bluuebunny/crossref_metadata_2025 huggingface dataset:
Further studies of the action of disulfiram and 2,2′-dithiodipyridine on the dehydrogenase and esterase activities of sheep liver cytoplasmic aldehyde dehydrogenase
Authors: Kitson T M
Published: August 2015

1. Pre-modification of cytoplasmic aldehyde dehydrogenase by disulfiram results in the same extent of inactivation when the enzyme is subsequently assayed as a dehydrogenase or as an esterase. 2. 4-Nitrophenyl acetate protects the enzyme against inactivation by disulfiram, particularly well in the absence of NAD+. Some protection is also provided by chloral hydrate and indol-3-ylacetaldehyde (in the absence of NAD+). 3. When disulfiram is prevented from reacting at its usual site by the presence of 4-nitrophenyl acetate, it reacts elsewhere on the enzyme molecule without causing inactivation. 4. Enzyme in the presence of aldehyde and NAD+ is not at all protected against disulfiram. It is proposed that, under these circumstances, disulfiram reacts with the enzyme-NADH complex formed in the enzyme-catalysed reaction. 5. Modification by disulfiram results in a decrease in the amplitude of the burst of NADH formation during the dehydrogenase reaction, as well as a decrease in the steady-state rate. 6. 2,2′-Dithiodipyridine reacts with the enzyme both in the absence and presence of NAD+. Under the former circumstances the activity of the enzyme is little affected, but when the reaction is conducted in the presence of NAD+ the enzyme is activated by approximately 2-fold and is then relatively insensitive to the inactivatory effect of disulfiram. 7. Enzyme activated by 2,2′-dithiodipyridine loses most of its activity when stored over a period of a few days at 4 degrees C, or within 30 min when treated with sodium diethyldithiocarbamate. 8. Points for and against the proposal that the disulfiram-sensitive groups are catalytically essential are discussed.
Modulation of collagen production by fibroblasts. Effects of chronic exposure to agonists that increase intracellular cyclic AMP
Authors: Saltzman L E , Moss J , Berg R A , Hom B , Crystal R G
Published: August 2015

Cultured human lung fibroblasts were evaluated for their responsiveness to isoprenaline (isoproterenol) or prostaglandin E2 before and after chronic incubation with the agonist. Cells incubated for 6 h with either agonist were suppressed in terms of collagen production and exhibited increased intracellular cyclic AMP. Cells incubated for 72 h with the agonist and then re-challenged for 6 h with the same agonist did not demonstrate suppressed collagen production or increased cyclic AMP. Cells incubated for 72 h with isoprenaline still responded to prostaglandin E2 when challenged for 6 h; however, when the order of agonist exposure was reversed, cells incubated with prostaglandin E2 did not respond to a challenge by isoprenaline. If cells were allowed to recover for 48 h without the agonist after a 72 h chronic incubation, they recovered their responsiveness to the agonist. The results indicate that, although cultured fibroblasts may become desensitized to one agonist, they may retain their sensitivity to a second agonist and chronic suppression of collagen production may be achieved by alternate exposure to isoprenaline and prostaglandin E2.
Regulation of adenylate cyclase and cyclic AMP phosphodiesterase by 5-hydroxytryptamine and calcium ions in blowfly salivary-gland homogenates
Authors: Litosch I , Fradin M , Kasaian M , Lee H S , Fain J N
Published: August 2015

Salivary-gland homogenates contain 5-hydroxytryptamine-stimulated adenylate cyclase. Half-maximal stimulation was obtained with 0.1 microM-5-hydroxytryptamine in the presence of added guanine nucleotides. Gramine antagonized the stimulation of cyclase caused by 5-hydroxytryptamine. In the presence of hormone, guanosine 5′-[gamma-thio]triphosphate produced a marked activation of adenylate cyclase activity. Stimulation of adenylate cyclase by forskolin or fluoride did not require the addition of guanine nucleotides or hormone. In the presence of EGTA, Ca2+ produced a biphasic activation of cyclase activity. Ca2+ at 1-100 microM increased activity, whereas 2000 microM-Ca2+ inhibited cyclase activity. The neuroleptic drugs trifluoperazine and chlorpromazine non-specifically inhibited adenylate cyclase activity even in the absence of Ca2+. The cyclic AMP phosphodiesterase activity in homogenates was not affected by Ca2+ or exogenous calmodulin. This enzyme was also inhibited by trifluoperazine in the absence of Ca2+. These results indicate that Ca2+ elevates adenylate cyclase activity, but had no effect on cyclic AMP phosphodiesterase of salivary-gland homogenates.
Subcellular localization of phospholipid changes in response to muscarinic stimulation of perfused bovine adrenal medulla
Authors: Azila N , Hawthorne J N
Published: August 2015

The effects of carbachol on catecholamine secretion and [32P]Pi incorporation into phospholipids was studied in perfused bovine adrenal medulla. After a labelling period, the gland was stimulated with carbachol in the absence of 32P. Subcellular fractions were then prepared from the medulla. Carbachol roughly halved the specific radioactivities of phosphatidylinositol and phosphatidate in microsomal, chromaffin-granule, mitochondrial and plasma-membrane fractions. With Ca2+-free perfusion medium, catecholamine secretion was abolished but the phospholipid changes remained. Stimulation of secretion by KCl was not accompanied by phospholipid changes. The results are not consistent with the theory relating phosphatidylinositol hydrolysis and Ca2+ gating.
Biosynthesis of intestinal microvillar proteins. Translational evidence in vitro that aminopeptidase N is synthesized as a Mr-115000 polypeptide
Authors: Danielsen E M , Norén O , Sjöström H
Published: August 2015

A crude RNA fraction, prepared from pig small intestine, was found to be more efficient than a fraction enriched in polyadenylated RNA in directing translation of polypeptides with Mr greater than 100000 in a rabbit reticulocyte lysate system. Aminopeptidase N (EC 3.4.11.2) synthesized in vitro was immunopurified from the translation mixture and analysed by sodium dodecyl sulphate/polyacrylamide-gel electrophoresis. It was found to have an apparent Mr of 115000 regardless of whether the translation was performed in the absence or presence of proteinase inhibitors. This result contradicts the possibility of aminopeptidase N being synthesized as a large single-chain precursor polypeptide.
A new Kupffer cell receptor mediating plasma clearance of carcinoembryonic antigen by the rat
Authors: Toth C A , Thomas P , Broitman S A , Zamcheck N
Published: August 2015

Native human carcinoembryonic antigen is rapidly removed from the circulation by the rat liver Kupffer cell after intravenous injection. The molecule is subsequently transferred to the hepatocyte in an immunologically identifiable form. Carcinoembryonic antigen has a circulatory half-life of 3.7 (+/- 0.8) min, and cellular entry is by receptor-mediated endocytosis. Non-specific fluid pinocytosis and phagocytosis can be excluded as possible mechanisms by the kinetics of clearance and failure of colloidal carbon to inhibit uptake. Substances with known affinity for the hepatic receptors for mannose, N-acetylglucosamine, fucose and galactose all fail to inhibit carcinoembryonic antigen clearance. After two cycles of the Smith degradation, carcinoembryonic antigen is still able to inhibit clearance of the native molecule. Receptor specificity is apparently not dependent on those non-reducing terminal sugars of the native molecule. Performic acid-oxidized carcinoembryonic antigen also inhibits clearance of carcinoembryonic antigen in vivo. Receptor binding is not dependent on tertiary protein conformation. Non-specific cross-reacting antigen, a glycoprotein structurally similar to carcinoembryonic antigen, is cleared by the same mechanism.
THe proton-per-electron stoicheiometry of ‘site 1’ of oxidative phosphorylation at high protonmotive force is close to 1.5
Authors: de Jonge P C , Westerhoff H V
Published: August 2015

The maximum redox potential difference between the NAD+/NADH couple and the succinate/fumarate couple generated during ATP-energized reduction of NAD+ by succinate in submitochondrial particles was measured, together with the electrochemical potential difference for protons (delta mu approximately H+). The presence of cyanide, the time-independence of the redox potential difference and the irrelevance of the initial redox state of the NAD+/NADH couple ensured that the experimental situation corresponded to a ‘static-head condition’ with delta mu approximately H+ as the input force and the redox potential difference as the output force, the flow of electrons having reached dynamic equilibrium. Consequently, the observed value of 1.6 for the ratio delta Ge/delta mu approximately H+ is interpreted as indicating that the leads to H+/e- stoicheiometry at ‘site 1’ is 1.5 and that therefore the mechanism of the proton pump at ‘site 1’ is not of the group-translocation type (no direct leads to e - leads to H+ coupling).
Failure of leucine to stimulate protein synthesis in vivo
Authors: McNurlan M A , Fern E B , Garlick P J
Published: August 2015

The effect of 100 mumol of leucine on protein synthesis in several tissues was assessed in the intact rat. Leucine had no immediate effect on protein synthesis in gastrocnemius muscle, heart, jejunal serosa, jejunal mucosa or liver in rats which were fed, starved for 2 days or deprived of dietary protein for 9 days. Leucine treatment for 1 h also failed to stimulate protein synthesis in tissues of 2-day-starved animals.
Complement proteins C2, C4 and factor B. Effect of glycosylation on their secretion and catabolism
Authors: Matthews W J , Goldberger G , Marino J T , Einstein L P , Gash D J , Colten H R
Published: August 2015

Tunicamycin, an inhibitor of N-acetylglucosaminylpyrophosphopolyisoprenol-dependent glycosylation, was used to study the effect of glycosylation on the synthesis, post-translational modification, secretion and function of the complement proteins that are associated with the major histocompatibility complex in humans, mice and guinea pigs. Tunicamycin blocked glycosylation of pro-C4, C2 and factor B and inhibited secretion of the corresponding native complement proteins synthesized by guinea-pig peritoneal macrophages in tissue culture. In addition, underglycosylated pro-C4 was more rapidly catabolized intracellularly than the corresponding fully glycosylated pro-complement protein. C4 protein secreted by cells incubated with tunicamycin had approximately the same specific biological activity as the protein obtained from control culture media, suggesting that carbohydrate is not required for its activity in immune haemolysis. Direct studies of carbohydrate incorporation and the tunicamycin effect suggested an unequal distribution of sugar among the C4 subunits, with maximal incorporation of carbohydrate into alpha-, and less into the beta-chain of the native protein.
Relationship between lipogenesis and glycogen synthesis in maternal and foetal tissues during late gestation in the rat. Effect of dexamethasone
Authors: Benito M , Lorenzo M , Medina J M
Published: August 2015

Treatment with dexamethasone enhanced 3H2O incorporation into liver and blood lipid, and also increased plasma glucose, insulin, non-esterified fatty acid and triacylglycerol concentrations during late gestation in the mother rat. An inverse relationship between glycogen and lipid synthesis in foetal liver and lung was observed in control rats. This relationship was also observed in foetal liver, but not in foetal lung, after treatment with dexamethasone.
Evolution of α2-macroglobulin. The structure of a protein homologous with human α2-macroglobulin from plaice (Pleuronectes platessa L.) plasma
Authors: Starkey Phyllis M. , Barrett Alan J.
Published: August 2015

The plaice (Pleuronectes platessa L.) papain-binding protein previously demonstrated to be homologous with human α2-macroglobulin, and designated plaice α2-macroglobulin homologue or αMh, was shown to be a glycoprotein of s20,w 11.86S. In polyacrylamide-gel pore-limit electrophoresis under non-denaturing conditions plaice αMh migrated to the same position as half-molecules of human α2-macroglobulin, and treatment with methylamine or a proteinase caused no change in its electrophoretic properties. Either denaturation in urea (4m) or mild reduction by dithiothreitol (1mm) partially dissociated plaice αMh into half-molecules. Denaturation with reduction further dissociated the protein into quarter-subunits. In sodium dodecyl sulphate/polyacrylamide-gel electrophoresis under reducing conditions plaice αMh dissociated into subunits of Mr 105000 (I) and 90000 (II). Approximately equal amounts of each subunit were formed, and peptide ‘mapping’ showed subunits I and II to be distinct polypeptide chains. Under alkaline denaturing conditions, a proportion of the I chains of αMh were cleaved into fragments of Mr about 60000 and 40000. This cleavage was favoured by reducing conditions and prevented by prior inactivation of the αMh with methylamine. [14C]Methylamine allowed to react with αMh became covalently linked to subunit I. These properties suggested the existence of an autolytic site on subunit I analogous to the autolytic site of human α2-macroglobulin. Reaction of αMh with a proteinase resulted in cleavage of a fragment of Mr 10000–15000 from subunit I. A proportion of the proteinase molecules trapped by αMh became covalently linked to the inhibitor. A scheme is proposed for the evolution of human α2-macroglobulin and plaice αMh from a common ancestral protein, which may also have been an ancestor of complement components C3 and C4.
Purification and properties of acetyl-CoA:l-glutamate N-acetyltransferase from human liver
Authors: Bachmann C , Krähenbühl S , Colombo J P
Published: August 2015

Acetyl-CoA:L-glutamate N-acetyltransferase (amino acid acetyltransferase, EC 2.3.1.1) was isolated from human liver mitochondria by precipitation with (NH4)2SO4 and chromatography on hydroxyapatite, DEAE-cellulose and Sephacryl 300. This gave a 360-fold purification. The molecular weight was estimated to be approx. 190 000. The kinetic properties in the absence of arginine are compatible with a rapid-equilibrium random Bi Bi mechanism. The estimated constants are: for the substrates Km, acetyl-CoA 4.4 mM, Ki, acetyl-CoA 4.7 mM, Km, glutamate 8.1 mM, Ki, glutamate 8.8 mM; for the products, Ki, acetylglutamate 0.28 mM, Ki, CoA 5.6 mM. The rate constant for the forward direction is 1.24s-1. If in vivo the constants are of the same order of magnitude as in vitro, the synthesis of N-acetylglutamate, an obligate activator of the first step of urea synthesis, can be expected to occur in the mitochondrion under conditions where the amino acid acetyltransferase is not saturated by its substrates. The regulation of the first step of urea synthesis could thus depend mainly on the intramitochondrial substrate and perhaps product concentrations of amino acid acetyltransferase.
Fractionation of membrane proteins on phenylboronic acid-agarose
Authors: Williams G T , Johnstone A P , Dean P D
Published: August 2015

Detergent-solubilized plasma-membrane proteins from lymphocytes of patients with chronic lymphocytic leukaemia were applied to phenylboronic acid-agarose. Some of the polypeptides were bound specifically and subsequently eluted with sorbitol. Immunoprecipitation analyses showed that no surface immunoglobulin M was bound, but that most of the histocompatibility antigens HLA-A, HLA-B, HLA-C and HLA-DR were.
Isolation and protein composition of gap junctions from rabbit hearts
Authors: Manjunath C K , Goings G E , Page E
Published: August 2015

We have modified a method for isolating gap-junctional membrane from mouse hearts [Kensler & Goodenough (1980) J. Cell Biol. 86, 755-764] to isolate gap junctions of comparable purity from rabbit hearts more rapidly, with better yield, and without resort to non-ionic detergents. Purification was monitored by electron microscopy of thin-sectioned membrane pellets and by sodium dodecyl sulphate/polyacrylamide-gel electrophoresis. Gap junctions were obtained as vesicles whose mean surface area approximated that of junctions in intact myocardial cells. About 10-20% of the vesicles were ferritin-impermeable. Approx. 125 micrograms of membrane protein was obtained per 8 g of rabbit heart. Sodium dodecyl sulphate/polyacrylamide-gel electrophoresis of purified gap junctions showed five major protein bands of mol.wts. 46 000, 44 000, 33 000, 30 000 and 28 500 that co-purified with the junctions. This protein composition was nearly identical with that published for gap junctions of mouse hearts, and differed markedly from the protein composition of gap junctions from non-excitable cells (lens and liver). The constancy of junctional protein composition between hearts of two different species and its non-identity with that from liver and lens suggest that, although gap-junctional structure in mammalian tissues seems to be remarkably similar by electron-microscopic techniques, junctional-channel protein composition actually varies from tissue to tissue and may be adapted to the permeability requirements of the tissue.
Purification and immunofluorescent localization of rat submandibular mucin
Authors: Fleming Norman , Brent Michael , Arellano Ramiro , Forstner Janet F.
Published: August 2015

Rat submandibular mucin (RSM) was purified by acid precipitation, then alcohol precipitation of the 30000g supernatant of gland homogenate, followed by column chromatography on Sephadex G-200. The mucin, which was eluted in the void volume, had an amino acid profile typical of a salivary mucus glycoprotein with high proportions of threonine, serine and proline (48.8% of total amino acids), and low proportions of aromatic and basic amino acids. It consisted of 63% (w/w) carbohydrate, which was shown by g.l.c. analysis to contain N-acetylglucosamine, N-acetylgalactosamine, galactose, sialic acid and fucose in the proportions 1.0:3.4:2.6:3.1:1.2. After staining of the mucin with periodic acid/Schiff reagent, analytical equilibrium ultracentrifugation in a CsCl density gradient produced a symmetrical peak of buoyant density 1.449g/ml, without evidence of protein contaminants. Sedimentation velocity centrifugation revealed a major periodate/Schiff-positive component (S020,w 5.06) with an associated shoulder of slower sedimenting material, suggesting polydispersity in the size of the mucin. Our findings suggest that the RSM purified in these studies has a molecular weight between 200000 and 1×106. Antibody to RSM was prepared in a rabbit and produced a single precipitin line on immunoelectro-osmophoresis with the mucin. Immunofluorescence studies showed that the antibody localized only to submandibular acinar cells and confirmed that these cells were the source of RSM. The antibody was not directed towards the blood-group-A determinant (terminal N-acetylgalactosamine) present in the mucin.
Lectin affinity chromatography of glycopeptides and oligosaccharides from normal and lectin-resistant Chinese-hamster ovary cells
Authors: Hunt L A
Published: August 2015

The [3H]mannose-labelled glycopeptides from two lectin-resistant lines of Chinese-hamster ovary cells were fractionated by chromatography on lentil lectin-Sepharose and concanavalin A-agarose columns and subsequently analysed by gel filtration in comparison with the glycopeptides of the parental cell line. Essentially all of the [3H]mannose-labelled asparaginyl-oligosaccharides from the ‘single-mutant’ cells selected for resistance to phytohaemagglutinin and the ‘double-mutant’ cells selected for additional resistance to concanavalin A were not bound to lentil lectin, whereas approximately one-sixth of the parental-cell glycopeptides were bound and specifically eluted with alpha-methyl mannoside. These bound and eluted glycopeptides represented a specific subset of the complex acidic-type asparaginyl-oligosaccharides. The percentage of radiolabelled glycopeptides and oligosaccharides from each cell line that were specifically bound to concanavalin A was consistent with the relative sensitivities of the three cell lines to this lectin. The major radiolabelled species in the endoglycosidase digest of the ‘double-mutant’-cell glycopeptides (Man4GlcNAc1-size neutral oligosaccharides) were not bound to concanavalin A, whereas essentially all of the other neutral-type oligosaccharides were bound. In addition, the larger neutral-type oligosaccharides (Man8-9GlcNAc1) were more strongly bound to concanavalin A than were either the smaller neutral-type or the di-antennary acidic-type structures.
A quick method for the determination of inhibition constants
Authors: Waley S G
Published: August 2015

The inhibition constant Ki in the common case of competitive inhibition can be obtained by simple comparison of progress curves in the presence and in the absence of inhibitor. The difference between the times taken for the concentration of substrate to fall to the same value is used to obtain Ki. The procedure to use when the product inhibits is described. When there is mixed inhibition, reactions at different substrate concentrations are used to obtain both inhibition constants.
Limitations of commonly used spectrophotometric assay methods for phosphoenolypyruvate carboxykinase activity in crude extracts of muscle
Authors: Duff D A , Snell K
Published: August 2015

Phosphoenolpyruvate carboxykinase activity in crude extracts of muscle has frequently been determined by using a continuous spectrophotometric method, which is shown to grossly overestimate enzyme activity. NADH oxidation attributed to phosphoenolpyruvate carboxykinase activity in the assay is due to lactate production. Under the normal assay conditions. Na+ ions stimulate pyruvate kinase, providing pyruvate for lactate formation by lactate dehydrogenase and sufficiently to account for most of the observed NADH oxidation.
Studies, with a luminogenic peptide substrate, on blood coagulation Factor X/Xa produced by mouse peritoneal macrophages
Authors: Lindahl Ulf , Kolset Svein O. , Bøgwald Jarl , Østerud Bjarne , Seljelid Rolf
Published: August 2015

The formation and secretion of coagulation Factor X/Xa by mouse peritoneal macrophages was studied with a luminogenic peptide substrate (S-2613; t-butyloxycarbonylisoleucylglutamyl-γ-piperidylglycylarginylisoluminol). Amidolysis was quantified by measuring the light emitted during oxidation of isoluminol, released by Factor Xa. A lower detection limit of about 0.5ng of Factor Xa was established; the assay was linear with enzyme concentration up to at least 100ng/ml. Factor X was determined after treatment with the Factor X-activating component of Russell's-viper (Vipera russelli) venom. Macrophages, cultured in the absence of serum, released Factor X/Xa into the culture medium. The concentration of coagulation enzyme in the medium increased in an essentially linear fashion over a period of at least 3 days, at a rate corresponding to 6–8ng produced/24h per 106cells. The ratio of Factor Xa/X+Xa varied from about 60 to 100%, showing that activation of Factor X to Xa is not prerequisite to release of the enzyme from the cells. Factor Xa activity was suppressed in the presence of warfarin [3-(α-acetonylbenzyl)-4-hydroxycoumarin; 12.5μg/ml of medium], but could be restored by adding vitamin K (0.1μg/ml) along with the warfarin. Cultures to which Sepharose beads containing covalently bound anti-(Factor X) antibodies had been added showed decreased amounts of free Factor X/Xa in the culture medium. The missing activity could be demonstrated by incubating the recovered conjugate with the substrate peptide S-2613. Factor Xa produced by the macrophages was efficiently inactivated by heparin in the presence of antithrombin, heparin with high affinity for antithrombin being more effective than the corresponding low-affinity species.
Respiration of the rumen ciliate Dasytricha ruminantium Schuberg
Authors: Yarlett N , Lloyd D , Williams A G
Published: August 2015

The endogenous respiration of the rumen ciliate Dasytricha ruminantium maintained under an O2 tension of 2kPa (approximately 0.02 atm) was partially inhibited by KCN (40% inhibition) and NaN3 (58% inhibition). The organisms lack cytochromes, and sensitivity of respiration to KCN, NaN3, chloroquine and quercetin suggest that the operation of flavoprotein-iron-sulphur-mediated electron transport. As in Tritrichomonas foetus, hydrogenosomal respiration can be stimulated by the addition of CoA in the presence of 0.025% Triton X-100; stimulation by ADP was not detected. Stimulation of pyruvate-supported O2 uptake by Pi suggests that acetate is produced via acetyl phosphate.
Processing of calcitonin and epidermal growth factor after binding to receptors in human breast cancer cells (T 47D)
Authors: Findlay D M , Ng K W , Niall M , Martin T J
Published: August 2015

125I-labelled calcitonin and 125I-labelled epidermal growth factor (EGF) bound to T 47D breast cancer cells at 37 degrees C in a manner that became increasingly resistant to removal by acid pH. Bound 125I-labelled EGF became resistant to acid removal more rapidly than did bound 125I-labelled calcitonin. The shift from acid accessibility to acid inaccessibility was energy-dependent since it was not seen at 4 degrees C and was inhibited in the presence of cell metabolic inhibitors. Radioactivity removed by acid represented intact hormone as assessed by trichloroacetic acid precipitation, whereas radioactivity released spontaneously by the cells was trichloroacetic acid-soluble. Inclusion of 10 mM-NH4Cl in the incubation medium resulted in an accumulation of cell-associated radioactivity without affecting the shift to acid inaccessibility. The accumulated radioactivity was relatively more trichloroacetic acid-precipitable in comparison with that associated with control cells. These data are consistent with internalization of receptor-bound EGF and a similar though slower mechanism of processing for receptor-bound calcitonin. The predominant route of hormone release from cells seems to occur via intracellular degradation rather than dissociation from cell-surface receptors.
Acute effects of glucagon on citrulline biosynthesis
Authors: Rabier D , Briand P , Petit F , Parvy P , Kamoun P , Cathelineau L
Published: August 2015

Mitochondria isolated from livers of rats fed on different diets showed altered capacity to synthesize citrulline. Glucagon, 15 min after injection, increases citrulline biosynthesis, except after the high-protein diet. A significant correlation between citrulline biosynthesis and N-acetylglutamate content with and without glucagon treatment was shown when rats were fed on a standard or a carbohydrate diet. Different diets modified carbamoyl phosphate synthetase I (EC 6.3.4.16) and N-acetylglutamate synthase (acetyl-CoA:L-glutamate N-acetyltransferase, EC 2.3.1.1) activities. Glucagon did not modify these activities.
Sendai virus causes a rise in intracellular free Ca2+ before cell fusion
Authors: Hallett Maurice B. , Fuchs Pinhas , Campbell Anthony K.
Published: August 2015

1. Sendai virus caused a large increase in the concentration of free Ca2+ within human erythrocyte ghosts detected by the Ca2+-activated photoprotein obelin. 2. The increase in intracellular [Ca2+] preceded fusion. However, fusion could also be observed in the absence of a detectable rise in intracellular free [Ca2+]. 3. It was concluded that the increase in intracellular free [Ca2+] was not an absolute requirement for cell fusion, but may be necessary to produce fusion at the maximum rate.
The autoreducible cytochromes c of the methylotrophs Methylophilus methylotrophus and Pseudomonas AM1
Authors: Beardmore-Gray M , O'Keeffe D T , Anthony C
Published: August 2015

The two types of soluble cytochrome c (cytochrome cH and cytochrome cL) found in methylotrophs are completely distinct proteins; one type is not a dimer or degradation product of the other. Free thiol groups are probably not involved in the unusually rapid autoreduction of the cytochromes at high pH. The axial ligands to the haem iron, histidine and methionine, are the same as in other low-spin cytochromes c. The methionine ligand is displaced at high pH by an alternative strong-field ligand. This displacement does not occur on reduction of cytochrome cL by methanol dehydrogenase, but this does not rule out the possibility that the autoreduction mechanism is involved in the interaction of the dehydrogenase and cytochrome c.
Interaction of β-iodopenicillanate with the β-lactamases of Streptomyces albus G and Actinomadura R39
Authors: Frère J M , Dormans C , Duyckaerts C , De Graeve J
Published: August 2015

The beta-lactamases of Streptomyces albus G and Actinomadura R39 are inactivated by beta-iodopenicillanate. However, in contrast with the beta-lactamase I from Bacillus cereus, they also efficiently catalyse the hydrolysis of the inactivator; with the S. albus G enzyme, kcat. is larger than 25s-1 and the number of turnovers before inactivation is 515. With the A. R39 enzyme, kcat. is larger than 50s-1 and the number of turnovers before inactivation is 80. After hydrolysis of the beta-lactam amide bond, the product rearranges into 2.3-dihydro-2,2-dimethyl-1,4-thiazine-3,6-dicarboxylate, which exhibits an absorption maximum at 305 nm.
Purification and characterization of rat adipose tissue lipoprotein lipase
Authors: Parkin S M , Speake B K , Robinson D S
Published: August 2015

Lipoprotein lipase (EC 3.1.1.34) extracted from adipose tissue of glucose-fed rats with 5 mM-sodium barbital, pH 7.5, containing 20% (v/v) glycerol and 0.1% (v/v) Triton X-100, was partially purified by affinity chromatography on heparin linked to Sepharose 4B. Sodium dodecyl sulphate/polyacrylamide-gel electrophoresis of the partially purified enzyme preparation revealed the presence of two major Coomassie-staining bands (mol.wts. 62 000 and 56 000) as well as a number of minor bands. Treatment of partially purified enzyme with [1,3-3H]di-isopropyl fluorophosphate resulted in the incorporation of radiolabel into the band of mol.wt. 56 000, but not into the band of mol.wt. 62 000. Both the amount of the 56 000-mol.wt. polypeptide and the incorporation of [1,3-3H]di-isopropyl fluorophosphate into this band were greatly reduced in the enzyme preparations isolated from adipose tissue of 48 h-starved rats. whereas the amount of the 62 000-mol.wt. polypeptide was unaffected by starvation. Purification of lipoprotein lipase from adipose tissue of glucose-fed rats was also carried out using affinity chromatography on Sepharose 4B linked to heparin with low affinity for antithrombin-III. This procedure resulted in the presence of a single band of mol.wt. 56 000 on sodium dodecyl sulphate/polyacrylamide-gel electrophoresis. These results suggest that the polypeptide of mol.wt. 56 000 corresponds to the subunit of lipoprotein lipase, whereas the 62 000-mol.wt. polypeptide probably represents antithrombin-III.
Properties of the ribosome-inactivating proteins gelonin, Momordica charantia inhibitor, and dianthins
Authors: Falasca A , Gasperi-Campani A , Abbondanza A , Barbieri L , Stirpe F
Published: August 2015

The amino acid and sugar compositions of four ribosome-inactivating proteins (gelonin, Momordica charantia inhibitor, dianthin 30 and dianthin 32) were determined. The proteins are all basic glycoproteins (pI greater than 8) containing mannose (more abundant in gelonin), glucose, xylose, fucose (absent from gelonin) and glucosamine. The ribosome-inactivating properties of the proteins examined are not modified by pretreatment with N-ethylmaleimide. Precipitating and inactivating antibodies can be raised against ribosome-inactivating proteins; a weak cross-reaction was observed only between dianthin 30 and dianthin 32.
Characterization of the c-type cytochromes of Nitrosomonas europaea with the aid of fluorescent gels
Authors: Miller David J. , Wood Paul M.
Published: August 2015

When a total soluble extract of Nitrosomonas europaea was denatured with dodecyl sulphate, subjected to dodecyl sulphate/polyacrylamide-gel electrophoresis and illuminated with near-u.v. light, eight bands of protein fluorescence were observed. All but one of these bands were red in colour, a property characteristic of c-type cytochromes. Standard techniques were used to purify soluble c-type cytochromes from this organism, and it was then possible to assign all but two very minor bands to specific c-type cytochromes, namely hydroxylamine oxidase, cytochrome c-554, cytochrome c-552 and a cytochrome c-550 not previously described. The eight band had fluorescence peaking in the green region of the spectrum, probably caused by covalently bound flavin, and co-purified with hydroxylamine oxidase. The following physical properties were determined for these components: isoelectric point, molecular weights according to gel filtration and mobility on dodecyl sulphate/polyacrylamide gels, and α-band spectra at room temperature and 77K. Redox potentials were measured as follows: cytochrome c-554, Em,7 = +20mV; cytochrome c-552, Em,7 = +230mV; cytochrome c-550, Em,7 = +140mV. When washed membranes were applied to dodecyl sulphate/polyacrylamide gels in the same way, a number of fluorescent bands were observed that could be matched by soluble proteins. In addition, there was one band that could not be detected in supernatants, migrating with an apparent molecular weight of 24000. This species is probably coincident with a c-type cytochrome having Em,7 = +170mV found in redox titration of these membranes. In future studies, gel fluorescence should form a useful complement to spectroscopy for analysis of cytochrome composition in active cell-free preparations or semi-purified material.
Actin and tubulin of normal and leukaemic lymphocytes
Authors: Atkins H , Anderson P J
Published: August 2015

Double labelling and the isolation of actin- and tubulin-derived peptides were used to determine the amounts of these proteins in peripheral lymphocytes from normal donors and from patients with chronic lymphocytic leukaemia. As a precaution against proteolysis, samples were boiled before assay. The actin content of chronic-lymphocytic-leukaemia (CLL) lymphocytes was 8.1 +/- 2.1% of total protein, which was lower (P less than 0.05) than the amount (12.8 +/- 3.0%) of actin found in normal lymphocytes. The tubulin content of CLL lymphocytes was 4.4 +/- 1.5% of total protein, which was also significantly less (P less than 0.05) than that of normal lymphocytes, which was found to be 6.1 +/- 1.1%.
Superoxide dismutase-inhibitible reduction of cytochrome c by the alloxan radical. Implications for alloxan cytotoxicity
Authors: Winterbourn C C
Published: August 2015

Cytochrome c was reduced when superoxide was generated from xanthine oxidase in the presence of alloxan, and by the reaction of alloxan and with reduced glutathione. In each case, most of the reduction was inhibited by superoxide dismutase, but considerably more enzyme was required than with superoxide alone. This indicates that the superoxide dismutase-inhibitible cytochrome c reduction was mainly due to a direct reaction with the alloxan radical, and implies that other reactions that are inhibited by superoxide dismutase could be due to either alloxan radicals or superoxide.
Type-specific collagen degradation by eosinophils
Authors: Hibbs M S , Mainardi C L , Kang A H
Published: August 2015

Despite the association of eosinophils with wound-healing and fibrotic processes, their collagenolytic ability has been poorly defined. By using highly purified eosinophil preparations (greater than 95% eosinophils) obtained from guinea-pigs by peritoneal lavage, we have examined type-specific collagen degradation by eosinophils using sensitive detection methods. The results show that eosinophils contain a metalloprotein that degrades types I and III collagens. This activity was apparent only after the addition of 4-aminophenylmercuric acetate to the reaction mixture, a finding similar to that for latent collagenases described from other sources. No collagenolytic activity against types IV and V collagens could be detected. Thus eosinophils may play a role in the alterations in connective-tissue matrices seen in physiological and pathological states.
Transcriptional control of ribosome production in regenerating rat liver
Authors: Dabeva M D , Dudov K P
Published: August 2015

Kinetic experiments of labelling in vivo with [14C]orotate of cellular free UMP and/or UTP, nucleolar, nucleoplasmic and cytoplasmic rRNA in normal and 12 h-regenerating rat liver were performed. The specific-radioactivity curves obtained were analysed by computer and the rates of synthesis of precursor rRNA (45S pre-rRNA) and cytoplasmic 28S and 18S rRNA calculated. (a) The rates of synthesis of 45S pre-rRNA in normal and regenerating rat liver are 1400 and 3700 molecules/min per nucleus respectively; (b) the average rates of formation of mature 28S and 18S rRNA are identical with the rates of synthesis of 45S pre-rRNA in both normal and regenerating rat liver. Thus the synthesis of rRNA in 12h-regenerating rat liver is activated 2.7-fold. The analysis of rRNA synthesis in isolated nucleoli also shows a 2.7-fold stimulation of transcription in regenerating liver. It is concluded that all the 45S pre-rRNA molecules synthesized are processed and transferred as 28S and 18S rRNA in the cytoplasm, i.e. degradation (wastage) of newly synthesized ribosomes in the nucleus does not occur in both normal and regenerating rat liver. Thus the enhanced production of ribosomes in regenerating rat liver is regulated only at the transcriptional level.
Determination and comparative analysis of the catalytic subunit of adenosine 3′,5′-cyclic phosphate-dependent protein kinase by an enzyme-linked immunosorbent assay
Authors: Schwoch G , Hamann A
Published: August 2015

A specific antiserum against bovine heart catalytic subunit was used for the determination of the catalytic subunit in an enzyme-linked immunosorbent assay. Under the conditions elaborated the assay has a lower detection limit for catalytic subunit of 0.25 pmol/ml. In crude bovine heart extracts the concentration of catalytic subunit was determined by this method to be 0.18 +/- 0.02 mumol/kg wet wt. The immunochemical comparison of various animal species and cells, including organisms like amoebae and yeast, shows the broad applicability of the assay and provides evidence that the catalytic subunit is a highly conserved molecule.
Na+-gradient-dependent stimulation of renal transport of ρ-aminohippurate
Authors: Sheikh M I , Møller J V
Published: August 2015

Transport of rho-aminohippurate was studied by the use of a preparation of rabbit kidney basolateral-membrane vesicles and in rat kidney-cortex slices under anaerobic conditions. With both preparations clear evidence of Na+-gradient stimulation of rho-aminohippurate transport (‘overshoot’) was obtained. These results thus indicate that a significant aspect of active rho-aminohippurate transport is by co-transport with Na+, and they appear to resolve previous disagreements concerning the role of Na+. Vesicle studies with a potential-sensitive dye suggested that rho-aminohippurate may be transported electroneutrally, i.e. in a 1:1 complex with Na+.
Transformation-specific cell killing by a cancer-associated galactosyltransferase acceptor and cellular binding
Authors: Podolsky Daniel K. , Isselbacher Kurt J.
Published: August 2015

Cancer-associated galactosyltransferase acceptor (CAGA glycoprotein), a small glycoprotein purified from human malignant effusion that selectively kills transformed cells, was tritiated by reductive methylation in the presence of NaB3H4. CAGA-glycoprotein-sensitive cells (baby-hamster kidney cells transformed by polyoma virus and chick-embryo fibroblasts infected with Ts68 temperature-sensitive mutant of Rous sarcoma virus grown at 37°C, the permissive temperature) bound 3–5-fold more 3H-labelled CAGA glycoprotein than did their CAGA-glycoprotein-resistant non-transformed counterparts. The Rous-sarcoma-virus-infected chick-embryo fibroblasts grown at non-permissive temperature (41°C) bound an intermediate amount of 3H-labelled CAGA glycoprotein; however, this intermediate amount appeared to be sufficient to induce inhibition of cell growth when the infected chick-embryo fibroblasts treated at 41°C were switched to 37°C. Binding of 3H-labelled CAGA glycoprotein was time- and temperature-dependent and was not inhibited by monosaccharide. Binding was completely inhibited by the oligosaccharide liberated by endoglucosaminidase H treatment or by exhaustive Pronase digestion of intact CAGA glycoprotein. However, the isolated oligosaccharide failed to demonstrate the growth-inhibition characteristics of the intact glycopeptide. Binding of 3H-labelled CAGA glycoprotein was unaffected by co-incubation with the peptide core released by endoglucosaminidase H treatment. 3H-labelled CAGA glycoprotein bound to intact cells could be removed by trypsin treatment up to 4h after addition of the glycoprotein but not thereafter. This time course paralleled the decreasing reversibility of growth inhibition. However, all 3H-labelled CAGA glycoprotein was found in the supernatant when cells were first disrupted by sonication followed by trypsin treatment for up to 12h. 3H-labelled CAGA glycoprotein linked to Sepharose 4B failed to cause growth inhibition in CAGA-glycoprotein-sensitive cells. These findings suggest that binding of CAGA glycoprotein occurs via its oligosaccharide moiety. Binding appears to be a necessary but not sufficient condition to induce cell killing. Growth inhibition appears to depend on internalization of the glycoprotein and the presence of a transformation-specific cell process.
Cellular content and biosynthesis of polyamines during rooster spermatogenesis
Authors: Oliva R , Vidal S , Mezquita C
Published: August 2015

The natural polyamines spermine and spermidine, and the diamine putrescine, were extracted from rooster testis cells separated by sedimentation at unit gravity, and from vas-deferens spermatozoa. The ratios spermine/DNA and spermidine/DNA were kept relatively constant throughout spermatogenesis, whereas the ratio putrescine/DNA rose in elongated spermatids. The cellular content of spermine, spermidine and putrescine decreased markedly in mature spermatozoa. Two rate-limiting enzymes in the biosynthetic pathway of polyamines, ornithine decarboxylase and S-adenosyl-L-methionine decarboxylase, showed their highest activities at the end of spermiogenesis and were not detectable in vas-deferens spermatozoa. A marked reduction in cell volume during spermiogenesis without a parallel decrease in the cellular content of polyamines suggests the possibility that the marked changes in chromatin composition and structure occurring in rooster late spermatids could take place in an ambience of high polyamine concentration.
Zinc release and the sequence of biochemical events during triggering of Bacillus megaterium KM spore germination
Authors: Johnstone K , Stewart G S A B , Scott I R , Ellar D J
Published: August 2015

Zinc release is the first quantitatively significant event detected during the triggering of Bacillus megaterium KM spore germination. Of the total spore Zn2+ pool 25% is released from non-heat-activated spores within 4 min of triggering germination. During this period only 10% of the spore population becomes irreversibly committed to germinate. The investigation of a putative role for Zn2+ in the germination trigger mechanism has established a relationship between the rate and extent of Zn2+ release and the stimulation of spore germination by heat activation. Furthermore, a correlation can be demonstrated between the extent of zinc release from spore populations and the time required to obtain 50% commitment of these populations to germinate over a wide temperature range. These findings have been used to expand a recently published model for the triggering of bacterial spore germination.
Inhibition of phosphatidylcholine synthesis by vasopressin and angiotensin in rat hepatocytes
Authors: Alemany S , Varela I , Mato J M
Published: August 2015

The addition of 1 microM-vasopressin or -angiotensin to isolated rat hepatocytes induced a fast transient inhibition of the rate of incorporation of [Me-3H]choline into phosphatidylcholine. The cationophore A23187 induced a similar inhibition of phosphatidylcholine synthesis. The addition of micromolar Ca2+ to rat liver microsomes inhibited the activity of CDP-choline: 1,2-diacylglycerol cholinephosphotransferase. This inhibition is due a decrease in the Vmax. of the enzyme without affecting the Km for CDP-choline. It is concluded that Ca2+ regulates phosphatidylcholine synthesis in rat liver.
Cation transport mechanisms in Mycoplasma mycoides var. Capri cells. Na+-dependent K+ accumulation
Authors: Benyoucef M , Rigaud J L , Leblanc G
Published: August 2015

We have studied some features of K+ accumulation by glycolysing Mycoplasma mycoides var. Capri cells. We report that when Na+ is absent from the external medium, K+ accumulates up to the level predicted by the amplitude of the transmembrane electrical potential, delta psi m, measured by Rb+ and methyltriphenylphosphonium cation (TPMP+) distribution. Therefore, under these experimental conditions, the coupling mechanism of K+ uptake consists of a delta psi m-driven uniport. More important, when Na+ is present in the external medium, the level of K+ accumulation by glycolysing Mycoplasma cells is far too steep to be equilibrium with delta psi m (-120 mV for delta muK+ compared with −90mV for delta muRb+ or delta muTPMP+). Our results clearly indicate the presence in Mycoplasma of an active K+-transport system specifically stimulated by Na+. Furthermore, by controlling the amplitude of the energy-dependent delta muH+, we obtain strong evidence that this specific Na+-stimulated K+ transport is modulated by the transmembrane electrical potential. Finally, we show that ATP is consumed when such a transport system is in activity.