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M Llanillo

Publications and source records attributed to M Llanillo.

At least 19 recordsLinked to original sources
PubMed1997

Characterization of phosphotyrosine phosphatase activity in sheep platelets: amphiphilic and hydrophilic forms.

Using O-phosphotyrosine as a substrate, we characterized the phosphotyrosine phosphatase (PTPase; protein-tyrosine-phosphate-phosphohydrolase, EC 3.1.3.48) activity from sheep platelets. PTPase was found to be located in three particulate subcellular fractions and in the cytosol, with K(m) values in the millimolar range. PTPase was strongly inhibited by vanadate, molybdate and HgCl2 and only weakly inhibited by Zn2+. Other divalent cations and NaF had no significant effect on the activity associated with the membrane fraction but were slightly stimulatory as regards cytosolic activity. Heparin inhibited cytosolic activity 2-fold more than membrane-bound activity and dithiothreitol only inhibited cytosolic PTPase. Polycationic compounds were seen to be weak stimulators of all the PTPase activity. Solubilization of the PTPase from membranes always required a detergent. When subjected to Triton X-114 phase partitioning, PTPase was recovered in the detergent-rich (35%) and in the detergent-poor (65%) phases. Sedimentation analysis of the cytosolic PTPase showed a peak of 3.2S that remained unmodified when Triton X-100 or Brij 97 sucrose gradients were used. Sedimentation analysis of the membrane-associated PTPase showed 6S and 3.7S peaks unchanged in Triton X-100 or Brij 97 gradients together with 7.5S and 10.3S shoulders that shifted to smaller sedimentation coefficients in Brij 97 sucrose gradients. These results support the view that sheep platelets contain amphiphilic and hydrophilic forms of PTPase.

Animals↗
PubMed1997

Decrease in platelet, erythrocyte and lymphocyte acetylcholinesterase activities due to the presence of protease inhibitors in the storage buffer.

The effect of the presence of protease inhibitors in the storage buffer on acetylcholinesterase (AChE) activity was studied in crude membrane preparations from sheep platelets before and after solubilization of the membranes with Triton X-100. Although sensitive to the action of trypsin, the biological activity of AChE remained unchanged for as long as 6 days at 4 degrees C in a protease-inhibitor-free medium. At 10(-5) M final concentration PMSF reduced AChE activity to 50% after 24 hours of storage. This reduction was abolished in mixtures in which PMSF was present together with 3 mM EGTA, 5 mM EDTA and 0.1 mg/mL trypsin inhibitor. Nevertheless, under these conditions, storage periods longer than 24 hours still drastically reduced AChE activity. A mixture of EDTA, EGTA and trypsin inhibitor also produced a decrease in AChE activity after 24 hours. A more complex cocktail of inhibitors including several commonly used peptides decreased AChE activity only if PMSF or EDTA were present in the mixtures. Similar results were obtained with sheep erythrocytes or lymphocytes, and bovine erythrocytes.

Acetylcholinesterase↗
PubMed1996

Enzymatic determination of phosphatidylcholine, sphingomyelin and phosphatidylglycerol in lipid dispersions, blood cell membranes and rat pulmonary surfactant.

A new micromethod for the determination of sphingomyelin in samples suspended in aqueous solutions, and modified micromethods for determining phosphatidylcholine and phosphatidylglycerol were used to determine phosphatidylcholine and sphingomyelin (detection limits of 1.8 mumol/l), and phosphatidylglycerol (detection limit of 2.3 mumol/l) in lipid dispersions, membranes from sheep erythrocytes and platelets, and pulmonary surfactants from rats of different ages and rats maintained under normobaric hyperoxia for 2 days prior to their sacrifice. The procedures are easy to perform, accurate, require less sample than conventional methods and can also be applied directly to aqueous samples. Phospholipase C and sphingomyelinase were used to release phosphorylcholine from phosphatidylglycerol and sphingomyelin, respectively. The choline released from phosphorylcholine by alkaline phosphatase is reconverted to phosphorylcholine by ATP and choline kinase. In the phophatidylglycerol determination, phospholipase D was used to release glycerol and phosphatidate. The glycerol formed was converted to glycerolphosphate using ATP and glycerol kinase. In all cases, the ADP thus formed was determined by following the enzymatic conversion of NADH to NAD at 340 nm in an coupled pyruvate kinase/lactate dehydrogenase system. Significant variations in the phospholipid composition of rat pulmonary surfactant were found during development; in particular there was an increase in the phosphatidylglycerol content of adult rats as compared with younger rats. Hyperoxia produced changes in the phosphatidylglycerol content of surfactant from adult rats, but not from 2-day old rats.

Animals↗
PubMed1995

Effects of dietary oil related to the toxic oil syndrome on the lipids of guinea pig liver microsomes.

The potential effects of oil specimens related to cases of toxic oil syndrome (TOS) on the liver microsomal lipid composition from guinea pigs were investigated. For four weeks, animals were fed diets supplemented with either "case oil" (oil related to cases of TOS) or "control oil" (oil unrelated to cases of TOS), either previously heated or not. Results were compared with those from guinea pigs fed the same diet with no oil. The administration of case oil produced changes in liver microsomal lipid composition. Statistically significant differences were also found between heated case and heated control oils. The cholesterol/phospholipid molar ratios and the major phospholipid class distribution were unaffected under these diet conditions. However, increases in the relative contents of linoleic and arachidonic acids and, simultaneously, a reduction in palmitic and palmitoleic acid levels were observed by diet effects. Heated oil administration decreased the saturated/unsaturated ratios in all cases. Our data suggest that changes observed in the fatty acid composition are attributable to the free fatty acid contents of administered oils. The toxic constituents of case oil seem to be able to alter the liver microsomal lipid composition.

Aniline Compounds↗
PubMed1995

Biochemical characterization of sheep platelet acetylcholinesterase after detergent solubilization.

The biochemical characterization of detergent-solubilized acetylcholinesterase (AChE) from subcellular particles of sheep platelets and the effects of different effectors on AChE activity from solubilized platelet crude membranes have been undertaken and studied. Solubilization of AChE with detergent increased the thermal stability of the enzyme from all particulate fractions. Solubilized AChE from the mitochondria-granule fraction was the most thermostable at 55 degrees C. The Km values against acetylthiocholine chloride and the Arrhenius plot obtained were very similar for the AChE from all the solubilized fractions. There were no differences in the ability of solubilized AChE from different subcellular fractions to bind concanavalin A (Con A). In solubilized platelet crude membranes, benzyl alcohol was a potent AChE inhibitor at a concentration of 10(-2) M, whereas ethanol was not. Mg2+ cations and, to a lesser extent, Ca2+ and Mn2+ cations, activated AChE at concentrations higher than 1 mM. Serine hydrolase inhibitors and cholinesterase-specific inhibitors were very effective in the inactivation of AChE, whereas EDTA and EGTA had no effect. Of all the monosaccharides tested, only N-acetylneuraminic acid exerted an inhibitory effect on AChE activity. Immobilized-lectin binding studies demonstrated the interaction of solubilized crude membrane-bound AChE with Con A, lentil lectin and wheat germ agglutinin. Taken together, these data suggest the presence of a unique form of the membrane-bound AChE which has at least alpha-mannose and N-acetylglucosamine residues in the glycan chain.

Acetylcholinesterase↗
PubMed1995

Inhibition of prostaglandin E2-responsive adenylyl cyclase in embryonal human kidney 293 cells by phorbol esters.

A clonal primary embryonal human kidney cell line, 293, increased cAMP production in response to prostaglandin E2 (PGE2) (0.02-2 microM). The purpose of this study was to show the effects of tumor-promoting phorbol esters (e.g., 4 beta-phorbol 12-myristate 13-acetate, PMA) on PGE2-stimulated cAMP production. Pretreatment with PMA (0.2-200 nM) for 30 min markedly reduced PGE2-stimulated cAMP production in the presence of 0.5 mM isobutylmethylxanthine. The reduction by PMA was dose- and time-dependent. PMA seems to attenuate the increase in cAMP accumulation elicited by PGE2 primarily, if not entirely, by inhibiting adenylyl cyclase activity, since we were unable to demonstrate an effect of PMA on the degradation half-life of cAMP in intact 293 cells. The action of PMA had some specificity for the agonist used; thus, PMA inhibited PGE2-activated adenylyl cyclase but had no effect on the forskolin-activated enzyme. Co-pretreatment with PMA and H-7, an inhibitor of protein kinase C (PKC), partially prevented the PMA-induced attenuation of the PGE2-stimulated cAMP accumulation, and 1-oleoyl-2-acetylglycerol, a synthetic diacylglycerol analog, partially mimicked the PMA action. Thus, PMA appeared to decrease cAMP production by a PKC-mediated mechanism, inhibiting adenylyl cyclase activity at a point other than the catalytic subunit of the enzyme in the kidney 293 cell line.

Adenylyl Cyclase Inhibitors↗
PubMed1995

Phospholipid and fatty acid composition in stored sheep erythrocytes of different densities.

The mammalian erythrocyte is an ideal model for studies of membrane aging under conditions of storage. The present study describes the variations in the membrane lipid composition of three density groups (light, 1.110 < d < 1.125; intermediate, 1.125 < d < 1.130, and dense, 1.130 < d < 1.140) of sheep erythrocytes separated by centrifugation in a discontinuous Ficoll density gradient after storage at 4 degrees C in a nutrient medium for up to 6 days. The only changes in phospholipid composition took place in the erythrocyte light fraction where sphingomyelin (SM) and phosphatidic acid increased (p < 0.05), whereas phosphatidylethanolamine (PE) decreased (p < 0.05). Moreover, polyunsaturated fatty acids (20:4 and 22:6) decreased during storage, whereas lipid fluorescence increased (p < 0.01) after 24 hours of storage in all the fractions separated. These observations suggest a lipid peroxidation process in all three erythrocyte groups during storage.

Animals↗
PubMed1993

Changes in the fatty acid composition of stored erythrocytes from sheep of different ages.

This study examined the profile in sheep erythrocyte fatty acids from animals of different ages during storage at 4 degrees C in a nutritive medium for up to 6 days. The changes found in the fatty acyl profile were a decrease (P < 0.01) in the percentage of arachidonic acid and an increase (P < 0.01) in the percentage of minor fatty acids (representing < 2% in each case; 20:2, 22:0 and 22:1) with respect to fresh erythrocytes in all age groups. However, the saturated/unsaturated ratios and unsaturated index started almost constant in all cases. The changes observed occurred after 24-48 h of storage, with significant increases (P < 0.01) in the fluorescence detected in the lipid extracts from stored erythrocytes during this period. The above findings suggest peroxidative damage and changes in the erythrocyte lipid membrane during storage.

Aging↗
PubMed1991

Ganglioside and phospholipid composition of forebrain, cerebellum, and brain stem from adult and newborn rats.

The aim of this study was to elucidate whether sex or pregnancy state might affect the content and/or pattern of gangliosides from the forebrain, cerebellum and brain stem of rats. Adult male, mother (1-day after offspring) and nonpregnant rats of similar age were analyzed. Non-significant differences in ganglioside concentrations and patterns were found for the respective neural area of adult male and female rats except for a decrease in cerebellum and brain stem content from mothers and 12.0 months-old males, respectively. Thus, it seems that neither sex nor pregnancy hormones affect these parameters. By contrast, significant differences were found for pattern and ganglioside contents between adult (male and female) rats and newborns (1 day-old). Newborns showed a significant decrease in their forebrain (2.5-fold), cerebellum (2.0-fold) and brain stem (2.0-fold) ganglioside content when compared with adult (male and female) rats. Significant increases (p less than 0.001) were found in the phospholipid and cholesterol contents in the different brain areas in mothers versus their newborns. The phospholipid pattern also showed significant changes in all brain areas, with an increase (p less than 0.001) in phosphatidylethanolamine percentage in adult animals, among the main variations. An explanation for these facts is suggested.

Animals↗
PubMed1991

Fatty acid composition of subcellular particles from sheep platelets and topological distribution of phosphatidylethanolamine fatty acids in the plasma membrane.

The fatty acid composition of individual phospholipids in subcellular fractions of sheep platelets and the asymmetrical distribution of phosphatidylethanolamine (PE) fatty acyl chains across the plasma membrane were examined. The main fatty acids of total lipid extracts were oleic (18:1; 32-41%), linoleic (18:2, 10-17%), stearic (18:0; 13-15%), palmitic (16:0; 11-15%) and arachidonic (20:4; 8-12%) acids, with a saturated/unsaturated ratio of about 0.4. Each phospholipid class had a distinct fatty acid pattern. Sphingomyelin (SM) showed the highest degree of saturation (50%), with large proportions of behenic (22:0), 18:0 and 16:0 acids. The main fatty acid in PE, phosphatidylserine (PS) and phosphatidylcholine (PC) was 18:1n-9. Our findings suggest that fatty acids are asymmetrically distributed between the choline versus the non-choline phospholipids, and also between plasma membranes and intracellular membranes. The transbilayer distribution of PE fatty acids in plasma membranes from non-stimulated sheep platelets was investigated using trinitrobenzene-sulfonic acid (TNBS). A significant degree of asymmetry was found, which is a new observation in a non-polar cell. The PE molecules from the inner monolayer contained higher amounts of 18:2 and significantly less 18:1 and 20:5 than those found in the outer monolayer, although no major differences were detected in the transbilayer distribution of total unsaturated versus saturated PE acyl chains.

Animals↗
PubMed1990

Subcellular distribution and characterization of acetylcholinesterase activities from sheep platelets: relationships between temperature-dependence and environment.

Acetylcholinesterase is a key enzyme in cholinergic neurotransmission for hydrolyzing acetylcholine and has been shown to possess arylacylamidase activity in addition to esterase activity. The enzyme is found at various loci, where its functional significance remains to be clarified, and it exists in multiple molecular forms. Sheep platelets have been shown to exhibit acetylcholinesterase activity associated with plasma membrane (Bp), endoplasmic reticulum (Cp), mitochondria granules (Dp), and soluble (As) fractions. These activities show differences in some physicochemical and kinetic properties. The soluble acetylcholinesterase is the most thermostable, and the enzyme from the Cp fractions shows the lowest affinity for the acetylthiocholine substrate and the strongest inhibition by fluoride. In all cases a noncompetitive inhibition of the enzyme by this ion is found. When membrane-bound acetylcholinesterases were assayed at temperatures between 12 degrees C and 33 degrees C, the Arrhenius plots of all activities exhibited a break point at about 17 degrees C. This discontinuity was abolished by addition of detergent to the assay medium (0.02% Triton X-100, final concentration). Their Hill coefficients were calculated in the presence of fluoride, showing unitary values in all cases, which points to a noncooperative effect and nonallosteric behavior in the particulate enzyme. These results suggest that the sheep platelet acetylcholinesterase associated with membrane-bound systems is modulated by the physical state of its environment, despite the fact that the enzyme might be lipid- or nonlipid-dependent.

Acetylcholinesterase↗
PubMed1990

Changes in the phospholipid and fatty acid composition in normal erythrocytes from sheep of different ages. Aminophospholipid organization in the membrane bilayer.

Development and aging processes in mammals are associated with changes in several physiological parameters. The aim of the present study was to investigate the changes in erythrocyte lipid composition during sheep development. In all the age groups studied, cholesterol/phospholipid ratios remained constant, at close to unity, while phospholipid patterns (sphingomyelin: 45-51%, phosphatidylethanolamine: 26-33%, phosphatidylserine: 13-19% and phosphatidylcholine: less than 2%) changed during development, with a statistically significant decrease (P less than 0.01) in phosphatidylserine and an increase in sphingomyelin content. These data suggest an increase in the rigidity of the erythrocyte lipid bilayer in adult sheep when compared with 1-month-old animals due to a decrease in the phosphatidylserine/sphingomyelin ratio. Fatty acid profiles consistently showed 5 main acids: oleic (52-54%), stearic (17-18%), linoleic (9-15%), palmitic (8.5-11%) and arachidonic acid (2-3%), mainly with significant variations (P less than 0.01) in palmitic and linoleic acid contents, respectively reaching the highest and lowest percentages in the youngest sheep. However, the developmental process seems to have no influence on the aminophospholipid topology of erythrocytes. This study suggests that the animals' developmental process has a marked effect on the lipid composition of erythrocyte membranes, which could affect cell functions.

Aging↗
PubMed1989

Aminophospholipid translocase in the plasma membrane of Friend erythroleukemic cells can induce an asymmetric topology for phosphatidylserine but not for phosphatidylethanolamine.

The ATP-dependent translocation of phospholipids in the plasma membrane of intact Friend erythroleukemic cells (FELCs) was studied in comparison with that in the membrane of mature murine erythrocytes. This was done by following the fate of radiolabeled phospholipid molecules, previously inserted into the outer monolayer of the plasma membranes by using a non-specific lipid transfer protein. The transbilayer equilibration of these probe molecules was monitored by treating the cells--under essentially non-lytic conditions--with phospholipases A2 of different origin. Rapid reorientations of the newly introduced aminophospholipids in favour of the inner membrane leaflet were observed in fresh mouse erythrocytes; the inward translocation of phosphatidylcholine (PC) in this membrane proceeded relatively slow. In FELCs, on the other hand, all three glycerophospholipids equilibrated over both halves of the plasma membrane very rapidly, i.e. within 1 h; nevertheless, an asymmetric distribution in favour of the inner monolayer was only observed for phosphatidylserine (PS). Lowering the ATP-level in the FELCs caused a reduction in the rate of inward translocation of both aminophospholipids, but not of that of PC, indicating that this translocation of PS and phosphatidylethanolamine (PE) is clearly ATP-dependent. Hence, the situation in the plasma membrane of the FELC is rather unique in a sense that, though an ATP-dependent translocase is present and active both for PS and PE, its activity results in an asymmetric distribution of PS, but not of PE. This remarkable situation might be the consequence of the fact that, in contrast to the mature red cell, this precursor cell still lacks a complete membrane skeletal network.

Adenosine Triphosphate↗
PubMed1988

Externalization of aminophospholipids in platelet liposome bilayers by supplementation with sphingomyelin.

Negatively charged phospholipids are mainly located on the inner leaflet of platelet liposomes. Using a specific reagent for amino groups, trinitrobenzenesulfanilic acid, we have shown that in large artificial vesicles prepared with total lipids from sheep platelets supplemented with increased amounts of exogenous sphingomyelin, an alteration in the amino-phospholipid topology occurs, with a progressive appearance of these lipid species -in particular phosphatidylserine- in the outer membrane bilayer.

Blood Platelets↗
PubMed1987

Rearrangement of aminophospholipids in bilayers from sheep platelet plasma membranes and platelet liposomes by increasing their cholesterol levels.

Phospholipid orientation in platelet plasma membranes and other blood cells, such as erythrocytes, appears to be rather similar. The negatively charged phospholipids are almost exclusively located on the inner leaflet of the bilayer. No phosphatidylserine is present on the outer membrane bilayer. The results of the present study, using a specific reagent for amino groups, trinitrobenzenesulfanilic acid, showed that in sheep platelet plasma membranes enriched with free exogenous cholesterol, an alteration in the aminophospholipid topology occurs, with a portion of phosphatidylserine moving from the inner to the outer side. A progressive appearance of aminophospholipids in the outer membrane bilayer was also observed in artificial vesicles prepared with total lipids from sheep platelets supplemented with increased amounts of free cholesterol.

Animals↗
PubMed1987

Isolation, characterization and phospholipid composition of lamellar bodies and subcellular fractions from dog lung.

1. Lamellar body fractions from dog lung can be separated by a procedure based on differential centrifugation before ultracentrifugation onto a discontinuous sucrose gradient. This fraction yields about 1% of total protein from the homogenate. 2. The different fractions obtained in the isolation were assayed for the measurement of four subcellular marker enzymes: beta-N-acetylglucosaminidase, acid phosphatase, 5'-nucleotidase and succinate dehydrogenase. 3. Lamellar bodies were not contaminated by mitochondria (0.7 succinate dehydrogenase relative specific activity), whereas high specific hydrolase activities were found (beta-N-acetylglucosaminidase and 5'-nucleotidase were enriched 1.8- and 2.8-fold, respectively). 4. The chemical criterion was established by measuring the specific components of lamellar bodies. The lamellar bodies have the highest phospholipid/protein ratio (0.35); cholesterol/protein ratio (0.15) and the highest phosphatidylglycerol percentages (7.9%). 5. The phospholipid composition of lamellar bodies is distributed among phosphatidylcholine (64.5%), phosphatidylethanolamine (11%), phosphatidylglycerol (7.9%), sphingomyelin (4%), phosphatidylserine and phosphatidylinositol (3%), respectively. The remainder were considered as trace amounts (less than 1%).

Animals↗