bluuebunny/crossref_metadata_2025 huggingface dataset:
Dynamic structure of the lower density lipoproteins. I. Incorporation of high levels of labelled lipids into very low and low density lipoproteins
Authors: Chana Ravinder S. , Treleaven W. Dale , Cushley Robert J. , Steinbrecher Urs P.
Published: December 2009

Selectively labelled lipids have been incorporated into the surface monolayer of human serum low density lipoprotein (LDL) and very low density lipoprotein (VLDL). From 3 to 17 mol% of phosphatidylcholine, selectively deuterated at various positions along the sn-2-acyl chain, was transferred from unilamellar vesicles to VLDL using a partially purified phosphatidylcholine transfer protein. Selectively deuterated palmitic acids were incorporated into LDL (6–20 mol%) and into VLDL (7–10 mol%). Electron microscopy, light scattering, and 31P nuclear magnetic resonance indicated that particle size remained unchanged. Gel exclusion chromatography and chemical analysis showed no difference in hydrodynamic properties and only slight alteration to particle component ratios. Biological activity of labelled VLDL was measured from the rate of cholesterol esterification by cultured J774A.1 cells. Effect of labelling LDL was evaluated by monitoring LDL uptake and degradation by cultured human skin fibroblasts. In all cases the lipoproteins containing labels were indistinguishable from their native counterparts.Key words: deuterium incorporation, cell culture studies, light scattering, lipoproteins, electron microscopy.
Structures of polar lipids from the thermophilic, deep-sea archaeohacterium Methanococcus jannaschii
Authors: Ferrante Giulio , Richards James C. , Sprott G. Dennis
Published: December 2009

Cells of Methanococcus jannaschii, grown at 65 °C in a defined medium, contained 7% of lipid composed of 87% polar and 13% neutral components. Within the polar fraction 16 lipids were resolved by thin-layer chromatography, 4 of which were present in trace amounts. Staining reactions demonstrated that the more abundant lipids were glycolipids, aminophospholipids, and an aminophosphoglycolipid. Most of the polar fraction (82%) consisted of five diether lipids, which were purified and their structures were resolved largely through nuclear magnetic resonance, mass spectrometry, and optical rotation methods. Macrocyclic diethers had the head groups phosphoethanolamine-(1 → 6)-β-D-glucopyranose, β-D-glucopyranose, and β-D-glucopyranosyl-(1 → 6)-β-D-glucopyranose. Phosphoethanolamine was identified as a head group for both the noncyclized and macrocylic diether core lipids. The neutral lipids were mainly acyclic C30 isoprenoids, predominantly dihydro-, hexahydro-, and octahydro-squalenes.Key words: macrocyclic diether lipids, structures, Methanococcus jannaschii.
Binding of glucagon to lipid bilayers
Authors: Oomen Raymond P. , Kaplan Harvey
Published: December 2009

At physiological pH and temperature, glucagon binds to liposomes composed of egg phosphatidylcholine and cholesterol (2:1 mol/mol) in a highly specific manner. The chemical reactivities of the functional groups were determined over the concentration range of 1.0 × 10−6 – 3.0 × 10−8 M by the method of competitive labelling with 1-fluoro-2,4-dinitrobenzene as the labelling reagent. At concentrations above 3 × 10−7 M, the amino terminal histidine and the two tyrosine residues showed a marked decrease in reactivity in the presence of liposomes, but the reactivity of the Lys-12 Nε-amino group was unaltered. At lower concentrations the Lys-12 reactivity also decreased markedly, owing to a change in the environment of this group. These results indicated that two different forms of glucagon existed over the concentration range studied. Both in the absence and presence of liposomes the Lys-12 Nε-amino groups showed a transition in reactivity at 1.8 × 10−7 M. In the presence of liposomes the other functional groups also showed a transition in reactivity at 2 × 10−7 M but the change was much smaller. The pattern of reactivities were consistent with the X-ray crystallographic structure of the type 2 glucagon trimer being the predominant species at 10−6 M, with free monomeric glucagon occurring at 3 × 10−8 M. A trimerization constant of 4 × 1013 M−2 at pH 7.5 and 37 °C was determined. It is concluded that trimeric glucagon binds to phosphatidylcholine–cholesterol bilayers primarily with the amino-terminal segment of the polypeptide chain and that additional regions of the molecule are involved in binding of the free monomeric unit.Key words: glucagon, liposomes, binding, reactivity.
Synthesis of acyl-CoA thioesters
Authors: Mangroo Dev , Gerber Gerhard E.
Published: December 2009

An improved synthesis of fatty acyl coenzyme A has been developed that permits the synthesis of highly radioactive fatty acyl coenzyme A derivatives and thus can be used for the synthesis of high specific activity photoaffinity labels. Conditions were developed to solubilize the coenzyme A in anhydrous solvent for the acylation. The complete activation of fatty acid to the imidazolide is described and the acylation of the coenzyme A under anhydrous conditions was shown to result in the conversion of the fatty acid to the fatty acyl coenzyme A derivative. The synthetic product was shown by its chemical and biochemical reactivity to be the pure thioester of coenzyme A. The purification of the fatty acyl coenzyme A by reverse-phase chromatography is described. The yield of pure fatty acyl coenzyme A was essentially quantitative.Key words: acyl coenzyme A, N,N′-carbonyldiimidazole, high pressure liquid chromatography, imidazolide, synthesis.
Synthesis of optically active l-acyl-2-O-alkyl-sn-glycero-3-phosphocholine via 1-O-benzyl-sn-glycerol 3-arenesulfonate
Authors: Ali Shaukat , Bittman Robert
Published: December 2009

A stereocontrolled route to 1-palmitoyl-2-O-hexadecyl-sn-glycero-3-phosphocholine from (R)-glycidyl tosylate is described. This method gives very high enantioselectivity (93–96% enantiomeric excess) and can be used to prepare 3-acyl-2-O-alkyl-sn-glycero-1-phosphocholines from (S)-glycidyl tosylate. The key step is the preparation of 1-O-benzyl-sn-glycerol 3-tosylate by the boron trifluoride etherate catalyzed regio- and stereo-specific opening of the epoxide ring with excess benzyl alcohol. The alkyl group is introduced using alkyl trifluoromethanesulfonate in the presence of excess 2,6-di-tert-butyl-4-methylpyridine. Debenzylation gives 2-O-alkyl-sn-glycerol 3-arenesulfonate, which is acylated and then converted into the phosphocholine. The use of chiral glycidyl derivatives as starting materials for the synthesis of glycerophospholipids is discussed.Key words: acylalkylglycerophospholipids, phospholipid synthesis, glycidyl derivatives in phospholipid synthesis, epoxides as precursors of phospholipids.
Effects of halocin H6 on the morphology of sensitive cells
Authors: Torreblanca Marina , Meseguer Inmaculada , Rodriguez-Valera F.
Published: December 2009

The halobacteria, extremely halophilic archaebacteria, produce bacteriocins that are globally termed halocins. In the present work we have studied the effect of halocin H6 on the morphology of sensitive cells. Halocin H6 produced deformation and lysis of sensitive cells, leaving empty "ghosts" in which the cell envelopes seemed to be intact. With massive doses of halocin, the effect was much more rapid and produced the same result. Halocin H6 also induced lysis of stable spheroplasts, and therefore the target could be located at the membrane level.Key words: bacteriocin, halocin, halobacteria.
Drug and metabolite-induced perturbations in nuclear structure and function: a review
Authors: Brasch Klaus
Published: December 2009

Clarification of large-scale organization in cell nuclei is central to a full understanding of the mechanisms which underlie replication, transcription, and interaction with the cytoplasmic compartment. Many drugs and natural biological agents affect such processes directly, through inhibitory or stimulatory action, and often reveal structural and functional aspects in nuclei not otherwise apparent. There is increasing evidence that the pore complex – lamina of the nuclear matrix is a primary structural element during interphase, which is pivotal to most nuclear processes. This communication reviews evidence based on studies with drugs, inhibitors, and natural biological processes, that the nuclear matrix concept is indeed valid. It is further shown that despite diversity in mode of action and affected target cells, many compounds induce related or complementary changes in nuclei and that such uniformity in pattern is attributable to the nuclear matrix. A general model of the functional organization of the nucleus is presented, to account for the perturbations observed after mass interference with either transcription, replication, or protein synthesis.Key words: nuclear structure, nuclear function, nuclear matrix.
Role of acid proteases in brown adipose tissue atrophy caused by fasting in mice
Authors: Desautels M. , Michalska E. , Mozaffari B.
Published: December 2009

The lysosomal proteolytic capacity of mouse brown adipose tissue (BAT) and its role during fasting were evaluated. The specific activities of acid phosphatase and cathepsins B, D, H, and L were measured in BAT of mice acclimated at 33, 21, and 4 °C and in BAT undergoing different rates of protein loss during a 24- to 48-h fast. The specific activities of lysosomal proteases in BAT did not vary with the acclimation status of the animals. Mice acclimated at 33 °C showed no significant atrophy of BAT after a fast. In mice kept at 21 °C, protein loss from BAT was observed after a fast without change in tissue DNA content. Protein loss from BAT was partially reduced by injection of the acidotropic agent chloroquine. Furthermore, tyrosine release from BAT during fasting was also reduced by injections of chloroquine or leupeptin, a thiol-protease inhibitor. Tyrosine release from BAT was maximum within 24 h and returned to prefast values by 36 h, suggesting rapid activation followed by inhibition of the tissue proteolytic activity. However, there was no change in acid protease specific activities, suggesting that these enzymes were not limiting for protein degradation. When cold-acclimated mice were fasted at 21 °C, BAT protein loss was markedly enhanced and increases in cathepsin D and L activities were observed, but there was no change in cathepsin B and H and acid phosphatase specific activities. These results indicate that BAT contains an important lysosomal proteolytic pathway that is involved in the rapid reduction of the tissue thermogenic capacity during a fast.Key words: temperature regulation, protein degradation, lysosomes, brown fat.
Evidence for an essential sulfhydryl group at the substrate binding site of the A-system transporter of Ehrlich cell plasma membranes
Authors: McCormick John , Johnstone R. M.
Published: December 2009

Plasma membrane suspensions of Ehrlich ascites cells solubilized with cholic acid were used to study the effects of sulfhydryl reagents on Na+-dependent amino acid transport. These suspensions were treated with the sulfhydryl binding agents p-chloromercuribenzenesulfonic acid or N-ethylmaleimide prior to reconstitution for the assay of transport activity. The proteoliposomes formed from dissolved membranes treated with p-chloromercuribenzenesulfonic acid showed no Na+-dependent α-aminoisobutyric acid transport, while N-ethylmaleimide pretreated membranes retained ~ 90% of the original activity. To avoid interference by the N-ethylmaleimide component, further studies were carried out with membranes pretreated with 200 μM N-ethylmaleimide prior to p-chloromercuribenzenesulfonic acid treatment. A concentration of 25 μM p-chloromercuribenzenesulfonic acid inhibited Na+-dependent α-aminoisobutyric acid transport by 50%. The degree of inhibition was dramatically reduced in the presence of substrates specific for the A transport system. Using an inhibition index to address the efficacy of inhibition in presence and absence of substrates, it could be shown that an index of 1.0 in presence of p-chloromercuribenzenesulfonic acid was reduced to 0.84 with (methylamino)isobutyric acid alone and 0.05 in the presence of 100 mM Na+ and 5 mM (methylamino)isobutyric acid. Na+ alone offered no protection. The results show that sulfhydryl group(s) on the amino acid carrier may be directly involved in substrate binding and that substrate binding sites are functional in the disaggregated membrane state. Furthermore, Na+ directly affects (methylamino)isobutyrate binding, since the degree of protection by the amino acid analogue against p-chloromercuribenzenesulfonic acid inhibition was influenced by the presence of Na+. Transport activity was also inhibited by treatment of solubilized membranes with dithiothreitol, suggesting that the amino acid carrier contains an essential disulfide bridge. p-Chloromercuribenzenesulfonic acid inhibited transport activity could be partly recovered by 2-mercaptoethanol or 2,3-dimercapto-1-propanol, with a maximum recovery of 65% of the original activity at 20 mM of the dithiol.Key words: reconstitution, Na+-dependent amino acid transport, p-chloromercuribenzenesulfonic acid inhibition, substrate protection.
Glucose uptake in human and animal muscle cells in culture
Authors: Sarabia Vivian , Ramlal Toolsie , Klip Amira
Published: December 2009

Human muscle cells were grown in culture from satellite cells present in muscle biopsies and fusion-competent clones were identified. Hexose uptake was studied in fused myotubes of human muscle cells in culture and compared with hexose uptake in myotubes of the rat L6 and mouse C2C12 muscle cell lines. Uptake of 2-deoxyglucose was saturable and showed an apparent Km of about 1.5 mM in myotubes of all three cell types. The Vmax of uptake was about 6000 pmol/(min∙mg protein) in human cells, 4000 pmol/(min∙mg protein) in mouse C2C12 muscle cells, and 500 pmol/(min∙mg protein) in L6 cells. Hexose uptake was inhibited ~90% by cytochalasin B in human, rat, and mouse muscle cell cultures. Insulin stimulated 2-deoxyglucose uptake in all three cultures. The hormone also stimulated transport of 3-O-methylglucose. The sensitivity to insulin was higher in human and C2C12 mouse myotubes (half-maximal stimulation observed at 3.5 × 10−9 M) than in rat L6 myotubes (half-maximal stimulation observed at 2.5 × 10−8 M). However, insulin (10−6 M) stimulated hexose uptake to a larger extent (2.37-fold) in L6 than in either human (1.58-fold) or mouse (1.39-fold) myotubes. It is concluded that human muscle cells grown in culture display carrier-mediated glucose uptake, with qualitatively similar characteristics to those of other muscle cells, and that insulin stimulates hexose uptake in human cells. These cultures will be instrumental in the study of human insulin resistance and in investigations on the mechanism of action of antidiabetic drugs.Key words: glucose uptake, insulin action, primary muscle cultures, L6 cells, C2 cells.
Reduction in VLDL, but not HDL, in plasma of rats deficient in choline
Authors: Yao Zemin , Vance Dennis E.
Published: December 2009

We have analyzed plasma lipoprotein levels in young male rats fed a choline-deficient diet for 3 days. We confirmed previous studies that choline deficiency promotes 6.5-fold accumulation of triacylgycerol in the liver (23.9 ± 6.0 versus 3.69 ± 0.92 μmol/g liver) and reduction of triacylglycerol concentration in plasma by 60% (0.17 ± 0.04 versus 0.46 ± 0.10μmol/mL plasma). Agarose gel electrophoresis showed that the plasma very low density lipoprotein (VLDL) levels were reduced in choline-deficient rats, but the concentration of plasma high density lipoproteins (HDL) was not affected. Sodium dodecyl sulfate – polyacrylamide gel electrophoresis of fractionated plasma lipoproteins revealed that the concentrations of apolipoproteins (apo) BH, BL, and E in VLDL from choline-deficient rats were 37.1, 11.0, and 37.2% of normal levels, respectively. In contrast, the amount of apo A-I, the major one in HDL, was almost unchanged. Correspondingly, there were decreased lipid (mainly phosphatidylcholine and triacylglycerol) levels in VLDL from choline-deficient rats, but no change in the levels of phosphatidylcholine, cholesterol, and cholesterol ester in HDL. There were similar levels of apo B and E (components of VLDL) in homogenates of livers from normal and choline-deficient rats, as determined by immunoblotting. These results support the hypothesis that choline deficiency causes reduction of VLDL, but not HDL, levels in plasma as a consequence of impaired hepatic VLDL secretion.Key words: choline deficiency, very low and high density lipoproteins, apolipoproteins, rat plasma.
Interaction of N-phenyl-1-naphthylamine with phospholipid monolayers: a fluorescence investigation
Authors: Teissié J.
Published: December 2009

Binding isotherms of N-phenyl-1-naphthylamine to different lipid monolayers are obtained by use of fluorescence spectroscopy for different surface pressures. In the case of dipalmitoyl phosphatidylcholine, the affinity is high in the liquid expanded state, but drops in the liquid condensed state. The affinity for the negatively charged dilauryl phosphatidylglycerol is very low. In the case of phosphatidylethanolamine extracted from Escherichia coli, a strong decrease in the affinity is detected about 20 mN/m. This can be explained by an increase of interactions between the molecules of the lipid matrix. These data lead to the conclusion that the packing is not uniform in sonicated vesicles.Key words: monolayer, phospholipid, fluorescence, phase transition.
Phospholipid asymmetry in the plasma membrane of malaria infected erythrocytes
Authors: Moll G. N. , Vial H. J. , Bevers E. M. , Ancelin M. L. , Roelofsen B. , Comfurius P. , Slotboom A. J. , Zwaal R. F. A. , Kamp J. A. F. Op den , Deenen L. L. van
Published: December 2009

The transbilayer distribution of glycerophospholipids in the plasma membrane of Plasmodium knowlesi infected erythrocytes was studied by using lysine-116-ε-N-palmitoyl amidinated pancreatic phospholipase A2. As a consequence of its superior membrane penetrating capacities, this modified enzyme rapidly degrades its substrates in the outer membrane leaflet of intact erythrocytes, a property that makes the enzyme an excellent tool to study the malaria parasitized red cell. The modified phospholipase A2 caused a nonlytic hydrolysis of up to 12–15% of the phosphatidylethanolamine and none of the phosphatidylserine in the red cell membrane, irrespective of whether the cells harboured trophozoite and schizont stages of parasites or no parasites at all. The absence of phosphatidylserine at the exterior surface of Plasmodium infected erythrocytes was confirmed by applying the prothrombinase assay on Plasmodium falciparum infected human erythrocytes. Consequently, the results from these and previous studies indicate that the plasma membrane of Plasmodium infected erythrocytes exhibit a normal transbilayer phospholipid asymmetry.Key words: malaria, plasma membrane, phospholipid asymmetry, erythrocyte, prothrombinase assay.
Calcium binding proteins. Elucidating the contributions to calcium affinity from an analysis of species variants and peptide fragments
Authors: Marsden Brian J. , Shaw Gary S. , Sykes Brian D.
Published: December 2009

This paper describes the sequence homology of calcium-binding proteins belonging to the troponin C superfamily. Specifically, this similarity has been examined for 276 twelve-residue calcium-binding loops. It has been found that, in the calcium-binding loop, several residues appear invariant, regardless of the species of origin or the affinity of the protein. These residues are Asp at position 1 (+ X of the coordinating position of the calcium), Asp or Asn at position 3 (+ Y), Gly at position 6, Ile at position 8, and Glu at position 12 (− Z). It has also been found that conservation of certain residues can vary in similar sites in similar proteins. For example, position 3 (+ Y) in site 3 of troponin C is always an Asn, whereas in calmodulin the residue is always Asp. This study also examined the calcium-binding affinities of peptide fragments comprising the loop, helix–loop, loop–helix, and helix–loop–helix. These were compared with larger enzymatic or chemically generated protein fragments in an effort to understand the various contributions to the calcium-binding affinity of a single-site versus a two-site domain as found in troponin C and calmodulin. Based on free energy differences, it was found that a 34-residue helix–loop–helix peptide represents about 60% of the binding affinity found in the intact protein. Cooperativity with a second calcium binding site accounted for the remaining 40% of the affinity.Key words: calcium-binding proteins, sequence homology, peptide fragments, species variants, calcium affinity.
Interaction of sodium molybdate with the thyroid hormone receptor
Authors: Faure Robert , Dussault Jean H.
Published: December 2009

The 3,5,3′-triiodothyronine (T3) binding activity of solubilized nuclear proteins from rat liver was decreased when molybdate (10 mM) was present in the incubation medium in the absence of thiol reagents. The equilibrium affinity constant was reduced by 40%. The rate of degradation of T3-receptor complexes at 37 °C remained unchanged, but when the extracts were further reincubated in the presence of β-mercaptoethanol, molybdate had a protective effect after 5 h incubation at 37 °C. In contrast, the thyroxine (T4) binding activity was not affected by heating at 37 °C or by molybdate. Ion-exchange chromatography confirmed the existence of a molybdate–receptor interaction: the T3–receptor complexes shifted from elution at 0.22 to 0.20 M NaCl with the progressive appearance of a small leader peak, whereas the T4-receptor complexes eluted in a large and split peak (0.22–0.4 M NaCl). The destabilizing effect on T3 binding induced by exogenous dephosphorylation is more efficiently reversed by β-mercaptoethanol when the extracts were pretreated by molybdate. In controls, the loss of saturable T3 binding activity was recovered by 50% at a 10 mM concentration of β-mercaptoethanol, but in the presence of molybdate, the loss of T3 binding activity was recovered by 50% at a 5 mM concentration of β-mercaptoethanol. This molybdate–receptor interaction is similar to that with nuclear receptor models in term of (i) stabilization of hormone binding, (ii) dependency on a thiol, and (iii) reversibility of the destabilizing effect by exogenous dephosphorylation.Key words: thyroid hormone receptor, binding activity, sodium molybdate, alkaline phosphatase.
The acylation of 1-acyl-sn-glycero-3-phosphate by neuronal nuclei and microsomal fractions of immature rabbit cerebral cortex
Authors: Baker R. Roy , Chang H.-Y.
Published: December 2009

The acylation of 1-acyl-sn-glycero-3-phosphate to form phosphatidic acid was studied using a neuronal nuclear fraction N1 and microsomal fractions P3, R (rough), S (smooth), and P (neuronal microsomes from nerve cell bodies) isolated from cerebral cortices of 15-day-old rabbits. The assays contained this lysophospholipid, ATP, CoA, MgCl2, NaF, dithiothreitol, and radioactive palmitate, oleate, or arachidonate. Of the subfractions, N1 and R had the highest specific activities (expressed per micromole phospholipid in the fraction). The rates with oleate were two to four times the values seen for phosphatidic acid formation from sn-[3H]glycero-3-phosphate and oleoyl-CoA. Using oleate or palmitate, fraction R had superior specific rates to N1 at low lysophosphatidic acid concentrations. With increasing lysophospholipid concentrations the specific rates of N1 and R came closer together and maintained at least a twofold superiority over fraction P. Fraction S had the lowest specific rates of phosphatidic acid formation. Fractions N1, R, and P showed a preference for palmitate and oleate over arachidonate, particularly at low concentrations of lysophosphatidic acid. For N1 and R, the preference was also more marked at higher concentrations of fatty acid. Thus a selectivity for saturated and monounsaturated fatty acids was shown in the formation of phosphatidic acid, as was a concentration of acylating activity in the neuronal nucleus and the rough endoplasmic reticulum.Key words: 1-acyl-sn-glycero-3-phosphate, acylation, neuronal nuclei, microsomes, cerebral cortex.
Geometric and positional fatty acid isomers interact differently with desaturation and elongation of linoleic and linolenic acids in cultured glioma cells
Authors: Cook H. W. , Emken E. A.
Published: December 2009

A range of geometrical and positional isomers of 18-carbon acyl chains are potential components of diets containing fats from processed vegetable oils or ruminant animals. We have examined seventeen 18-carbon fatty acids, with cis or trans double bonds in positions 8–15, for their effects on desaturation and chain elongation of [1-14C]18:2(n − 6) or [1-14C]18:3(n − 3), primarily to 20:4(n − 6) or 20:5(n − 3) respectively, in cultured glioma cells. All trans monoenoic positional isomers inhibited Δ5 desaturation by approximately 60% when 18:2(n − 6) was the substrate, with no positional discrimination evident; with 18:3(n − 3) as substrate, only the 11- and 12-trans isomers were effective inhibitors of formation of 20:5(n − 3). All cis positional monoene isomers, except for 12-cis 18:1, produced 25–30% inhibition of conversion of 18:2(n − 6) to 20:4(n − 6), but had little effect on the conversion of 18:3(n − 3) to 20:5(n − 3). For dienoic isomers, the presence of a 12-trans bond inhibited formation of both 20:4(n − 6) and 20:5(n − 3) and this was enhanced markedly when the other bond in the dienoic acid was 9-trans. Presence of a 9-trans or 15-trans bond with 12-cis gave little effect except for a slight inhibition of 20:4(n − 6) formation by the 12-cis,15-trans 18:2 isomer. All-cis 20:3(n − 9) blocked Δ5 desaturation, increasing 20:3(n − 6) accumulation from 18:2(n − 6) and suggesting that formation of 20:3(n − 9) from 18:1(n − 9) during essential fatty acid deficiency may further exacerbate the already compromised formation of 20:4(n − 6). Further, the differential effects of various cis and trans isomers on the metabolism of 18:2(n − 6) and 18:3(n − 3) suggest that either Δ5 desaturation is not common in both pathways of conversion of 18:2(n − 6) or 18:3(n − 3) to their primary products or that selective interactions of trans and cis isomers occur when the essential fatty acid substrates are different.Key words: trans isomers, essential fatty acids, desaturation, glioma cells, fatty acid metabolism.
Cholesterol-mediated regulation of HMG-CoA reductase in microsomes from human skin fibroblasts and rat liver
Authors: George R. , Davis P. J. , Luong L. , Poznansky M. J.
Published: December 2009

3-Hydroxy-3-methylglutaryl-CoA (HMG-CoA) reductase activity was determined in microsomes from human skin fibroblasts and rat liver that had been variously manipulated in vivo or in tissue culture to up- and down-regulate the enzyme. The cholesterol content of these microsomal preparations was then altered by depletion to or enrichment from either cholesterol-free or cholesterol-rich lipid vesicles. Microsomes from human skin fibroblasts responded to cholesterol depletion by increasing HMG-CoA reductase activity and by decreasing it in response to cholesterol enrichment. This was independent of the initial enzyme activity or the tissue culture conditions. Alterations in cholesterol content of rat liver microsomes in vitro failed to demonstrate any significant changes in HMG-CoA reductase activity whether the microsomes started with low enzyme activity (cholesterol-fed rats) or with high enzyme activity (cholestyramine-treated rats). The results are discussed in relation to previously published data and in respect to differences in the control of the human skin fibroblast and rat liver enzymes.Key words: cholesterol, HMG-CoA reductase, microsomes, fibroblasts, rat liver.
A possible mechanism for the in vitro activation of L-serine deaminase activity in Escherichia coli K12
Authors: Newman E. B. , Walker Caroline , Ziegler-Skylakakis K.
Published: December 2009

L-Serine deaminase is inactive in crude extracts of Escherichia coli K12, but can be activated by incubation with iron and dithiothreitol. This activation requires oxygen, and is inhibited by free radical scavengers and by diethylene triamine pentaacetic acid, which prevents Fe cycling. We suggest that in vitro activation of L-serine deaminase is catalyzed by an oxidant (perhaps hydroxyl radicals). Also, activation may be accompanied by a decrease in molecular weight and involve both a cleavage of the polypeptide chain and a reversible reduction of the molecule.Key words: enzyme activation, serine degradation.
Gangliosides and glycophorin inhibit T-lymphocyte activation
Authors: Sharom Frances J. , Chiu Anita L. H. , Ross T. Elaine
Published: December 2009

Increased levels of gangliosides in the serum have been linked to tumour-induced immunosuppression in vivo. Both bovine brain gangliosides and human erythrocyte glycophorin were potent inhibitors of concanavalin A, periodate, and phorbol ester – ionomycin induced activation of murine T-lymphocytes. Structurally complex gangliosides were more inhibitory, while simpler glycolipids caused less inhibition. Lymphocytes exposed to these molecules for up to 24 h could still proliferate after washing. Substantial inhibition was observed only when gangliosides and glycophorin were present during the first 18 h of activation. Studies using Quin-2 showed that gangliosides did not block the initial rapid rise in cytoplasmic Ca2+ following mitogen stimulation. Interleukin-2 (IL-2) production by ganglioside- and glycophorin-treated lymphocytes was unchanged. After treatment with gangliosides for 24 h, lymphocytes proliferated normally in response to added IL-2. These results suggest that the first round of signal transduction in response to mitogen was unaffected by gangliosides. Addition of gangliosides to activated lymphocytes in the presence of IL-2 resulted in complete inhibition of proliferation. Immunosuppression by gangliosides and glycophorin thus appears to occur at the IL-2-dependent stage of proliferation and may be partially due to IL-2 binding to these molecules. However, high levels of IL-2 failed to reverse inhibition and IL-2-dependent cell lines were much less sensitive to ganglioside inhibition than T-lymphocytes, suggesting that more than one mechanism of inhibition likely exists.Key words: gangliosides, glycophorin, T-lymphocyte, interleukin-2, interleukin-2 receptor.
Dual coenzyme activities of high-Km aldehyde dehydrogenase from rat liver mitochondria
Authors: Tsai C. Stan , Senior D. J.
Published: December 2009

Various kinetic approaches were carried out to investigate kinetic attributes for the dual coenzyme activities of mitochondrial aldehyde dehydrogenase from rat liver. The enzyme catalyses NAD+- and NADP+-dependent oxidations of ethanal by an ordered bi-bi mechanism with NAD(P)+ as the first reactant bound and NAD(P)H as the last product released. The two coenzymes presumably interact with the kinetically identical site. NAD+ forms the dynamic binary complex with the enzyme, while the enzyme-NAD(P)H complex formation is associated with conformation change(s). A stopped-flow burst of NAD(P)H formation, followed by a slower steady-state turnover, suggests that either the deacylation or the release of NAD(P)H is rate limiting. Although NADP+ is reduced by a faster burst rate, NAD+ is slightly favored as the coenzyme by virtue of its marginally faster turnover rate.Key words: aldehyde dehydrogenase, coenzyme preference.
Acidic extracellular environment induces only a subset of heat-shock proteins in primary mouse kidney cell cultures
Authors: Khandjian Edward W.
Published: December 2009

Exposure of primary mouse kidney cell cultures to acidic medium (pH 5.5) induced the expression of a 70 kilodalton (kDa) protein. This protein was identified as the major inducible heat-shock protein 70 (hsp70) by immunoprecipitation with anti-hsp70 serum and Northern blot analysis with a hsp70 cDNA probe. Maximum induction of the 70-kDa protein at pH 5.5 after 240 min was about 30% of that observed after 60 min of thermal treatment at 43 °C. In addition, there was an apparent induction of the glucose-regulated proteins (GRPs) of 76–78 and 98–100 kDa, but not of the other hsps. This subset induction of the heat-shock response by acidic medium suggests that different mechanisms are responsible for the induction of the various families of hsps.Key words: heat-shock proteins, stress proteins, acidic induction, viral infection, mouse kidney cells.
Glutaraldehyde effect on hemoglobin: evidence for an ion environment modification based on electron paramagnetic resonance and Mossbauer spectroscopies
Authors: Chevalier Alain , Guillochon Didiers , Nedjar Naima , Piot Jean Marie , Vijayalakshmi Mokambes Waran , Thomas Daniel
Published: December 2009

Glutaraldehyde is a widely used reagent for hemoglobin cross-linking in blood substitutes research. However, hemoglobin polymerization by glutaraldehyde involves modifications of its functional properties, such as oxygen affinity, redox potentials, and autoxidation kinetics. The aim of this article is to investigate, by electron paramagnetic resonance and Mossbauer spectroscopies, the changes that occur in the iron environment after glutaraldehyde cross-linking. Spectrometric studies were performed with native hemoglobin and hemoglobin cross-linked as soluble and insoluble polymers. Spectrometry data comparison with glutaraldehyde-modified hemoglobin functional properties allows to interpret from a structural point of view that glutaraldehyde action occurs as a decrease of the O—N(F8His) distance, an increase of the Fe—N(F8His) bond length, and the decrease of the distal-side steric hindrance.Key words: hemoglobin, glutaraldehyde, Mossbauer spectroscopy, electron paramagnetic resonance, blood substitute.
Purification and properties of different isoforms of bovine cathepsin B
Authors: Deval Christiane , Bechet Daniel , Obled Alain , Ferrara Marc
Published: December 2009

A rapid purification procedure is described for cathepsin B from bovine liver. After preparation of crude lysosomal extracts, the method only involves DEAE Zeta-Prep-Disk chromatography, gel filtration, and fast protein liquid chromatography on Mono-S column. Two active peaks (P1 and P2) of cathepsin B were distinguished. Both presented uncleaved (relative mass (Mr) 30 000) and cleaved (Mr 25 000 + Mr 5000) chains, but different isoforms as revealed by isoelectrofocusing. These two different populations of cathepsin B isoforms nevertheless exhibited similar enzymatic properties. Km and kcat were 114 μM and 52 s−1, and 125 μM and 75 s−1, for hydrolysis of Z-Arg-Arg-NMec by P1 and P2, respectively. Both were rapidly inhibited by low concentrations of E-64 or leupeptin, but were unaffected by cathepsin-L-specific inhibitor Z-Phe-Phe-CHN2.Key words: protein/enzyme purification, cathepsin B, isoforms, lysosomes.
Influence of carbohydrates on the antigenic structure of gonadotropins: distinction of agonists and antagonists
Authors: Sairam M. R. , Linggen J. , Sairam J. , Bhargavi G. N.
Published: December 2009

The biological properties of glycosylated (native) and deglycosylated gonadotropins are different. The immunological characteristics of antibodies prepared against deglycosylated lutropin and human chorionic gonadotropin were investigated. Distinct antibodies of rabbit polyclonal antisera against deglycosylated lutropin and deglycosylated chorionic gonadotropin were separated by affinity chromatography on divinylsulfonyl-Sepharose-immobilized hormone or antagonist columns, respectively, in successive runs. Antibodies that could discriminate between agonist and antagonistic forms of the hormones could thus be obtained. In radioimmunoassays using 125I-labeled antagonists and respective antagonist antibodies, only the deglycosylated hormones or their deglycosylated α-subunits showed preferential reaction. Based on recombinations using different deglycosylated subunits, it was concluded that the loss of antennary sugars in the α-subunits was mainly responsible for the changes that led to the formation of antagonist-specific antibodies. Only the agonist-specific antibody could neutralize hormone action. Thus, the type and extent of glycosylation appears to influence the antigenic structure of these secreted glycoproteins.Key words: gonadotropins, glycoproteins, antigenic structure, affinity chromatography, agonist, antagonist.
A model to explain the osmotic pressure behavior of hemoglobin and serum albumin
Authors: Cameron Ivan L. , Fullerton Gary D.
Published: December 2009

Previously published osmotic pressure data on hemoglobin and bovine serum albumin were used to determine the osmotically unresponsive solvent volume per unit dry mass of protein. A model is presented that accounts for the osmotic pressure of globular proteins based on a surface-associated osmotically unresponsive solvent volume. The model also accounts for changes in the osmotically unresponsive solvent volume owing to changes in pH, cosolute salt concentration, protein conformation, and protein aggregation.Key words: hemoglobin, serum albumin, osmotic pressure, water of hydration, salt, pH, model of protein structure.
Differential release of proteins from bovine fat globule membrane
Authors: Malin Edyth L. , Basch Jay J.
Published: December 2009

Alkaline phosphatase and 5′-nucleotidase are covalently linked to phosphatidylinositol in bovine fat globule membrane, as demonstrated by their release following treatment with phospholipase C specific for phosphatidylinositol. The failure of this treatment to liberate phosphodiesterase I may indicate that it has a variant linkage resistant to release. In a test of exposure at the membrane surface, alkaline phosphatase and phosphodiesterase I, but not 5′-nucleotidase, were released from fat globule membrane by treatment with proteinase K. These apparent differences in accessibilities of membrane surface proteins suggest that attachment to phosphatidylinositol does not necessarily impart greater exposure to proteins with which it is linked.Key words: alkaline phosphatase, 5′-nucleotidase, phosphodiesterase I, phosphatidylinositol, phospholipase C, fat globule membrane.
Biochemical aspects of free radicals
Authors: Basaga H. S.
Published: December 2009

Toxic free radicals can be produced by many reactions required for the maintenance of normal metabolism and the production of energy in the cell. The reactivity of both primary and secondary radicals with biomolecules and in whole tissue systems is of interest, not only because of their importance in radiobiology but also because of the role these species play in toxicity and various disorders. Oxidant stress is known to increase the production of free radicals. In the presence of metals, especially iron, these radicals are converted into more damaging species. Trace elements play an important role in many systems that have evolved to deal with free radicals. The dietary status of the cell can affect the preventative antioxidant constituents of the cell. The chain-breaking antioxidant status can clearly be influenced by the dietary content of substances such as vitamins E and C.Key words: free radicals, oxidant stress, metal ions, antioxidants.
Characterization of an insect ferritin subunit synthesized in a cell-free system
Authors: Ketola-Pirie C. A.
Published: December 2009

Ferritin, an iron-sequestering and -binding protein, is localized to the vacuolar system in Calpodes ethlius larvae. The amount of iron-loaded ferritin in intact larval midgut can be increased by pretreatment with iron. When poly(A)+ RNA from control or iron-treated larvae was translated in vitro, a 24 kilodalton (kDa) protein was a major translation product. If the cell-free system was supplemented with dog pancreatic microsomes, the 24-kDa protein was not detectable: the major translation product was 28–30 kDa. The 24-kDa and 28- to 30-kDa proteins were identified as ferritin subunits by immunoprecipitation with anti-Manduca ferritin antibodies. Proteinase K digestion of the translation products showed that the 28- to 30-kDa subunit was targeted into the lumen of, and protected by, the microsomes. The change in molecular mass of the ferritin monomer was attributed to glycosylation of the 24-kDa subunit within the lumen of the microsomes. This was demonstrated by (i) the ability of the 28- to 30-kDa subunit, but not the 24-kDa subunit, to bind concanavalin A on Western blots and (ii) inhibition of the change in molecular mass from 24 to 28–30 kDa if tunicamycin is added to the microsomes. The results indicate that the Calpodes ferritin subunit was synthesized, targeted to microsomes, and glycosylated within their lumen in a rabbit reticulocyte cell-free system primed with midgut poly(A)+ RNA extracted from control or iron-treated larvae.Key words: insect ferritin, cell-free synthesis, glycosylation.
Adrenocorticotropin- and β-endorphin-like substances in brains of the freshwater snake Ptyas mucosa
Authors: Ng T. B. , Ng Alfred S. L. , Wong C. C.
Published: December 2009

Snake (Ptyas mucosa) brains (400 g) were extracted with a mixture of acetone, water, and hydrochloric acid. The precipitate (5.6 g) that formed upon addition of five volumes of acetone to the extract, designated acid–acetone powder, was subjected to gel filtration on Sephadex G-25. A large unretarded peak (SB-1) with molecular weight greater than 5000 and a small retarded peak (SB-2) with molecular weight smaller than 5000 were obtained. They were then separately subjected to ion-exchange chromatography on CM-cellulose. Adrenocorticotropic activity was detected in the fractions by their ability to stimulate isolated rat adrenal cells to produce corticosterone. Opiate activity was detected in the fractions by their ability to inhibit the binding of (D-Ala2,D-Leu5)-[tyrosyl-3,5-3H]enkephalin to rat brain membranes and their cross-reactivity in a β-endorphin radioimmunoassay. Adrenocorticotropic and opiate activities were found to be concentrated in fractions strongly adsorbed on CM-cellulose, which were eluted by combined pH and ammonium acetate concentration gradients. There appeared to be a separation between adrenocorticotropic and opiate activities, suggesting that they were due to separate molecular entities.Key words: adrenocorticotropin, β-endorphin, snake brains.
Molecular characterization of three mutations in katG affecting the activity of hydroperoxidase I of Escherichia coli
Authors: Loewen Peter C. , Switala Jacek , Smolenski Mark , Triggs-Raine Barbara L.
Published: December 2009

Hydroperoxidase I (HPI) of Escherichia coli is a bifunctional enzyme exhibiting both catalase and peroxidase activities. Mutants lacking appreciable HPI have been generated using nitrosoguanidine and the gene encoding HPI, katG, has been cloned from three of these mutants using either classical probing methods or polymerase chain reaction amplification. The mutant genes were sequenced and the changes from wild-type sequence identified. Two mutants contained G to A changes in the coding strand, resulting in glycine to aspartate changes at residues 119 (katG15) and 314 (katG16) in the deduced amino acid sequence of the protein. A third mutant contained a C to T change resulting in a leucine to phenylalanine change at residue 139 (katG14). The Phe139-, Asp119-, and Asp314-containing mutants exhibited 13, < 1, and 18%, respectively, of the wild-type catalase specific activity and 43, 4, and 45% of the wild-type peroxidase specific activity. All mutant enzymes bound less protoheme IX than the wild-type enzyme. The sensitivities of the mutant enzymes to the inhibitors hydroxylamine, azide, and cyanide and the activators imidazole and Tris were similar to those of the wild-type enzyme. The mutant enzymes were more sensitive to high temperature and to β-mercaptoethanol than the wild-type enzyme. The pH profiles of the mutant catalases were unchanged from the wild-type enzyme.Key words: catalase, hydroperoxidase I, mutants, sequence analysis.
Kinetic characterization of dihydrofolate reductase from Drosophila melanogaster
Authors: Rancourt Susan L. , Walker Virginia K.
Published: December 2009

The kinetic characteristics of a purified insect dihydrofolate reductase (DHFR) have been described. The Km values for the substrate dihydrofolate and the cofactor NADPH have been estimated by primary and secondary Hanes plots to be 0.3 and 5.2 μM, respectively. Drosophila melanogaster DHFR can use folate and NADH at acidic pH values, but at a much lower rate than the preferred substrate and cofactor. Folic acid is a partial competitive inhibitor of Drosophila DHFR (Ki = 0.4 μM) and trimethoprim is a complete competitive inhibitor (Ki = 5.4 μM). Methotrexate binds less tightly to the Drosophila enzyme than to many other DHFRs (Kd = 0.9 nM). Drosophila DHFR is inhibited by KCl and organic mercurials and is slightly activated by urea. These data indicate that Drosophila DHFR has some characteristics which are typical of vertebrate DHFRs and others which are typical of prokaryotic DHFRs. The study of this enzyme, therefore, should aid in the definition of the structural features that are responsible for the kinetic characteristics in different DHFRs.Key words: dihydrofolate reductase, Drosophila melanogaster, methotrexate.
G–protein linked polyphosphoinositide phospholipase C activity in cell membranes from clonally derived and ras-transformed Madin–Darby canine kidney cells
Authors: Waite Jerene J. , Insel Paul A.
Published: December 2009

Membranes prepared from clone D1 of Madin–Darby canine kidney (MDCK) cells contain activity that can be attributed to Gp, a guanine nucleotide binding protein linked to phosphatidylinositol 4,5-bisphosphate dependent phospholipase C. Polyphosphoinositides are produced by addition of GTP, nonhydrolyzable GTP analogs, or fluoroaluminate. This production is inhibited by guanosine 5′-(β-thiodiphosphate). While Ca2+ at 1 μM or more can generate high yields of inositol phosphates, guanine nucleotide activation of Gp can potentiate this Ca2+-dependent yield at resting levels of the cation. Membranes from cells expressing large amounts of ras-p21 exhibit small differences in guanine nucleotide induced polyphosphoinositide quantities. The greatest difference between normal and ras membranes was seen with AlF4− incubation. Of the three inositol phosphates measured, only the inositol bisphosphate yield was greatly increased in ras membranes compared with membranes from both parental and the D-1 clone of MDCK cells. From these data, we conclude that the presence of ras-p21 may affect production of polyphosphoinositides in MDCK cell membranes by some means other than direct participation in phospholipase C activation.Key words: guanine nucleotide binding protein, phospholipase C, inositol phosphate, ras, Madin–Darby canine kidney cells.
Cellular functions of ascorbic acid
Authors: Padh Harish
Published: December 2009

It has long been suspected that ascorbic acid is involved in many cellular reactions. This is evident from the multitude of seemingly unrelated symptoms seen in scurvy. However, until recently, our understanding of its involvement was confined to its role in the synthesis of collagen. Studies in the past few years have unveiled mechanisms of its actions in collagen formation and many other enzymatic reactions. In addition, numerous physiological responses are reportedly affected by ascorbic acid. From the well-characterized enzymatic reactions involving ascorbic acid, it has become clear that in animal cells the ascorbate does not seem to be directly involved in catalytic cycles. Rather its major function seems to keep prosthetic metal ions in their reduced form. The role of ascorbate as a reductant in these enzymatic reactions complements its other antioxidant functions which have been recently appreciated, including that as a scavenger of free radicals. Therefore, it seems that the major function of ascorbate is to protect tissues from harmful oxidative products and to keep certain enzymes in their required reduced forms. However, it remains unclear how the deficiency of ascorbate leads to the pathological symptoms found in scurvy.Key words: ascorbic acid, vitamin C, biochemical functions, antioxidant, recommended dietary allowances, hydroxylation.