Effects of 1-palmitoyl-sn-glycero-3-phosphorylcholine on the properties of a solubilized sialyltransferase activity from mouse liver.

Asialomucin-sialyltransferase (CMP-N-acetylneuraminate:D-galactosyl-glycoprotein N-acetylneuraminyltransferase, EC 2.4.99.1) activity was solubilized from mouse liver microsomes by sonication. The catalytic activity was markedly inhibited by a series of lysophosphatidylcholines, particularly 1-palmitoyl-sn-glycero-3-phosphorylcholine. This lysophospholipid did not alter optimal conditions for enzyme activity. In contrast, it was found that affinities for binding of Mn2+, desialylated mucin and CMP-sialic acid were decreased by adding the lipid. A reasonable interpretation of these data is that the presence of phospholipid modifies the enzyme conformation.

Breaks in arrhenius plots of reactions involving membrane-bound and solubilized sialyltransferases, due to temperature dependence of kinetic parameters.

Temperature dependence of asialomucin-sialyltransferase (CMP-N-acetylneuraminate:D-galactosyl-glycoprotein N-acetylneuraminyltransferase, EC 2.4.99.1) activity is investigated. Discontinuities in Arrhenius plots are observed, whether the enzyme is membrane-associated or solubilized. These discontinuities cannot be firmly correlated with the phase-transition temperatures of either endogenous or exogenous phospholipids. Arrhenius plots of the kinetic parameters also exhibit sharp discontinuities, so that it is concluded that a significant change in Km and Vmax values occurs with varying temperature. Our results suggest that the biphasic behavior of Arrhenius plots may be attributed to the temperature dependence of the kinetic parameters for both membrane-associated and solubilized sialyltransferase activities.

Purification and properties of a mannosyltransferase solubilized from mitochondrial outer membranes.

The enzyme GDPmannose: dolichyl monophosphate mannosyltransferase has been solubilized and purified from mice liver mitochondrial outer membranes. The purification combines detergent extraction of purified outer membranes using Nonidet P-40, with subsequent ion-exchange chromatography on DEAE-cellulose. At this stage, a 400-fold purification is obtained. The partially purified mannosyltransferase is activated by choline-containing lipids such as phosphatidylcholine, lysophosphatidylcholine and sphingomyelin. The reaction is dependent upon the addition of exogenous dolichyl monophosphate. The sole reaction product has been identified as dolichyl phosphate-mannose. The partially purified mannosyltransferase exhibits a Km of 1.33 microM for GDPmannose. Enzyme activity, eluted from DEAE-cellulose, could be further purified after incorporation into sphingomyelin vesicles containing dolichyl monophosphate followed by a sucrose density gradient centrifugation. The mannosyltransferase activity is completely associated with the liposomes at the top of the gradient. Significant stabilization and purification (approx. 1600-fold) of enzyme activity associated with these liposomes is obtained. Furthermore, the reconstitution of this purified enzyme within specific liposomes provides a good model membrane to investigate the molecular requirement of this mitochondrial mannosyltransferase.

[Mitochondrial sialyltransferase (author's transl)]. / Sialyl-transferase mitochondriale.

A sialyl transferase activity is found in purified mitochondria. It is not due to residual contamination and this enzymatic system is located in the outer mitochondrial membrane. This proves mitochondrial autonomy in regard to glyconconjugate sialylation.

[Mitochondrial N-acetylglucosaminyl transferase connected with viral infection]. / N-acétylglucosaminyl-transférase mitochondriale en relation avec l'infection virale.

A higher level of N-acetylglucosamine incorporation by proteinic and polyprenic endogenous acceptors is observed after infection by Myxovirus. This phenomenon occurs in whole mitochondria as in outer mitochondrial membranes, where it is particularly obvious with proteinic endogenous acceptors. Under viral infection, no new N-acetylglucosaminylated polyprenol is detected. In the case of infected animals as in the case of control animals, compounds P1 (extracted by chloroform/methanol 2:1) are identified by thin layer chromatography as a N-acetylglucosamyl-pyrophosphoryl-dolichol and a N, N'-diacetylchitobiosyl-pyrophosphoryl-dolichol. In the case of infected animals, biosynthesis of proteinic acceptors and dolichol is not modified; therefore the increase of N-acetylglucosamine incorporation is not due to a modification of the endogenous acceptors level. By the use of exogenous dolichol-monophosphate we demonstrate that the increased transfer of [14C] N-acetylglucosamine into polyprenic acceptors is the result of a higher activity of the mitochondrial N-acetylglucosaminyltransferase after viral infection.

Temperature dependence of membranous and solubilized sialyltransferase activities in the presence of 1-palmitoyl-sn-glycero-3-phosphorylcholine and fatty acids.

The temperature dependence of sialyltransferase (CMP-N-acetylneuraminate: D-galactosyl-glycoprotein N-acetyl-neuraminyltrasferase, EC 2.4.99.1) inhibition is described when 1-palmitoyl-sn-glycero-3-phosphorylcholine, or a saturated fatty acid (lauric, myristic or palmitic acid) or an equimolar mixture of the two components are added. Lysophospholipid and fatty acids have no appreciable effect on the optimal temperature for sialyltransferase activity. In the presence of lysophospholipid, the membranous sialyltransferase activity is decreased for all the temperature range tested. In contrast, the solubilized sialyltransferase activity is decreased for temperatures exceeding 29 degrees C. In the presence of saturated fatty acids, the membranous activity is decreased above a chain-length dependent temperature: 22 degrees, 25 degrees and 30 degrees C for lauric, myristic and palmitic acids, respectively. In contrast, the solubilized activity remains unchanged. In the presence of equimolar mixtures of lysophospholipid and fatty acid, the membranous activity is decreased above the same critical temperature as that described for fatty acids added alone. In contrast, the solubilized activity is decreased above 29 degrees C. From these observations, it is suggested that lysophospholipid inhibits the solubilized enzyme when the temperature exceeds the critical micellar temperature of this lipid. The fatty acids inhibit the microsomal enzyme probably by incorporating into the membrane. It is also suggested that equimolar mixtures of lysophospholipid and fatty acid give rise to molecular analogs of 1,2-dipalmitoyl-sn-glycero-3-phosphorylcholine.

Topological investigations. Study of the trypsin sensitivity of the N-acetylglucosaminyl and mannosyl-transferase activities located in the outer mitochondrial membrane.

The trypsin sensitivity of the mitochondrial N-acetylglucosaminyl and mannosyltransferase activities involved in the N-glycoprotein biosynthesis through dolichol intermediates as well as the N-acetylglucosaminyl-transferase activity involved in direct N-glycosylation were examined in mitochondria and isolated outer mitochondrial membrane preparations. The trypsin action on mitochondrial membrane was checked by measuring the activities of marker enzymes (rotenone-insensitive NADH cytochrome c reductase, adenylate kinase, and monoamine oxidase). Glycosyl-transferase activities of both N-glycosylation pathways were insensitive to trypsin action and consequently were located in the outer mitochondrial membrane. Based on the activator effect of the trypsin on these enzyme activities, the results suggested two distinct orientations of their active sites. As regards the N-glycoprotein biosynthesis pathway through dolichol intermediates, the dolicholphosphoryl-mannose and dolichol-pyrophosphoryl-di-N-acetylchitobiose synthases would be oriented outside while the oligomannosyl-synthase and the oligomannosyl-transferase would be rather oriented inside in the outer membrane. The N-acetylglucosaminyl-transferase involved in the direct transfer of N-acetylglucosamine from its nucleotide donor to a proteinic acceptor would be oriented outside in the outer membrane.

Rapid and sensitive discriminating determination of acetylcholinesterase activity in amniotic fluid with a choline sensor.

A simple method for the separate determination of acetylcholinesterase and butyrylcholinesterase activities in amniotic fluid is reported. This determination is performed with an enzyme electrode involving an immobilized choline oxidase membrane associated with the amperometric detection of hydrogen peroxide. Acetylcholine or butyrylcholine, in the presence of samples containing acetylcholinesterase or butyrylcholinesterase are specifically hydrolyzed, the formation of choline being detected vs time by the sensor with no need for a selective inhibitor. The dynamic linear ranges for acetylcholinesterase and butyrylcholinesterase are respectively 100 microU to 10 mU and 30 microU to 3 mU per ml sample.

Effects of defined synthetic phospholipids on glycosylation processes. Modifications of membrane-bound N-acetylglucosaminyl-transferase and solubilized sialyl-transferase activities.

In this paper, we study effects of phosphatidylcholine synthetic derivatives on two different glycosyltransferasic systems, a membranous mitochondrial N-acetylglucosaminyl-transferase and a solubilized microsomal sialyl-transferase. From all our results, we proved the strong inhibitory effect of lysophosphatidylcholines in these glycosylation processes. We investigated then the relationship between this inhibitory effect and the chemical structure of the studied phospholipids. From this work, it became obvious that the most important inhibitory effect is obtained with a molecule containing a long aliphatic side chain in sn-1 of glycerol, a carbon atom devoid of any large group in sn-2 and an hydrophilic part in sn-3 (specially choline).

Effects of chronic ethanol administration on plasma-membrane-bound glycosyltransferase activities.

Physiological and membranous modifications induced by a 4-week ethanol administration in mouse liver plasma membrane were studied. Galactosyl- and glucosyltransferase activities were stimulated in the presence of dolichylphosphate alone or with phosphatidyl-choline. The galactosyltransferase activity was inhibited by chronic ethanol administration. These enzymes were modulated by different phospholipids. The phosphatidic acid was the most efficient activator. Ethanol provoked an inhibition of the galactosyltransferase activity whatever the phospholipid used, as well as an inhibition of the glucosyltransferase activity, chiefly in presence of phosphatidyl-inositol. The preincubation of control or treated mouse liver plasma membranes with liposomes loaded by dolichylphosphate and cholesterol greatly enhanced the enzymatic activities without removing the inhibition by ethanol treatment.

Influence of phospholipidic microenvironment on the performance of a polypyrrole enzyme electrode.

Enzyme loaded phosphatidylcholine vesicles have been introduced as a new component in the design of sensing layers for the direct coating of an amperometric transducer. For this purpose, choline oxidase was chosen as a model enzyme, and the resulting vesicles were mixed with an amphiphilic pyrrole derivative [12-(pyrrol-1-yl)dodecyltriethylammonium tetrafluoroborate]. A minute amount of this mixture was deposited and dried on a platinum electrode, then electropolymerized. Compared to a similar preparation omitting phospholipid, the sensitivity of the enzyme electrode increased from 0.06 to 0.85 mA .1. mol(-1). The response, obtained in less than 20 sec, was linearly related to the choline concentration within a broader range, extending from 2.5. 10(-7) to 3. 10(-5)M.

Glucosyltransferase activities in liver mitochondria. I. Biosynthesis of dolichyl[14C]glucosyl phosphate and [14C]glucosylceramide.

Mitochondria, and specially outer mitochondrial membranes, incorporate D-[14C]glucose from UDP-D-[14C]glucose into products extracted with organic solvents and into a residual precipitate, with a pH optimum of about 6.5 in (2-N-morpholino-ethane)-sulfonic acid (MES) buffer. The chloroform/methanol (2:1, v/v) extract contains two products. The major [14C]glucolipid is stable to mild alkali, but releases [14C]glucose upon mild acid hydrolysis. It is retained on DEAE-cellulose (acetate form) and is eluted with the same ionic strength as an hexosyldolichyl monophosphate diester. This [14C] glucolipid has the same chromatographic behaviour as dolichyl-mannosylphosphate in neutral, acidic and basic solvent systems; and its biosynthesis is greatly increased by exogenous dolichylmonophosphate. The other [14C]glucolipid is stable upon mild acid hydrolysis and is not retained on DEAE-cellulose. On silicic acid it is eluted with acetone. The biosynthesis of this compound is stimulated by exogenous ceramide. This glucolipid has the same chromatographic mobility in different solvent systems as glucosylceramide isolated from the liver of a patient with Gaucher's disease. Biosynthesis of these two glucolipids is inhibited by UDP, but only biosynthesis of dolichylglucosyl monophosphate is reversible with this nucleotide. The biosynthesis of these different glucosylated derivatives is stimulated by the addition of divalent cations (Mn2+, Mg2+). the effect of these two metal ions on dolichylglucosyl monophosphate and glucosylceramide formation is studied in different conditions.

[Demonstration of glucosyltransferases in the outer membrane of mitochondria]. / Mise en évidence de glucosyl-transférases dans la membrane externe des mitochondries.

Outer mitochondrial membranes catalyze the transfer of glucose into polyprenic, lipidic and proteinic endogenous acceptors. A glucosyl-phosphoryl-dolichol and a glucosyl-ceramide are identified and biosynthesis of these two products is studied.

Biochemical evidence for the role of alkyl-lysophospholipids on liver sialyltransferase.

Antineoplastic alkyl-lysophospholipids were found to exert a strong inhibitory effect on membranous or solubilized asialomucin-sialyltransferase (CMP-N-acetylneuraminate: D-galactosyl-glycoprotein N-acetylneuraminyltransferase, EC 2.4.99.1) activity. This inhibitory effect was dependent on the presence of the choline moiety in position 3 of the glycerol molecule, as well as on the presence of long ether-linked aliphatic side chain in position 1 and the absence of any large substituent in position 2. As an example, 1-octadecyl-2-O-methyl-glycero-3-phosphorylcholine acted as a mixed-type inhibitor. Such an inhibitory process on sialyltransferase activity might be an additional factor in the tumor cell destructive effect of alkyl-lysophospholipids.

[Demonstration of N-acetylglucosaminyl-transferases in the outer membrane of liver mitochondria]. / Mise en évidence de N-acétyl-glucosaminyl-transférases dans la membrane externe des mitochondries de foie.

Outer mitochondrial membranes catalyze the transfer of N-acetyl-glucosamine into polyprenic and proteinic endogenous acceptors. Different effectors (nucleotides, EGTA) are used, suggesting that there are two separate metabolic ways.