bluuebunny/crossref_metadata_2025 huggingface dataset:
Nuclear acceptor sites for androgen-receptor complexes in seminal-vesicle epithelium
Authors: Weinberger M J , Veneziale C M
Published: August 2015

An assay method in vitro was developed and applied to quantify acceptor binding of steroid-receptor complexes in nuclei from isolated epithelium of guinea-pig seminal vesicle. Steroid-receptor complex prepared from 1-day-castrated animals was incubated with purified nuclei from 1-28 day-castrated animals in a medium containing 0.15 M-KCl. Free and bound steroid-receptor complexes were measured and the data were submitted to Scatchard analysis. With nuclei from 1-day-castrated animals the Kd for binding of cytosolic [3H]dihydrotestosterone-receptor complexes was found to be 0.83 × 10(-10) M and the capacity for binding was 0.35 pmol/mg of nuclear DNA. Scatchard analysis consistently disclosed only a single line of constant slope and gave the same kinetic constants for nuclei obtained from animals castrated up to 28 days before assay. Administration of 2 mg of dihydrotestosterone, 3 alpha-androstanediol or androsterone or 100 microgram of oestradiol-17 beta 1 h before killing of the 1-day-castrated animals that provided the nuclei resulted in a significant decrease in nuclear acceptor binding of the steroid-receptor complex compared with untreated animals. Thus our assay method disclosed nuclear acceptor sites that may be involved in responses to androgens (and oestrogens) in vivo. We conclude that there is a class of nuclear accept or sites of high affinity and limited capacity that may be occupied by steroid-receptor complexes in vivo.
Antiserum against the catalytic subunit of adenosine 3′:5′-cyclic monophosphate-dependent protein kinase. Reactivity towards various protein kinases
Authors: Schwoch G , Hamann A , Hilz H
Published: August 2015

An antiserum against the catalytic subunit C of cyclic AMP-dependent protein kinase, isolated from bovine heart type II protein kinase, was produced in rabbits. Reaction of the catalytic subunit with antiserum and separation of the immunoglobulin G fraction by Protein A-Sepharose quantitatively removed the enzyme from solutions. Comparative immunotitration of protein kinases showed that the amount of antiserum required to eliminate 50% of the enzymic activity was identical for pure catalytic subunit, and for holoenzymes type I and type II. The reactivity of the holoenzymes with the antiserum was identical in the absence or the presence of dissociating concentrations of cyclic AMP. Most of the holoenzyme (type II) remains intact when bound to the antibodies as shown by quantification of the regulatory subunit in the supernatant of the immunoprecipitate. Titration with the antibodies also revealed the presence of a cyclic AMP-independent histone kinase in bovine heart protein kinase I preparations obtained by DEAE-cellulose chromatography. Cyclic AMP-dependent protein kinase purified from the particulate fraction of bovine heart reacted with the antiserum to the same degree as the soluble enzyme, whereas two cyclic AMP-independent kinases separated from the particle fraction neither reacted with the antiserum nor influenced the reaction of the antibodies with the cyclic AMP-dependent protein kinase. Immunotitration of the protein kinase catalytic subunit C from rat liver revealed that the antibodies had rather similar reactivities towards the rat liver and the bovine heart enzyme. This points to a relatively high degree of homology of the catalytic subunit in mammalian tissues and species. Broad applicability of the antiserum to problems related to cyclic AMP-dependent protein kinases is thus indicated.
Chemical structure and biodegradability of halogenated aromatic compounds. Halogenated muconic acids as intermediates
Authors: Schmidt E , Remberg G , Knackmuss H J
Published: August 2015

Substituted muconic acids were prepared from the corresponding catechols by pyrocatechase II from Pseudomonas sp. B13. The stabilities of substituted muconic acids were compared under different pH conditions. 3-Substituted cis, cis-muconic acids cycloisomerized readily in slightly acidic solutions, whereas 2-chloro- and 2-fluoro-cis, cis-muconic acids were stable under these conditions and could be isolated as crystalline compounds. They were isomerized to the cis, trans-form in highly acidic solution (pH 1), particularly when heated to 80 degrees C. Cycloisomerization of 2-chloro-cis, cis-muconic acid in 75% (v/v) H2SO4 yields 4-carboxymethyl-2-chloro-but-2-en-4-olide (4-chloro-2,5-dihydro-5-oxo-3H-furan-2-ylacetic acid). THe cis, cis-configuration of 2-chloromuconic acid was certified by 1H n.m.r. spectroscopy and by enzymic cycloisomerization. Although the cis, cis-configuration of 2-fluoromuconic acid was confirmed by corresponding spectroscopic data, it was not cycloisomerized by crude extracts or cycloisomerase II preparations from Pseudomonas sp. B13.
Isolation of α-subunits of factor F1 from submitochondrial particles and the reconstitution of active ATPase from isolated α-subunits and β-subunits bound to the mitochondrial membrane
Authors: Kozlov I A , Milgrom Y M , Tsybovski I S
Published: August 2015

The alpha-subunits of factor-F1 ATPase are removed by extraction of submitochondrial particles with 1.75 M-LiCl, with the consequent loss of ATPase activity. ATPase activity is reconstituted by incubation of LiCl-extracted particles with purified alpha-subunits, and the reconstituted ATPase activity is oligomycin-sensitive. Reconstitution is enhanced by maintenance of the alpha-subunits in reduced form by dithiothreitol or NaBH4 and by modification of the alpha-subunits by p-chloromercuribenzoate, iodoacetic acid or N-ethylmaleimide. Experiments with the mixed anhydride of ATP and mesitylene-carboxylic acid, which was previously shown to interact with the F1 active site, localized on the beta-subunits, indicate that the active site of ATPase is shielded by the alpha-subunits.
The latent collagenase and gelatinase of human polymorphonuclear neutrophil leucocytes
Authors: Murphy G , Bretz U , Baggiolini M , Reynolds J J
Published: August 2015

Two metallo-proteinases of human neutrophil leucocytes, collagenase and gelatinase, were studied. Collagenase specifically cleaved native collagen into the TCA and TCB fragments, whereas gelatinase degraded denatured collagen, i.e. gelatin, and the TCA fragments produced by collagenase. On subcellular fractionation by zonal sedimentation, collagenase was found to be localized in the specific granules, separate from gelatinase, which was recovered in smaller subcellular organelles known as C-particles. Neither enzyme was present in the azurophil granules, which contain the two major serine proteinases of neutrophils, elastase and cathepsin G. Collagenase and gelatinase were separated by gel filtration from extracts of partially purified granules. Both enzymes were found to occur in latent forms and were activated either by trypsin or by 4-aminophenylmercuric acetate. Gelatinase was also activated by cathepsin G, which, however, destroyed collagenase. Both enzymes were destroyed by neutrophil elastase. Activation resulted in a decrease by 25 000 in the apparent mol. wt. of both latent metallo-proteinases.
Biosynthesis of glycoproteins by membranes of Acer pseudoplatanus. Incorporation of mannose and N-acetylglucosamine
Authors: Barr J , Nordin P
Published: August 2015

Membrane preparations from Acer pseudoplatanus suspension cultures were demonstrated to incorporate radioactivity from GDP-[U-14C]mannose and UDP-N-acetyl-[6-(3)H]glucosamine into high-molecular-weight polymers characterized as glycoprotein. From 20 to 25% of the 14C was incorporated as fucose with the remainder as mannose, whereas 90% of the 3H was incorporated as N-acetylglucosamine with the remainder as N-acetylgalactosamine. Pronase digestion yielded radioactive glycopeptides that were separated into four fractions by gel-permeation chromatography and paper electrophoresis. The isolated glycopeptides differed in molecular weight and isotopes incorporated, as well as in amino-acid and monosaccharide composition. The membrane preparation also incorporated radioactivity from the added nucleotides into chloroform/methanol (2:1, v/v)- and chloroform/methanol/water (10:10:3, by vol.)-soluble lipids, and into an insoluble pellet.
Measurement of the synthesis rates of collagens and total protein in rabbit muscle
Authors: Palmer R M , Robins S P , Lobley G E
Published: August 2015

1. Collagen- and total-protein-synthesis rates were determined in rabbit muscle by continuous infusion of radioactive proline. 2. The precursor pool of free proline used for collagen synthesis was defined by measuring the specific radioactivity of hydroxy-proline in isolated type I procollagen. The specific radioactivities of type I procollagen were about 40% of those for free proline in the homogenate. 3. The mean ratio (+/- S.E.M.) between the fractional synthesis rates of muscle collagen and total protein was 0.99 +/- 0.10, where the total protein values were based on specific radioactivities of the homogenate free proline pools. 4. Types I, III and V collagen were solubilized by pepsin and isolated by fractional precipitation with NaCl. The fractional synthesis rates of types I and III collagens were very similar. Type V collagen samples had higher specific radioactivities than the other collagens, but this was not necessarily indicative of a higher rate of synthesis because of uncertainty about the cellular origin of this collagen and, hence, the specific radioactivity of its precursor proline pool.
Selective permeability of rat liver mitochondria to purified malate dehydrogenase isoenzymes in vitro
Authors: Passarella S , Marra E , Doonan S , Quagliariello E
Published: August 2015

1. The mitochondrial malate dehydrogenase from rat liver has been purified to a state of homogeneity as judged by starch-gel electrophoresis and the cytoplasmic isoenzyme has been obtained in a partically purified state. 2. Inhibition of the isoenzymes by sulphite has been studied. 3. In mitochondria loaded with sulphite, the catalytic activity of the (partially inhibited) internal malate dehydrogenase has been measured by addition of oxaloacetate to the suspension medium and observation of the consequent decrease in fluorescence of NADH. 4. Addition of mitochondrial malate dehydrogenase to suspensions of mitochondria loaded with sulphite resulted in an increase in the level of intramitochondrial enzymic activity as measured by the above technique. Addition of the cytoplasmic isoenzyme did not result in such an increase. 5. These results show that mitochondria in suspension are permeable to the mitochondrial malate dehydrogenase but not to the cytoplasmic isoenzyme. 6. This conclusion has been confirmed by direct measurement of a decrease of enzyme activity in solution and an increase inside the mitochondria after incubation of organelles in solutions containing mitochondrial malate dehydrogenase. No such effect was observed with the cytoplasmic isoenzyme. 7. Some features of the permeation process have been studied.
Interrelationship of glycogen metabolism and lactose synthesis in mammary epithelial cells of mice
Authors: Emerman J T , Bartley J C , Bissell M J
Published: August 2015

Glycogen metabolism in mammary epithelial cells was investigated (i) by studying the conversion of glucose into glycogen and other cellular products in these cells from virgin, pregnant and lactating mice and (ii) by assaying the enzymes directly involved with glycogen metabolism. We find that: (1) mammary epithelial cells synthesized glycogen at rates up to over 60% that of the whole gland; (2) the rate of this synthesis was modulated greatly during the reproductive cycle, reaching a peak in late pregnancy and decreasing rapidly at parturition, when abundant synthesis of lactose was initiated; (3) glycogen synthase and phosphorylase activities reflected this modulation in glycogen metabolism; (4) lactose synthesis reached a plateau during late pregnancy, even though lactose synthase is reported to increase in the mouse mammary gland at this time. We propose that glycogen synthesis restricts lactose synthesis during late pregnancy by competing successfully for the shared UDP-glucose pool. The physiological advantage of glycogen accumulation during late pregnancy is discussed.
Calcium-activated thiol-proteinase activity in the fusion of rat erythrocytes induced by benzyl alcohol
Authors: Ahkong Q F , Botham G M , Woodward A W , Lucy J A
Published: August 2015

1. Rat erythrocytes were fused by incubation with benzyl alcohol and Ca2+. 2. Cell fusion was inhibited by EGTA, N-ethylmaleimide, tetrathionate, iodoacetamide, cystamine, Tos-Lys-CH2Cl, and to a lesser extent by Tos-Phe-CH2Cl. Phenylmethanesulphonyl fluoride, Tos-Arg-OMe and histamine did not inhibit cell fusion. 3. Gel electrophoresis of membrane proteins from “ghosts” of the erythrocytes treated with benzyl alcohol showed that a high-molecular-weight polymer was present: this was consistent with the entry into the cells of Ca2+ and the activation of a transglutaminase enzyme. 4. In the treated cells the proteins corresponding to bands 2 and 3 in human erythrocytes were decreased, and a polypeptide with a slightly greater mobility than band 3 was produced. 5. These changes were inhibited by EGTA, N-ethylmaleimide, tetrathionate, iodoacetamide, cystamine, and Tos-Lys-CH2Cl, but not by phenylmethanesulphonyl fluoride, Tos-Arg-OMe, or histamine. 6. The intramembraneous particles of the P-fracture face of cells treated with benzyl alcohol to induce fusion were decreased in number and were susceptible to cold-induced aggregation; both of these phenomena were markedly inhibited to EGTA, and partially inhibited by Tos-Lys-CH2Cl and N-ethylmaleimide. 7. These several observations indicate that a Ca2+-activated thiol-proteinase, which acts to degrade membrane proteins and to give freedom of lateral movement to intramembranous particles, may be essential feature of membrane fusion in this system. 8. It is suggested that this proteinase may act to degrade spectrin-binding proteins that attach band-3 protein to the erythrocyte cytoskeleton.
NADH- and NADPH-dependent lipid peroxidation in bovine heart submitochondrial particles. Dependence on the rate of electron flow in the respiratory chain and an antioxidant role of ubiquinol
Authors: Takayanagi R , Takeshige K , Minakami S
Published: August 2015

Malondialdehyde formations by bovine heart submitochondrial particles supported by NADH or NADPH in the presence of ADP and FeCl3 was studied. The NADH-dependent reaction was maximal at very low rate of electron input from NADH to the respiratory chain and it decreased when the rate became high. The reaction was stimulated by rotenone and inhibited by antimycin A when the input was fast, whereas it was not affected by the inhibitors when the input was slow. The input rate of the electrons from NADPH was also so low that the reaction supported by NADPH was not affected by the inhibitors. Most of the endogenous ubiquinone in the particles treated with antimycin A was reduced by NADH even in the presence of ADP-Fe3+ chelate, but uniquinone was not reduced by NADPH when ADP-Fe3+ was present. Succinate strongly inhibited both NADH- and NADPH-dependent lipid peroxidation. The inhibition was abolished when uniquinone was removed from the particles, and it appeared again when uniquinone was reincorporated into the particles. Reduced uniquinone-2 also inhibited the peroxidation, but duroquinol, which reduces cytochrome b without reducing endogenous uniquinone, did not. Thus the malondialdehyde formation appeared to be inversely related to the extent of the reduction of endogenous uniquinone. These observations suggest that both NADH- and NADPH-dependent liquid-peroxidation reactions are closely related to the respiratory chain and that the peroxidation is controlled by the concentration of reduced ubiquinone.
Sulphate transport by H+ symport and by the dicarboxylate carrier in mitochondria
Authors: Saris N E L
Published: August 2015

1. Swelling of mitochondria was induced in non-respiring mitochondria by 30 mM or more Na2SO4 or by respiration in the presence of K2SO4. Respiration-drive swelling resulted in loss of respiratory control. Sulphate, when present at 10 mM concentration, promoted the release of accumulated Ca2+. 2. Swelling was prevented by N-ethylmaleimide and formaldehyde, known inhibitors of the phosphate carrier. Sulphate-induced swelling was more sensitive to the inhibitors than was phosphate-induced swelling. At lower concentration of sulphate, 5 mM, an alkalinisation of the medium was observed in addition of sulphate, indicating H+-sulphate symport. There was competition between sulphate and phosphate for transport by this mechanism. It is suggested that sulphate may be transported, though at a comparatively slow rate, by the phosphate carrier. 3. Uptake of sulphate was stimulated when preceded by energy-dependent accumulation of Ba2+, especially when acetate was also present, indicating precipitation of BaSO4 in the matrix. Using this system the influx of sulphate was studied at lower concentrations, 10 mM or less. the contributions of the H+ symporter (sensitive to N-ethylmaleimide) and the dicarboxylate carrier (sensitive to butylmalonate) could then be studied. The dicarboxylate carrier had a lower Km and was mainly responsible for sulphate transport at lower concentration range. At 10 mM-sulphate the transport rates by the two systems appeared to be similar; at still higher concentrations the H+ symporter may become more important.
Autolytic fragmentation of complement components C3 and C4 under denaturing conditions, a property shared with α 2-macroglobulin
Authors: Sim R B , Sim E
Published: August 2015

The alpha polypeptide chain of the complement protein C3 splits into two fragments of 74 000 and 46 000 apparent mol.wt. under certain conditions used to prepare the protein for SDS (sodium dodecyl sulphate)/polyacrylamide-gel electrophoresis. The cleavage reaction occurs over a wide range of temperatures and from pH 4.6 to 10.6 in the presence of denaturants such as urea, SDS and guanidine hydrochloride. It is also induced by heat-denaturation of C3 in the absence of chemical denaturants. The reaction occurs only with haemolytically active C3, and is not observed with hydroxylamine-inactivated C3 or with C3b. A similar cleavage of the alpha-chain of complement component C4 occurs under the same conditions, forming fragments of 53 000 and 41 000 apparent mol.wt. This reaction is again specific for haemolytically active C4, and does not occur with C4b or hydroxylamine-inactivated C4. The complement component C5, although structurally similar to C3 and C4, does not undergo a reaction of this type. The characteristics of the denaturation-induced cleavage of C3 and C4 match those described for the ‘heat-induced’ cleavage of alpha 2-macroglobulin [Harpel, Hayes & Hugli (1979) J. Biol. Chem. 254, 8669-8678]. Cleavage of alpha 2-macroglobulin is also specific for the active form of the protein, and does not occur with chemically inactivated or proteinase-cleaved forms. The unusual conditions and specificity of the peptide-bond cleavage in all three proteins suggest that it is an autolytic process rather than being the result of trace proteinase contamination. The active forms of C3, C4 and alpha 2-macroglobulin have the transient ability to form covalent bonds after activation. The autolytic cleavage reaction is likely to be related to the covalent-bond-forming reactions of these proteins.
On the reactivity of metallothioneins with 5,5′-dithiobis-(2-nitrobenzoic acid)
Authors: Li T Y , Minkel D T , Shaw C F , Petering D H
Published: August 2015

Rat liver and horse kidney metallothioneins react with 5,5′-dithiobis-(2-nitrobenzoic acid) (Nbs2) to release 5-thio-2-nitrobenzoate and metal ions. The reactions are slow and exhibit biphasic kinetics with each process having an empirical rate law of the form: rate - k[RSM] X [Nbs2] + k'[RSM], where RSM represents mental-bound thiolate groups. The pseudo-first-order rates are insensitive to pH but are modified in guanidine hydrochloride solution. Rat liver metallothioneins of variable zinc, copper and cadmium composition react similarly and give observable thiol/total metal ratios in good agreement with stoichiometries of SH/(Cd + Zn) of 3 and SH/Cu of 1. A model complex cadmium-2,3-dimercaptopropanol, resembles the proteins in its reaction with Nbs2.
The simultaneous binding of lanthanide and N-acetylglucosamine inhibitors to hen egg-white lysozyme in solution by 1H and 13C nuclear magnetic resonance
Authors: Perkins S J , Johnson L N , Phillips D C , Dwek R A
Published: August 2015

Lanthanide ions and the N-acetylglucosamine (GlcNAc) sugars are able to bind simultaneously to hen egg-white lysozyme (EC 3.2.1.17). The present study characterizes the properties of the ternary complexes with lysozyme, which involve up to seven paramagnetic lanthanides and two diamagnetic lanthanides, together with alpha GlcNAc, beta GlcNAc, alpha MeGlcNAc and beta MeGlcNAc. pH titrations and binding titrations of the GlcNAc sugars with lysozyme-La(III) complexes show that the GlcNAc sugars bind to at least two independent sites and that one of them competes with La(III) for binding to lysozyme. Given the known binding site of lanthanides at Asp-52 and Glu-35, the competitive binding site of GlcNAc is identified as subsite E. A simple analysis of the paramagnetic-lanthanide-induced shifts shows that the GlcNAc sugar binds in subsite C, in accordance with crystallographic results [Perkins, Johnson, Machin & Phillips (1979) Biochem. J. 181, 21-36]. This finding was refined by several computer analyses of the lanthanide-induced shifts of 17 proton and carbon resonances of beta MeGlcNAc. Good fits were obtained for all the signals, except for two that were affected by exchange broadening phenomena. No distinction could be made between a fit for a two-position model of Ln(III) binding with axial symmetry to lysozyme, according to the crystallographic result, or a one-position model with axial symmetry where the Ln(III) is positioned mid-way between Asp-52 and Glu-35. Although this work establishes the feasibility of lanthanide shift reagents for study of protein-ligand complexes, further work is required to establish the manner in which lanthanides bind to lysozyme in solution.
Calcium-binding constants of trypsin and trypsinogen. A reassessment
Authors: Cliffe S G R , Grant D A W
Published: August 2015

The Ca2+-binding constants for trypsin and trypsinogen have been reassessed by using enzyme that has been purified by affinity chromatography and measuring the distribution of 45Ca2+ between the protein and a cation exchanger. The pKCa2+ value of 4.5 for the high-affinity site on trypsin was 1 logarithmic unit greater than that previously reported.
The stability and aggregation properties of human liver acid β-d-galactosidase
Authors: Heyworth C M , Neumann E F , Wynn C H
Published: August 2015

1. A method is described for following continuously the action of beta-galactosidase on 4-methylumbelliferyl beta-D-galactoside at pH 4.5, in which 4-methylumbelliferone production is measured at fluorescence excitation and emission wavelengths of 324 and 444nm respectively. 2. Initial-rate studies show that the presence of salt activates beta-galactosidase up to 100 mM, but is inhibitory above that concentration. The enzyme is very unstable at 37 degrees C and low ionic strength, but stability increases with ionic strength. 3. The stability of the enzyme at 37 degrees C decreases markedly with rising pH in the range 5.9–8.0. 4. Gel-filtration patterns demonstrate that there is a marked tendency to polymerization with increasing ionic strength. The gel-filtration pattern shows decreasing amounts of dimer with increasing pH. 5. The correlation between activity, stability and molecular form of beta-galactosidase is discussed. It is suggested that the dimeric form of the enzyme is the most stable and active form. The implications of this finding for the assay of beta-galactosidase under physiological conditions for prenatal diagnosis are discussed. 6. Evidence for the possible occurrence of a 36 000-mol.wt. from of beta-galactosidase is presented. 7. A computer program for the calculation of initial rates has been deposited as Supplementary Publication SUP 50114 (4 pages) at the British Library Lending Division, Boston Spa, Wetherby, West Yorkshire LS23 7BQ, U.K., from whom copies can be obtained on the terms indicated in Biochem. J. (1981) 193, 5.
Isolation, properties and amino acid sequence of a long-chain neurotoxin, Acanthophis antarcticus b, from the venom of an Australian snake (the common death adder, Acanthophis antarcticus)
Authors: Kim H S , Tamiya N
Published: August 2015

The venom of an Australian elapid snake, the common death adder (Acanthophis antarcticus), was chromatographed on a CM-cellulose CM52 column. One of the neurotoxic components, Acanthophis antarcticus b (toxin Aa b) was isolated in about 9.4% (A280) yield. The complete amino acid sequence of toxin Aa b was elucidated. Toxin Aa b is composed of 73 amino acid residues, with ten half-cystine residues, and has a formula weight of 8135. Toxin Aa b has no histidine or methionine residue in its sequence. The amino acid sequence of toxin Aa b is homologous with those of other neurotoxins with known sequences, although it is novel in having a valine residue at its N-terminus and an arginine residue at position-23, where a lysine residue is found in almost all the so-far-known neurotoxins. Irrespective of the latter replacement, the toxin Aa b is fully active, with an LD50 value (in mice) of 0.13 microgram/g body weight on intramuscular injection.
A cryptobiosis-specific 19S protein complex of Artemia salina gastrulae
Authors: De Herdt E , De Voeght F , Clauwaert J , Kondo M , Slegers H
Published: August 2015

The postribosomal supernatant of Artemia salina cryptobiotic embryos contains a large quantity of a 19S protein complex. An amount of 3.6 mg/g of cysts is measured by immunoprecipitation with anti-(19S protein complex) antibody. The quantity of this complex decreases during further development to nauplius larvae to only 15% of the quantity present in cryptobiotic embryos. The complex was no longer detectable after 7 days of growth. The 27000-Mr protein subunit of the 19S complex is not synthesized by mRNA isolated from cryptobiotic embryos. The cryptobiosis-specific complex has Mr 573000 and 610000 as calculated from light-scattering and sedimentation-diffusion measurements respectively. The 19S homocomplex contains 20-23 27000-Mr proteins and has no function in the translation of homologous mRNA. From hydrodynamic data a hydration of 1.25 g of water/g of protein is calculated. The abundant presence of the 19S protein complex in cryptobiotic embryos and the absence of synthesis during development to nauplius larvae indicate a functional role during the cryptobiotic process in early embryogenesis. A role in maintaining the water content of the cytoplasm above a critical threshold during desiccation is suggested.
Effect of sodium n-butyrate on induction of prostaglandin synthase activity in cloned mastocytoma P-815 2-E-6 cells
Authors: Koshihara Y , Kawamura M , Senshu T , Murota S
Published: August 2015

Treatment of a cloned mastocytoma P-815 cell line (2-E-6) with 1 mM-sodium n-butyrate for 40h induced prostaglandin synthase activity and arrested cell growth. The induction of enzyme activity by n-butyrate was not associated with suppression of DNA synthesis, since hydroxyurea had no effect on prostaglandin synthase induction. The effect of sodium n-butyrate was reversible. Experiments with cycloheximide and actinomycin D showed that the induction of prostaglandin synthase activity involved synthesis de novo of protein and RNA. The time of half-maximal induction of the newly synthesized RNA for prostaglandin synthase activity was estimated as about 15h, which is similar to the generation time of the cells. Selective induction of fatty acid cyclo-oxygenase activity by sodium n-butyrate was suggested by the following two pieces of experimental evidence. (1) There was no significant difference between treated and untreated cells in the activities of radioactive prostaglandin H2 conversion into individual prostaglandins. (2) The incorporation of [3H]acetylsalicylic acid into the fraction equivalent to protein of mol.wt. approx. 75000 of sodium n-butyrate-treated cells was higher than that of untreated cells, on analysis of cell particulate fraction by sodium dodecyl sulphate/polyacrylamide-gel electrophoresis. A high concentration (5 mM) of sodium propionate also induced prostaglandin synthesis, but other short-chain fatty acids, such as isobutyrate and sodium acetate, had no effect.
Ca2+-dependent and Ca2+-independent degradation of phosphatidylinositol in rabbit vas deferens
Authors: Egawa K , Sacktor B , Takenawa T
Published: August 2015

The effects of Ca2+ and acetylcholine on the degradation and synthesis of phosphatidylinositol in rabbit vas deferens was studied in vitro by a pulse–chase technique and by measuring the content of the phospholipid in the tissue. Ca2+-dependent degradation of phosphatidylinositol was found in slices and homogenates prelabelled with myo-[2-3H]inositol. The phosphatidylinositol content of the slices also decreased by a Ca2+-dependent mechanism. On the other hand, removal of intracellular Ca2+ with the ionophore A23187 and EGTA increased the amount of phosphatidylinositol. These results indicate that the intracellular Ca2+ concentration has an important role in regulating the phosphatidylinositol content of the tissue. Increasing the extracellular K+ concentration, which causes an increase in plasma-membrane Ca2+ permeability, did not enhance phosphatidylinositol breakdown nor decrease its tissue content. However, phosphatidylinositol synthesis was clearly inhibited. After stimulation of the smooth muscle with acetylcholine, degradation of phosphatidylinositol was enhanced. Furthermore, the content of phosphatidylinositol in the tissue also decreased. These phenomena were evident even in the absence of Ca2+. The acetylcholine-induced degradation of phosphatidylinositol was blocked by the muscarinic antagonist atropine, but not by the nicotinic antagonist (+)-tubocurarine. The acetylcholine-induced decrease in the phosphatidylinositol content of the tissue led to the compensatory synthesis of phosphatidylinositol. Synthesis was separated from degradation in the same tissue. Compensatory synthesis was inhibited by acetylcholine. The degradation of phosphatidylinositol induced by acetylcholine was not inhibited by 8-bromoguanosine 3′:5′-cyclic monophosphate, indicating that the degradative process was not mediated by an increase in the cyclic nucleotide.
Phosphatidylinositol metabolism in rat hepatocytes stimulated by vasopressin
Authors: Kirk C J , Michell R H , Hems D A
Published: August 2015

In isolated rat hepatocytes, vasopressin evoked a large increase in the incorporation of [32P]Pi into phosphatidylinositol, accompanied by smaller increases in the incorporation of [1-14C]oleate and [U-14C]glycerol. Incorporation of these precursors into the other major phospholipids was unchanged during vasopressin treatment. Vasopressin also promoted phosphatidylinositol breakdown in hepatocytes. Half-maximum effects on phosphatidylinositol breakdown and on phosphatidylinositol labelling occurred at about 5 nM-vasopressin, a concentration at which approximately half of the hepatic vasopressin receptors are occupied but which is much greater than is needed to produce half-maximal activation of glycogen phosphorylase. Insulin did not change the incorporation of [32P]Pi into the phospholipids of hepatocytes and it had no effect on the response to vasopressin. Although the incorporation of [32P]Pi into hepatocyte lipids was decreased when cells were incubated in a Ca2+-free medium, vasopressin still provoked a substantial stimulation of phosphatidylinositol labelling under these conditions. Studies with the antagonist [1-(beta-mercapto-beta, beta-cyclopentamethylenepropionic acid),8-arginine]vasopressin indicated that the hepatic vasopressin receptors that control phosphatidylinositol metabolism are similar to those that mediate the vasopressor response in vivo. When prelabelled hepatocytes were stimulated for 5 min and then subjected to subcellular fractionation. The decrease in [3H]phosphatidylinositol content in each cell fraction with approximately in proportion to its original phosphatidylinositol content. This may be a consequence of phosphatidylinositol breakdown at a single site, followed by rapid phosphatidylinositol exchange between membranes leading to re-establishment of an equilibrium distribution.
The effects of N-alkylmaleimides on the activity of rat liver glucose 6-phosphatase
Authors: Vakili B , Banner M
Published: August 2015

A series of N-alkylmaleimides has been synthesized and used to investigate the thiol groups that are essential for the activity of rat liver microsomal glucose 6-phosphatase. All of the N-alkylmaleimides inactivated glucose 6-phosphatase when preincubated with microsomes (microsomal fractions) at pH 6.5 and 30 degrees C. When enzyme activity was assayed in intact microsomes, the inactivation was non-linear with respect time, showing an initial rapid phase followed by a slower secondary phase. During the initial rapid phase the inactivation may apparently be completely reversed by disrupting the microsomal membrane with detergent. However, after longer exposure to N-alkylmaleimides the reversal is no longer complete. This observation was explained by the results obtained from studying the inactivation in detergent-disrupted microsomes. In this case glucose 6-phosphatase was also completely inactivated, but much more slowly than was seen in intact microsomes, and the process was linear with respect to time. When assayed in both intact and detergent-disrupted microsomes, glucose 6-phosphatase inactivation was dependent on the number of carbon atoms in the alkyl side chain of the N-alkylmaleimides; this dependence was much more marked in disrupted microsomes. Analysis of the data showed that in neither case was there a saturating effect at high concentrations of maleimide. The data have been interpreted to suggest that there are are least two thiol groups essential for activity located in two separate non-polar regions of the membrane-enzyme system. The conclusions are discussed in the light of the current model for the microsomal glucose 6-phosphatase system.
The relationship of phosphatidylinositol turnover to receptors and calcium-ion channels in rat parotid acinar cells
Authors: Weiss S J , Putney J W
Published: August 2015

To help elucidate the possible role of phosphatidylinositol in the regulation of membrane permeability to Ca2+, the relationship in the rat parotid gland of phosphatidylinositol turnover to hormone receptor binding and to the hormone-mediated increase in K+ permeability (a Ca2+-dependent phenomenon) was investigated. The concentrations of adrenaline and substance P required to stimulate phosphatidylinositol turnover were found to be similar to those required for the Ca2+-mediated change in K+ permeability and for ligand binding. However, in the case of muscarinic (cholinergic) receptor stimulation, the phosphatidylinositol response was better correlated to the increase in membrane permeability to Ca2+, as determined by the change in K+ permeability, than to receptor occupation. Consistent with this relationship between the phosphatidylinositol response and Ca2+-channel activation were results obtained by simultaneous administration of maximal or submaximal concentrations of muscarinic and alpha-adrenergic agonists. The extent of 32P incorporation when stimulated by maximal concentrations of two agonists did not summate, but, rather, was intermediate between the response of either agonist alone. One interpretation for these observations is that the phosphatidylinositol response may not be related to receptor occupation or activation, but may be involved in the Ca2+-gating mechanism itself.
The activation of pyruvate dehydrogenase in the perfused rat heart by adrenaline and other inotropic agents
Authors: McCormack J G , Denton R M
Published: August 2015

Adrenaline resulted in a reversible 4-fold increase in the amount of pyruvate dehydrogenase in its active non-phosphorylated form in the perfused rat heart within 1 min. The increase was less in extent in hearts from starved or diabetic rats or in hearts from control rats oxidizing acetate, unless pyruvate was added to the perfusion medium. Increases could also be induced by other inotropic agents, supporting the hypothesis that increases in cytoplasmic Ca2+ can be relayed into mitochondria and influence oxidative metabolism.
Factors controlling the expressed activity of histidine ammonia-lyase in the epidermis and the resulting accumulation of urocanic acid
Authors: Scott I R
Published: August 2015

The synthesis of urocanic acid by histidine ammonia-lyase in guinea-pig epidermis was investigated in various ways. 1. In epidermal homogenates the enzyme obeys Michaelis-Menten kinetics and shows marked dependence of its activity of pH, such that below pH 6 it is inactive. 2. Part-thickness skin samples cultured with radioactive histidine do not accumulate detectable radioactive urocanic acid during 3 days in culture. 3. Very little histidine ammonia-lyase activity can be detected in the living cells of the epidermis. The enzyme is almost completely restricted to the dead cells of the stratum corneum. 4. Radioactive histidine injected into living animals does not result immediately in the accumulation of radioactive urocanic acid. By 3 days after the injection, however, both radioactive urocanic acid and histidine appear, apparently at the expense of radioactive proteins, 5. In isolated stratum corneum, the residual histidine can be converted into urocanic acid by the histidine ammonia-lyase in the tissue only if the natural acidity of the tissue is neutralized. It is concluded from these observations that the biosynthesis of urocanic acid occurs naturally only in the stratum corneum, which contains only dead cells. The amount of urocanic acid accumulated is limited by the availability of free histidine produced by proteolysis of residual protein in these cells and by the penetration into the stratum corneum of the ‘acid mantle’ of the skin.
Synthesis and secretion of alkaline phosphatase in vitro from first-trimester and term human placentas
Authors: Galski H , Fridovich S E , Weinstein D , De Groot N , Segal S , Folman R , Hochberg A A
Published: August 2015

The synthesis and secretion of alkaline phosphatases in vitro by human placental tissue incubated in organ culture were studied. First-trimester placenta synthesizes and secretes two different alkaline phosphatase isoenzymes (heat-labile and heat-stable), whereas in term placenta nearly all the alkaline phosphatase synthesized and secreted is heat-stable. The specific activities of alkaline phosphatases in first-trimester and term placental tissue remain constant throughout the time course of incubation. In the media, specific activities increase with time. Hence, alkaline phosphatase synthesis seems to be the driving force for its own secretion. The rates of synthesis de novo and of alkaline phosphatases were measured. The specific radioactivities of the secreted alkaline phosphatases were higher than the corresponding specific radioactivities in the tissue throughout the entire incubation period. The intracellular distribution of the alkaline phosphatase isoenzymes was compared.
Elimination and replenishment of tricarboxylic acid-cycle intermediates in myocardium
Authors: Nuutinen E M , Peuhkurinen K J , Pietiläinen E P , Hiltunen J K , Hassinen I E
Published: August 2015

1. The contribution of Co2 fixation to the anaplerotic mechanisms in the myocardium was investigated in isolated perfused rat hearts. 2. K+-induced arrest of the heart was used to elicit a transition in the concentrations of the intermediates of the tricarboxylic acid cycle. 3. Incorporation of 14C from [14]bicarbonate into tricarboxylic acid-cycle intermediates was measured and the rates of the reactions of the cycle were estimated by means of a linear optimization program which solves the differential equations describing a simulation model of the tricarboxylic acid cycle and related reactions. 4. The results showed that the rate of CO2 fixation is dependent on the metabolic state of the myocardium. Upon a sudden diminution of cellular ATP consumption, the pool size of the tricarboxylic acid-cycle metabolites increased and the rate of label incorporation from [14C]bicarbonate into the cycle metabolites increased simultaneously. The computer model was necessary to separate the rapid equilibration between bicarbonate and some metabolites from the potentially anaplerotic reactions. The main route of anaplerosis during metabolite accumulation was through malate + oxaloacetate. Under steady-state conditions there was a constant net outward flow from the tricarboxylic acid cycle via the malate + oxaloacetate pool, with a concomitant anaplerotic flow from metabolites forming succinyl-CoA (3-carboxypropionyl-CoA).
Insulin inhibition of α-adrenergic actions in liver
Authors: Dehaye J P , Hughes B P , Blackmore P F , Exton J H
Published: August 2015

The effects of insulin on alpha-agonist (phenylephrine)- and [Arg8]vasopressin-induced Ca2+ and glucose release and mitochondrial Ca2+ fluxes in isolated perfused rat livers were examined. Insulin (6 nM) inhibited the ability of phenylephrine (1 and 0.5 microM) to elicit Ca2+ and glucose release, whereas it was without effect on vasopressin (10 and 2.5 nM) actions. Correspondingly, insulin inhibited the action of phenylephrine to induce a stable increase in mitochondrial Ca2+ uptake, but it did not affect the alteration caused by vasopressin. Phenylephrine and vasopressin caused transient increases in hepatocyte respiration. Insulin inhibited the effect of phenylephrine on this parameter, but not that of vasopressin. Insulin added alone did not alter any of the above parameters. It is concluded from these data that insulin does not alter cellular Ca2+ fluxes and respiration themselves, but selectively inhibits alpha-adrenergic stimulation of these processes. It is proposed that insulin acts either to inhibit binding of alpha-agonists to their specific plasma-membrane receptors or to alter generation and/or degradation of the putative alpha-adrenergic ‘second messenger’. If this latter possibility is the case, then the alpha-adrenergic ‘second messenger’ must be different from the ‘second messenger’ of vasopressin.
Phosphorylation in vivo of non-ribosomal proteins from native 40 S ribosomal particles of Krebs II mouse ascites-tumour cells
Authors: Schuck J , Reichert G , Issinger O G
Published: August 2015

Four non-ribosomal proteins from native 40 S ribosomal subunits with mol.wts. of 110 000, 84 000, 68 000 and 26 000 were phosphorylated in vivo when ascites cells were incubated in the presence of [32P]Pi. The 110 000-, 84 000- and 26 000-dalton proteins are identical with phosphorylated products from native 40 S subunits after phosphorylation in vitro by a cyclic nucleotide-independent protein kinase. Phosphoserine was the major phosphorylated amino acid of the proteins phosphorylated in vivo and in vitro.
Ribosomal core-particles as the target of ricin
Authors: Zamboni M , Battelli G , Montanaro L , Sperti S
Published: August 2015

Core-particles and split-proteins were prepared by treatment with ethanol and NH4Cl of control and ricin-treated Artemia salina ribosomes. No modifications of the ricin-treated split-proteins was detected by polyacrylamide-gel electrophoresis. Moreover, the split-proteins from ricin-treated ribosomes complemented control core-particles in poly(U)-directed phenylalanine polymerization. Conversely, ricin-treated core-particles remained totally inactive when supplemented with control split-proteins.
An inhibitor of collagenase from human amniotic fluid. Purification, characterization and action on metalloproteinases
Authors: Murphy G , Cawston T E , Reynolds J J
Published: August 2015

1. An inhibitor of collagenase of apparent mol.wt. 28000 was isolated from term human amniotic fluid. 2. It is active against mammalian collagenases from a number of species and tissues as well as other mammalian metalloproteinases, but has no activity against bacterial metalloproteinases. 3. Activity is destroyed by treatment with either trypsin or 4-aminophenylmercuric acetate, by heat, and by reduction and carboxymethylation. 4. All the properties observed suggest that it is similar to the synthesized tissue inhibitor of metalloproteinases.
Alkaline O→N-transacylation. A new method for the quantitative deacylation of phospholipids
Authors: Clarke N G , Dawson R M C
Published: August 2015

1. Quantitative O-deacylation of phospholipids has been achieved by incubation with a reagent containing monomethylamine, methanol and water. The reaction is primarily an O leads to N-transacylation with N-methyl fatty acid amides being formed. 2. The reagent can be removed easily by volatilization and under defined conditions no secondary decomposition of the phosphorus-containing deacylation products occurs. 3. The water-soluble phosphorus compounds derived by deacylation of mammalian tissue O-diacylated phospholipids have been completely separated by a single-dimensional paper ionophoresis with a volatile pH9 buffer. 4. The O-deacylated alkyl and alkenyl phospholipids have been examined by t.l.c. before and after catalytic hydrolysis with Hg2+. 5. A complete analysis of rat brain phospholipids by the above methods agrees closely with that obtained by other procedures.
[Identification and characterization of two phospholipase A2 activities in resident mouse peritoneal macrophages.]
Authors: Wightman P D , Humes J L , Davies P , Bonney R J
Published: August 2015

Resident mouse peritoneal macrophages synthesize and release large amounts of prostaglandins in response to inflammatory stimuli. Release of prostaglandin E2 and 6-oxoprostaglandin F1 alpha occurs at a rate of 1 nmol/h per mg of cell protein. The mechanisms by which substrate arachidonic acid is released have yet to be established. We have therefore initiated studies to characterize those enzymes that can catalyse its release from phospholipid and may be of significance at the cellular level. We report initially the characterization of two phospholipase A2 activities in homogenates of mouse peritoneal macrophages. The first is active at pH 4.5 and is not dependent on Ca2+. The second is Ca2+-dependent and is optimally active at pH 8.5. Either phospholipase A2 activity is capable of hydrolysing [14C] arachidonic acid from [14C] arachidonic acid-labelled phospholipids in quantities sufficient to account for the amounts of prostaglandins by macrophages in culture. Phospholipid substrates are prepared from mouse LM fibroblasts in serum-free Higuchi medium containing radiolabelled phospholipid precursors. Single-labelled phospholipids bear the 14C label in the arachidonic acid moiety. Dual-labelled phospholipids bear a 14C label in the polar head group and a 3H label in the arachidonic acid moiety. Experiments with dual-labelled substrates establish that both phospholipase activities are of the A2 type as indicated by the equimolar recovery of [3H] arachidonic acid and [14C] lysophospholipid. Studies with aqueous sonicated dispersions of purified [14C] arachidonic acid-labelled phospholipid or mixed liposomal substrates formed from mixtures of cellular polar lipids reveal that the pH 4.5 activity hydrolyses phosphatidylethanolamine and phosphatidylcholine more efficiently when they are present in a mixture of other polar lipids. The pH 8.5 activity, however, hydrolyses the purified phospholipids more efficiently.