Enrique Belocopitow

A phosphatase acting on dolichyl phosphate in membranes from neuronal perikarya

Since the isolation of Del-P-Glc in 1970 [I] significant progress has been made in the elucidation ofhow the carbohydrate moieties of the asparagine-type glycoproteins are synthesized. Clycosylation occurs via sugar lipid intermediates [2]. Some of the properties of most of the enzymes in this pathway have been studied [2]. There is however scant amount of information* available about Dol-P and Dol-P-P phosphatases. The control mechanism operating in the dolichol pathway is yet to be elucidated. It has been proposed that the endogenous level of Dol-P could play a regulatory role in protein glycosylation [3]. By analogy with procaryotic systems [4-61, Dol-P, Dol.P-P phosphatases and dolichoi kinase may be implicated in the control bf the endogenous level of Dol-P. Enzymes that split the phosphate bonds from prenyl phosphates have been described in mammalian systems [7--91, although in these cases Dol-P was not used as substrate and Mgzf were always included in the incubation mixture. Here we report an enzymatic activity present in membranes from neuronal perikarya which removes the pl~osphate from Dol-P and requires no divalent cations.

Glycosyl transfer to an acceptor lipid from insects

Abstract Insect extracts were found to contain a lipid which becomes glycosylated when incubated with uridine diphosphate glucose or uridine diphosphate N-acetylglucosamine and microsomal enzymes of rat liver. The behaviour of the lipid on column or thin-layer chromatography and its stability to acid were equal to those of dolichol monophosphate. The glycosylated compounds were acid labile. Treatment with alkali of the acetylglucosaminyl compound produced a substance that migrated like a hexose phosphate on electrophoresis and that liberated acetylglucosamine on treatment with alkaline phosphatase. The behaviour of the insect glucosylated lipid on thin-layer chromatography and its stability to phenol were similar to dolichol monophosphate glucose and different from ficaprenyl monophosphate glucose. It is concluded that the insect glycosyl acceptor lipid is an α saturated polyprenyl phosphate.

Biosynthesis of polyprenol phosphate sugars by Ceratitis capitata extracts.

Polyprenol-phosphate-sugars have been found to occur in bacteria [1], fungi [2], plants [3] and vertebrate tissues [3 -5 ] . In a previous paper we described a lipid phosphate which we called IGAL (insect glycosyl acceptor lipid) extracted from insect tissues [6]. This compound, in the presence of rat liver microsomes, accepts sugars from sugar nucleotides. It behaves as a prenolophos. phate with an a-saturated isoprene unit [6]. This makes it similar to rat liver DolMP* [4]. We now want to describe an insect enzyme which catalyzes similar reactions. UDP-Glc, GDP-Man and UDP-GlcNac were used as sugar donors and IGAL and DolMP as sugar acceptors.

The effect of dolichol on the permeability properties of phosphatidylcholine bilayers

The permeability of liposomes to water, glucose, Ca2+ and alkaline cations was monitored by recording the change in absorbance at 450 nm using a rapid reaction stopped-flow spectrophotometer. Liposomes were prepared with egg phosphatidylcholine and concentrations of dolichol ranging from 0.1% to 9% (w/w). Net permeability of phosphatidylcholine bilayers to alkaline cations was induced by the incorporation of dolichol. This effect was not observed in the case of non-charged solutes like glucose or in that of alkaline earth cations such as calcium. Permeation of K+ was significantly increased above the phase transition temperature. These results suggest that dolichols may play a role in biological membranes, besides the well-known glycosyl carrier function in the biosynthesis of glycoproteins.

ENZYMATIC SYNTHESIS OF POLYPRENOL MONOPHOSPHATE MANNOSE IN INSECTS

SummaryThe microsomal fraction of insects was found to contain an enzyme which transfers mannose from guanosine diphosphate mannose to an endogenous or exogenous insect lipid and to other acceptors such as dolichol monophosphate or ficaprenol monophosphate. This activity depended on the presence of Triton X-100 and magnesium ions, the optimal concentration of the latter being 10mM. The optimal temperature of the reaction was 25 °C and the maximal activity was obtained at pH 7.9. The mannolipid formed behaved as a monophosphodiester when chromatographed on DEAE-cellulose. Weak acid treatment of the product liberated mannose. Its behaviour both on thin layer and Sephadex G-150 chromatography would indicate the presence of a number of isoprenyl units similar to the dolichol and different from the ficaprenol derivative. Stability to phenol treatment indicated that the lipid fraction of the mannolipid is an±-saturated polyprenol phosphate similar to dolichol monophosphate.

Glycogen metabolism in muscle homogenates: I. The effect of potassium ions on glycogen synthesis☆

Abstract The incorporation of 14 C-glucose into glycogen by pigeon breast muscle homogenates was studied. The extent of glycogen labeling depended on the addition of boiled extract, oxygen supply, and potassium ions, and was stimulated by the addition of uridine nucleotides. The incorporation was influenced by the concentration of glucose in the medium and was abolished by the addition of unlabeled glucose 6-phosphate, glucose 1-phosphate, or uridine diphosphate glucose. Potassium ions depressed the uptake of radioactive phosphate into non-nucleotidic phosphoric esters and increased the synthesis of radioactive adenosine triphosphate. At the same time, the respiration of the homogenate was slightly depressed by the addition of potassium ions. The possible correlation between the effects of potassium on phosphate and glycogen metabolism is discussed.

A specific method for the quantitative determination of β-glucose-1-phosphate

Abstract A specific spectrophotometric determination of β-glucose-1-phosphate has been devised. It allows β-glucose-1-phosphate to be measured in the presence of α-glucose-1-phosphate and of a one hundred-fold excess of glucose-6-phosphate. Phosphoglucomutase for β-glucose-1-phosphate obtained from cells of Euglena gracilis var. bacillaris must be prepared for the assay.

Dolichyl phosphate phosphatase in rat liver microsomes: avoidance of the use of detergent in testing the effect of phospholipids on dolichyl phosphate phosphatase

A system was developed for testing the effect of phospholipids on dolichyl phosphate phosphatase, a membrane-associated enzyme. This enzyme was solubilized, delipidated, stabilized and concentrated in such a way that minimal quantities of Triton X-100 were carried by enzyme extracts to the incubation mixture. Its substrate, dolichyl phosphate, could be kept in aqueous medium as suspended particles without addition of detergent. When dolichyl phosphate phosphatase was assayed using the substrate in this detergent-free form, values for Km, pH optimum and temperature optimum were different from those obtained with detergent-solubilized substrate. This assay of dolichyl phosphate phosphatase almost free of detergent allowed testing of the effect of specific phospholipids on enzyme activity with minimal interference produced by endogenous phospholipids or exogenous detergent. Sphingomyelin, phosphatidylethanolamine or phosphatidylcholine (zwitterionic phospholipids) acted as activators, whereas phosphatidic acid and phosphatidylinositol, negatively-charged phospholipids, were inhibitors of dolichyl phosphate phosphatase.

Assay of dolichol monophosphate and dolichol diphosphate phosphatases: Thin-layer chromatography in a nonequilibrated system

A simple and rapid method for determining the activity of dolichol monophosphate phosphatase and dolichol diphosphate phosphatase is reported. This procedure is based on the separation of the substrates, [3H]dolichol monophosphate and [3H]dolichol diphosphate, from their reaction products, [3H]dolichol and [3H]dolichol monophosphate, by thin-layer chromatography on silica gel plastic plates. The chromatography chamber is of a nonconventional type, as it has a thin slot from which the last millimeter of the plate emerges. This special chamber allows the solvent to evaporate as it reaches the top of the plate. As a consequence, the mobile compounds tend to concentrate at the edge of the plate. The dolichol monophosphate phosphatase and dolichol diphosphate phosphatase substrates, which are more negatively charged than the corresponding reaction products, remain at the origin whereas the products migrate to the edge of the plate when appropriate solvents are used. The measurement of the radioactivity present at both the origin and the edge of the plate allows rapid estimation of the enzymatic activities.