bluuebunny/crossref_metadata_2025 huggingface dataset:
Glutamate receptor binding to cat central nervous system membranes
Authors: Head Richard A. , Tunnicliff Godfrey , Matheson G. Keith
Published: December 2009

L-[3H]Glutamate exhibited specific binding to fresh membranes of cat CNS under physiological conditions of pH and temperature. This binding occurred in the absence of sodium ions. Kinetic analysis of the data for cerebellum suggested the presence of two distinct binding sites: a high-affinity process (Kd = 0.33 μM) with a capacity of 15 pmol/mg protein and a low-affinity process (Kd = 1.8 μM) which had a capacity of 65 pmol/mg protein. Several structural analogues of glutamic acid were able to appreciably inhibit the binding of [3H]glutamate. The distribution of glutamate binding between 12 regions of the CNS was measured. The amygdaloid complex exhibited the highest binding followed by hippocampus > hypothalamus ≡ visual cortex ≡ thalamus ≡ caudate nucleus > olfactory bulb ≡ tectum ≡ cerebellum > dorsal pons ≡ medulla > cervical spinal cord. These findings are consistent with the binding of [3H]glutamate being to its receptor.
Catalysis of exchange of terminal phosphate groups of ATP and ADP by purified nitrogenase proteins
Authors: Miller R. W. , Robson R. L. , Yates M. G. , Eady R. R.
Published: December 2009

A crude Azotobacter nitrogenase complex contained a highly active adenylate kinase which caused rapid equilibration of AMP, ADP, and ATP. Purified molybdenum–iron protein preparations also contained measurable adenylate kinase activity which could be removed by adsorption and elution from hydroxylapatite. Independent of adenylate kinase, the highly purified molybdenum–iron protein from both Klebsiella pneumoniae and Azotobacter chroococcum catalyzed the exchange of [32P]orthophosphate with the terminal phosphate of ATP or ADP. The exchange labeling of ATP was stimulated by ferricyanide ion due to the inhibition of ATP hydrolysis linked to substrate reductions which cannot occur in the absence of reductant. This exchange reaction is responsible for reported ATP synthesis by crude nitrogenase preparations.Binding of ATP labeled with 32P in the terminal phosphate group was measured directly with concentrated solutions of the molybdenum–iron nitrogenase protein from K. pneumoniae by the column gel filtration method. The protein was saturated with ATP at a calculated ratio of 4.0 ± 0.3 mol ATP/mol protein; half-saturation of 220 μM protein occurred at 600 ± 100 μM.The interactions between adenine nucleotides and the molybdenum–iron protein suggest the involvement of the nucleotides in a role distinct from the established reactions with the iron protein of the nitrogenase complex. A dual role for ATP in the reduction of dinitrogen by isolated nitrogenase proteins would be consistent with the existence of binding sites for ATP on both the iron and molybdenum–iron proteins.
Lipid metabolism during embryonic and early postembryonic development of Xenopus laevis
Authors: Mes-Hartree Mary , Armstrong John B.
Published: December 2009

Xenopus laevis embryos were analyzed for their lipid content from fertilization to feeding stage. The amounts of the major lipids did not change until after hatching, but at the feeding stage the amounts of phosphatidylcholine (PC), phosphatidylethanolamine (PE), and sterol esters (SE) increased approximately twofold. Prior to hatching, [14C]acetate label was incorporated primarily into the fatty acyl chains of PE. After hatching, increasing amounts of label were found in other phospholipids, in their glycerol backbones, and in nonglycerol lipids. The most marked changes occurred just before the onset of feeding, and pulse–chase experiments suggest that nonlipid reserves are mobilized at this stage for de novo lipid synthesis.
Reconstitution of lipoproteins. I. Lipid–protein interaction of high density apoproteins, purified apoA-I and apoA-II with dimyristoyl-lecithin and dimyristoyl-lecithin:cholesterol vesicles studied by isomeric spin-labelled lecithins
Authors: Vaughan D. J. , Breckenridge W. C. , Stanacev N. Z.
Published: December 2009

Reconstitution of high density lipoprotein-like particles from lecithin, cholesterol, and high density lipoprotein apoproteins was studied. Sonicated mixtures of sn-3-dimyristoyl-lecithin and cholesterol (molar ratio 10:1) were incubated with total human plasma high density apoproteins and purified apoprotein A-I and apoprotein A-II in the presence of isomeric (5′-, 12′-, and 16′-(N-oxyl-4″,4″-dimethyloxazolidine)stearoyl) spin-labelled lecithin probes. The supernatant obtained after centrifugation of the incubation mixture at 12 000× g for 5 min contained reconstituted lipoproteins with an average protein:lipid weight ratio of 0.52–0.53. To determine the position of apoprotein A-I and apoprotein A-II with respect to the lipid bilayer, the thermotropic behaviour of reconstituted lipoproteins was monitored by electron spin resonance spectroscopy using isomeric spin-labelled lecithin probes. Comparison of experimental spectral parameters (transition temperature, temperature range, and cooperative unit) obtained from reconstituted lipoproteins with liposomes of dimyristoyllecithin and dimyristoyllecithin:cholesterol (10:1) showed a relative increase of transition temperature and broadening of the transition range with 5- and 12-(N-oxyl-4′,4′-dimethyloxazolidine)stearic acid containing lecithin probes when reconstituted lipoproteins contained either apoprotein A-I or apoprotein A-II. With 16-(N-oxyl-4′,4′-dimethyloxazolidine)stearic acid containing lecithin probe, the transition temperatures and the temperature range relative to those values measured with 5′- and 12′-(N-oxyl-4″,4″-dimethyloxazolidine) lecithin probes were higher and narrower, respectively, with a tendency towards values obtained for liposomes not containing apoproteins. The cooperative unit in dimyristoyl-lecithin:apoprotein recombinants monitored with 5′- and 12′-(N-oxyl-4″,4″-dimethyloxazolidine) lecithin probes was smaller than or approximately equal to those measured with 16′-(N-oxyl-4″,4″-dimethyloxazolidine) lecithin probes when these systems contained either apoprotein A-I or apoprotein A-II. These and other results indicate that apoproteins A-I and A-II in lipoprotein recombinants of the composition used in our study, are partially embedded within the hydrocarbon portion of the lipid bilayer of reconstituted lipoproteins but do not span the entire lipid bilayer. Relevance of these results to the mechanism of lecithin:cholesterol acyltransferase activation is discussed.
Conformations of calf thymus and rye histones H3 and H4 in aqueous solution by laser Raman spectroscopy
Authors: Pézolet M. , Savoie R. , Guillot J.-G. , Pigeon-Gosselin M. , Pallotta D.
Published: December 2009

The Raman spectra of aqueous solutions of histones H3 and H4 from calf thymus and from rye reflect the high degree of conservation from species to species of the primary and secondary structures of these proteins. The amount of β-sheet structure is estimated at 40 ± 5% in H4 and at 33 ± 5% in H3 from the intensities of the amide I and amide III bands at 1663 and 1241 cm−1, respectively, in the spectra. These values are independent of the salt concentration of the solutions, most likely because of the high histone concentration (~3 mM) required to obtain the spectra, which results in some aggregation of the proteins. The intensity ratio of the tyrosine doublet at 852 and 826 cm−1 indicates that the four tyrosine residues in H4 are relatively exposed to the solvent or weakly bound to positively charged groups of basic amino acids, whereas in H3 at least one tyrosine is buried inside the protein and tightly bound to a carboxylate group. The results also show that the secondary structure of H3 is slightly influenced by the state of oxidation of the two cysteine residues it contains.
Cleavage of protected amino acids and peptides from benzyl ester resins by transesterification with potassium cyanide in benzyl alcohol
Authors: Moore Graham J. , Kwok Yan C.
Published: December 2009

The nature of the protected amino acid or peptide attached to the Merrifield resin influences the rate of KCN-catalyzed transesterification in benzyl alcohol. Although crown ether complexes of KCN do not expedite the reaction, sterically hindered amino acids are readily transesterified in the presence of saturating concentrations of KCN at elevated temperatures.
Inhibition by orthophosphate esters of glucose-6-phosphatase
Authors: Walls Michael A. , Lygre David G.
Published: December 2009

Inhibition of rat liver microsomal glucose-6-phosphatase (D-glucose-6-phosphate phosphohydrolase, EC 3.1.3.9) by orthophosphate and organophosphate esters was examined at pH 6.0 and 7.5 with and without enzyme pretreatment with 0.2% (w/v) deoxycholate. Inhibition by orthophosphate and monoethyl phosphate was competitive with respect to glucose-6-P while inhibition by mono- and di-phenyl phosphate was of the mixed type. Monoalkyl phosphates were more effective inhibitors than the analogous di- and tri-alkyl phosphates and deoxycholate potentiated the inhibitory effects. Mono- and di-phenyl phosphates were more effective inhibitors than triphenyl phosphate, and deoxycholate decreased these inhibitory effects. The results are interpreted in terms of inhibitor and deoxycholate interactions with the enzyme.
Purine ribonucleoside and deoxyribonucleoside kinase activities in thymocytes. Specificity and optimal assay conditions for phosphorylation
Authors: Lukey Trevor , Snyder Floyd F.
Published: December 2009

The optimal assay conditions and specificity for the principal reactions of purine nucleoside phosphorylation were studied in mouse thymocytes. The following relative activities were obtained for the nucleoside substrates: adenosine, 100; deoxyguanosine, 24; and deoxyadenosine, 14. The phosphorylation of adenosine, 45 μM, was optimal between pH 5.8 and 6.0 with a millimolar Mg:ATP ratio of 1:5. This activity was insensitive to inhibition by other nucleosides and dCTP. Optimal phosphorylation of deoxyguanosine, 350 μM, occurred at pH 8.4 with a millimolar Mg:ATP ratio of 10:3.5. Phosphorylation of 80 μM deoxyguanosine was inhibited approximately 90% by 10 μM deoxycytidine or dCTP and was inhibited 70% by 200 μM deoxyadenosine but unaffected by adenosine. Deoxyadenosine, 450 μM, phorphorylation was optimal between pH 6.5 and 8.5 with a millimolar Mg:ATP ratio of 5:1. Phosphorylation of deoxyadenosine, 100 μM, was partially inhibited by 200 μM adenosine, 34%; 200 μM deoxyguanosine, 10%; and 100 μM deoxycytidine or dCTP, 33%. Only deoxyadenosine phosphorylation was inhibited by 200 μM deoxyinosine, 10%. These results and those obtained from isokinetic sucrose density gradient analysis are consistent with there being a specific adenosine kinase, a faster sedimenting deoxycytidine kinase of broad specificity which also catalyzes the phosphorylation of deoxyguanosine and deoxyadenosine, and a specific deoxyguanosine kinase sedimenting more rapidly than either of the other activities.
ADP-ribosylation of rye histones
Authors: Laroche A. , Plante J. , Beaumont G. , Poirier G. G.
Published: December 2009

Chromatin from etiolated rye seedlings synthesized protein-bound, acid-insoluble material from [3H]NAD, presumably poly(ADP-ribose). [3H]ADP-ribosylated histone fractions were isolated from crude chromatin and characterized by gel electrophoresis and exclusion chromatography. It was found that histone H2B was the main acceptor, that H2A and H1 were modified to a lesser extent, and that H3 and H4 were only slightly modified. The average chain length on purified histones was 2.5 units of polymer.
A comparison of the citrate synthases of Escherichia coli and Acinetobacter anitratum
Authors: Morse David , Duckworth Harry W.
Published: December 2009

Citrate synthase has been purified to homogeneity from a strain of the Gram-negative aerobic bacterium Acinetobacter anitratum in a form which retains its sensitivity to the allosteric inhibitor NADH. In subunit size, amino acid composition, and antigenic reactivity the enzyme shows a marked structural resemblance to the citrate synthase of the Gram-negative facultative anaerobe Escherichia coli. Whereas the E. coli enzyme is subject to a strong, hyperbolic inhibition by NADH (Hill's number n = 1.0, K1 = 2 μM), the A. anitratum enzyme shows a weak, sigmoid response (n = 1.6, I0.5 = 140 μM) to this nucleotide. With E. coli, NADH inhibition is competitive with acetyl-CoA, and noncompetitive with oxaloacetate; with A. anitratum, NADH is noncompetitive with both substrates. Acinetobacter anitratum citrate synthase shows hyperbolic saturation with acetyl-CoA (n = 1.8). The finding of Weitzman and Jones (Nature (London) 219, 270 (1968)) that NADH inhibition of the enzyme from Acinetobacter spp. is reversible by AMP, while that from E. coli is not, is explained by the much greater affinity of the E. coli enzyme for NADH. Unlike E. coli citrate synthase, the A. anitratum enzyme does not react with the sulfhydryl reagent 5,5′-dithiobis(2-nitrobenzoic acid) (DTNB) in the absence of denaturation. With a second sulfhydryl reagent, 4,4′-dithiodipyridine (4,4′-PDS), the A. anitratum enzyme reacts with 1 equiv. of subunit; this modification induces a partial activity loss (attributable to a rise in the Km for acetyl-CoA) and an increase in the sensitivity to NADH. With the E. coli enzyme, 4,4′-PDS causes complete inactivation. Acinetobacter anitratum citrate synthase is much more resistant to urea denaturation than the E. coli enzyme is; the resistance of both enzymes to urea is greatly improved in the presence of 1 M KCl. It is suggested that the amino acid sequences of the subunits of the citrate synthases of these two bacteria are about 90% homologous, and that the 10% differences are in key residues, perhaps largely in the subunit contact regions, which account for the differences in allosteric properties.
Glucagon from the bovine fetal pancreas: chromatographic and electrophoretic characterizations of high molecular weight immunoreactive species
Authors: Tung A. K. , Ruse J. L. , Cockburn E.
Published: December 2009

Fetal bovine pancreas was extracted for glucagon using (A) ethanol–HCl after trichloroacetic acid (TCA) treatment of the pancreas, (B) ethanol–HCl and (C) urea–acetic acid. Fractionation of the acetic acid soluble proteins via Sephadex G-50 columns yielded glucagon immunoreactivity in the void volume, high molecular weight glucagon immunoreactivities (HMW-IRGs), "proglucagon" [Formula: see text], and true glucagon (3.5 K Δ) regions. HMW-IRGs were obtainable using all three methods of extraction. The material obtained from the ethanol–HCl–TCA method appeared stable on Sephadex G-100 (1 M acetic acid) rechromatography. Sodium dodecyl sulfate (SDS) polyacrylamide gel electrophoresis analysis showed immunoreactive species corresponding to [Formula: see text] and [Formula: see text]. HMW-IRGs did not bind to concanavalin A (Con A) – agarose. SDS–polyacrylamide gel electrophoresis of the Con A – agarose filtered IRG again showed a major immunoreactive peak of [Formula: see text]. Dose–response RIA studies indicated that the HMW-IRGs from both the gel filtration and SDS–polyacrylamide gel experiments were immunochemically indistinguishable from glucagon. HMW-IRGs bind to antiglucagon antibody agarose, further indicating their reactivity towards glucagon antibodies. When HMW-IRGs are incubated with guanidinium hydrochloride and gel filtered in the same system, a significant fraction of HMW-IRG (representing up to 25% of the total IRG analysed) was found to resist disruption. Our data support the contention that a significant portion of the HMW-IRGs (molecular weight > 20 K Δ) extracted from fetal bovine pancreas are composed of glucagon covalently linked to larger protein unit(s).
N-Trifluoroacetyladriamycin-14-valerate and adriamycin induced DNA damage in the RPMI-6410 human lymphoblastoid cell line
Authors: Brox L. , Gowans B. , Belch A.
Published: December 2009

The adriamycin- and N-trifluoroacetyladriamycin-14-valerate (AD-32) induced DNA cross-linking and breakage in human RPM1-6410 cells was compared using the alkaline elution technique of Kohn and co-workers. At comparable growth-inhibitory concentrations both adriamycin and AD-32 caused DNA cross-linking. Treatment with proteinase-K showed this cross-linking to be mainly DNA–protein in character. Proteinase-K treatment also revealed that both drugs caused either single-strand DNA breaks or increased alkaline sensitivity. With adriamycin the degree of cross-linking and breakage was dose related over the range studied (0.05–0.4 μg/mL), whereas with AD-32 there appeared to be a saturation of both effects at concentrations in excess of 3 μg/mL. With both drugs the extent of cross-linking and breakage was maximal at the end of the drug exposure. This work suggests that AD-32 or some metabolite of it binds to DNA and this binding leads to DNA damage that is similar to that caused by adriamycin. These AD-32 results are somewhat surprising in light of earlier model studies showing that AD-32 does not bind to isolated DNA.
Oxidation of methionine residues in lutropin
Authors: Llosa Pedro de la , Abed Amor El , Roy Marguerite
Published: December 2009

Bovine lutropin and its subunits were submitted to oxidation by sodium periodate or chloramine T. Methionine residues were easily oxidized but partial destruction of fucose was observed. After oxidation treatment most of the lutropin exhibits the same elution volume in gel filtration as the native hormone. Sucrose gradient sedimentation or gel filtration experiments show however that the oxidation of isolated subunits is accompanied by aggregation or conformational changes even in the case of porcine β subunit which contains only one methionine residue. Oxidized bovine lutropin was inactive. The recombination product of oxidized porcine β subunit and intact α subunit exhibit very low residual activity.
Amplification of the put genes and identification of the put gene products in Escherichia coli K12
Authors: Wood Janet M. , Zadworny David
Published: December 2009

The utilization of L-proline as carbon or nitrogen source for the growth of Escherichia coli K12 requires the activities of an L-proline porter (PP-I) and a bifunctional L-proline dehydrogenase – Δ1-pyrroline carboxylate dehydrogenase. PP-I is inactivated by mutations at putP and the bifunctional dehydrogenase is encoded in the adjacent locus, putA, at 22 min on the chromosome map. Two additional loci, proP (at 92 min) and proT (at 82 min), have also been implicated in L-proline transport. We have studied four ColE1/E. coli K12 hybrid plasmids from the plasmid bank prepared by Clarke and Carbon. Each of these plasmids was shown previously to complement an L-proline transport defect in E. coli. Genetic complementation analysis and biochemical assays of L-proline transport and L-proline dehydrogenase activity show that three of these hybrid plasmids bear the putPA region of the E. coli chromosome (plasmids pLC4-45, pLC10-29, and pLC43-41). The fourth plasmid, pLC35-38, specifically enhances the L-proline transport activity of its host bacteria but not their L-proline dehydrogenase activity. It probably encodes putP. We have used these plasmids in an E. coli minicell system to identify the putA and putP gene products.
Interaction of four local anesthetics with phospholipid bilayer membranes: permeability effects and possible mechanisms
Authors: Singer Michael A. , Jain Mahendra K.
Published: December 2009

Sodium 22 efflux was measured, at various temperatures, in the presence of different local anesthetics (dibucaine, tetracaine, benzocaine, procaine) in multilamellar liposomes composed of saturated phosphatidylcholines and dicetyl phosphate. These liposomes display a permeability maximum in the temperature region of their respective phase transitions. Local anesthetics cause this permeability maximum to occur at a lower temperature and also increase its magnitude. Binding of the anesthetics to these liposomes displays a biphasic temperature curve with a maximum in the vicinity of the transition temperature. In addition there appears to be a linear relationship between aqueous anesthetic concentrations causing equal permeability effects and corresponding membrane concentrations. All of the anesthetics decrease the cooperativity of the lipid phase transition and this is probably an important factor underlying induced permeability effects. However several observations suggest that additional factors are also important and we have postulated that one such factor is the preferential binding of the anesthetics to phase boundary regions.
The effect of cholesterol on the interaction of glucagon with the gel state of dimyristoylphosphatidylcholine
Authors: Epand Richard M.
Published: December 2009

Cholesterol has a large effect on the binding of glucagon to dimyristoylphosphatidylcholine (DMPC) and to dipalmitoylphosphatidylcholine (DPPC). At cholesterol concentrations of 20 mol% or greater there is virtually no interaction of glucagon with phospholipid. Glucagon can, however, solubilize DMPC or DPPC with 10% cholesterol. In the case of DPPC the solubilized lipid is depleted of cholesterol relative to the starting material. These results suggest that glucagon excludes cholesterol from its surroundings.It is shown from solubility, light scattering, and glucagon fluorescence measurements that the glucagon–DMPC complex containing 10% cholesterol is stable only in the region of the phase transition temperature. Scatchard analysis of lipid binding to glucagon indicates a decrease in the amount of bound lipid below 22 °C or above 24 °C. The effect of the phase transition on the interaction of glucagon with lipids is compared with that found for other membrane proteins. Several aspects of the effect of cholesterol on glucagon–lipid interactions are analogous to effects which have been observed with serum apolipoproteins.
The stimulation of rat liver microsomal CDP-diacylglycerol formation by guanosine triphosphate
Authors: Liteplo Robert G. , Sribney Michael
Published: December 2009

GTP has been found to markedly enhance the formation of CDP-diacylglycerol in rat liver microsomes. Neither GDP, GMP nor the nonhydrolyzable analogues of GTP increased the synthesis of the liponucleotide. The GTP stimulation of phosphatidate cytidylyltransferase activity is inhibited by EDTA and NaF. GTP enhances the activity of the enzyme in a concentration-, time-, and temperature-dependent manner and preincubation of rat liver microsomes with GTP produces a persistently activated phosphatidate cytidylyltransferase. GTP reduces the Km for phosphatidic acid, but has no effect on either the Km for CTP or the Vmax of the reaction. GTP, by stimulating the activity of the phosphatidate cytidylyltransferase, enhances the formation of phosphatidylinositol from CTP, phosphatidic acid, and inositol. Evidence is presented suggesting that the mechanism by which GTP stimulates the activity of the phosphatidate cytidylyltransferase involves a covalent modification of the enzyme itself or a protein intimately associated with the phosphatidate cytidylyltransferase.
Heterogeneous catalysis by phospholipase A2: mechanism of hydrolysis of gel phase phosphatidylcholine
Authors: Tinker David O. , Low Rosita , Lucassen Maria
Published: December 2009

Hydrolysis of gel phase dipalmitoylphosphatidylcholine (DPPC) at 37 °C catalysed by Crotalus atrox phospholipase A2 (PLA) is described extremely well by the "path 1" kinetic mechanism of Tinker and Wei (1979) (Can. J. Biochem. 57, 97–106), if reversible adsorption is allowed as a side reaction. Progress curves show an initial rapid phase, the initial velocity being a Michaelis–Menten function dependent on the catalytic properties of the enzyme ([Formula: see text], [Formula: see text]), then level off to a slower rate determined by the desorption equilibrium constant [Formula: see text] and desorption rate constant [Formula: see text]. The relaxation time, τ, for the transition to the desorption-limited reaction is ~0.5 min; this large value of τ probably arises from a slow conversion of active, dimeric enzyme to an inactive protein species adsorbed to the lipid surface. At later times in the reaction there is an increase in the rate of hydrolysis, attributed to a stimulation of desorption by the products. The desorption equilibrium constant KD is a quadratic function of the surface concentration of products and increases 20- to 30-fold when all accessible substrate is hydrolysed. Both lyso-phosphatidylcholine (lyso-PC) and fatty acid were found to stimulate the desorption, but lyso-PC was also found to be a competitive inhibitor of the catalysis. Adsorption of PLA to DPPC and egg PC vesicles was directly measured using a gel partition technique. Strong binding to egg PC was observed, which was not dependent on the presence of calcium ion (essential for catalysis); PLA inhibited by acylation of up to four lysine residues per mole of monomeric enzyme with ethoxyformic anhydride was equally strongly adsorbed, indicating that lipid binding is not dependent on catalytic activity. Reaction products greatly weakened the binding of PLA to the lipid surface as expected. Cholesterol had two effects on the hydrolytic reaction: there was a striking decrease in the rate of the slower, desorption-limited phase, the rate of which decreased to almost zero at 15 mol% cholesterol, but there was also evidence for the formation of a complex with stoichiometry 1 cholesterol: 2 DPPC in which DPPC is no longer a substrate for the enzyme. Implications of the proposed mechanism for specificity and control of surface catalysis by PLA are discussed.
Friend erythroleukemia cell membrane transferrin receptors
Authors: Wilczynska A. , Schulman H. M.
Published: December 2009

We have compared the uptake of transferrin by murine Friend erythroleukemia cells with the uptake of transferrin by murine reticulocytes. Friend cells which had been induced to erythroid differentiation by dimethyl sulfoxide took up transferrin in a manner qualitatively and quantitatively similar to the uptake of transferrin by reticulocytes, while uninduced Friend cells took up only negligible amounts of transferrin. Specific transferrin-binding activity could be demonstrated in detergent extracts of membranes from induced cells and this activity was isolated from membrane extracts by the use of antibody to transferrin. The isolated membrane component(s) with transferrin-binding activity migrated electrophoretically as a single protein on sodium dodecyl sulfate gels and had similar properties to a transferrin-binding protein isolated previously from reticulocytes.
Fluorescence polarization studies of rat liver microsomes with altered phospholipid desaturase activities
Authors: Pugh E. L. , Kates M. , Szabo A. G.
Published: December 2009

Phospholipid desaturase activity of rat liver microsomes can be varied by dietary manipulation: rats starved and refed a "fat-free" diet have two to three times the activity of normal controls whereas starved rats have no detectable activity. These changes were accompanied by changes in fatty acid composition of the microsomal phospholipids, resulting in a double bond:saturated fatty acid mole ratio (moles double bonds per mole saturated fatty acid) of 5.7 in control and starved rats and 4.7 in starved–refed rats. Fluorescence polarization ratio [Formula: see text] of cis- and trans-parinaric acid (PnA) showed no significant differences in physical state of the three microsomal preparations. However, the isolated microsomal phospholipids with trans-PnA as probe showed differences in the temperature at which onset of a change in polarization ratio occurred (starved–refed > normal > starved rats). With the cis-PnA as probe, the polarization ratio showed no change in the range 10–40 °C but was significantly higher (1.8) in starved–refed rats than in normal and starved rats (1.6 in both cases). These data indicate that the microsomal phospholipids of starved–refed animals were in a less fluid state than those from control and starved rats and that this decrease in fluidity was correlated with an increase in phospholipid desaturase activity.
Pathways of cytochrome c oxidation by soluble and membrane-bound cytochrome aa3
Authors: Nicholls P. , Hildebrandt V. , Hill B. C. , Nicholls F. , Wrigglesworth J. M.
Published: December 2009

In media of low ionic strength, membraneous cytochrome c oxidase, isolated cytochrome c oxidase, and proteoliposomal cytochrome c oxidase each bind cytochrome c at two sites, one of low affinity (1 μM > Kd′ > 0.2 μM) and readily reversible and the other of high affinity (0.01 μM > Kd) and weakly reversible. When cytochrome c occupies both sites, including the low affinity site, the maximal turnover measured polarographically with ascorbate and N,N,N′,N′-tetramethyl-p-phenylenediamine (TMPD) is independent of TMPD concentration, and lies between 250 and 400 s−1 (30 °C, pH 7.4) for fully activated systems. The apparent affinity of the enzyme for cytochrome c is, however, TMPD dependent. When cytochrome c occupies only the high-affinity site, the maximal turnover is closely dependent upon the concentration of TMPD, which, unlike ascorbate, can reduce bound cytochrome c. As TMPD concentration is increased, the maximal turnover approaches that seen when both sites are occupied. The lower activity of isolated cytochrome aa3 is due to the presence of inactive or inaccessible enzyme molecules. Incorporation of isolated enzyme into phospholipid vesicles restores full activity to all the subsequently accessible cytochrome aa3 molecules. Negatively charged (asolectin) vesicles show a higher cytochrome c affinity at the low-affinity sites than do the other enzyme preparations. A model for the cytochrome c – cytochrome aa3 complexes is put forward in which both sites, when occupied, are fully catalytically competent, but in which occupation of the "tight" site by a catalytically functional cytochrome c molecule is required for overall oxidation of cytochrome c via the "loose" site.
Nitroxide spin-probe study of amphotericin B – ergosterol interaction in egg phosphatidylcholine multilayers
Authors: Oehlschlager A. C. , Laks P.
Published: December 2009

The interaction between the polyene macrolide antibiotic, amphotericin B, and ergosterol in egg phosphatidylcholine multilayers was investigated using head group and acyl chain nitroxide spin-labelled phosphatidylcholine as probes. At physiological concentrations of less than 15 mol% sterol in egg phosphatidylcholine multilayers amphotericin B accumulates near the head group region until an amphotericin B: ergosterol ratio of approximately 0.7 is achieved. As the proportion of amphotericin B is increased above this value, formation of an acyl chain disordering complex occurs which has an approximate antibiotic:sterol ratio of unity.Dicetyl phosphate was used to increase the solubility of ergosterol past its normal limit in pure egg phosphatidylcholine (~15 mol%). At concentrations of ergosterol higher than 15 mol% a complex of two ergosterol molecules and one amphotericin B was postulated when there was insufficient antibiotic to form a 1:1 complex.
Hepatotrophic effects of insulin on glucose, glycogen, and adenine nucleotides in hepatocytes isolated from fed adult rats
Authors: Jeejeebhoy Khursheed N. , Ho Joseph , Mehra Rajni , Bruce-Robertson Alan
Published: December 2009

In vivo observations have suggested that there is an hepatotrophic effect of insulin. By contrast, subsequent in vitro work, using the isolated perfused liver system, showed no effect or indeterminate effects of insulin on the transport of glucose into the hepatocyte. However because this system may not have endured long enough to show such an influence we explored the transport of glucose using a 48-h suspension culture of hepatocytes isolated from young adult fed rats, the suspension being infused continuously with insulin at a rate approximating the maximum entering portal blood in the fed state. (In a separate study phloridzin was added after 2 h of incubation.) DNA, intracellular glucose and its inward transport, glycogen, and the adenine nucleotides were measured at intervals. By comparison with control or untreated cells, insulin-treated cells showed significantly more DNA and intracellular glucose, and the differences were abolished by phloridzin. Glucose transport rates fell to low values in untreated controls and still lower with insulin plus phloridzin. but the initial rate was maintained to the end (48 h) by insulin alone. Results for glycogen were similar to those for intracellular glucose. There was a close correlation (r = 0.96) between these two. The total adenine nucleotide pool and the concentration of ATP were maintained for about 24 h and fell to half their initial values by 48 h. Insulin had increased these concentrations significantly by 6 h. Although concentrations of ADP and AMP decreased gradually in all groups of cells, insulin enhanced the level of ADP by 12 h but had no measurable effect on that of AMP. The energy charge increased slightly throughout incubation but more so (by 6 h) in the presence of insulin. In conclusion the data support the concept that in the longer term (> 12 h) insulin in the portal circulation maintains the characteristic free permeability of the hepatocyte to glucose and this permits a variety of effects related to glucose entry into the hepatocyte.
Demonstration with monoclonal antibody of the glycoprotein nature of Thy-1.2 alloantigen
Authors: Letarte Michelle , Addis Jane , MacDonald Marcy E. , Bernstein Alan , Lake Phil
Published: December 2009

Thy-1 antigen, present in large amounts on the brain and thymus of mice, exists in two allelic forms, either Thy-1.1 or Thy-1.2. Previous results have shown that Thy-1.1 alloantigen is expressed on a glycoprotein of molecular weight 25 000, therefore referred to as the Thy-1 glycoprotein. However the presence of Thy-1.2 alloantigen on the purified Thy-1 glycoprotein could not be established unequivocally. The present paper demonstrates that Thy-1.2 alloantigen, identified by F7D5 monoclonal antibody, is localized on the Thy-1 glycoprotein. The limited neutralization by the glycoprotein of the reactivity of AKR anti-C3H allosera to thymocytes is explained by the fact that the antiserum contains antibodies to determinants other than Thy-1.2. The alloantisera are shown to react with viral proteins encoded for by endogenous murine leukemia virus and expressed at the surface of Friend cells and thymocytes.
Effects of acute hepatic ischemia on the electrophoretic patterns of the polypeptides and phosphopolypeptides of the microsomal membrane fraction of rat liver
Authors: Behar-Bannelier Monique , Pinteric Les , Murray Robert K.
Published: December 2009

The purpose of this study was to establish when alterations of the electrophoretic patterns of the polypeptides and phosphopolypeptides of the microsomal membrane fraction of the livers of rats become observable after initiation of acute hepatic ischemia. Ischemia was initiated by clamping the vascular supply to the left and median lobes of the livers of adult male rats. The animals were killed at various times thereafter (up to 6 h, and in certain instances, 24 h) and microsomal membrane fractions were prepared from each. The patterns of the polypeptides and phosphopolypeptides of these fractions were analysed by sodium dodecyl sulfate – polyacrylamide gel electrophoresis, using staining with Coomassie blue to analyse the polypeptides and radioautography to analyse 32P-labelled phosphopolypeptides. Alterations of the polypeptide pattern were apparent in the fractions from animals killed at 4 h and after; prior to this time point, subtle alterations, at most, could be distinguished. Effects of acute ischemia on the pattern of phosphopolypeptides of the microsomal membrane fraction were studied after phosphorylation in vivo (produced by intraperitoneal injection of [32P]phosphoric acid) and in vitro (using [γ-32P]ATP as phosphate donor). No marked changes in the phosphopolypeptide pattern produced by phosphorylation in vivo were observed until 6 h after clamping, by which time a diminution of the radioactivity in the majority of the phosphopolypeptides was evident. However, noteworthy alterations of the pattern of phosphopolypeptides produced by phosphorylation in vitro were observable in the membrane fractions from animals subjected to 2 h of ischemia. Overall the study provides a base line delineating the time sequence during which alterations of the electrophoretic patterns of the polypeptides and phosphopolypeptides of rat liver microsomal membranes become evident following the onset of acute hepatic ischemia and reveals that gross alterations of the polypeptide patterns of these membranes and of certain other subcellular fractions are not an early occurrence following this severe type of injury. The possible utility of the application of phosphorylation in vitro for detecting early alterations in membrane structure following cell injury is suggested.
Properties of a dolichol phosphokinase activity associated with rat liver microsomes
Authors: Rip Jack W. , Carroll Kenneth K.
Published: December 2009

Rat liver microsomes show a capacity to synthesize [1-3H]dolichyl phosphate from [1-3H]-dolichol. Formation of [1-3H]dolichyl phosphate increased continuously over 15 min although the reaction rate was never completely linear. Product formation was directly proportional to microsomal protein concentration between 1.1 mg/mL and the highest concentration tested, 5.5 mg/mL. The reaction rate was linear with respect to the dolichol content of the assay mixture to a saturation point (120 μM). An apparent Km of 50 μM was established for dolichol. The normal phosphate donor for the reaction is CTP and not ATP. The optimum concentration of CTP was 10 mM, and an apparent Km of 4 mM was calculated for this nucleoside triphosphate. The reaction was totally dependent on divalent metal ion, magnesium being more effective than calcium. The optimum concentration of magnesium ion and CTP were the same (10 mM), suggesting that MgCTP2− is utilized as the normal enzyme substrate. Activity measured in the absence of Triton X-100 was only 5% of the activity observed at the optimum (0.5% w/v) detergent concentration. The measurable levels of dolichol phosphokinase could be doubled by the inclusion of 10–15 mM NaF as phosphatase inhibitor. Optimal enzymatic activity was obtained between pH 7.0 and pH 7.5 and could be inhibited by EDTA. The sulfhydryl reagent DTT was slightly stimulatory while the product of the reaction, dolichyl phosphate, was noninhibitory at the highest concentration tested (13.8 μM). The second reaction product (CDP) inhibits the enzymatic phosphorylation of dolichol.
A Na+,K+ co-transport assay for essential hypertension
Authors: Dagher Georges , Garay Ricardo P.
Published: December 2009

In erythrocytes from essential hypertensive patients and some of their normotensive offspring, the Na+,K+ co-transport system is unable to transport Na+ in an uphill direction against the electrochemical Na+ gradient. Recent studies of the kinetic properties of this system have permitted us to further characterize the abnormal co-transport in hypertension.Thirty-four essential hypertensive patients were characterized by a two-to three-fold decrease in the maximal rate of the outward Na+,K+ co-transport and a partial uncoupling of this system as compared with 22 normotensive controls. On the other hand, a normal co-transport was found in five secondary hypertensives with no family history of hypertension, thus indicating that the abnormal co-transport is not the consequence of high blood pressure per se. An abnormal co-transport was also found in around 50% of 19 young normotensives born of one hypertensive parent, suggesting genetic transmission.This study allows us to propose the use of a specific Na+,K+ co-transport assay for the differential diagnosis between essential and secondary hypertension and for the detection of those normotensives born of hypertensive parents with a potential risk to develop high blood pressure.
Mitochondrial importation of lipids and liponucleotides from microsomes independent of and facilitated by purified cytosol proteins
Authors: Stuhne-Sekalec L. , Stanacev N. Z.
Published: December 2009

The mitochondrial importation of microsomal lipids and liponucleotides in the presence and in the absence of partially purified cytosol protein(s) isolated from guinea pig liver was studied by the aid of isomeric (5-, 12-, and 16-(N-oxyl-4′,4′-dimethyloxazolidine)stearoyl) spin-labelled radioactive phosphatidic acid, phosphatidylcholine, neutral lipids, and CDP-diglycerides. Using a conventional procedure for the protein purification, cytosol protein(s) was purified approximately 1000-fold in respect to its ability to catalyze the translocation of isomeric spin-labelled lipids and liponucleotides from the microsomal to mitochondrial membranes. The highest activity of this protein was exhibited with biosynthesized spin-labelled lipids and liponucleotides bound to the microsomal membranes as substrates and the lowest, with the synthetic liponucleotides and derived lipids bound to the microsomal membranes. The partially purified protein was active in catalyzing the mitochondrial import of phospholipids from microsomes after heat treatment up to 90 °C.In addition to the cytosol protein catalyzing mechanism of mitochondrial import of lipids and liponucleotides from microsomal membranes, another cytosol protein independent mechanism of the mitochondrial importation of the same lipids and liponucleotides was also demonstrated in an agreement with our previous reports on the existence of cytosol protein independent intermembranous translocation of phospholipids. These experimental findings are discussed in terms of possible physiological significance and reaction mechanisms involved in the mitochondrial import of lipids and liponucleotides from the microsomal membranes of guinea pig liver.
Membrane orientation of sheep spectrin
Authors: Prokopchuk P. , Sargent A. U.
Published: December 2009

Based primarily on studies of human erythrocytes, current theories of the structure and organization of erythrocyte membranes localize spectrin to the membrane cytoplasmic surface. Affinity purified anti-sheep spectrin antibodies were used in indirect immunofluorescence studies of intact erythrocytes from various vertebrate species and inside-out and right-side-out impermeable sheep erythrocyte vesicles. This investigation detected i0mmunologically reactive external and potentially transmembranal determinant(s) of the sheep erythrocyte spectrin "assembly." Parallel studies using anti-sheep and anti-human spectrin antibodies, as well as 125I surface-labelling studies of intact sheep and human erythrocytes, indicated that this particular membrane orientation of spectrin was evident in sheep but not in human erythrocytes. Antisera containing antibodies to the external portion of this spectrin "assembly" demonstrated external fluorescence to a variable degree on some, but not all, vertebrate erythrocytes surveyed, confirming that the sheep erythrocyte was not the only exception. It is suggested that there may be subtle species variability in the intermolecular associations of the spectrin "assembly" with(in) the erythrocyte membrane not requiring alterations of the spectrin molecule itself.
Evidence that Sindbis virus infects BHK-21 cells via a lysosomal route
Authors: Talbot Pierre J. , Vance Dennis E.
Published: December 2009

Chloroquine and NH4Cl, potent inhibitors of lysosomal function, decreased the production of infectious Sindbis virus particles in BHK-21 cells by 10- and 12-fold, respectively. There were no apparent toxic effects on cells exposed to these lysosomotropic agents. These chemicals did not alter the rate of cellular protein synthesis, with the exception of a reversible twofold inhibition by chloroquine. No additive effects of chloroquine and NH4Cl were observed when the cells were saturated with these weak bases, which suggests that their effect is exerted via the same mechanism, most likely as a result of an increase in lysosomal pH. The reduction in the formation of Sindbis virions was monitored by incorporation of [35S]methionine and shown to be fourfold by chloroquine or NH4Cl at 5 h postinfection but negligible at 11 h postinfection. These results strongly suggest that a productive Sindbis virus infection requires functional lysosomes. Thus, endocytosis is probably the main infectious mechanism for penetration of this virus into BHK-21 cells.
The influence of chronic ethanol feeding to rats on liver mitochondrial membrane structure and function
Authors: Hosein E. A. , Lee Hung , Hofmann Ilan
Published: December 2009

Arrhenius plots were generated on the activity of rat liver mitochondrial cytochrome c oxidase from Metrecal–sucrose fed controls and Metrecal–alcohol fed experimentals. Chronic alcohol feeding resulted in diminished specific activity of cytochrome c oxidase and abolition of the discontinuity temperature at 17.5 °C found in the controls. Twenty-four hours after alcohol withdrawal, a discontinuity temperature reappeared at 14.4 °C; at 48 h it increased to 22.6 °C and returned to normal (17.4 °C) at 72 h. Such liver mitochondria also showed a decreased capacity to oxidize the acetyl group of acetyl carnitine immediately following prolonged alcohol feeding. When the assay was performed following withdrawal from alcohol 24 h later, oxidation was enhanced and this effect persisted for another 48 h. These latter results revealed a diminished capacity of such mitochondria to oxidize short chain fatty acids during alcohol feeding and the reverse during alcohol withdrawal.These results, complemented by thermographic data obtained through differential scanning calorimetry (DSC) reinforced the view that chronic alcoholic feeding induced adaptive changes in the fluidity of rat liver mitochondrial membrane lipids. Moreover, they demonstrated that in the microenvironment of the membrane-bound enzymes on withdrawal from ethanol, the membrane readapts to the new conditions without alcohol. This involved modulation of membrane structure and function and at the same time demonstrated a role for the membrane in the expression of tolerance and functional dependence on alcohol.
Chloramphenicol-resistant variants of Pseudomonas aeruginosa defective in amino acid transport
Authors: Irvin Jean E. , Ingram Jordan M.
Published: December 2009

High-level chloramphenicol (CM) resistant variants of Pseudomonas aeruginosa were isolated after culture of the wild-type (WT) strain in broth containing high concentrations of the drug. These variants exhibit reduced ability to accumulate several amino acids. The extent of reduction in transport capacity is a function of the concentration of CM in which the variants are grown. Respiratory activity is not reduced in these strains. Amino acid uptake is not affected by the presence of CM during assay. An isogenic strain carrying a plasmid coding for CM resistance does not show this response to CM. Transport capacity is restored to the WT level in CM-sensitive revertants. These results suggest that the acquisition of CM resistance in P. aeruginosa is associated with a fundamental alteration in membrane permeability which is regulated by metabolism in the presence of the drug. The ramifications of this for the study of CM action and resistance are discussed.
Phosphatidylglycerol formation via transphosphatidylation by rat brain extracts
Authors: Chalifour Robert J. , Taki Takao , Kanfer Julian N.
Published: December 2009

Phosphatidylglycerol formation by a transfer of the phosphatidyl moiety of phosphatidylcholine to free glycerol was demonstrated in rat brain preparations. Identification of the product as phosphatidylglycerol was accomplished by chromatography, chemical derivatization, and enzymatic hydrolysis. The optimum pH for the reaction was 6.0 with an apparent Km for glycerol of 0.2 M and for phosphatidylcholine of 3.5 mM. Divalent ions were not required and when added caused some inhibition. Phosphatidylcholine was the most effective phosphatidyl donor tested. Similarities to certain properties of phospholipase D also present suggests that these two activities may reside in the same enzyme.