John W. Jensen

New assay for uronosyl 5-epimerases☆

Simple assays have been developed for the two uronosyl 5-epimerases which participate in the biosynthesis of heparin and dermatan sulfate (heparosan N-sulfate D-glucuronosyl 5-epimerase and chondroitin D-glucuronosyl 5-epimerase, respectively). Following previously published procedures, substrates labeled with tritium in the C-5 positions of the D-glucuronosyl and L-iduronosyl residues were prepared enzymatically by incubation of O-desulfated heparin and dermatan with 3H2O and crude epimerase preparations from bovine liver and human skin fibroblasts, respectively. In the new assays, 3H2O generated from these substrates during the epimerase reactions was quantitated by the method of Pollard et al. (Anal. Biochem. (1981) 110, 424-430). In this procedure, 3H2O in the aqueous reaction mixture is extracted into a toluene-based organic phase containing 25% isoamyl alcohol, while the polysaccharide substrate remains in the aqueous phase and does not generate scintillations. This procedure is much simpler than that used previously which involves distillation of each reaction mixture and quantitation of the radioactivity in the distillate. The new assays have been validated by the demonstration that conditions of linearity with time and enzyme concentration can be established for both epimerase reactions. Assays of this type should be useful in the study of any enzymatic reaction where 3H2O is formed from a 3H-labeled substrate and the unreacted substrate is not appreciably soluble in the organic phase.

Bilayer membrane destabilization induced by dolichylphosphate

Small vesicles containing the fluorescent probe calcein were used to investigate the effect of dolichyl phosphate (Dol-P) on phospholipid bilayer stability. In the absence of Dol-P, phospholipid vesicles retained the fluorescent probe upon the addition of divalent cations. Small vesicles containing Dol-P, however, exhibited calcein leakage when incubated in the presence of divalent cations. This effect was observed in liposomes composed of a mixture of phosphatidylethanolamine (PE), phosphatidylcholine (PC) and Dol-P, but not in PC/Dol-P liposomes. The rate of calcein leakage was proportional to divalent cation concentration and to temperature, but was independent of vesicle concentration. These results demonstrate that Dol-P has significant effects on the stability of PE containing phospholipid bilayers. Vesicle leakage was also promoted by the addition of rat liver Dol-P-mannose synthase (EC 2.4.1.83) to intact PE/PC/Dol-P vesicles. Enzyme induced leakage from phospholipid vesicles required the presence of both unsaturated PE and Dol-P. The phospholipid composition of leaky vesicles could be correlated with the lipid matrix required for maximal transferase activity of the rat liver synthase. The destabilizing effects of Dol-P on phospholipid bilayers may therefore be involved in the translocation of activated sugars across biological membranes.

Characterization of mannosyl-transfer reactions catalyzed by dolichyl-mannosyl-phosphate-synthase

Evidence suggesting that a single enzyme catalyzes mannosyl transfer from GDP-mannose to both dolichyl phosphate and to phenyl phosphate was obtained as follows: (a) The two activities were coeluted from columns of DEAE-cellulose and Sepharose CL-6B, (b) both reactions demonstrated similar kinetic constants for the glycosyl donor and for guanosine nucleoside inhibitors, (c) both reactions were sensitive to inhibition by low concentrations of nonionic detergents, and (d) both activities were found to be thermally inactivated at similar rates upon incubation at 55 degrees. The reaction conditions required for optimal mannosyl transfer by the purified enzyme preparation to the hydrophobic and water soluble acceptors, however, were found to be quite different. Whereas mannosyl transfer from GDP-mannose to dolichyl phosphate occurred at maximal rates only in the presence of specific phospholipids, the rate of mannosyl transfer to phenyl phosphate was essentially unaffected by the addition of phospholipid. These results indicate that dolichyl-mannosyl-phosphate-synthase, which has some of the properties of an intrinsic membrane protein, does not have an absolute requirement for phospholipid for catalytic activity, but rather that phospholipid is required for interaction of the enzyme with the long chain polyisoprenol substrate dolichyl phosphate.

Biosynthesis of heparin. A new substrate for heparosan-N-sulfate-d-glucopyranosyluronate 5-epimerase

Abstract New substrates have been prepared for heparosan- N -sulfate- d -glucopyranosyluronate 5-epimerase, which catalyzes formation of l -iduronic acid residues in the course of heparin biosynthesis. Heparin and heparan sulfate were chemically modified by desulfation in aqueous dimethyl sulfoxide, deacetylation by hydrazinolysis, and N -sulfation with sulfur trioxide-trimethylamine complex. The modified polysaccharides were incubated with partially purified epimerase from bovine liver in the presence of tritium oxide to incorporate tritium into both d -gluco- and l -ido-pyranosyluronate residues. Incubation of the labeled polysaccharides with liver epimerase released tritium. The complete release of radioactivity after exhaustive incubation indicated that the tritium atom was located at C-5 of the uronate residues. Under appropriate conditions, the release was linear with time and enzyme concentration; K m values of ~0.2m m (expressed as uronic acid concentration) were determined for both the heparin- and the heparan sulfate-derived substrates. In contrast to the modified polysaccharides, unmodified heparin did not incorporate significant amounts of radioactivity when exposed to tritium oxide in the presence of epimerase.

Biosynthesis of Heparin: Tritium Incorporation into Chemically Modified Heparin Catalyzed by C-5-Uronosylepimerase

Publisher Summary The epimerization of D-glucuronic to L-iduronic acid residues in the course of heparin biosynthesis is accompanied by the exchange of C-5 hydrogen atom. This feature of the reaction serves as the basis for an assay of epimerase activity, in which the release of tritium into the water of the incubation medium is measured with a D-[5-3H]glucosyluronic acid-labeled precursor polysaccharide as a substrate. This chapter discusses an experiment to study the enzymatic incorporation of radioactivity from T2O into chemically modified heparin, yielding a product suitable as a substrate in the epimerase assay. In the assay, heparin from hog mucosa was purified by repeated precipitation with cetylpyridinium chloride from 1.4 M NaCl essentially. The chapter illustrates the result by a graph providing the time course of tritium incorporation into modified heparin.