyroxine (40 mg/kg body wt. during 6 days), the incorporation of both radioactive precursors into phosphatidylcholine was significantly decreased compared with normal controls, whereas in the total homogenate and in the microsomal fraction the incorporation was similar in the experimental and control groups. In subcellular fractions isolated from brain, the incorporation of precursors was similar in L-thyroxine-treated and normal animals. 3. Liver mitochondria isolated from normal animals incubated in vitro with CDP-choline, in the presence of different concentrations of L-thyroxine, showed also a marked decrease in the incorporation of label into phosphatidylcholine, whereas no significant changes were found in the total homogenate and in the microsomal fraction compared with control experiments. 4. The differential effect of L-thyroxine on the incorporation of radioactive precursors into phosphatidylcholine of isolated liver subcellular fractions gives further support to the hypothesis that liver mitochondria can independently synthesize part of their own phospholipids. 5. Possible mechanisms of the action of the hormone at the mitochondrial level are discussed.