bluuebunny/crossref_metadata_2025 huggingface dataset:
A three step purification procedure for human liver ferritin
Authors: Pagé M. , Lagueux J. , Gauthier C.
Published: December 2009

We describe a method for the purification of normal human liver ferritin by ultrafiltration, gel filtration on Sephacryl S-300, and affinity chromatography on DEAE-Affi Gel Blue. The purity of the ferritin obtained was verified by immunoelectrophoresis, Ouchterlony immunodiffusion, polyacrylamide gel electrophoresis, and electrofocusing. This rapid method yields 32% of the original ferritin.
Cytotoxic activity of unsaturated fatty acids to lymphocytes
Authors: Kageyama K. , Nagasawa T. , Kimura S. , Kobayashi T. , Kinoshita Y.
Published: December 2009

Cytotoxic activities of unsaturated fatty acids against lymphocytes were studied by demonstrating the inhibition of RNA synthetic activity of incubated lymphocytes. In general, the cytotoxic activity of fatty acids increases with decreasing number of carbon atoms and increasing number of double bonds. An empirical formula predicting the degree of impairment of lymphocytes by various fatty acids was proposed. Furthermore, protective effects of serum on cytotoxic actions of unsaturated fatty acids to lymphocytes were studied and the significance of these findings was discussed.
ADP-ribosylation of pancreatic histone H1 and of other histones
Authors: Poirier G. G. , Savard P.
Published: December 2009

Incubation of pancreatic nuclei with high NAD concentrations resulted in increased ADP-ribosylation of histone H1. Interaction of [3H]ADP-ribosylated histone H1 with chromatin was significantly different from unmodified histone H1. The presence of a protein which is eluted at a lower salt concentration and which is ADP-ribosylated was also noticed. Pancreatic histones were isolated by column chromatography and their degree of ADP-ribosylation evaluated both by gel electrophoresis and by chromatography: histone H1 was the main acceptor while the core histones H3, H2B, and H2A were lightly labelled. Histones H1 and H10 have a differential binding to pancreatic chromatin and histone H10 is not ADP-ribosylated.
M-line protein preparations from frog skeletal muscle: isolation and localization of an M-line protein and a 105 000 dalton polypeptide contaminant
Authors: Dhanarajan Z. C. , Atkinson Burr G.
Published: December 2009

A 165 000 dalton polypeptide was purified and characterized from high salt extracted crude M-line protein preparations of frog skeletal muscle. It has an isoelectric point of 5.8–6.2 and an amino acid composition similar to that reported for the putative M-line protein in chicken muscle (termed component I) and rabbit muscle. Antibodies prepared against the 165 000 dalton polypeptide do not react with purified frog debranching enzyme, and indirect immunofluorescent studies localize this polypeptide wholly in the middle of the A band (presumably the M line) of frog skeletal muscle. We conclude that this 165 000 dalton polypeptide is a basic constituent of the M line in frog skeletal muscle.Crude M-line protein preparations from frog muscle invariably contained a 105 000 dalton polypeptide contaminant. In crude M-line protein preparation from chicken muscle, a polypeptide with a similar molecular mass was identified as phosphorylase b. Characterization of the 105 000 dalton polypeptide isolated from crude M-line protein preparations establishes that in frog muscle extracts this component has an amino acid composition which is different from phosphorylase b, but similar to frog α-actinin. Since it also exhibits immunochemical cross-reactivity with α-actinin and not with purified phosphorylase b, and is localized wholly at the Z line, we conclude that the 105 000 dalton polypeptide contaminant in crude M-line protein preparations from frog muscle is α-actinin.
Regulation of ethanolamine and choline phosphatide biosynthesis in isolated rat intestinal villus cells
Authors: O'Doherty P. J. A.
Published: December 2009

The effects of ethanolamine, choline, and different fatty acids on phospholipid synthesis via the CDP-ester pathways were studied in isolated rat intestinal villus cells. The incorporation of [14C]glucose into phosphatidylethanolamine was stimulated severalfold by the addition of ethanolamine and long-chained unsaturated fatty acids, while the addition of lauric acid inhibited the incorporation of radioactivity into phosphatidylethanolamine. At concentrations of ethanolamine higher than 0.2 mM, phosphoethanolamine accumulated, but the concentration of CDP-ethanolamine and the incorporation of radioactivity into phosphatidylethanolamine did not increase further. The incorporation of [14C]glucose into phosphatidylcholine responded in a way similar to that of phosphatidylethanolamine, except that a 10-fold higher concentration of choline was required for maximal stimulation. CCC inhibited the incorporation of choline into phosphatidylcholine. In contrast with hepatocytes, villus cells did not form phosphatidylcholine via phospholipid N-methylation. The data indicate that, in intestinal villus cells, the cytidylyltransferase reactions are rate limiting in the synthesis of phosphatidylethanolamine and probably also of phosphatidylcholine. The availability of diacylglycerol and its fatty acid composition may also significantly affect the rate of phospholipid synthesis.
Purification of human plasma lecithin:cholesterol acyltransferase and its activation by metal ions
Authors: Suzue Ginzaburo , Vézina Camilla , Marcel Yves L.
Published: December 2009

Lecithin:cholesterol acyltransferase (LCAT) has been purified from human plasma by sequential preparative ultracentrifugation, ion-exchange chromatography on DEAE-Sephacel, and affinity chromatography on HDL-Sepharose and on wheat germ agglutinin-Sepharose. After the final step, which included preparative electrophoresis or alternatively, chromatography on hydroxylapatite, a purification of about 24 000-fold was obtained. The LCAT preparation was pure according to alkaline polyacrylamide and SDS–polyacrylamide gel electrophoresis and did not react against antisera to apo AI, AII, and D. The LCAT preparation obtained by preparative electrophoresis was stimulated by Cu2+, Ni2+, Co2+, and Zn2+ at both stages of the reaction, phospholipase reaction and cholesterol esterification. This stimulatory effect was abolished by EDTA.
Fatty acid metabolism in skeletal muscle mitochondria from two strains of dystrophic mice
Authors: Martens M. E. , Lee C. P.
Published: December 2009

Several aspects of fatty acid metabolism have been examined in skeletal muscle mitochondria from both strain 129 dystrophic (dy/dy) and myodystrophic (myd/myd) mice. Skeletal muscle mitochondria from dy/dy mice showed significantly decreased state 3 respiratory rates with both palmityl- and acetyl-carnitine + malate as substrates when compared with their normal littermate controls. A similar, though less severe impairment in acylcarnitine oxidation by mitochondria from myd/myd skeletal muscle has also been shown by us in a previous study. In the present study, kinetic measurements revealed decreased activities of the reverse carnitine palmityltransferase (palmitylcarnitine + CoASH as substrates) in intact mitochondria from dy/dy muscle, and of citrate synthase in myd/myd muscle mitochondria. However, neither of these reactions appeared to be rate limiting for acylcarnitine oxidation in mouse skeletal muscle mitochondria. All other enzyme activities or cofactor contents measured were either comparable to those of controls or were higher. The results reported here indicate that neither of the impairments in acylcarnitine oxidation by skeletal muscle mitochondria from dy/dy or myd/myd mice is due to deficiencies in either carnitine palmityltransferase, carnitine acetyltransferase, citrate synthase, coenzyme A, or substrate-reducible flavoprotein.
Yeast cell wall, membrane, and soluble marker polypeptides identified by comparative two-dimensional electrophoresis
Authors: Robertson A. J. , Gerlach J. H. , Rank G. H. , Fowke L. C.
Published: December 2009

Yeast cell wall, plasma membrane, total spheroplast, and total soluble protein fractions were isolated from exponentially growing Saccharomyces cerevisiae batch cultures. The cell wall, plasma membrane, and soluble protein fractions were obtained by mechanical disruption of intact yeast cells under identical osmotic conditions. Electron micrographs of purified wall fractions appeared free of vesicular membrane contamination and micrographs of plasma membrane vesicles were free of cell wall contamination. Various stages of cell wall purification were monitored by electron microscopy and comparative two-dimensional sodium dodecyl sulfate – polyacrylamide gel electrophoresis. This resulted in the identification of a glycopeptide designated 16 w in the cell wall fraction, with an apparent isoelectric point of 5.0 and an apparent molecular weight of 25 000. Protein analyses of soluble and plasma membrane protein fractions failed to detect component 16w. Two-dimensional protein analyses of total cellular homogenates were capable of resolving the cell wall glycopeptide 16w. However, protein separations of spheroplasts formed by glusulase degradation of the cell wall complex did not detect 16w. These observations suggest that component 16w is unique to the cell wall fraction. In addition, comparison of two-dimensional gels of soluble and plasma membrane proteins, with a total cellular homogenate, tentatively identified several polypeptides unique to each of the soluble and plasma membrane fractions.
Reconstitution of lipoproteins. II. Lipid–protein interaction between dimyristoyl-lecithin and dimyristoyl-lecithin:cholesterol vesicles and purified apolipoprotein C-I and C-III2 studied by isomeric spin-labelled lecithins
Authors: Vaughan D. J. , Breckenridge W. C. , Stanacev N. Z.
Published: December 2009

The reconstitution of purified apolipoprotein C-I and C-III2 with sn-3-dimyristoyl-lecithin and sn-3-dimyristoyl-lecithin:cholesterol (10:1) vesicles was studied by electron spin resonance spectroscopy using isomeric 5′-, 12′-, and 16′-(N-oxyl-4″,4″-dimethyloxazolidine)stearoyl spin-labelled lecithin probes. Results obtained from the temperature-induced changes of lipoprotein recombinants showed the hydrophilic nature of the lipid–protein interactions. The temperature-induced phospholipid phase transition, as measured by 5′-(N-oxyl-4″,4″-dimethyloxazolidine)stearoyl spin-labelled lecithin probe in recombinants containing apoprotein C-1 or apoprotein C-III2, is very broad and has a small cooperative unit indicative of extensive lipid–protein interactions occurring at the head group region of the phospholipid bilayer. When 12′- and 16′-(N-oxyl-4″,4″-dimethyloxazolidine)stearoyl spin-labelled lecithins are used as probes in the same system, similar sharper and more cooperative lipid phase changes are detected. These results indicate a surface location for both apoprotein C-I and apoprotein C-III2 with respect to the phospholipid bilayer in lipoprotein recombinants with and without cholesterol.
Inosine synthesis and phosphoribosylpyrophosphate availability in rat liver cells in the presence of allopurinol
Authors: Lalanne Marcel , Lafleur Francine
Published: December 2009

In the presence of allopurinol, apparent phosphoribosylpyrophosphate (PP-ribose-P) availability as measured by adenine incorporation into ribonucleotides was decreased in rat liver cells, hypoxanthine incorporation into ribonucleotides was increased, and there was a large synthesis of inosine from hypoxanthine. Inosine was formed directly by the reversal of the purine nucleoside phosphorylase reaction which was very rapid in liver cells. We tested the hypothesis that utilization of ribose 1-phosphate for inosine synthesis could decrease PP-ribose-P availability. Our results indicate that the apparent decrease of PP-ribose-P availability in the presence of allopurinol was due to competition between adenine and hypoxanthine salvage pathways into nucleotides, and not to the synthesis of inosine.
The fatty acid composition of lipids of neuronal and glial nuclear fractions isolated from 15-day-old rabbit cerebral cortex
Authors: Baker R. Roy , Chang Huu-Yi
Published: December 2009

A neuronal nuclear fraction (N1) and a glial nuclear fraction (N2) have been isolated from 15-day-old rabbit cerebral cortex using the Thompson procedure. More than 56% of the homogenate DNA was recovered in the two nuclear fractions, with N1 being the larger by about eightfold. Fractions N1 and N2 had very similar phospholipid distributions, with phosphatidyl-inositol being a larger component than phosphatidylserine. Fatty acid analyses demonstrated that phosphatidylethanolamine and phosphatidylinositol, individually, had similar fatty acid profiles in fractions N1 and N2, and also in nuclear and microsomal fractions derived from homogenates of nerve cell bodies isolated from cerebral cortex of 15-day-old rabbits. In contrast, the nuclear phosphatidylcholines had lower levels of palmitate and higher levels of arachidonate than did microsomal phosphatidylcholines. Molecular species analyses indicated that monoenes (41 mol%), tetraenes (20 mol%), and saturates (13 mol%, composed chiefly of palmitate) were the principal classes of N1 phosphatidylcholines, while the diacyl species of phosphatidylethanolamine of this fraction were characterized by high levels of tetraenes (44 mol%), pentaenes (17 mol%), and hexaenes + polyenes (24 mol%). The neutral glycerides of fraction N1 occurred collectively at a level of 0.05 mol/mol phospholipid. Prominent fatty acids of diacylglycerols included palmitate (31%), oleate (20%), arachidonate (14%), and stéarate (13%). Triacylglycerols showed a similar pattern but with relatively high levels of linoleate (11%), while monoacylglycerols consisted almost entirely of palmitate (33%), stearate (35%), and oleate (24%).
Characterization of the oligosaccharides of liver Z variant α1-antitrypsin
Authors: Hercz Albert , Harpaz Noam
Published: December 2009

The plasma α1-antitrypsin (AT) of normal individuals has been reported to have four oligosaccharides per molecule; these oligosaccharides are all of the complex type, containing only three D-mannose (Man) residues per oligosaccharide as well as several residues of galactose, sialic acid, and N-acetyl-D-glucosamine (GlcNAc) (Chan, S. K., Rees, D. C., Li, S.-C. &Li, Y.-T. (1976) J. Biol. Chem. 251, 471–476; Hodges, L. C., Laine, R. &Chan, S. K. (1979) J. Biol. Chem. 254, 8208–8212). Analysis of the carbohydrates of purified liver Z variant AT was consistent with the presence of three moles of Man-rich oligosaccharide per mole protein with an average of seven Man and two GlcNAc residues per oligosaccharide and a complete absence of galactose and sialic acid (Hercz, A., Katona, E., Cutz, E., Wilson, J. R., &Barton, M. (1978) Science 201, 1229–1232). We have now carried out a more definitive structural analysis of the oligosaccharides of liver Z variant. Glycopeptides were prepared from purified liver Z variant by digestion with pronase. Treatment of a sample of the glycopeptides with endo-β-N-acetylglycosaminidase CII (endo CII) resulted in the formation of three oligosaccharides. These were identified by gel permeation chromatography on a Biogel P-4 column, calibrated with a series of ovalbumin and Sindbis virus oligosaccharides, as GlcNAcMan5, GlcNAcMan6, and GlcNAcMan7. Treatment of a sample of the glycopeptides with endo-β-N-acetylglycosaminidase D gave rise only to a single oligosaccharide product, GlcNAcMan5. Upon incubation of the glycopeptides with UDP[U-14C]GlcNAc in the presence of purified bovine colostrum UDPGlcNAc:α-D-mannoside α-2-N-acetylglucosaminyltransferase I, AsnGlcNAc2Man5 quantitatively disappeared and a new product consistent with the structure AsnGlcNAc2Man5[14C]GlcNAc was formed. Incubation of the oligosaccharides, formed with endo CII, with α-mannosidase gave rise to a 90% homogeneous product with the probable structure of Manβ1 → 4GlcNAc. These findings suggest that the precursors of the complex oligosaccharides of the secretory glycoprotein, plasma AT, are Man-rich oligosaccharides. It appears also that the glycosidases which process the precursor to the AsnGlcNAc2Man5 stage are present and active in the rough endoplasmic reticulum of Pi (protease inhibitor) ZZ individuals; however, for an as yet undetermined reason, most of the Z variant AT appears unable to migrate at a normal rate to the smooth endoplasmic reticulum and Golgi apparatus for eventual secretion from the liver.
The binding sites of rabbit skeletal troponin-I on troponin-T
Authors: Pearlstone Joyce R. , Smillie Lawrence B.
Published: December 2009

Various fragments derived from rabbit skeletal muscle troponin-T (Tn-T) by chemical and (or) proteolytic cleavage were mixed with whole troponin-I (Tn-I) and applied to a Sephadex G-75 gel filtration column in order to determine the binding site of Tn-I on Tn-T. This site of interaction was found to span two distinct regions of Tn-T. The first site involves the highly acidic NH2-terminal fragment CB3 (residues 1–70 of Tn-T). A second separate site is located in the region of residues 152–209 of Tn-T. The present study, in conjunction with our earlier work on tropomyosin – Tn-T binding and Tn-T – troponin-C binding, depicts Tn-T as being a functionally efficient molecule composed of several distinct domains of specialized amino acid sequence, each of which carries out a role in the binding of a different protein.
The formation of phosphatidylethanolamine from diglyceride by extracts of Escherichia coli
Authors: Proulx P. , Tomalty C. , Landry F. , Milczarek J. J.
Published: December 2009

Extensively dialyzed cell-free homogenates or washed particulate fractions, of Escherichia coli in the presence of added CTP, Mg2+, serine, and rac-glycerol 3-phosphate, incorporated [32P]phosphatidic acid into phosphatidylglycerol and cardiolipin but not into phosphatidylethanolamine. [14C]Phosphatidic acid could be incorporated by these preparations into phosphatidylethanolamine in the absence of added CTP and Mg2+. CDP-diglyceride did not significantly affect the formation of [14C]phosphatidylethanolamine from [14C]phosphatidic acid. 14C-labeled diglyceride was also readily incorporated into phosphatidylethanolamine in the absence of added cofactors by both homogenate and particulate fraction. Serine stimulated the incorporation somewhat whereas CTP + Mg2+ diminished this conversion slightly because of concurrent [14C]phosphatidic acid and [14C]phosphatidylglycerol formation. CDP-diglyceride did not significantly affect the conversion of 14C-labeled diglyceride to phosphatidylethanolamine. Dialyzed 14C-labeled cytosol fractions of E. coli, obtained from cells grown in medium containing [14C]serine, transferred some of their label to phosphatidylethanolamine in the presence of fresh particulate fraction. This transfer was stimulated by added diglyceride. The results indicate that phosphatidylethanolamine can be synthesized from exogenous diglyceride by a pathway which does not require CDP-diglyceride as an intermediate but which likely makes use of an endogenous cofactor supplying phosphorylethanolamine and (or) phosphorylserine.
Enzymatic sulfation of steroids. XI. The extensive purification and some properties of hepatic sulfotransferase III from female rats
Authors: Singer Sanford S. , Bruns Lawrence
Published: December 2009

Our earlier studies showed that livers from female rats contained three glucocorticoid sulfating enzymes we named sulfotransferases I, II, and III, (STI, STII, and STIII, respectively). In this report STIII from female Charles River CD rats was purified 1010- to 1300-fold compared with liver homogenates. The most highly purified STIII fraction electrophoresed as a single protein band. The molecular weight of STIII was 68 300 ± 4900. Its pH optimum for cortisol sulfation was pH 6.0 ± 0.1. However, it was routinely assayed at pH 6.8 for reasons enumerated in the text. Cortisol sulfation by STIII proceeded by either an ordered sequential mechanism or by an Iso Theorell-Chance mechanism at pH 6.8. The Km's for cortisol and the reaction coenzyme, 3′-phosphoadenosine-5′-phosphosulfate were 6.48 ± 0.78 and 6.78 ± 1.26 μM, respectively. Comparison of the ability of the enzyme to sulfate 40 μM cortisol, estradiol-17β, testosterone, deoxycorticosterone, and dehydroepiandrosterone, showed that the glucocorticoid was sulfated preferentially. Interestingly, its cortisol sulfotransferase activity was inhibited by a number of steroids. p-Hydroxymercuribenzoate inhibition studies indicated the presence of essential sulfhydryl groups in STIII. The enzyme was activated by divalent Ba, Ca, Co, Cr, Mg, Mn, and Ni salts. It was inactivated by Zn2+ and Cd2+ salts. The text compares STIII from female rats with other steroid sulfotransferases including the major glucocorticoid sulfotransferase from male rats.
Diffusional limitations in liquid-phase reactions catalyzed by enzymes immobilized on porous supports: sucrose inversion by β-fructooxidase immobilized on IRA-93 resin
Authors: Toro Luigi , Gaudioso Domenico
Published: December 2009

An experimental procedure has been proposed to study liquid-phase reactions catalyzed by enzymes immobilized on porous supports. The kinetic behavior can be assessed by experimental runs which allow a separate evaluation of the internal diffusion, the surface kinetics, and the fluid-particle mass transfer. The method has been applied in the study of sucrose inversion by β-fructooxidase immobilized on IRA-93 resin.
Peroxidase biosynthesis as part of protein synthesis by cultured peanut cells
Authors: Stephan D. , Huystee R. B. Van
Published: December 2009

Membrane-bound and free ribosomes from cultured peanut cells were separated by differential centrifugation. The former ribosomes were liberated from the 27 000 × g pellet with 1% Triton X100. Purification of both polysome populations was obtained by passage through a 1.5 M sucrose cushion. Both populations have a high rate of protein synthesis in vitro in the presence of a wheat germ S30 fraction. Comparisons of protein synthesis in vitro with that in vivo, and also with labelled proteins released by these cells into the medium did not provide significant information regarding peroxidase biosynthesis. However, immunoprecipitation of the products formed in vitro with antibodies raised against a purified cationic peroxidase fraction, resulted in isolating one major radioactive product. The higher molecular weight of this isolated product compared with the molecular weight of the purified peroxidase fraction suggests the occurrence of a signal peptide for peroxidase commensurate with its being a secreted protein.
Correlation of chromatin composition with metabolic changes in nuclei of primitive erythroid cells from chicken embryos
Authors: Urban Michael K. , Neelin James M. , Betz Thomas W.
Published: December 2009

Changes in levels of biosynthesis of DNA, RNA, and histones were compared with relative proportions of each histone class during primitive erythropoiesis in embryonic chicks. We confirmed that erythrocyte-specific histone 5 (H5) was substantial in the earliest accessible, erythroblast-enriched stage and that it doubled in relative amount between polychromatic and orthochromatic stages to about 1 mol per 2 mol of each nucleosomal histone, still considerably less than in adult definitive erythrocytes. No other histones changed during primitive erythropoiesis, but the molar proportion of histone 1 (H1) always exceeded that of H5 in these cells, unlike definitive erythrocytes. The increase in content of H5 was accompanied by continued incorporation of lysine into this histone in orthochromatic erythrocytes in contrast to the decline in synthesis of the other histones and DNA. The accumulation of H5 during development appears to occur in steps corresponding to the maturation of the primitive and definitive erythroid cell lines. Lysine-rich histones were more easily extracted from nuclei of the erythroblast-enriched stage than from later stages; this change accompanied an early decrease in RNA synthesis in whole cells and in isolated nuclei.
Partial purification of diphosphatidylglycerol synthetase from liver mitochondrial membranes
Authors: McMurray W. C. , Jarvis E. C.
Published: December 2009

The enzyme responsible for the conversion of phosphatidylglycerol to diphosphatidylglycerol (cardiolipin) in the presence of cytidine diphosphate diacylglycerol is firmly associated with mitochondrial membranes and is not extracted with hypotonic or hypertonic media or with nonionic detergents. Some solubilization was obtained with bile salt solutions, but the zwitterionic detergent, Miranol H2M, was most effective in extracting the enzyme. The Miranol extracts were fractionated by column chromatography on Bio-Gel A-1.5 m. The solubilized enzyme is considerably more active in converting unsaturated than saturated phosphatidylglycerols, but shows little preference for the cytidine diphosphate diacylglycerols with different fatty acyl substituents. There is an absolute dependence upon divalent cations with the order of effectiveness: [Formula: see text]. In the presence of optimal levels of Co2+ other divalent cations are inhibitory with the order of inhibition: Cd2+ > Zn2+ > Ca2+ > Ba2+ > Cu2+ > Hg2+ > Ni2+. The solubilized enzyme exhibited no requirement for added phospholipids and several phospholipids inhibited the reaction in the order: diphosphatidylglycerol > phosphatidylethanolamine > phosphatidylserine > phosphatidylinositol.
Hydrocarbon specificity for transport on the tricarboxylate transporter of rat liver mitochondria
Authors: Cheema-Dhadli S. , Halperin M. L. , Duperrouzel P. R. , Leznoff C. C.
Published: December 2009

The purpose of these studies was to evaluate the relative importance of the C-6 carboxyl group on citrate and of the hydroxyl group on citrate and L-malate analogues. Specific methyl esters of citrate and tricarballylate were synthesized and their structures verified by 1H and 13C nuclear magnetic resonance spectra. The formation of an α- or β-methyl ester markedly reduced the ability of the citrate analogue to be a substrate or inhibitor for the citrate transporter. The hydroxyl group was important only for dicarboxylates and in these compounds, it could not be replaced by a keto group. Stereospecificity was also very important for this transport system. In addition, the intercarboxyl distance appeared to play an important role in determining whether the compound could counter transport with citrate or L-malate.
The influence of various lipids on the activity of bovine milk galactosyltransferase
Authors: Mitranic M. M. , Moscarello M. A.
Published: December 2009

Purified bovine milk galactosyltransferase was combined with liposomes of different lipid composition. The activity was markedly affected by the nature of the lipid used. Thus phosphatidylcholine, phosphatidylethanolamine, and phosphatidylglycerol stimulated the activity, while phosphatidic acid and phosphatidylserine inhibited the activity of the transferase. Phosphatidylcholine, phosphatidylglycerol, and phosphatidic acid had identical fatty acid compositions, yet phosphatidylcholine and phosphatidylglycerol stimulated the activity while phosphatidic acid inhibited the activity. The effect on the enzyme was probably related to the nature of the head group since the inhibition by phosphatidic acid could be converted to stimulation by methylating the phosphatidic acid. The properties of several of the head groups is discussed. The physical state of the lipid was shown to affect the activity markedly. When the enzyme was combined with dimyristylphosphatidylcholine the activity was markedly stimulated when the lipid was in the liquid-crystalline state i.e., above the phase transition.
Inhibition of mitochondrial carnitine–acylcarnitine translocase by sulfobetaines
Authors: Parvin Rehana , Goswami Tapas , Pande Shri V.
Published: December 2009

Sulfobetaines (N-alkyl-N,N-dimethyl-3-ammonio-1-propanesulfonates) have been identified as relatively specific and selective inhibitors of mitochondrial carnitine–acylcarnitine translocase. Thus, sublytic concentrations of sulfobetaines (alkyl = octyl to tetradecyl) inhibit the respiration of rat heart mitochondria supported by added acylcarnitines or pyruvate plus malonate and carnitine. Both exchange efflux and unidirectional net efflux of mitochondrial carnitine are also inhibited; the half-maximal inhibition of the former occurs at micromolar concentrations of sulfobetaines and the inhibitory effect is reversible and competitive with respect to carnitine. As a stop-inhibitor, 20 mM sulfobetaine8, (alkyl = octyl), is useable at near 0 °C but is less effective than 2 mM mersalyl when transport rates are very rapid as at higher temperatures especially with liver mitochondria. The loss of mitochondrial carnitine that normally occurs owing to the progress of net efflux during the isolation of mitochondria is prevented by the inclusion of 20 mM sulfobetaine8 in the isolation medium and this enables a better estimate of the mitochondrial carnitine content. Sulfobetaines inhibit the activities of mitochondrial carnitine acetyltransferase and carnitine palmitoyltransferase but only at concentrations severalfold higher than those inhibitory for the translocase. This observation supports the belief that carnitine–acylcarnitine translocase is an entity distinct from that of carnitine acyltransferases.
Increased cationic fluxes in stimulated lymphocytes of the mouse: response of enriched B- and T-cell subpopulations to B- and T-cell mitogens
Authors: Owens Trevor , Kaplan J. Gordin
Published: December 2009

Suspensions of mouse (Balb/c) splenic lymphocytes took up the potassium congener 86Rb at a net rate (calculated as K+) of 4.6 ± 0.2 fmol cell−1 h−1 before stimulation with the mitogen concanavalin A (Con A) and of 8.5 ± 0.4 fmol cell−1 h−1 after stimulation. These net transport rates were of similar magnitude to those previously reported in the case of resting and stimulated human peripheral blood lymphocytes. One difference between the mouse and human systems was the increased transport noted within minutes of stimulation in the case of the latter that required 6 h before becoming evident in the former. When similar transport studies were performed using splenic lymphocytes of the homozygous, congenitally athymic. nude mouse, Con A, a T-cell mitogen, caused neither a blastogenic response (operationally defined as increased uptake of labeled thymidine into DNA after 48-h culture in presence of mitogen) nor the increase in cation influx; lipopolysaccharide (LPS), a B-cell mitogen, caused both increased transport and blastogenesis. As it has been shown that both mitogens bind equally well to B cells and to T cells, it follows that the increased cation pumping characteristic of activated lymphocytes is not caused by the binding of mitogen per se but is a consequence of the proliferative response. Attempts to demonstrate the same correspondence between B- and T-cell specificity of mitogen and increased cation fluxes in Balb/c splenic lymphocytes purified by the wheat germ agglutinin (WGA) technique yielded equivocal results. The T cell enriched population, although greatly purified as judged by membrane markers (fluorescent goat antimouse immunoglobulin staining), showed no purification with respect to enhanced cation influx and little or none with respect to the incorporation of labeled thymidine induced by Con A. This mitogen also induced both increased Rb+ transport and thymidine incorporation in a sub-population enriched for B cells by the WGA technique.86Rb efflux from Con A treated, unfractionated Balb/c splenocytes was observed to rise after stimulation to a rate 40% higher than that from resting cells. It was calculated on the basis of reasonable assumptions that this observed imbalance between the magnitude of increase in influx (100%) and efflux could account for the increased number of potassium ions required to be transported into the cell water in order that concentration of this ion be kept constant during the enormous expansion of cell volume characteristic of blastogenesis.
Peptide models for protein-mediated cation transport
Authors: Deber Charles M.
Published: December 2009

Substances which can perturb the transmembrane cation balance in a predictable manner have wide-ranging uses in the study of cellular processes. We have undertaken to examine transmembrane calcium transport on the molecular level through the design and synthesis of a series of ionophoric peptides as models for protein-mediated calcium transport. General mechanisms for carrier-mediated membrane transport are discussed. Cation transport profiles are presented for transport by synthetic peptides of structure cyclo(Glu(OR)-Sar-Gly-(N-R1)-Gly)2, where R = benzyl ester or H; R1 = n-decyl or cyclohexyl. Transport of physiologically abundant cations across "liquid membranes" in Pressman cells mediated by cyclo(Glu-Sar-Gly-(N-decyl)Gly)2 was observed to be essentially calcium specific, as long as calcium ions were present in the system. Multilamellar and unilamellar phosphatidylcholine vesicles were each found to be emptied of internal 45Ca2+ ions upon addition of cyclo(Glu(OBz)-Sar-Gly-(N-cyclohexyl)Gly)2 to the vesicle suspension. The results are compared with the naturally occurring calcium ionophore A23187.
The effect of cholesterol on measured interaction and compressibility of dipalmitoylphosphatidylcholine bilayers
Authors: Rand R. P. , Parsegian V. A. , Henry J. A. C. , Lis L. J. , McAlister M.
Published: December 2009

We have examined the phase diagram of dipalmitoylphosphatidylcholine (DPPC) – cholesterol – water mixtures at low cholesterol content, and report phase separation between 3 and 10 mol% cholesterol. The two lamellar phases at equilibrium in this region appear to be pure DPPC and 11 mol% cholesterol in DPPC. For these two lamellar phases, which are made up of alternating layers of water and bimolecular lipid leaflets, we have measured the forces of interaction between leaflets and the lateral pressure and compressibility of the leaflets. Both bilayers experience a strong repulsive force when forced together only a few ångströms (1 Å = 0.1 nm) closer than their maximum separation in excess water. However, the presence of 11 mol% cholesterol causes the bilayers to move apart to 35-Å separation from the 19-Å characteristic of pure DPPC in excess water. This swelling may result from a decrease in van der Waals attraction between bilayers or from an increase in bilayer repulsion. Differences in bilayer interaction can be a cause for phase separation. More importantly these differences can cause changes in the composition of regions of membranes approaching contact. At 11 mol%, cholesterol substantially increases the lateral compressibility of DPPC bilayers leading to higher lateral density fluctuations and potentially higher bilayer permeability.
The use of bee venom melittin to assess the topography of membrane vesicles derived from Paracoccus denitrificans
Authors: Pik Jeanette R. , Lawford Hugh G.
Published: December 2009

There exists considerable controversy regarding membrane topography in vesicles derived by osmotic lysis of spheroplasts of Gram-negative bacteria. It has been reported by others that bee venom can be used to quantitate the portion of a heterogeneous vesicle population with an inside-out orientation by determining the degree of loss of crypticity of NADH dehydrogenase activity. We have demonstrated that a major component of bee venom, melittin, causes an increase in the activity of several different respiratory enzymes in isolated membrane vesicles of Paracoccus denitrificans. The degree of stimulation produced by melittin is dependent upon (i) the nature of the respiratory substrates, (ii) the pH, (iii) the presence of Mg2+, (iv) the melittin: membrane protein ratio, and (v) the growth history of the cells from which the membrane vesicles were derived. Melittin-induced enhancement of TMPD: ascorbate and cytochrome c oxidase activities cannot be accounted for by increased accessibility of non-permeant substrate to the interior of the vesicle. The stimulatory effect of melittin may rely in part on its ability to alter the proton permeability of the membrane thereby abolishing respiratory control. Collectively these observations call into question the usefulness of bee venom melittin in quantitative analyses of membrane topography. These results are consistent with the postulated existence of a homogeneous vesicle population in which the topography of the NADH dehydrogenase is different from that of the intact cell.
Selenium-independent glutathione peroxidase activity in rabbit liver
Authors: Morrissey Paul , O'Brien Peter J.
Published: December 2009

The reduction of linoleic acid hydroperoxide catalyzed by rat liver cytosol was previously shown to be catalyzed by a selenium-dependent glutathione peroxidase. In contrast, the activity in rabbit liver cytosol could also be attributed to a selenium-independent glutathione peroxidase present in an approximately equal amount to the selenium-dependent peroxidase. The selenium-independent peroxidase copurified with glutathione transferase B and was completely inhibited by antitransferase B antiserum and transferase substrates. These results suggest that glutathione transferase B in rabbit liver cytosol is involved in the intracellular decomposition of lipid peroxide and could explain the lower selenium requirement of rabbits in comparison with other species.
Adenylate cyclase in lean and obese (ob/ob) mouse epididymal white adipocytes
Authors: Bégin-Heick Nicole
Published: December 2009

The results presented in this study indicate that the defect in catecholamine-stimulated adenylate cyclase which is characteristic of the ob/ob mouse is associated with a decrease in the sensitivity of the system to guanine nucleotides (guanosine 5′-[βγ-imido]triphosphate and guanosine 5′-triphosphate). No difference in the β-adrenergic receptor activity was found between the lean and obese mice on the basis of their capacity to bind the β-adrenergic antagonist [3H]dihydroalprenolol. The data suggest that a defect in the activation of the adenylate cyclase by β-adrenergic agents may reside in the guanyl nucleotide binding site(s).
Intracellular (Golgi) receptors for insulinlike peptides in rat liver
Authors: Posner Barry I. , Gonzalez Roberto M. , Guyda Harvey J.
Published: December 2009

An insulinlike peptide (ILAs) has been isolated in our laboratory from human serum. The binding of 125I-labeled ILAs was studied in subcellular fractions from rat liver and found to be much greater in microsomes than purified plasmalemma. The high level of microsomal binding was due to a particular enrichment of binding sites in Golgi elements. Binding to Golgi was time and temperature dependent and was augmented by an increase of either subcellular fraction or 125I-labeled ILAs in accordance with a mass action process. Degradation of 125I-labeled ILAs was greatest in the Golgi vesicle fraction and was reduced by incubation at 4 °C. Bound 125I-labeled ILAs could be eluted and was found to retain integrity. Binding was pH dependent with a broad optimum at pH 7.7–8.5. Dissociation of bound 125I-labeled ILAs was time and temperature dependent. It was greater at 37 than 4 °C, and was uninfluenced by unlabeled ILAs. The ILAs receptor was stable at 4 °C but was markedly decreased by preincubation at 37 °C. The binding of 125I-labeled ILAs was inhibited by unlabeled ILAs and related insulinlike peptides (the insulinlike growth factors, IGF-1 and IGF-2) in a dose-dependent manner. Insulin and its analogues had only a partially inhibitory effect, and structurally unrelated peptides were without inhibitory efficacy. In contrast ILAs and IGF-1 and IGF-2 inhibited 125I-labeled insulin binding to its receptors in a dose-dependent fashion.These observations identify a receptor for insulinlike peptides in the Golgi elements of rat liver. It is distinct from the insulin receptor previously observed in these elements. The dual interaction of ILAs and other insulinlike peptides with both the insulin receptor and their own unique receptor constitutes the presumed biochemical basis for the two types of action effected by this family of peptides, namely, an effect on metabolism comparable to insulin and an effect on cellular anabolism and growth.
Phospholipids and membrane transport
Authors: Cullis P. R. , Kruijff B. de , Hope M. J. , Nayar R. , Schmid S. L.
Published: December 2009

The ability of membrane lipids to adopt nonbilayer configurations suggests dynamic roles for lipids in many functional abilities of biological membranes. In this work evidence supporting the involvement of lipids in three types of membrane transport process is presented and discussed. These transport processes include facilitated transbilayer transport of polar molecules, transport mechanisms involving fusion events, and transport possibilities arising from alternative membrane morphology. In particular it is shown that lipids such as cardiolipin, which adopt the hexagonal H11 phase in the presence of Ca2+, may be logically proposed to facilitate Ca2+ transport across membranes via an inverted micellar intermediate. Alternatively, in transport processes such as exocytosis the ability of Ca2+ to generate membrane instabilities favouring nonbilayer alternatives suggests a crucial role of phospholipid in the fusion event vital to exocytotic release. Finally, nonbilayer lipid structures may be suggested to favour formation of isolated compartments connected by a continuous membrane where lateral diffusion processes can lead to transport. These various possibilities are summarized in a "metamorphic mosaic" model of biological membranes.
Studies on incorporation of mannose into rat α1-acid glycoprotein: evidence for precursor forms in rough membranes
Authors: Friesen A. D. , Jamieson J. C.
Published: December 2009

Rats were given pulse injections of D-[14C]mannose and were killed at various times up to 60 min after injection. Rough, smooth, and Golgi fractions were prepared from liver, and α1-acid glycoprotein was isolated from Lubrol extracts of the fractions. The kinetics of incorporation of D-[14C]mannose into total protein, Lubrol protein, and α1-acid glycoprotein showed that proteins associated with rough fractions had particularly high specific radioactivities at early times of incorporation. One explanation for the kinetic data is that glycoproteins contain a high mannose content at early times of assembly of oligosaccharide chains. This idea was confirmed in the case of α1-acid glycoprotein by isolation of a high mannose containing precursor species of α1-acid glycoprotein from rough fractions of liver. This species contained 56 residues of hexose (mainly mannose) compared with 35 residues of hexose (roughly equal amounts of mannose and galactose) which are found in the native protein. It is proposed that the high mannose precursor is a form of α1-acid glycoprotein that exists at an early stage in assembly of the glycoprotein and which contains largely unprocessed carbohydrate chains. In addition, evidence is presented from amino acid analyses and gel electrophoresis of the high mannose precursor and another fraction from which it is formed by limited tryptic treatment, that pro-forms of α1-acid glycoprotein with extensions of the polypeptide chain may also exist.
Membrane changes induced by early myocardial ischemia in the dog
Authors: Vasdev S. C. , Biro G. P. , Narbaitz R. , Kako K. J.
Published: December 2009

Sarcolemmal and mitochondrial phospholipids were extracted from normally perfused and ischemic regions of the dog heart and the composition of these extracts was analyzed. Relatively pure sarcolemmal fraction obtained from myocardium subjected to 3 h of ischemia exhibited a significantly lower concentration of phospholipids than that obtained from normal myocardium. In particular, phosphatidylcholine and phosphatidylethanolamine were reduced by approximately 33%. The fatty acid content in the sarcolemmal phospholipid fraction was also reduced by approximately 30% without any change in the relative composition. In the mitochondrial fraction, the relative phospholipid composition was also altered by ischemia; the major components (phosphatidylcholine, phosphatidylethanolamine, and cardiolipin) being reduced by approximately 15–20%. An attempt was made to correlate these biochemical changes with ultrastructural lesions observed electron microscopically. These observations revealed extensive regional variability and a wide heterogeneity in the extent of ultrastructural damage evident in the different organelles even in a single cell. This may suggest that ischemic damage, in the early stages, may advance at widely varying rates in different regions. Our findings demonstrate that significant biochemical and structural disorganization occurs during 3 h of ischemia in the myocardium and raise the possibility that one of the initiating events is the activation of sarcolemmal and mitochondrial phospholipases.
Temperature-induced physical changes in fungal plasma membranes
Authors: Miller R. W.
Published: December 2009

Ordered structures suggesting phase separation of lipids were observed in freeze-fracture replicas of plasma membranes of Fusarium sulphureum after free protoplasts had been fixed at 0 °C. Areas representing up to 50% of the protoplast plasma membrane inner fractured face assumed a lateral, hexagonally ordered structure from which the normally randomly distributed 25-nm membrane protein complexes had been excluded. Intact macroconidia only rarely showed small regions of lateral ordering of this type when fixed at the same, nonlethal temperature.The normal impermeability of the macroconidial plasma membrane to divalent cations such as Ni2+ declined rapidly when the cells were incubated at temperatures 5 °C or more above the physiological growth temperature range of the organism. Admission of divalent cations and death of the cells was associated with an irreversible thermotropic change in the fluidity in the membrane lipids as indicated by lipophilic spin probes.Partitioning spin probe and freeze-fracture techniques were best suited to the observation of thermotropic physical changes in the fungal plasma membrane which were induced at supra- and sub-physiological temperatures, respectively.
Resolution of the phosphoenolpyruvate: fructose phosphotransferase system of Escherichia coli into two components; enzyme IIfructose and fructose-induced HPr-like protein (FPr)
Authors: Waygood E. Bruce
Published: December 2009

A protein that substitutes for histidine-containing protein (HPr) in the phosphoenolpyruvate: fructose phosphotransferase system has been found in Escherichia coli grown on fructose. The impure preparation of the fructose-induced HPr-like protein (FPr) appears to be an extrinsic membrane protein which differs from HPr on the basis of its apparent molecular weight (45 000 vs. 9600, respectively), its affinity for DEAE-cellulose and its ability to promote sugar phosphorylation which is specific for fructose, rather than for glucose.