Luis R. Maréchal

β-1,3-oligoglucan: Orthophosphate glucosyltransferases from Euglena gracilis: I. Isolation and some properties of a β-1,3-oligoglucan phosphorylase

1. 1.|A new enzyme has been found in cell free extracts of Euglena gracilis which catalyzes the reversible phosphorolytic cleavage of β-1,3-oligoglucans according to the following reaction: (β-1,3-glucose)n + Pi ⇋ (β-1,3glucose)n−1 + α-D-glucose 1-phosphate This enzyme was distinguished from laminaribiose phosphorylase, which catalyzes the same reaction. They could also be separated using calcium phosphate gel. 2. 2.|In the direction of synthesis, glucose could serve as good an acceptor as β-1,3-oligoglucans but with a Km approx. 10–20 times higher. Other β-glucosyl derivatives were also good acceptors, with the exception of laminarin and paramylon. On the other hand, several sugar phosphates could not substitute for α-glucose 1-phosphate. 3. 3.|The novel enzyme showed a nearly absolute requirement for sulfhydryl groups, a property especially observed in aged preparations. 4. 4.|The methods used for the identification of laminaritriose, one of the products formed using laminaribiose as acceptor, showed that the glucosyl moiety of α-glucose 1-phosphate was attached to the non-reducing end of disaccharide. 5. 5.|Some kinetic properties of the enzyme and the stoichiometry of the reaction are also reported.

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.

β-1,3-Oligoglucan:orthophosphate glucosyltransferases from Euglena gracilis: II. Comparative studies between laminaribiose- and β-1,3-oligoglucan phosphorylase

1. 1. Further studies have been carried out on the specificity of laminaribiose- and β-1,3-oligoglucan phosphorylase. Both enzymes, present in extracts of Euglena gracilis, could be separated either by calcium phosphate gel or by DEAE-cellulose column chromatography. 2. 2.|From the data presented it can be concluded that the two enzymes catalyze the same reaction but with different quantitative specificity. The β-1,3-oligoglucan phosphorylase was found to phosphorolyze laminaritriose and higher homologues at a greater rate than laminaribiose while the opposite behavior was observed with laminaribiose phosphorylase. 3. 3.|Another, and probably more striking, difference between the two enzymes was the fact that aged preparations of β-1,3-oligoglucan phosphorylase showed an absolute requirement of sulfhydryl donors while laminaribiose phosphorylase was only slightly activated. 4. 4.|The possibility that the qualitative similarity might be due to a cross-contamination of the enzymatic fractions was discarded by competition experiments between substrates and by the elution pattern of the DEAE-cellulose chromatography.

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.

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.