bluuebunny/crossref_metadata_2025 huggingface dataset:
Lectin-binding proteins in central-nervous-system myelin. Binding of glycoproteins in purified myelin to immobilized lectins
Authors: Quarles Richard H. , McIntyre Laurence J. , Pasnak Carol F.
Published: August 2015

The capacities of immature and mature rat brain myelin, bovine myelin and human myelin to be agglutinated by soya-bean agglutinin, Ricinus communis agglutinin, wheatgerm agglutinin, and Lotus tetragonolobus agglutinin were examined. The first two lectins, which are specific for galactose and N-acetylgalactosamine, strongly agglutinated immature and mature rat myelin, weakly agglutinated bovine myelin, but did not affect human myelin. The other myelin and lectin combinations resulted in very weak or no agglutination. [3H]Fucose-labelled glycoproteins of purified adult rat brain myelin were solubilized with sodium dodecyl sulphate and allowed to bind to concanavalin A–Sepharose and each of the other lectins mentioned above, which had been immobilized on agarose. About 60% of the radioactive fucose was in glycoproteins that bound to concanavalin A–Sepharose and these glycoproteins could be eluted with solutions containing methyl α-d-mannoside and sodium dodecyl sulphate. Periodate/Schiff staining or radioactive counting of analytical gels showed that most of the major myelin-associated glycoprotein (apparent mol.wt. approx. 100000) bound to the concanavalin A, whereas the glycoproteins that did not bind were mostly of lower molecular weight. Preparative polyacrylamide-gel electrophoresis of the glycoprotein fraction that was eluted with methyl α-d-mannoside yielded a relatively pure preparation of the myelin-associated glycoprotein. Similar results were obtained with each of the other lectins, i.e. the myelin-associated glycoprotein was in the fraction that bound to the immobilized lectin. Double-labelling experiments utilizing [3H]fucose-labelled glycoproteins from adult myelin and [14C]fucose-labelled glycoproteins from 14-day-old rat brain myelin did not reveal any difference in the binding of the mature and immature glycoproteins to any of the immobilized lectins. The results in this and the preceding paper [McIntyre, Quarles & Brady (1979) Biochem. J.183, 205–212] suggest that the myelin-associated glycoprotein is one of the principal receptors for concanavalin A and other lectins in myelin, and that this property can be utilized for the purification of this glycoprotein.
Evidence for association-activation effects in reactions of papain from studies on its reactivity towards isomeric two-protonic-state reactivity probes
Authors: Brocklehurst K , Herbert J A L , Norris R , Suschitzky H
Published: August 2015

4-(N-Aminoethyl 4-pyridyl disulphide)-7-nitrobenzo-2-oxa-1,3-diazole was synthesized and evaluted as a two-protonic-state reactivity probe by kinetic study of its reactions with papain (EC 3.4.22.2) and with benzimidazol-2-ylmethanethiol. Evidence is presented to suggest that: (i) both this probe molecule and its 2-pyridyl isomer bind to papain; (ii) the binding is followed by a change in the environment of the thiol group of cysteine-25; (iii) the striking rate maximum in neutral media observed in the reaction of papain with the 2-pyridyl isomer but not with the 4-pyridyl isomer arises from association of the 2-pyridyl leaving group with the imidazolium ion of histidine-159.
Chemiluminescence of Acanthamoeba castellanii
Authors: Lloyd D , Boveris A , Reiter R , Filipkowski M , Chance B
Published: August 2015

1. Chemiluminescence of Acanthomoeba castellanii in the presence of O2 was of similar intensity in organisms harvested early or late during exponential growth [when cyanide (1 mM) stimulates or inhibits respiration respectively]. 2. Cyanide (up to 1.5 mM) stimulated photoemission in both types of organism by 250–300 photons/s per 10(7) cells above the value observed under aerobic conditions. 3. ‘Dibromothymoquinone’ (2,5-dibromo-6-isopropyl-3-methyl-p-benzoquinone) (up to 80 microM) further increased chemiluminescence. 4. Similar responses were also demonstrated in whole homogenates and in subcellular fractions; 36% of the chemiluminescence was provided by a fraction sedimenting at 100000g-min, and 20% in that fraction that was non-sedimentable at 200000g-min. 5. Mitochondrial substrates (succinate, 2-oxoglutarate, NADH) in the presence or absence of ADP and Pi or peroxisomal substrates (glycollate, urate or ethanol) gave no increases in light emission by whole homogenates or in any of the fractions. 6. It is suggested that reactions responsible for production of chemiluminescence are those primarily producing superoxide anions and leading to lipid peroxidation and singlet-oxygen formation. Photoemission enhancement and superoxide dismutase inhibition showed similar cyanide concentration-dependencies.
The effects of food deprivation and re-feeding on bovine adipose-tissue glycogen synthase
Authors: Eichner R D , Arnold R J
Published: August 2015

Bovine adipose-tissue glycogen metabolism was studied during food deprivation and re-feeding. Changes in the specific activity of adipose-tissue glycogen synthase paralleled changes in tissue glycogen content: both parameters increased during food deprivation and remained so during the first 10 days of re-feeding. The values for the A0.5 (activation constant) for glucose 6-phosphate of the freshly isolated enzyme from adipose tissue from fed and starved steers were 2.9 +/- 0.1 mM and 0.90 +/- 0.05 mM respectively. Additionally, whereas incubation of adipose-tissue extracts from fed steers did not activate endogenous glycogen synthase (through a presumed phosphoprotein phosphatase mechanism), the enzyme from starved or re-fed (up to 3 days re-feeding) steers was reversibly activated as measured by changes in the value for the A0.5 for glucose 6-phosphate. Thus activation of bovine adipose-tissue glycogen synthase during food deprivation appears to be related to expression of glycogen synthase phosphatase activity. These effects of food deprivation on bovine glycogen metabolism contrast markedly with the effects observed in rat adipose tissue.
Initiation and processing in vitro of the primary translation products of guinea-pig caseins
Authors: Craig R K , Perera P A J , Mellor A , Smith A E
Published: August 2015

1. Guinea-pig caseins synthesized in a mRNA-directed wheat-germ cell-free protein-synthesizing system represent the primary translation products, even though they appear to be of lower molecular weight when analysed by sodium dodecyl sulphate/polyacrylamide-gel electrophoresis in parallel with caseins isolated from guinea-pig milk. 2. Identification of the N-terminal dipeptide of the primary translational product of caseins A, B and C and alpha-lactalbumin showed that all shared a common sequence, which was identified as either Met-Arg or Met-Lys. 3. Procedures utilizing methionyl-tRNAfMet or methionyl-tRNAMet in the presence or absence of microsomal membranes during translation provide a rapid method of distinguishing between N-terminal processing of peptides synthesized in vitro and other post-translational modifications (glycosylation, phosphorylation), which also result in a change in mobility of peptides when analysed by sodium dodecyl sulphate/polyacrylamide-gel electrophoresis. 4. The results demonstrate that guinea-pig caseins, in common with most other secretory proteins, are synthesized with transient N-terminal ‘signal’-peptide extensions, which are cleaved during synthesis in the presence of microsomal membranes.
Relationship of prostaglandin secretion by rabbit alveolar macrophages to phagocytosis and lysosomal enzyme release
Authors: Hsueh Wei , Kuhn Charles , Needleman Philip
Published: August 2015

The phospholipids of rabbit alveolar macrophages were pulse-labelled with [14C]-arachidonic acid, and the subsequent release of labelled prostaglandins was measured. Resting macrophages released measurable amounts of arachidonic acid, the prostaglandins E2, D2 and F2α and 6-oxoprostaglandin F1α. Phagocytosis of zymosan increased the release of arachidonic acid and prostaglandins to 2.5 times the control value. In contrast, phagocytosis of inert latex particles had no effect on prostaglandin release. Indomethacin inhibited the release of prostaglandin, and, at high doses (20μg/ml), increased arachidonic acid release. Analysis of the cellular lipids showed that after zymosan stimulation the proportion of label was decreased in phosphatidylcholine, but not in other phospholipids or neutral lipids. Cytochalasin B, at a dose of 2μg/ml, inhibited the phagocytosis induced by zymosan but increased prostaglandin synthesis to 3.4 times the control. These data suggest that the stimulation of prostaglandin synthesis by zymosan is not dependent on phagocytosis. Exposure to zymosan also resulted in the release of the lysosomal enzyme, acid phosphatase. Furthermore, cytochalasin B augmented the zymosan-stimulated release of acid phosphatase at the same dose that stimulated prostaglandin synthesis. However, indomethacin, at a dose that completely inhibited prostaglandin synthesis, failed to block the lysosomal enzyme release. Thus despite some parallels between the release of prostaglandins and lysosomal enzymes, endogenous prostaglandins do not appear to mediate the release of lysosomal enzymes. The prostaglandins released from the macrophages may function as humoral substances affecting other cells.
Evidence for the rapid direct control both in vivo and in vitro of the efficiency of oxidative phosphorylation by 3,5,3′-tri-iodo-l-thyronine in rats
Authors: Palacios-Romero R , Mowbray J
Published: August 2015

1. Examination of the distribution of L-tri-iodothyronine among rat liver tissue fractions after its intravenous injection into thyroidectomized rats focused attention on mitochondria at very short times after administration. By 15 min this fraction contained 18.5% of the tissue pool; however, the content had decreased sharply by 60 min and even further over the next 3 h. By contrast, the content in all other fractions was constant or increased over 4 h. About 60% of tissue hormone was bound to soluble protein. 2. Mitochondria isolated from thyroidectomized rats showed P/O ratios that were about 50% of those found in normal controls, with both succinate and pyruvate plus malate as substrates. There was no evidence of uncoupling; the respiratory-control ratio was about 6. 3. Mitochondria isolated 15 min after injection of tri-iodothyronine into thyroidectomized rats showed P/O ratios and respiratory-control ratios that were indistinguishable from those obtained in mitochondria from euthyroid animals. The oxidation rate was, however, not restored. 4. Incubation of homogenates of livers taken from thyroidectomized animals injected with L-tri-iodothyronine before isolation of the mitochondria restored the P/O ratio to normal; by contrast, direct addition of hormone to isolated mitochondria had no effect. The role of extramitochondrial factors in rapid tri-iodothyronine action is discussed. 5. Possible mechanisms by which tri-iodothyronine might rapidly alter phosphorylation efficiency are considered: it is concluded that control of adenine nucleotide translocase is unlikely to be involved. 6. The amounts of adenine nucleotides in liver were measured both after thyroidectomy and 15 min after intravenous tri-iodo-thyronine administration to thyroidectomized animals. The concentrations found are consistent with a decreased phosphorylation efficiency in thyroidectomized animals. Tri-iodothyronine injection resulted in very significant changes in the amounts of ATP, ADP and AMP, and in the [ATP]/[ADP] ratio, consonant with those expected from an increased efficiency of ADP phosphorylation. This suggests that the changes seen in isolated mitochondria may indeed reflect a rapid response of liver in vivo to tri-iodo-thyronine.
Metabolism of [14C]diethylstilboestrol epoxide by rat liver in vitro
Authors: Jellinck P H , Bowen J H
Published: August 2015

1. The trans-epoxide of diethylstilboestrol and its pinacolone were synthesized chemically and the pinacolone shown to be formed from the epoxide by a non-enzymic process. 2. [14C]Diethylstiboestrol epoxide was converted by rat liver microsomal fraction into 4'-hydroxypropiophenone by a new type of cleavage reaction involving mono-oxygenase. Conditions for the formation of this metabolite and also water-soluble products were investigated together with the effect of inhibitors. A sex-difference in the conversion of diethylstilboestrol epoxide into 4'-hydroxypropiophenone and to polar and water-soluble products was observed. 3. Diethylstilboestrol epoxide was found to be a relatively stable compound that did not form a glutathione conjugate readily without further microsomal activation. A purified preparation of epoxide hydratase did not enhance its rate of conversion into the pinacolone. 4. Diethylstilboestrol epoxide was found to have about one-tenth the oestrogenic activity of diethylstilboestrol as measured by the increase in uterine weight or the induction of peroxidase in immature rat uteri. It was inactive as a mutagen when tested for its ability to inhibit bacteriophage phi X174 DNA viral replication. 5. The possible role of diethylstilboestrol epoxide as an intermediate in the metabolism of diethylstilboestrol and in mediating the harmful effects of this synthetic estrogen is discussed.
Purification and characterization of mouse α-lactalbumin and preparation of its antibody
Authors: Nagamatsu Y , Oka T
Published: August 2015

alpha-Lactalbumin was purified to apparent homogeneity from mouse milk by combined use of gel filtration, chromatography on DEAE-cellulose and hydroxyapatite, and concanavalin A-Sepharose affinity chromatography. Mouse alpha-lactalbumin exists in several species with different charges and in two molecular-size forms. The smaller form, which constituted over 90% of total alpha-lactalbumin, included two major and two minor species, each of which showed different electrophoretic mobility on polyacrylamide-gel electrophoresis, but gave the same single band on sodium dodecyl sulphate/polyacrylamide-gel electrophoresis in two different buffer systems and over the range 10-15% acrylamide concentrations. The molecular weight was estimated as 14 100. The two major species of the smaller form had the same amino acid composition and contained no significant amount of carbohydrate. The larger form of alpha-lactalbumin, consisting of two species with different charges, was present in a small amount (less than 10%) in the milk and was isolated by its ability to interact with concanavalin A-Sepharose. Each of the two species also gave the same single band of apparent mol.w.t 18 500 on sodium dodecyl sulphate/polyacrylamide-gel electrophoresis. However, this value may be anomalous, since this larger form appears to be glycosylated, and glycoproteins can behave anomalously on sodium dodecyl sulphate/polyacrylamide gels by binding less sodium dodecyl sulphate. All species of mouse alpha-lactalbumin from milk were active in the lactose synthase reaction and showed identical immunological properties, as determined by the mono-specific antibody prepared against the small major species. The presence of both the larger and the smaller forms, each in a percentage concentration similar to that found in milk, was also demonstrated in alpha-lactalbumin induced by hormones in organ cultureof pregnant-mouse mammary gland.
Isolation of a serum component that stimulates the spreading of cells in culture
Authors: Whateley J G , Knox P
Published: August 2015

A foetal-calf-serum component has been isolated that stimulates cells in culture to adopt typical fibroblast morphology. This “spreading” factor is a glycoprotein with a mol. wt. of approx. 62000 and contains 12% carbohydrate; it has an isoelectric point of 4.8. The method described allows purification to homogeneity as judged by sodium dodecyl sulphate/polyacrylamide-gel electrophoresis and analytical isoelectric focusing. Under cell-culture conditions the factor is active at 18 micrograms/ml.
Unoccupied nuclear oestradiol-receptor sites in normal human endometrium
Authors: Lévy C , Mortel R , Eychenne B , Robel P , Baulieu E E
Published: August 2015

The existence of unoccupied nuclear oestradiol-receptor sites in normal human endometrium was investigated. Nuclei were prepared from endometrial samples obtained by curettage and exposed to [3H]oestradiol, which became maximmaly bound at 0 degrees C within 1 h. This result contrasted with the binding kinetics of oestradiol-receptor complexes, since the exchange of hormone took at least 3 h at 30 degrees C and no displacement occurred at 0 degrees C. Before concluding that the nuclear sites were unoccupied, the presence of endogenous low-affinity ligands was excluded, because the association rate of oestradiol was unchanged after nuclei were stripped from their putative ligands, and the displacement of oestrone bound to nuclear receptor by oestradiol was very slow at 0 degrees C. The available sites had high affinity for oestradiol (KD 1.3 nM) and binding-specificity characteristics of oestradiol receptors. Similar results were observed with crude and purified nuclear preparations. It was concluded that a significant proportion of nuclear oestradiol receptors in normal human endometrium is unoccupied by endogenous hormones.
Energy transduction in intact synaptosomes. Influence of plasma-membrane depolarization on the respiration and membrane potential of internal mitochondria determined in situ
Authors: Scott I D , Nicholls D G
Published: August 2015

A method is described, based on the differential accumulation of Rb+ and methyltriphenylphosphonium, for the simultaneous estimation of the membrane potentials across the plasma membrane of isolated nerve endings (synaptosomes), and across the inner membrane of mitochondria within the synaptosomal cytoplasm. These determinations, together with measurements of respiratory rates, and ATP and phosphocreatine concentrations, are used to define the bioenergetic behaviour of isolated synaptosomes under a variety of conditions. Under control conditions, in the presence of glucose, the plasma and mitochondrial membrane potentials are respectively 45 and 148mV. Addition of a proton translocator induces a 5-fold increase in respiration, and abolishes the mitochondrial membrane potential. The addition of rotenone to inhibit respiration does not affect the plasma membrane potential, and only lowers the mitochondrial membrane potential to 128mV. Evidence is presented that ATP synthesis by anaerobic glycolysis is sufficient under these conditions to maintain ATP-dependent processes, including the reversal of the mitochondrial ATP synthetase. Addition of oligomycin under non-respiring conditions leads to a complete collapse of the mitochondrial potential. Even under control conditions the plasma membrane (Na+ + K+)-dependent ATPase is responsible for a significant proportion of the synaptosomal ATP turnover. Veratridine greatly increases respiration, and depolarizes the plasma membrane, but only slightly lowers the mitochondrial membrane potential. High K+ and ouabain also lower the plasma membrane potential without decreasing the mitochondrial membrane potential. In non-respiring synaptosomes, anaerobic glycolysis is incapable of maintaining cytosolic ATP during the increased turnover induced by veratridine, and the mitochondrial membrane potential collapses. It is concluded that the internal mitochondria must be considered in any study of synaptosomal transport.
Effect of electrical stimulation post mortem of bovine muscle on the binding of glycolytic enzymes. Functional and structural implications
Authors: Clarke F M , Shaw F D , Morton D J
Published: August 2015

The extent of binding of glycolytic enzymes to the particulate fraction of homogenates was measured in bovine psoas muscle before and after electrical stimulation. In association with an accelerated glycolytic rate on stimulation, there was a significant increase in the binding of certain glycolytic enzymes, the most notable of which were phosphofructokinase, aldolase, glyceraldehyde 3-phosphate dehydrogenase and pyruvate kinase. From the known association of glycolytic enzymes with the I-band of muscle it is proposed that electrical stimulation of anaerobic muscle increases enzyme binding to actin filaments. Calculations of the extent of enzyme binding suggest that significant amounts of enzyme protein, particularly aldolase and glyceraldehyde 3-phosphate dehydrogenase, are associated with the actin filaments. The results also imply that kinetic parameters derived from considerations of the enzyme activity in the soluble state may not have direct application to the situation in the muscle fibre, particularly during accelerated glycolysis.
2-Oxocarboxylic acids and function of pancreatic islets in obese–hyperglycaemic mice. Insulin secretion in relation to 45Ca uptake and metabolism
Authors: Lenzen Sigurd , Panten Uwe
Published: August 2015

The effects of aliphatic 2-oxocarboxylic acids, at concentrations of up to 40mm, on the function of pancreatic islets from ob/ob (obese–hyperglycaemic) mice were investigated. 1. 2-Oxopentanoate, dl-3-methyl-2-oxopentanoate, 4-methyl-2-oxopentanoate and 2-oxohexanoate all induced insulin release by isolated incubated islets and a biphasic insulin-secretory pattern in perfused mouse pancreas. The last two substances were similar in potency to glucose. Pyruvate, 2-oxobutyrate, 3-methyl-2-oxobutyrate and 2-oxo-octanoate did not induce insulin release significantly. 2. 2-Oxocarboxylic acids with significant insulin-secretory potency also induced significant 45Ca uptake by isolated incubated islets. 3. The rates of decarboxylation of [1-14C]pyruvate, 3-methyl-2-oxo[1-14C]butyrate and 4-methyl-2-oxo[1-14C]pentanoate were twice as high as the rates of oxidation of the corresponding U-14C-labelled compounds. However, whereas the rates of metabolism of labelled pyruvate and 3-methyl-2-oxobutyrate steadily increased over the concentration range 1–40mm, those of labelled 4-methyl-2-oxopentanoate and d-[U-14C]glucose levelled off at concentrations above 10mm. 4. Omission of 40CaCl2 from the incubation medium reduced the rate of oxidation of the insulin secretagogue [U-14C]4-methyl-2-oxopentanoate, but left that of the non-(insulin secretagogue) [U-14C]3-methyl-2-oxobutyrate unaffected. 5. Only glucose, and not pyruvate, 3-methyl-2-oxobutyrate and 4-methyl-2-oxopentanoate, significantly inhibited oxidation of endogenous fatty acids. 6. It is suggested that stimulus–secretion coupling and the resulting exocytosis of insulin in pancreatic β-cells may modulate both fuel oxidation and 45Ca uptake.
Induction of peroxisomal β-oxidation in rat liver by high-fat diets
Authors: Neat C E , Thomassen M S , Osmundsen H
Published: August 2015

Liver peroxisomes were prepared by using a Percoll gradient in a vertical rotor. beta-Oxidation was measured in peroxisomes isolated from livers of rats fed on either high-(15% by wt.) or low- (5% by wt.) fat diets. The feeding of high-fat diets gave a 1.4-2.4-fold increase in total liver peroxisomal beta-oxidation, and a similar increase in specific activity. A 1.5-4.5-fold increase was seen in the specific activity of purified peroxisomal preparations. The reasons for these increases are discussed.
Calcium ion-stimulated phosphorylation of myelin proteins
Authors: Sulakhe P V , Petrali E H , Thiessen B J , Davis E R
Published: August 2015

Myelin isolated from the central and peripheral nervous system contains a Mg2+-dependent protein kinase that catalyses phosphorylation of myelin-specific proteins. This phosphorylation is markedly stimulated by Ca2+ but not by cyclic AMP. Evidence was obtained that suggested an involvement of calmodulin-like protein in the stimulatory effects of Ca2+ on myelin phosphorylation.
Subcellular localization of chicken kidney aryl acylamidase activity
Authors: Gaynor J J , Still C C
Published: August 2015

The intracellular localization of aryl acylamidase (aryl-acylamide amidohydrolase, EC 3.5.1.13) in chicken kidney was investigated. By separation on density gradients of the silica sol Ludox AM, the enzyme was localized in the mitochondrial fraction. This mitochondrial fraction was shown to be substantially free of lysosomal contamination. Subfractionation of the purified mitochondria indicates that the enzyme is located on the outer membrane, can be solubilized, and may be a suitable marker enzyme for kidney mitochondria.
Enzyme inactivation via disulphide–thiol exchange as catalysed by a rat liver membrane protein
Authors: Francis Geoffrey L. , Ballard F. John
Published: August 2015

1. The inactivation of cytosol enzymes by a rat liver membrane protein was studied with crude microsomal fraction, plasma membranes or a partially purified preparation of inactivation factor. 2. Complete inactivation of 125I-labelled glucose 6-phosphate dehydrogenase (EC 1.1.1.49) by membranes did not result in any detectable change in molecular weight when the products were analysed by gradient polyacrylamide-gel electrophoresis. 3. Inactivation of radioactive enzyme was not accompanied by extensive binding to the membrane surface. The maximum extent of binding was 15% of the total enzyme labelled, and bound radioactivity was released only slowly, mainly as trichloroacetic acid-insoluble material. 4. Treatment of membranes with dithiothreitol destroyed the inactivation capacity, whereas the thiol-alkylating agent iodoacetamide had no effect. Partial restoration of the inactivation capacity of reduced membranes after exposure to air was prevented by membrane alkylation with iodoacetamide. 5. Modification of enzyme thiol groups during inactivation was determined by measuring a decrease in iodoacetamide-reactive groups in purified glucose 6-phosphate dehydrogenase. 6. Incubation of membrane-inactivated glucose 6-phosphate dehydrogenase with dithiothreitol resulted in a partial restoration of enzyme activity. 7. As a result of these experiments it is concluded that inactivation proceeds by a disulphide–thiol exchange mechanism. The proposal that this reaction could be involved in the initial step of enzyme degradation is discussed.
Effects of ACTH (corticotropin) analogues on steroidogenesis and cyclic AMP in rat adrenocortical cells. Evidence for two different steroidogenically responsive receptors
Authors: Bristow A F , Gleed C , Fauchère J L , Schwyzer R , Schulster D
Published: August 2015

Comparative studies on the mechanism of action of ACTH1-39 and ACTH5-24 [corticotropin-(1-39)- and corticotropin-(5-24)-peptides] on isolated rat adrenal cells were performed. The relationship between stimulated steroidogenesis and cyclic AMP was very different, suggesting that cyclic AMP does not play the same role in mediating the action of the two agonists. Data from three separate experiments showed that the competitive antagonist ACTH6-24 [corticotropin-(6-24)-peptide] had mean inhibitor constants of 13.4 +/- 3.1 nM against ACTH1-39 and 3.4 +/- 1.0 nM against ACTH5-24, indicating that the steroidogenic actions of these two agonists are mediated by two different receptor types. We conclude that there are two possible mechanisms for corticotropin action, only one of which involves the necessary production of cyclic AMP.
Utilization of energy-providing substrates in the isolated working rat heart
Authors: Taegtmeyer H , Hems R , Krebs H A
Published: August 2015

1. An improved perfusion system for the isolated rat heart is described. It is based on the isolated working heart of Neely, Liebermeister, Battersby & Morgan (1967) (Am. J. Physiol. 212, 804-814) and allows the measurement of metabolic rates and cardiac performance at a near-physiological workload. The main improvements concern better oxygenation of the perfusion medium and greater versatility of the apparatus. Near-physiological performance (cardiac output and aortic pressure) was maintained for nearly 2 h as compared with 30 min or less in the preparations of earlier work. 2. The rates of energy release (O2 uptake and substrate utilization) were 40-100% higher than those obtained by previous investigators, who used hearts at subphysiological workloads. 3. Values are given for the rates of utilization of glucose, lactate, oleate, acetate and ketone bodies, for O2 consumption and for the relative contributions of various fuels to the energy supply of the heart. Glucose can be replaced to a large extent by lactate, oleate or acetate, but not by ketone bodies. 4. Apart from quantitative differences there were also major qualitative differences between the present and previous preparations. Thus insulin was not required for maximal rates of glucose consumption at near-physiological, in contrast with subphysiological, workloads when glucose was the sole added substrate. When glucose oxidation was suppressed by the addition of other oxidizable substrates (lactate, acetate or acetoacetate), insulin increased the contribution of glucose as fuel for cardiac energy production at high workload. 5. In view of the major effects of workload on cardiac metabolism, experimentation on hearts performing subphysiologically or unphysiologically is of limited value to the situation in vivo.
Production of thiol groups and retention of calcium ions by cardiac mitochondria
Authors: Harris E J , Baum H
Published: August 2015

Efflux of Ca2+ from previously Ca2+-loaded heart mitochondria was measured after inhibiting respiratory activity. The efflux was increased by p-chloromercuribenzoate, methylmercuric chloride, Cu2+, Fe2+, 4,5,6,7-tetrachloro-2-trifluoromethylbenzimidazole (uncoupler). 1,1,1-trifluoro-3-(2-thienylacetone and indomethacin; after such increase it could be diminished by dithiothreitol. The induced loss of the Ca2+ was accompanied by a loss of endogenous adenine nucleotide. Methylmercuric chloride was particularly effective, since it was active at ratios of about 1 nmol/mg of mitochondrial protein. The non-respiring mitochondria were found to regenerate bound thiol groups after their original complement had reacted with thiol-blocking reagent. This regeneration was diminished by the Ca2+-efflux stimulatig agents that were not themselves thiol-blocking reagents, such as thyroxine, uncoupler, trifluorothienylacetone and indomethacin. The external exposure of thiol groups was also diminished by thyroxine, uncoupler and trifluorothienylacetone. The results support the proposal made previously that the membrane is maintained in a state of low permeability by adenine nucleotide and Mg2+ being bound to thiol-dependent sites.
Binding of insulin to bovine liver plasma membrane. Use of insulin analogues modified at the A1 residue
Authors: Rösen Peter , Simon Martina , Reinauer Hans , Friesen Heinz J. , Diaconescu Cornelia , Brandenburg Dietrich
Published: August 2015

Bovine liver plasma membranes [Rösen, Ehrich, Junger, Bubenzer & Kühn (1979) Biochim. Biophys. Acta587, 593–605] show similar insulin-binding characteristics, as evaluated by Scatchard analysis, to those of membrane systems from other species. However, the dissociation rate of bound insulin cannot be accelerated by the addition of insulin, in contrast with membranes isolated from rat liver. The dissociation rate is strongly dependent on the pH. Although dependent on temperature, the total capacity of binding sites is minimally changed, but the number of high-affinity sites is increased 2–3-fold, by lowering the incubation temperature. These data might be interpreted by assuming a single population of receptors whose distribution between different affinity states depends on temperature. In competition studies, most of the modified insulins examined show a close correlation between binding, determined in plasma membranes from bovine liver, and biological activity, measured in adipocytes. The hypothesis that a positive charge on the A1 residue may be favourable for binding is supported by experiments with an isosteric pair of insulins modified at this residue ([carbamoyl-GlyA1]- and [amidino-GlyA1]insulin) and with modified insulins carrying one or more positive charges on the A1 residue ([Arg-GlyA1]-, [Arg-Arg-GlyA1]-, [Arg-Arg-Arg-GlyA1]- and [Lys-Arg-GlyA1]insulin). The latter insulin derivatives show a higher binding activity for plasma membranes from bovine, porcine and rat liver than expected from their biological activities in adipocytes.
Role of Ca2+ and cyclic nucleotides in the control of prostaglandin E production in the rat anterior pituitary gland
Authors: Betteridge A
Published: August 2015

In an attempt to relate changes in the intracellular concentration of prostaglandin E to the secretion process, two agents known to increase cyclic nucleotide concentrations and hormone release were added to dispersed rat anterior pituitary cells. They caused increases in teh intracellular prostaglandin E concentrations. Increasing the K+ concentration in the medium (which stimulates hormone release) caused a rapid rise in prostaglandin E concentrations. The addition of the Ca2'onophore A23187 had a similar effect. The effects of changes in the K+ and Ca2+concentrations and the addition of EDTA were measured on the redistribution of radioactivity in pituitary glands prelabelled with [3H]arachidonic acid. Elevated K+ concentrations stimulated the transfer of label to prostaglandins and free arachidonic acid, suggesting an increased phospholipase A activity. On the other hand, the absence of extracellular CaCl2 and the addition of EDTA had the opposite effect, which could be cancelled by the addition of sufficient amounts. of CaCl2. It is concluded that the addition of agents that increase membrane permeability to bivalent cations probably results in an influx of Ca2+ and this appears to result in increased phospholipase A activity, which in turn leads to an increase in prostaglandin production.
Glycoproteins from the cell wall of Phaseolus coccineus
Authors: O'Neill M A , Selvendran R R
Published: August 2015

1. The use of a modified sodium chlorite/acetic acid delignification procedure for the solubilization of a hydroxyproline-rich glycoprotein fraction from the depectinated cell walls of Phaseolus coccineus is described. 2. The crude glycoprotein was associated with some pectic material; hydroxyproline and serine were the most abundant amino acids, and arabinose, galactose and galacturonic acid the predominant monosaccharides. 3. The bulk of the hydroxyproline is O-glycosidically substituted with tetra- and tri-arabinofuranosides. From methylation analysis the linkages in these arabinosides could be inferred. 4. Ion-exchange chromatography of the crude glycoprotein gave one major and two minor hydroxyproline-rich fractions, with similar amino acid but different monosaccharide composition. 5. In the major fraction, serine appears to be O-glycosidically substituted with a single galactopyranoside residue that can be removed by the action of alpha-galactosidase but not beta-galactosidase. Removal of arabinofuranoside residues by partial acid hydrolysis greatly enhanced the action of alpha-galactosidase. 6. Methylation followed by carboxy reduction with LiAl2H4 has shown the presence of (1 leads to 4)-linked galacturonic acid in the crude glycoprotein fraction but not in the major fraction from the ion-exchange column. Hence the bulk of the pectic material is not associated with the major glycoprotein component. It is suggested that the glycoprotein is held in the wall by phenolic cross-links. 7. Similarities with the glycopeptide moiety of potato lectin provides further evidence for a class of hydroxyproline-rich glycoproteins with common features.
N-acetylation of drugs. Observations on the properties of partially purified N-acetyltransferase from peripheral blood of rabbit
Authors: Drummond G S , Kelker H C , Weber W W
Published: August 2015

A method is described for the partial purification of N-acetyltransferase (EC 2.3.1.5) from peripheral blood of rabbit. This enzyme purified from both rapid- and slow-ioniazid-acetylator phenotype rabbits was examined with regard to stability, substrate specificity, cation effect, rate of inactivation, temperature and pH optimum. Data show that in the biochemical characteristics examined the enzyme is independent of acetylator phenotype. The possible significance of these findings to the evolutionary origin of drug-acetylating enzymes in rabbit blood and liver is discussed.
Separation of putrescine oxidase and spermidine oxidase in foetal bovine serum with the aid of a specific radioactive assay of spermidine oxidase
Authors: Gahl W A , Vale A M , Pitot H C
Published: August 2015

1. A sensitive and specific assay for spermidine oxidase is described. The method involves the separation of [14C]spermidine (substrate) from [14C]putrescine (product) and other 14C-labelled products on a Dowex 50 cation-exchange column: 92% of the putrescine applied to the column was eluted by 2.3 M-HCl, but this treatment left 96% of the spermidine bound to the column. Unchanged spermidine could be removed from the column by elution with 6 M-HCl. 2. By means of this assay, foetal and adult bovine serum were each shown to contain spermidine oxidase activity, putrescine being a major product of the oxidation of spermidine by the serum enzymes. 3. In foetal bovine serum, spermidine oxidase activity is separable from putrescine oxidase activity by chromatography on a cadaverine-Sephadex column, by gel filtration and by ion-exchange column chromatography. Putrescine oxidase was purified 1900-fold and spermidine oxidase 130-fold by these procedures. The former oxidized putrescine but not spermidine, and spermidine oxidase exhibited no activity with putrescine as substrate.
Titrations with ferrocyanide of japanese-lacquer-tree (Rhus vernicifera) laccase and of the type 2 copper-depleted enzyme. Interrelation of the copper sites
Authors: Morpurgo L , Graziani M T , Desideri A , Rotilio G
Published: August 2015

1. Redox titrations are reported of the metal centres in Japanese-lacquer-tree (Rhus vernicifera) laccase with ferrocyanide. 2. The redox potential of Type 1 Cu was found to increase with ferrocyanide concentration up to a limiting value similar to that for the Type 1 Cu in Type 2 Cu-depleted enzyme (which is independent of ferrocyanide concentration). 3. The redox potential of the two-electron acceptor (Type 3 Cu) is also independent of ferrocyanide concentration in Type 2 Cu-depleted enzyme and lower than values reported for the native enzyme. 4. The two-electron acceptor is present in the oxidized state in the Type 2 Cu-depleted enzyme, though the latter lacks the 330 nm absorption band. 5. The redox potential of Type 2 Cu also depends on ferrocyanide concentration, at least in the presence of azide. 6. The redox potentials are affected by freezing the solutions and/or addition of azide, the latter binding to Type 2 Cu with affinity dependent on the redox state of the two-electron acceptor.
Cross-linking preserves conformational changes induced in penicillinase by its substrates
Authors: Klemes Y , Citri N
Published: August 2015

Exopenicillinase of Bacillus cereus 569/H was cross-linked with toluene 2,4-diisocyanate in the presence of cephalothin, cloxacillin or no substrate. The derivatives show significant differences in susceptibility to inactivation by heat, urea, iodination or proteolysis. Such differences can be predicted from the contrasting effects of these substrates on the conformation of the enzyme.
Covalent binding of proteinases in their reaction with α2-macroglobulin
Authors: Salvesen G S , Barrett A J
Published: August 2015

Although it is known that most of the plasma proteinase inhibitors form complexes with proteinases that are not dissociated by SDS (sodium dodecyl sulphate), there has been disagreement as to whether this is true for alpha 2M (alpha 2-macroglobulin). We have examined the stability to SDS with reduction of complexes between alpha 2M and several 125I-labelled proteinases (trypsin, plasmin, leucocyte elastase, pancreatic elastase and papain) by gel electrophoresis. For each enzyme, some molecules were separated from the denatured alpha 2M chains, but amounts ranging from 8.3% (papain) to 61.2% (trypsin) were bound with a stability indicative of a covalent link. Proteolytic activity was essential for the covalent binding to occur, and the proteinase molecules became attached to the larger of the two proteolytic derivatives (apparent mol.wt. 111 000) of the alpha 2M subunit. We take this to mean that cleavage of the proteinase-susceptible site sometimes leads to covalent-bond formation between alpha 2M and proteinase. Whatever the nature of this bond, it does not involve the active site of the proteinase, as bound serine-proteinase molecules retain the ability to react with the active-site-directed reagent [3H]Dip-F (di-isopropyl phosphorofluoridate). Our conclusion is that the ability to form covalent links is not essential for the inhibitory capacity of alpha 2M. It may, however, help to stabilize the complexes against dissociation or proteolysis.
Lysis of erythrocytes from stored human blood by phospholipase C (Bacillus cereus)
Authors: Little C , Rumsby M G
Published: August 2015

The ability of phospholipase C (Bacillus cereus) to lyse erythrocytes from human blood that had been stored under Transfusion Service conditions for up to 16 weeks has been examined. When incubated at 20 degrees C with enzyme (0.03 mg/ml, 55 units/ml) for up to 1 h fresh erythrocytes were not lysed. After about 4 weeks of storage a population of very readily lysed erythrocytes appeared. The morphological changes in erythrocytes from blood stored up to 16 weeks were examined by scanning electron microscopy. The proportion of very readily lysed erythrocytes correlated well with the proportion of spheroechinocytes I. This morphological form was shown to be preferentially removed by phospholipase C and before lysis a transient appearance of smooth spheres occurred. The decrease in blood ATP concentrations on storage was measured and found to correlate with the disappearance of discoid erythrocyte forms, but not directly with the increased susceptibility of the erythrocytes to lysis by the enzyme. However, erythrocytes of up to at least 15 weeks of age could be made less susceptible to lysis by pre-incubation in a medium designed to cause intracellular regeneration of ATP. During the lysis of spheroechinocytes I by electrophoretically pure recrystallized phospholipase C a rapid degradation of phosphatidylcholine, phosphatidylethanolamine and phosphatidylserine + phosphatidylinositol) occurred together with a slower degradation of sphingomyelin.
Free pyrimidine nucleotide pool of Ehrlich ascites-tumour cells. Characteristics related to quantitative studies of RNA metabolism
Authors: Genchev Dobri D.
Published: August 2015

Ehrlich ascites-tumour cells incubated in a mineral medium supplemented with glucose and glutamine intensely incorporated labelled uridine into free nucleotides and RNA. Detailed kinetic studies of the labelling of total acid-soluble pools of uridine and cytidine nucleotides and of RNA under standard conditions and after chase with non-radioactive uridine were carried out. The relative distribution of the radioactivity between the individual uridine phosphates as well as their total cellular contents were also determined under standard and chase conditions. The labelling kinetics of the mononucleotides and RNA by radioactive uridine and orotate were compared, and some turnover parameters were estimated by mathematical analysis of a steady-state model. The following conclusions were drawn. (1) The observed chase effect of addition of non-radioactive uridine is due to a rapid expansion of the acid-soluble uridine nucleotide pool and consequently a severalfold lowering of its specific radioactivity. (2) The free uridine nucleotides are partly separated into a large and a small compartment, labelled preferentially by exogeneous orotate and uridine respectively. (3) The half-life of the mononucleotides, at least those located in the smaller compartment, is a few minutes only, owing to a rapid exchange with the uridylic acid residues of unstable RNA species. (4) This process of reutilization of RNA-degradation products accounts for 85% (a minimum estimate) of the overall turnover of the nucleotide pool in resting cells.
Phosphatidylethanolamine distribution and fluidity in outer and inner membranes of the gram-negative bacterium Erwinia carotovora
Authors: Shukla S D , Green C , Turner J M
Published: August 2015

1. The distribution of phosphatidylethanolamine, the major lipid of Erwinia carotovora, was investigated in intact bacteria, spheroplasts and outer- and inner-membrane preparations, with the amino-group reagent 2,4,6-trinitrobenzenesulphonic acid. Only 4% was found on the external surface of the outer membrane with 30% on the internal surface, whereas the inner membrane had 27 and 38% on its external and internal surfaces respectively. Some comparative studies were made with three other bacteria. 2. The fluidity of the membranes of E. carotovora was studied by using the fluorescent probe 1,6-diphenylhexa-1,3,5-triene. Results were consistent with the hydrocarbon region of the outer membrane bilayer being less fluid than that of the inner one. 3. On the basis of these and other results a model for the outer- and inner-membrane structures of E. carotovora is proposed.
Fractionation of rat ventricular nuclei
Authors: Jackowski G , Liew C C
Published: August 2015

Myocardial cells were isolated after treatment with collagenase (0.05%) and hyaluronidase (0.1%) by discontinuous-gradient centrifugation on 3% Ficoll. Nuclei derived from these myocardial cells were then fractionated on a discontinuous sucrose density gradient with the following steps: (I) 2.0M/2.3M, (II) 2.3M/2.4M, (III) 2.4M/2.5M, (IV) 2.5M/2.6M, and (V) 2.6M/2.85M. The myocardial nuclei were sedimented in the interfaces of gradient fractions (II) and (III). Nuclei from whole ventricles that had been treated with the enzymes before isolation sedimented into five major subsets of nuclei. These findings suggest that nuclei sedimented in the isopycnic gradient at fractions (II) and (III) are most probably derived from myocardial cells. However, this procedure is laborious and lengthy, and the recovery of myocardial-cell nuclei is low. An alternative method was developed to isolate an enriched fraction of myocardial-cell nuclei from whole ventricular tissue without exposing the tissues to enzyme digestion. These ventricular nuclei could be fractionated into five nuclear subsets by using the same discontinuous sucrose density gradient as that described above. The content of DNA, RNA and protein per nucleus for each band was determined. Although the DNA content per nucleus was constant (10pg), that of RNA varied from 1.5 to 4.5pg and that of protein from 16 to 24pg. Nuclei from each band were examined by light-microscopy: large nuclei occurred in the ligher regions whereas smaller nuclei were found in the denser regions of the gradient. From the size distribution pattern of myocardial-cell nuclei compared with that of total ventricular nuclei, it was found that nuclear subsets (II), (III), and (IV) were similar to myocardial nuclei. Electrophoretic analyses of the proteins solubilized in sodium dodecyl sulphate/phenol or Tris/EDTA/2-mercaptoethanol/phenol obtained from each nuclear subset indicate that these fractions are similar, with limited qualitative differences. These findings indicate that isolation of an enriched fraction of myocardial-cell nuclei could be achieved by discontinuous-sucrose-density-gradient centrifugation.