bluuebunny/crossref_metadata_2025 huggingface dataset:
Purine metabolism in human T lymphocytes: role of the purine nucleoside cycle
Authors: Cohen Amos , Barankiewicz Jerzy , Lederman Howard M. , Gelfand Erwin W.
Published: December 2009

Human intrathymic T lymphocytes were separated by a bovine serum albumin density gradient into a population of G1-phase small thymocytes and a population of S-phase-enriched large thymocytes. Purine metabolism was studied in these thymocyte populations, representing immature T lymphocytes, and compared with the metabolism of mature T lymphocytes isolated from the peripheral blood. De novo purine biosynthesis was highly cell cycle dependent; i.e., de novo purine biosynthetic activity was found only in large S-phase thymocytes, whereas both G1 T-cell populations lacked any significant activity. Thus G1-phase small thymocytes and G1-phase peripheral blood T lymphocytes have only salvage pathways to maintain their purine nucleotide pools. Despite the similarity of purine salvage activities in G1 thymocytes and in peripheral blood T lymphocytes, small thymocytes have fourfold lower levels of purine nucleoside triphosphates. The decreased levels of purine nucleotides in G1 thymocytes may be the result of increased purine efflux. It was found that an unusually large proportion (24–48%) of hypoxanthine incorporated by G1 thymocytes is excreted into the medium in the form of inosine.
Mechanisms of stimulus-calorigenesis coupling in brown adipose tissue
Authors: Bukowiecki Ludwik J.
Published: December 2009

The sequence of metabolic events leading to increased calorigenesis in brown adipose tissue has been reviewed. The first step of this sequence consists in the binding of norepinephrine to adrenergic receptors of the beta1 subtype. This results in the stimulation of adenylate cyclase and activation of lipolysis via the system of protein kinases. Hormone-sensitive lipases represent the "flux-generating" step regulating mitochondrial respiration. Fatty acids released from intracellular triglyceride droplets in consequence of lipase activation play a messenger role between lipolysis and mitochondrial respiration. They stimulate respiration by serving as substrates for beta oxidation (via carnitine-dependent pathways) and (or) by simultaneously increasing mitochondrial permeability to protons (physiological "loose coupling"). The control of brown adipose tissue respiration by lipolysis represents a self-regulatory process, as excessive concentrations of fatty acids retroinhibit lipolysis. At the mitochondrial level, fatty acids appear to interact with an "uncoupling" protein (thermogenin or 32 000 relative mass protein) localized in the inner membrane that confers upon brown adipose mitochondria a unique sensitivity for fatty acid uncoupling. This explains that, contrary to other tissues, respiration is principally controlled in brown adipose tissue by substrate supply (mainly long-chain fatty acids), rather than by the phosphorylation state ratio.
Purification and characterization of a chymosinlike protease from the gastric mucosa of harp seal (Pagophilus groenlandicus)
Authors: Shamsuzzaman K. , Haard N. F.
Published: December 2009

Four zymogens of acidic proteases A, B, C, and D were isolated from the gastric mucosa of harp seals by ion-exchange chromatography on a diethylaminoethyl-Sephadex A-50 column. The major zymogens were A and C, and the ratio of zymogen A to zymogen C was greater in extracts from 1-week-old animals than in extracts from adult animals. Zymogens A and C were further purified by affinity chromatography using carbobenzoxy-D-phenylalaninetriethylene tetramine Sepharose and gel filtration on a Sephadex G-100 column. Certain physical and catalytic properties of proteases A and C were compared with those of calf chymosin (EC 3.4.23.4) and porcine pepsin (EC 3.4.23.1). Zymogen C and the corresponding enzyme were homogeneous on analytical polyacrylamide gel electrophoresis. Zymogen A was homogeneous as judged by sodium dodecyl sulphate (SDS) – polyacrylamide gel electrophoresis and high performance liquid chromatography, but was heterogenous by polyacrylamide gel electrophoresis at pH 8.3. Zymogens A and C had molecular weights of 33 800 and 44 000, respectively, as estimated by SDS–polyacrylamide gel electrophoresis. Protease A had an isoelectric point of 4.90. Protease A was similar to calf chymosin with respect to several criteria. It had a higher ratio of milk-clotting to proteolytic activity than those of seal protease C and porcine pepsin and had a pH optimum of 2.2–3.5 for hemoglobin hydrolysis. It did not inactivate ribonuclease, had very low activity on N-acetyl-L-phenylalanyl-3,5-diiodo-L-tyrosine and lost activity in 6 M urea. These results indicate protease A is chymosinlike.
The thermodynamic properties of mixed phospholipid bilayers: a theoretical analysis
Authors: Mondat Maryse , Georgallas A. , Pink D. A. , Zuckermann M. J.
Published: December 2009

A theoretical model is presented with the intention of describing lateral phase separations in binary lipid mixtures in which the acyl chains of the components differ in their length. The model includes explicitly interactions between the acyl chains and between polar heads of the lipid molecules. Phase diagrams and thermodynamic properties of binary lipid mixtures were calculated using a wide range of interaction parameters. It is shown that the occurrence of immiscibility in the gel phase is related to the interactions between the polar heads of the lipid molecules. The calculated results for binary lipid mixtures are compared with the available experimental data. In particular, the calculated specific heat for dilauroyl phosphatidylcholine – distearoyl phosphatidylcholine is in reasonable agreement with experimental results obtained from differential scanning calorimetry measurements.
Effect of taxol on the interaction of tubulin with myosin filaments
Authors: Fujii Toshihiro , Suzuki Tatsuo , Hachimori Akira , Fujii Michiyo , Kondo Yoshiyuki , Ohki Kosuke
Published: December 2009

The interaction between polymerized tubulin from porcine brain and myosin from rabbit skeletal muscle was examined. The addition of myosin to the solution of tubulin polymerized by taxol resulted in a remarkable increase in turbidity within a few minutes at 37 °C, and a dense and stable precipitate was formed. The maximal molar ratio of tubulin bound to myosin was calculated to be about 4, while the value was about 2 when 6S tubulin was used. Both podophyllotoxin and colchicine suppressed the taxol-dependent increase of the binding of tubulin to myosin, but only when they were preincubated with tubulin prior to addition of taxol. 6S tubulin inhibited with aetin-activated Mg2+-ATPase activity of myosin, and polymerized tubulin inhibited the Mg-ATPase more than 6S tubulin. Dense precipitates of tubulin and myosin were observed by thin-section electron microscopy. Microtubules were observed to be entangled in myosin filaments and single microtubules were occasionally surrounded by five myosin filaments in a cross section, similar to actin–myosin arrays in muscle. After incubation of tubulin with myosin, taxol was able to induce tubulin polymerization in the same way as it polymerized microtubules in the absence of myosin.
The purification and properties of (1→4)-β-D-glucan cellobiohydrolase from Sclerotium rolfsii: substrate specificity and mode of action
Authors: Patil Rajkumar V. , Sadana Jai C.
Published: December 2009

A cellulolytic enzyme which shows high activity towards H3PO4 – swollen cellulose, but no viscosity-lowering activity towards carboxymethylcellulose, has been purified from the culture filtrates of Sclerotium rolfsii. The purified enzyme is homogeneous in disc gel electrophoresis, with and without sodium dodecyl sulfate, and in analytical isoelectric focusing in polyacrylamide gel. The enzyme is a glycoprotein containing 7.0% total carbohydrate and has a relative mass of 41 500 and an isoelectric point of 4.32. The enzyme is composed of a single polypeptide chain and has no cystine or half-cystine residues. It is specific for β-D-(1→4)-linkage. The principal product from each of the substrate was cellobiose (93–96%); glucose was also detected (4–7%). The rate of cellodextrin hydrolysis increased as the degree of polymerization increased. The enzyme has been identified as (1→4)-β-D-glucan cellobiohydrolase. The enzyme does not show transglycosylase or transferase activity.
A detailed investigation of the cytoplasmic cortisol-binding receptor of North American eel (Anguilla rostrata) tissues
Authors: DiBattista John A. , Mehdi A. Z. , Sandor Thomas
Published: December 2009

The in vitro binding of tritiated cortisol to ammonium sulfate precipitate (35% saturation) prepared from the gill and gut mucosal cytosol of the North American eel (Anguilla rostrata) was investigated. The sodium molybdate stabilized cytoplasmic preparations bound tritiated cortisol with the following parameters: gill, equilibrium dissociation constant (KD) = 3.7 ± 0.4 nM, (± SEM; n = 4), the maximum concentration of binding sites (Nmax) = 294 ± 26 fmol/mg protein; gut, KD = 5.2 ± 0.4 nM, Nmax = 1085 ± 288 fmol/mg protein. The [3H]cortisol–receptor complexes sedimented on linear (16–41% w/v) glycerol density gradients in single peaks at 6.7S–7.0S or 3.0S–3.6S in hypo- or hyper-tonic (± 0.4 M KCl) gradients, respectively. Sephacryl S-300 column chromatography of the hormone–receptor complex yielded the following hydrodynamic parameters: gill, relative mass (Mr) = 292 000 daltons, Stokes radius (Rs) = 78.7 Å(1 Å = 0.1 nm), frictional ratio (f/f0) = 1.79; gut, Mr = 242 000 daltons, Rs = 68.8 Å, f/f0 = 1.66. Competition studies revealed the following competitive hierarchies of radioinert steroids vis-à-vis the inhibition of [3H]cortisol binding to the receptor with both tissues: cortisol > 11-deoxycortisol > 21-deoxycortisol > 17α-hydroxyprogesterone [Formula: see text] corticosterone [Formula: see text] 11-deoxycorticosterone > 11β-hydroxyprogesterone. Aldosterone, cortisone, progesterone, or promegestone (R5020) hardly competed. These findings underline the importance of the C-17, C-21, and C-11 hydroxyl groups in receptor binding. Hydroxylation of progesterone in positions C-17, C-21, and C-11 contributed free energy changes (ΔG) of −5.8 to −6.2, −3.1 to −3.9, and −1.3 kJ/mol, respectively, to the binding of steroids to the eel glucocorticoid receptor. From these data we conclude that the piscine cortisol receptor is different from other vertebrate glucocorticoid receptors because of its physical and thermodynamic characteristics and its function in mediating electrolyte homeostatic action of a typical glucocorticoid in the transport epithelia. It is conceivable that the fish glucocorticoid receptor is an ancestral form of the glucocorticoid and (or) mineralocorticoid receptors of vertebrates.
Characteristics of skeletal muscle ecto-ATPase in situ
Authors: Manery J. F. , Dryden E. E. , Still J. S. , Madapallimattam G.
Published: December 2009

The properties of a cell surface nucleoside 5′-triphosphatase have been studied in small, intact, frog skeletal muscles, as a means of distinguishing the enzyme from other adenosine 5′-triphosphatases and of understanding its behaviour in the muscle membrane. The ectoenzyme in situ was shown to be a Ca2+- or Mg2+-activated ATPase liberating 7.5 ± 0.4 (mean ± SEM, n = 30) μmol of inorganic phosphate/g of muscle per 20 min, when the muscle was exposed to 2 mM ATP and 2 mM Ca2+ in Ringer's solution. The apparent Km for Mg2+ was 0.74 mM and for Ca2+ was 0.23 mM. A residual ATPase activity (20%) was found in the complete absence of divalent cations suggesting the existence of two ATPase types. A broad specificity toward nucleoside 5′-triphosphates was exhibited by the ecto-ATPase, but there was no nonspecific phosphatase activity. The enzyme was inhibited by La3+ and Cd2+, but was insensitive to ouabain, 2,4-dinitrophenol, oligomycin, and ruthenium red. Thus the ectoenzyme was not a Na+,K+-transport ATPase, was not an ATPase of mitochondrial origin, or a Ca2+-transport enzyme. Insulin had no effect. Inhibition by mersalyl, carbodiimide, and polar and cross-linking nonpolar nitrobenzene derivatives suggested that, for maximum activity, this membrane-bound enzyme required free sulfhydryl groups, certain free carboxyls, and an appreciable degree of hydrophobicity in its microenvironment.
The heme moiety in peanut peroxidase
Authors: Chibbar R. N. , Cella R. , Huystee R. B. Van
Published: December 2009

Heme is present in an equimolar ratio to the apoprotein in the major cationic fraction of peanut peroxidase. The removal of heme from the holoenzyme does not affect the physicochemical and immunological properties of the apoperoxidase, however peroxidase activity is completely lost. The indoleacetic acid (IAA) oxidase activity of the apoperoxidase is reduced to 1/20 of the original holoenzyme. Both the peroxidase and IAA-oxidase activity could partially be restored in the holoenzyme reconstituted with hemin. It is suggested that heme may also participate in the IAA-oxidase activity possibly by altering the active site.
Free-flow electrophoresis of the low-density structures that contain asialoglycoproteins in the rat liver
Authors: Debanne Maria T. , Bolyos Maria , Regoeczi Erwin
Published: December 2009

Rats were given an intravenous dose (1–2 μg/100 g) of iodine-labelled asialotransferrin, asialofetuin, or asialoorosomucoid either alone or in combinations, and the distribution of the radioactivity in the liver, removed 10–20 min after the injection, was analyzed by free-flow electrophoresis in an Elphor VaP 11 apparatus. Liver homogenates were prepared for electrophoresis according to an elaborate ultracentrifugation scheme that is outlined in detail with respect to conditions and yields. The scheme involved differential centrifugation, followed by density gradient centrifugation in a linear sucrose gradient and gel filtration using Sepharose 2B. Two ligand-containing fractions were obtained during differential centrifugation, each associated with a different complement of subcellular marker enzymes. On free-flow electrophoresis, the ligand present in either fraction exhibited a major and a minor peak. They were incompletely separated, the minor peak shouldering on the major one. The major peak had a higher electrophoretic mobility than the peaks of the acid phosphatase and phosphodiesterase I activities, but it had the same mobility as the sialyltransferase activity. The minor, less electronegative peak comigrated with the peaks of acid phosphatase and phosphodiesterase I activities and also with the major protein component of the subcellular fraction. It is concluded that the asialoglycoprotein-transporting subcellular vesicles are heterogeneous in regard to charge and that their complete separation from subcellular marker enzymes cannot be accomplished by free-flow electrophoresis.
Structural study on liver gelactin
Authors: Gruda Julian , Thérien Hélène-Marie
Published: December 2009

Electron microscopy, ultracentrifugation, gel filtration, and isoelectric focusing were carried out with gelactin, an actin-gelling protein from rabbit liver. Gelactin is a dimeric acidic protein (isoelectric point (pI) = 5.45), with a molecular weight of 190 000, a Svedberg constant of 6.25, and a Stoke's radius and length of 7.0 and 28 nm, respectively. While different from α-actinin by pI and amino acid composition, gelactin belongs by its dimensions to the class of α-actinins.
Pulmonary surfactant
Authors: Possmayer Fred , Yu Shou-Hwa , Weber J. Marnie , Harding Paul G. R.
Published: December 2009

The mammalian lung is stabilized by a specialized material, the pulmonary surfactant, which acts by reversibly reducing the surface tension at the air–liquid interface of the lung during breathing. Pulmonary surfactant contains approximately 90% lipid and 10% proteins. Dipalmitoyl phosphatidylcholine, the major lipid component, appears to be primarily responsible for the ability to reduce surface tension to near 0 dyn/cm (1 dyn = 10 μN). The other components of pulmonary surfactant promote the adsorption and spreading of this disaturated lecithin at the air–liquid interface. Surfactant activity can be accessed by physical and biological assays. Apparent discrepancies between the results obtained with the Wilhelmy plate surface balance and the pulsating bubble surfactometer have led to the suggestion that separate "protein-facilitated" (catalytic type) and "protein-mediated" (chemical type) processes may be involved in adsorption and (or) spreading at the different surfactant concentrations used with these two techniques. Artificial surfactants, which mimic the essential properties of the natural product with the pulsating bubble surfactometer, can be produced with synthetic lipids. Treatment of prematurely delivered infants suffering from the neonatal respiratory distress syndrome with lipid extracts of pulmonary surfactant leads to a marked improvement in gaseous exchange.
Diffusion and aggregation in biological membranes
Authors: Petersen Nils O.
Published: December 2009

Membrane lipids and proteins diffuse laterally in the membrane plane unless constrained by interactions with immobile or slowly moving components. Direct diffusion measurements help characterize the interactions among surface components and can provide information about the mechanism of physiologically interesting processes in membranes. Association of membrane proteins into aggregates may result from changes in intermolecular interactions and will affect the rate of diffusion of the protein. Quantitative measurements of aggregation are therefore becoming important both to aid interpretation of constraints of diffusion and to explore the role of aggregation in physiological events. In this paper, the popular fluorescence photobleaching measurements of diffusion are discussed and their relevance is illustrated with selected examples. Moreover, a variation on the theme of fluorescence correlation spectroscopy is presented as a new approach to measuring the state of aggregation of membrane components on surfaces of living cells
Chemistry, structure, and function of insulin-like growth factors and their receptors: a review
Authors: Perdue James F.
Published: December 2009

Human serum contains two classes of somatomedins (Sin) with similar three-dimensional structures and mass (i.e., ~7500 kdaltons (kd)), but that can be distinguished from each other by their isoelectric point (pI) values. Those with alkaline pI's include the presumably identical molecules of insulin-like growth factor I (IGF-I), SmA, SmC, and basic Sm. In humans, the neutral–acidic class of Sm is represented by IGF-II. The sera of rats and other animals contain similar classes of Sm. In normal children and adults, plasma levels of both IGF-I and -II are under growth hormone control, although only elevated concentrations of the former can be positively correlated with adolescent skeletal growth. Elevated blood levels of IGF-II or an embryonic form of it, however, are correlated with fetal development of rats, sheep, and possibly in humans. As evidenced from competitive radioreceptor binding studies and affinity-labelling techniques, receptors that bind insulin and IGF-I are immunologically, structurally, and functionally related: (a) monoclonal and polyclonal antibodies to insulin receptors recognize determinants on receptors that bind IGF-I; (b) both receptors have 140-, 95-, and possibly 45-kd subunits that are interchain disulfide-bonded to form glycoprotein complexes of relative masses (Mr) 300 000 – 400 000; and (c) occupancy of the 140-kd subunits by hormone or growth factor stimulates the phosphorylation of tyrosine residues on the 95-kd subunit by receptor-associated protein kinases. Receptors that bind IGF-II are very different from insulin or IGF-I receptors: (a) there is no evidence of immunological cross-reactivity to the insulin receptors or of intrinsic protein kinase activity and (b) estimates of the molecular weight of the detergent-solubilized IGF-II receptor from its hydrodynamic properties or from its electrophoretic mobility during quantitative or sodium dodecyl sulfate–polyacrylamide gel electrophoresis established it to be a single-chained glycoprotein of MT 220 000 held together by intrachain disulfide bonds.
The fate of the transferrin receptor during maturation of sheep reticulocytes in vitro
Authors: Johnstone R. M. , Adam M. , Pan B. T.
Published: December 2009

The transferrin receptor of sheep reticulocytes is excised from the cell during in vitro maturation to erythrocytes. The excised receptor may be recovered from the medium by centrifugation at 100 000 × g. Loss of transferrin-binding activity parallels the loss of binding of anti-receptor antibody, as well as RNA content. The released receptor retains the molecular size of the receptor isolated from the plasma membranes (93 000 monomer, 186 000 dimer), has an identical iodotyrosyl peptide map, and is still capable of binding transferrin, as well as an antibody directed against the receptor. The receptor is released in a vesicular form. The major peptides of the vesicles are the receptor and an unidentified peptide of 70 000 whose iodotyrosyl peptide map is distinct from that of the receptor. Although the transferrin receptor has been shown to undergo posttranslational modification (phosphorylation, acylation, and glycosylation) in cultured cells, it has not been established whether any of the transformations are retained in nongrowing cells. In the present communication, it is shown that isolated reticulocyte plasma membranes are capable of receptor phosphorylation, a process previously shown only with intact, cultured cells. The phosphorylating activity is retained in immunoprecipitates of the receptor, but is absent in the vesicles released during maturation. No evidence has been obtained for an effect of either transferrin or an anti-receptor antibody on receptor phosphorylation in intact cells or isolated membranes.
The assay and partial characterization of 3β-hydroxysteroid sulfotransferase of the hamster epididymis
Authors: Bouthillier M. , Bleau G. , Chapdelaine A. , Roberts K. D.
Published: December 2009

Using a partially purified enzyme preparation obtained from hamster epididymis, a simple assay has been developed to measure the sulfurylation of dehydroisoandrosterone (DHA) and desmosterol in the presence of 3′-phosphoadenosine 5′-phospho[35S]sulfate ([35S]PAPS). After stopping the enzymatic reaction with methanol and KCl, the 35S-labelled steroid sulfates are readily extracted into an organic phase. Optimal conditions for the sulfurylation of the two steroids were compared; optimum pH is 8.7 for DHA and 9.8 for desmosterol. Sulfoconjugation of desmosterol increases with magnesium concentrations up to 6 mM, while 40 mM concentrations of the divalent ion are required for the optimal sulfurylation of DHA. Maximum sulfurylation of these steroids requires the presence of 15 mM cysteine. Michaelis–Menten kinetics are observed with DHA which has an apparent Km of 32 μM, while desmosterol inhibits sulfotransferase activity at high concentrations. Saturation of the enzyme with PAPS results in an allosteric behaviour. Only the 3β-hydroxyl function of the steroid nucleus appears to be an appropriate sulfate acceptor for the epididymal hydroxysteroid sulfotransferase.
Changes in lipid composition of oat root membranes as a function of water-deficit stress
Authors: Liljenberg C. , Kates M.
Published: December 2009

The effect of repeated water-deficit stress on the lipid composition of root cells from 5-day-old oat (Avena sativa L. cv. Seger) seedlings was studied. The content of total acyl lipids was found to decrease with increasing degree of water-deficit stress, owing largely to decreases in free fatty acids, triglycerides, phosphatidylethanolamine (PE), wax esters, steryl esters, and acylated steryl glycosides. Major polar lipids both in total root cells and in the plasma membrane enriched fraction, as well as the microsomal membrane fraction, were PE, phosphatidylcholine (PC), digalactosyldiacylglycerol (DGDG), monogalactosyldiacylglycerol (MGDG), and polyglycolipid. Decreases in the degree of unsaturation of the fatty acids as a funtion of increased water-deficit stress were observed for the MGDG and polyglycolipid components of total root cells and for the MGDG, DGDG, and polyglycolipid of the plasma membrane fraction. Electron microscopy showed that stressed root tip cells had much smoother plasma membranes than those of control unstressed root cells. These results suggest that root cells of oat seedlings respond to water-deficit stress by reducing the total plasma membrane mass and degree of lipid fluidity, which would reduce the water permeability of the plasma membranes and help maintain cell turgidity.
Immunological studies of the uncoupling protein of brown adipose tissue
Authors: Afong Michael , Olynyk Kimberley A. , Patel Hasmukh V. , Arnold John , Liao Shuen-Kuei , Freeman Karl B.
Published: December 2009

The immunological relationship of the uncoupling protein from brown adipose tissue of several mammalian species was examined by using a rabbit antibody preparation against the rat protein. Complete cross-reactivity of the antibody to the protein from hamster, mouse, and rat was found, whereas the protein from rabbit cross-reacted only 25%. Cross-reactivity was also found with the human uncoupling protein, although the human protein was found to be about 1 kdalton smaller than the rat protein. No protein of the size of the uncoupling protein was detected in several tumor cell lines examined.
The binding of juvenile hormone to membranes of follicle cells in the insect Rhodnius prolixus
Authors: Ilenchuk T. T. , Davey K. G.
Published: December 2009

3H-labelled insect juvenile hormone, which has previously been shown to stimulate the Na,K-ATPase of membrane preparations of the follicle cells of the ovary of Rhodnius prolixus, exhibits saturable binding to microsomal preparations from the follicle cells of vitellogenic and previtellogenic follicles. No saturable binding is detectable in preparations from brain. Scatchard analysis shows that there is a greater density of binding sites on the membranes from vitellogenic, as opposed to previtellogenic, follicles. Sodium dodecyl sulfate – polyacrylamide gel electrophoresis of solubilized membrane preparations shows that the hormone binds to peptides in two regions of the gel, corresponding to 43 000 and 150 000 daltons. [3H]Ouabain also binds to a peptide in the 150 000 region, as well as to the 100 000 region. Juvenile hormone thus may be binding directly to at least one of the constituent peptides of the Na,K-ATPase complex.
Glucose 1,6-bisphosphate decline in human erythrocytes: possible involvement of phosphoglucomutase PGM2 isoenzymes
Authors: Ninfali Paolino , Piatti Elena , Accorsi Augusto , Palma Fulvio , Fazi Antonio , Tozzi Maria Grazia , Fornaini Giorgio
Published: December 2009

Human erythrocytes incubated with various sugars lower their glucose 1,6-bisphosphate (Glc-1,6-P2) content, as do haemolysates containing exogenous Glc-1,6-P2 incubated with sugar monophosphates (sugar-P). Experiments performed with isolated erythrocyte phosphoglucomutase (PGM) isoenzymes indicate that only definite isoenzymatic forms, namely PGM2, are able to consume Glc-1,6-P2 during the mutation of sugar-P other than glucose-P. In this process a phosphate group is released from Glc-1,6-P2 and can be partially recovered in the bisphosphate of the mutated sugar-P. The relevance of this mechanism of Glc-1,6-P2 degradation is discussed in regard to the physiological turnover of the bisphosphate.
Role of superoxide and hydrogen peroxide in cell lysis during irradiation in vitro of Ehrlich ascitic carcinoma cells in the presence of melanin
Authors: Menon I. Aravindakshan , Persad Surujdeen , Ranadive Narendranath S. , Haberman Herbert F.
Published: December 2009

The reactive species involved in the cell lysis during ultraviolet irradiation of Ehrlich ascitic carcinoma cells in the presence of red hair melanin (RHM) were investigated by determining 51Cr release from labeled cells. Cysteine at 1 mM in the presence of RHM increased the cell lysis during the incubation in the dark as well as during irradiation; this lysis was enhanced by superoxide dismutase (SOD). Catalase abolished the dark reaction and inhibited the cysteine-induced increase of cell lysis during irradiation. The cell lysis by the superoxide-generating xanthine oxidase system was not significantly increased by SOD, but was significantly decreased by nitroblue tetrazolium and completely abolished by catalase. The cell lysis induced by the supernatants obtained from the suspensions of RHM either irradiated alone or with cysteine was abolished by catalase. Sediments of irradiated RHM when incubated in the dark with the cells did not release 51Cr. Irradiation of the cells in the presence of the same sediments produced lysis which was not inhibited by catalase. These studies suggest that superoxide per se is not toxic to the cells, but the H2O2 formed by dismutation of superoxide produces cell lysis either directly or by generating ∙OH through Fenton-type reactions. A large part of the cell lysis seen during irradiation of cells in the presence of RHM is not due to H2O2, but may possibly be due to the melanin free radicals formed during irradiation.
Involvement of phospholipids in the modulation of a membrane-bound brain fucosyltransferase
Authors: Serres-Guillaumond M. , Broquet P. , Louisot P.
Published: December 2009

Microsomal fucosyltransferase isolated from sheep brain is strongly enhanced by charged lysophospholipids such as lysophosphatidylinositol and lysophosphatidic acid, while the corresponding phospholipids are inhibitive. Lysophosphatidylcholine (lyso-PC) also greatly increases the enzymatic activity and leads to its solubilization. Its stimulatory effect is related to the length of the fatty acyl chain involved in the lyso-PC structure: fatty acids C18 and C20 are less activating than the fatty acids C14–C16. Stimulation is restored when C18 fatty acids are unsaturated (e.g., C18:1–C18:3). Enzymatic activity enhancement is decreased when phosphatidylcholine structures are reformed by the addition of lyso-PC and the corresponding fatty acid. The physical state of these structures has no influence. These data provide evidence that bilayer structures do not modify enzymatic activity, while micellar structures formed by detergents and lysophospholipids lead to a strong increase in fucosyltransferase activity. However, lyso-PC does not interact in exactly the same way as Triton X-100. Although they both enhance the maximal velocity of fucosyltransferase for its two substrates, GDP-fucose and asialofetuin, the effect with lyso-PC is greater, and it clearly enables a better affinity for GDP-fucose. Endogenous phospholipids are also able to modify enzymatic activity. Hydrolysis of phosphatidylcholine by phospholipase A2 leads to an enzymatic stimulation.
Biochemical changes in progressive muscular dystrophy. XIII. Nucleic acids, proteins, and total nucleotides in the thymus and spleen of dystrophic mice
Authors: Srivastava Uma , Sebag Mikael , Thakur Manohar
Published: December 2009

Assessments were made of the thymus and spleen weights and the total nucleotide, nucleic acid, and protein content as well as the incorporation of [14C]leucine into protein and of [3H]orotate into RNA, in the thymus, spleen, liver, brain, kidney, lungs, heart, pancreas, and skeletal muscle of normal (+/+) and dystrophic (dy/dy) 129 ReJ mice aged 40, 60, or 90 days. The weights of the thymus and spleen were lower at all stages of dystrophy. Total nucleotide and RNA levels per thymus were reduced at 90 days, while total DNA content was decreased at 60 and 90 days. Protein concentrations per thymus were diminished at each stage of the disease. The specific activity of the free amino acid pool and total free nucleotide pool did not show any significant variations in the thymus at any phase of dystrophy. Incorporation of [14C]leucine into protein and of [3H]orotate into RNA was considerably lower in the thymus at each stage of the disease. Total nucleotide content per spleen was decreased at 40 days, with no change at 60 days and followed by an increase at 90 days in the dystrophic mice. DNA, RNA, and protein levels were all reduced in the spleen at each stage of the disease. The specific activity of the free amino acid pool and total free nucleotide pool, as well as the incorporation of [14C]leucine into protein and of [3H]orotate into RNA, showed similar changes in the spleen as noted in the thymus at each phase of dystrophy. These observations indicate that significant alterations in cellular growth occur not only in skeletal muscle and other nonlymphoid organs, but also in the lymphoid organs of dystrophic mice. Such changes in the cellular growth of lymphoid organs could be responsible for an impairment of immunologic responses reflecting thymic atrophy in murine muscular dystrophy.
Incorporation of glucose into glycogen in primary cultures of rat hepatocytes
Authors: Parniak M. , Kalant N.
Published: December 2009

Glucose may be incorporated into glycogen both by an indirect pathway that involves the metabolism of glucose to C3 intermediates prior to incorporation into glycogen and by a direct mechanism that utilizes the sequence glucose → glucose-6-P → glucose-1-P → UDP-glucose → glycogen. Studies were carried out to determine the major pathway in primary cultures of rat hepatocytes. When cells were incubated with medium containing [3-3H]- and [14C(U)]-glucose the ratio of 3H/14C in glycogen was 70–80% of that of the glucose in the medium. This ratio was unaffected by increases in glucose concentration or insulin, both of which promoted large increases in the incorporation of glucose into glycogen. Relative 3H/14C ratios in glycogen of 25–30% were observed when [2-3H]- and [14C(U)]-glucose was employed; this ratio was doubled in the presence of 2-deoxyglucose or sorbitol, each of which inhibits phosphohexose isomerase. It is concluded that about 75% of the glucose undergoes isomerization between glucose-6-P and fructose-6-P, while about 25% is further glycolysed to C3 intermediates, prior to incorporation. Lactate added to the medium was incorporated into glycogen to an extent of only 20% of that of glucose. However, the presence of lactate resulted in a large increase in the incorporation of glucose into glycogen. Little net deposition of glycogen was observed in these studies. It is concluded that cultured hepatocytes may be a model for the fed organism, and in this condition the direct pathway for the incorporation of glucose into glycogen was predominant.
Androgen cytosol binding during compensatory overload-induced skeletal muscle hypertrophy
Authors: Hickson R. C. , Kurowski T. T. , Galassi T. M. , Daniels D. G. , Chatterton Jr. R. J.
Published: December 2009

This study was undertaken to evaluate whether the increased androgen cytosol binding is an early or later event in the sequence of skeletal muscle hypertrophy induced by surgical overload. Following removal of the synergistic gastrocnemius and soleus muscles, plantaris muscle weights of overloaded hypophysectomized male rats were heavier than those in the controls by 29% at 2 days, 41% at 7 days, 38% at 14 days, and 47% at 35 days. Androgen cytosol receptor binding capacities (femtomoles per milligram protein), determined using a synthetic androgen, [3H]methyltrienolone (R1881), were higher than observed in muscles of controls at all points of muscle enlargement. At high concentrations of labeled ligand, Scatchard analyses became nonlinear and were resolved using a two-component binding model. Receptor capacity of the higher affinity "androgenic component" for methyltrienolone binding in plantaris muscles was lower at 2 days but 60–80% higher at 7, 14, and 35 days in the hypertrophied group than in the control group. The lower affinity "glucocorticoid component" was higher in the overloaded group at all points following surgery. Several glucocorticoids and estradiol-17β competed equally with androgens for methyltrienolone binding. However, when cytosol s were incubated with triamcinolone acetonide to block methyltrienolone binding to glucocorticoid receptors, the androgenic component was highly specific for androgens. These results show that total [3H]methyltrienolone cytosol concentrations increased in parallel with the muscle hypertrophy, yet the individual components of methyltrienolone binding attained greater concentrations in overloaded muscles by an apparently different sequence of events.
Chromosomol DNA fragments from mouse cells exposed to an intercalating agent contain a 175-kdalton terminal polypeptide
Authors: Ralph Ray K. , Hancock Ronald
Published: December 2009

A 175 kdalton (kDa) polypeptide is bound covalently to the chromosomal DNA fragments from mouse cells exposed to the intercalating agent 4′-[(9-acridinyl)-amino]methansulphon-m-anisidide. Electron microscopy shows a terminal protein on the DNA fragments, whose 5′-termini are blocked. Since the relative molecular mass of topoisomerase II polypeptide chains is also about 175 kDa and topoisomerase II inhibitors prevent intercalator-induced DNA fragmentation, we propose that the polypeptide bound covalently to the 5′-terminus of the DNA fragments is a polypeptide derived from frequently integrated topoisomerase II operating to normalize torsional stress resulting from intercalation.
Substrate binding properties of the human liver hexosaminidase A activator protein
Authors: Hechtman Peter , Isaacs Claudine , Smith-Jones Louise
Published: December 2009

The human liver hexosaminidase A activator protein has been shown to bind to the substrate GM2 ganglioside by cosedimentation in sucrose density gradients. Among other proteins tested only serum albumin forms a GM2 ganglioside – protein complex. Both activator protein and albumin bind to the monomeric form of GM2 ganglioside and not to the micellar form of the substrate. The GM2 ganglioside – activator protein complex can be recovered in a stable form. Storage at various temperatures or incubation with monosaccharides or with detergent does not result in dissociation of the complex. GM2 ganglioside in the activator–substrate complex is exchangeable with exogenous GM2 ganglioside. Hexosaminidase A, prepared from human liver, hydrolyzes GM2 ganglioside in the activator–substrate complex as efficiently as GM2 ganglioside supplied exogenously. The activator – Gm2 ganglioside complex forms at pH 3.0 and exhibits an optimum similar to the pH optimum of hexosaminidase A catalyzed hydrolysis of GM2 ganglioside in the presence of the activator; however, the ability of the activator to stimulate enzymic hydrolysis of substrate is rapidly lost after heating at 75 °C, whereas its ability to bind substrate is increased. The sphingolipids cerebroside sulfate and sphingomyelin show little or no binding to the hexosaminidase A activator protein nor do they inhibit activation of hexosaminidase A catalyzed hydrolysis of GM2 ganglioside. By contrast GM1 ganglioside inhibits both substrate binding and enzyme activation.
Insulin-related material in microbes: similarities and differences from mammalian insulins
Authors: Leroith Derek , Shiloach Joseph , Heffron Robert , Rubinovitz Chaim , Tanenbaum Robert , Roth Jesse
Published: December 2009

We have reported that extracts of Escherichia coli and Tetrahymena grown in synthetic media contained material that reacted specifically in the immunoassay and bioassay for insulin. One additional strain of Tetrahymena and four of E. coli yielded amounts of material similar to those reported previously. In addition to their behavior on Sephadex G-50, the immunoactive insulin-related materials from the microbial sources behaved like authentic vertebrate insulins in their ability to be adsorbed to and eluted from disposable octadecasilylsilica cartridges, DEAE-Sephadex, DEAE-cellulose, and one system of high-pressure liquid chromatography (HPLC). As with less purified microbial material, the "insulin" that had been purified on DEAE and HPLC, when tested for its bioactivity, had an immunoactivity:bioactivity ratio of approximately unity and the bioactivity was largely neutralized by anti-insulin antibody. Because the material from the microbes was so similar to authentic insulins, studies were undertaken to demonstrate that inadvertent contamination with vertebrate insulins was highly unlikely. Blanks carried through the entire procedure were always negative. Tetrahymena grown and extracted in another laboratory gave the same results. Tetrahymena that had been grown but then allowed to stand in the fermenter under adverse conditions and then carried through the entire procedure were devoid of insulin. Tetrahymena that were homogenized and subjected to acid hydrolysis were devoid of insulin. Further substantiation that exogenous contamination was highly unlikely was provided by two other types of experiments. In one of these, it was shown that the subcellular distribution of exogenously added porcine insulin or porcine 125I-labeled insulin was different from the distribution of endogenous insulin. In the second type of experiment, it was shown that during the log phase of growth of Tetrahymena or of E. coli the insulin content of the system increased multifold in a fashion that might be expected for living organisms but quite unexpected for exogenous contamination. (Interestingly, the insulin content of the E. coli medium far exceeded that which might be contributed by death of cells, estimated by the content in the medium of an intracellular enzyme.) When E. coli was grown and processed in four other laboratories having no contact with our own, similar levels of insulin-related material were recovered.
Lipoprotein metabolism and the role of apolipoproteins as metabolic programmers
Authors: Dolphin Peter J.
Published: December 2009

The plasma lipoproteins are large spherical macromolecular structures containing hydrophobic core lipids with phospholipids, cholesterol, and specific proteins (apoproteins) providing an amphipathic interface with the hydrophilic environment of the plasma. The major function of these particles, which are biosynthesized by the intestine and liver, is the transport of dietary or endogenously synthesized lipids to those tissues which utilize exogenous lipids for oxidative metabolism, storage, steroid hormone biosynthesis, or maintenance of their membrane integrity. The triacylglycerol-rich lipoproteins are biosynthesized as metabolically inert particles which are catabolically programmed by postsecretory addition of apoproteins which activate the major lipolytic enzymes, inhibit premature removal, and ensure the later interaction of the degraded particles with specific cellular receptors. During the course of lipolysis, those apoproteins which activate catabolic enzymes are lost from the lipoprotein particles and are transferred to the high-density lipoproteins from which they were initially acquired. High-density lipoprotein also mediates the removal of cholesterol deposited in peripheral tissues as a result of uptake of degraded triacylglycerol-rich lipoproteins. Acquisition of cellular cholesterol by high-density lipoproteins results in its apoprotein-stimulated esterification and the later addition of an apoprotein which mediates receptor recognition and removal of the particle from the plasma. The presence or absence of specific apoproteins on the surface of a lipoprotein particle is modulated by the lipid-binding properties of the apoprotein, the surface lipid composition, and the size of the particle. The nature and mass ratios of these surface lipids are themselves dependent upon the activity of apoprotein-stimulated catabolic enzymes and other proteins which mediate the exchange of surface lipids between lipoprotein particles. Thus the apoproteins are effective programmers of lipoprotein metabolism and fulfil their role as such by cycling, in a directed fashion, between nascent and existing plasma lipoproteins. Genetic defects resulting in a perturbation of this intricate mechanism can lead to premature and pronounced atherosclerosis.
Receptor interactions controlling lipoprotein metabolism
Authors: Weisgraber Karl H. , Innerarity Thomas L. , Rall Jr. Stanley C. , Mahley Robert W.
Published: December 2009

Lipoprotein receptors play a central role in lipoprotein metabolism and a major role in cholesterol homeostasis. The most completely characterized lipoprotein receptor is the LDL (low density lipoprotein) or apo-B,E(LDL) receptor. The apo-B,E(LDL) receptor is present on both hepatic and extrahepatic cells and is responsible for the metabolism of a major portion of plasma LDL. Binding and internalization of LDL particles by this receptor initiates a series of intracellular events, resulting in the regulation of cellular cholesterol metabolism. In addition to the apo-B on LDL interacting with the apo-B,E(LDL) receptor, the apo-E on apo-E-containing lipoproteins is also capable of interacting and regulating intracellular cholesterol metabolism. The liver has also been shown to contain a second distinct lipoprotein receptor that is specific for apo-E. This receptor has been demonstrated on hepatic membranes from humans, dogs, and swine and is referred to as the apo-E receptor. This receptor may be responsible for the clearance of chylomicron remnants from plasma by the liver and may participate in reverse cholesterol transport. Thus, apo-E is a major determinant in lipoprotein metabolism and cholesterol homeostasis. The receptor binding properties of apo-E are well characterized, and a series of structural variants, several with lipoprotein binding defects, have been identified. Studies of the binding activity of these receptor-defective apo-E variants have helped to define the receptor binding domain of apo-E. These results, in conjunction with receptor binding studies with various apo-E fragments and with an apo-E monoclonal antibody that inhibits receptor binding, have demonstrated that the receptor binding domain is located in the center of the molecule between residues 140 and 160.
Site-directed cleavage of immunoglobulin gene segments by lymphoid cell extracts
Authors: D'Agostaro Giacomo , Hevia Edith , Wu Gillian E. , Murialdo Helios
Published: December 2009

To study the enzyme(s) involved in the site-specific recombination of immunoglobulin (Ig) gene segments, we designed an assay to detect V–J joining in vitro. The DNA from a hybrid phage (λ VJCK) containing the VK41 gene segment separated by a 6-kilobase spacer region from the entire J–CK sequence was incubated with lymphoid cell extracts and packaged in vitro. Phages carrying genomic deletions were selected by screening for ethylenediaminetetraacetic acid resistance. Although no site-specific V–J fusion events were detected, the packaging efficiency of λ VJCK DNA was 102- to 103-fold lower than that of λ DNA. This suggested the presence in the cell extracts of an endonucleolytic activity with a specificity for the mouse DNA sequences. To detect the endonuclease cleavage products, plasmids containing VK or JK gene segments were used as a DNA substrate and the products of the in vitro reaction were visualized by autoradiography in Southern blots. Double-stranded cleavages were observed to occur near the 5′ end of each one of the five JK gene segments and near the 3′ end of a VK gene segment. A plasmid containing the mouse I-Aβ gene was found to be resistant to cleavage, thus confirming the specificity of the endonucleolytic activity for sequences associated with the mouse Ig gene segments.