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.

Effect of dolichyl monophosphate on the permeability properties of lipid membranes.

The effect of dolichyl monophosphate on the permeability properties of dimyristoylphosphatidylcholine bilayers to alkaline cations, Ca2+ and glucose has been determined by stop-flow spectrophotometry. The results show that, in contrast to free dolichol effects, the monophosphate derivative increased the permeability following a decreasing order of the permeating particle size. Phase diagrams indicate that dolichyl monophosphate is fully incorporated into the phosphatidylcholine bilayer around 0.75% weight/weight ratio. For these ratios, the permeation of ions is higher in the gel than in the liquid crystalline state.

Androgen dependence of protein N-glycosylation in rat epididymis.

The rat epididymis is known to produce and secrete glycoproteins which interact with spermatozoa during the maturation process. The synthesis of the protein core of these compounds is dependent on androgenic stimulation. As a consequence, we studied the possible androgenic control of the N-glycosylation process dependent on the dolichol (Dol) pathway. Glucosyl and mannosyl transferase activities in rat epididymal microsomes decreased by approximately 76% after only 2 days of castration with respect to intact controls. Depleted mannosyl transferase activity could be restored to control values by administration of 100 micrograms/day testosterone propionate (TP) for 4 days. The effect of 20 micrograms/day TP was blocked by the simultaneous administration of 500 micrograms/day of the antiandrogen cyproterone acetate. The addition of excess dolichyl phosphate (12 times the Michaelis-Menten constant (Km) value) to the incubation mixture did not eliminate the difference in mannosyltransferase activity between epididymal microsomes from castrated rats and these from control or testosterone-treated animals. Moreover, the endogenous pool of dolichyl phosphate was found unchanged in the different hormonal situations. Finally, the incorporation of [14C]mannose into lipid-bound oligosaccharides and into glycoproteins was decreased by approximately 60% as a result of castration and reinduced to control values by treatment with TP (50 micrograms/day for 4 days). The results demonstrate the androgen dependence of the initial steps of N-glycosylation in the rat epididymis and suggest that the hormonal regulation is exerted at the level of Dol-nucleotide sugar transferases, rather than upon the size of the endogenous Dol phosphate pool.

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.

Dolichyl-phosphate phosphatase and dolichyl-diphosphate phosphatase in rat-liver microsomes.

Dolichyl-phosphate phosphatase and dolichyl-diphosphate phosphatase activities of a liver-cell microsomal preparation were solubilized by treatment with Triton X-100. The 100,000 X g supernatant was then passed through a column of Sepharose-4B--concanavalin A. Both enzyme activities were found in the percolate. This treatment eliminated inhibition by ATP and glucose 6-phosphate in both phosphatase activities. In each case the activities were inhibited by higher concentrations of enzyme preparation due to the presence of phospholipids. The inhibitory effects of either phosphatidylcholine or phosphatidylethanolamine were due to competition for detergent. On the other hand, the effect produced by phosphatidic acid appeared to be different, since it did not change the optimal concentration of Triton X-100 for the two enzymes. Dolichyl-phosphate phosphatase was strongly inhibited by both Pi and PPi, whereas dolichyl-diphosphate phosphatase was only slightly inhibited by Pi and not at all by PPi. Dolichyl-diphosphate phosphatase was more inhibited by divalent cations than dolichyl-phosphate phosphatase. The apparent Km of dolichyl-phosphate phosphatase for dolichyl phosphate was 0.15 mM. Dolichol also inhibited dolichyl-phosphate phosphatase, but it produced a stronger inhibition on dolichyl-diphosphate phosphatase. The inhibitory effect of dolichol was not entirely due to detergent competition.

Lipid-bound oligosaccharides in insects.

Membrane preparations from immature stages of the fruit fly Ceratitis capitata catalyze the transfer of mannose from GDP-[14C]mannose into lipid-linked oligosaccharides. These compounds behave as polyprenyl derivatives and their formation is stimulated by the addition of an acidic glycolipid fraction isolated from insects. The mannose-labeled oligosaccharides are attached to the poly-isoprenol by a pyrophosphoryl linkage and can be released by mild acid hydrolysis. The trisaccharide lipid has been partially characterized. The results indicate that the compound is polyprenyl-pyrophosphate-N,N'-diacetylchitobiose-mannose. Incubation of dolichyl phosphate [14C]mannose or lower 14C-labeled oligosaccharide lipids with unlabeled GDP-mannose and the insect enzyme leads to the labeling of a higher lipid-bound oligosaccharide. When UDP-N-acetyl[14C]glucosamine was incubated with insect membranes a 14C-labeled chitobiosyl lipid was synthesized. If unlabeled GDP-mannose was also present, the 14C label appeared in the trisaccharide and higher oligosaccharide lipids. Preliminary evidence indicates that the insect polyprenyl oligosaccharides described here might participate in glycoprotein biosynthesis.

Chitin synthesis in Triatoma infestans and other insects.

In vivo and in vitro synthesis of chitin in Triatoma infestans was studied. For in vivo experiments, [14C] sugars were injected through the abdominal wall. Maximal incorporation of radioactivity into the cuticle was attained immediately after the ecdysis. The identification of in vivo synthesized chitin was performed by the enzymatic hydrolysis of the alkali-insoluble material from the cuticle with Helix chitinase. The main water-soluble compound obtained was N-acetylglucosamine as demonstrated by chromatographic procedures. In vitro synthesis of chitin was carried out with enzymatic crude extracts from Triatoma infestans, and UDP-N-acetylglucosamine was used as "source" of glycosyl moieties. Higher amounts of [14C] N-acetylglucosamine incorporation to chitin than those previously reported by others, were obtained. The identity of the product was confirmed in a similar way as that from in vivo synthesis. Radioactivity was also found in a liposoluble fraction concomitantly with chitin synthesis. This compound had an anionic behavior, was acid labile and had similar chromatographic properties as dolichol pyrophosphate N-acetylglucosamine obtained with pig liver extracts. Knowledge about dolichol phosphate sugars mediated glycoprotein synthesis in eukaryotes, suggests the involvement of this type of N-acetylglucosaminyl-phospholipid in macromolecule "building" even in insects.

Enzymatic synthesis of polyprenol monophosphate mannose in insects.

The 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 degrees 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 alpha-saturated polyprenol phosphate similar to dolichol monophosphate.

Identification of a major secretory glycoprotein from rat epididymis: interaction with spermatozoa.

A polypeptide with molecular mass of 17 kDa has been partially purified and identified as a major secretory glycoprotein in the rat epididymis. It is phosphorylated and contains high mannose-type oligosaccharides with 5 and 6 mannose units predominantly. These sugar residues are sufficiently exposed in the molecule to be released by endo-beta-N-acetylglucosaminidase H without prior denaturation or protease digestion. Specific binding of the glycoprotein to testicular spermatozoa was demonstrated with Ka 0.2 x 10(9) M-1 and 17,200 sites per cell, while no binding to epididymal spermatozoa was detectable. Direct labeling of surface proteins on cauda epididymis spermatozoa revealed the presence of a major band of 16.2 kDa, which may be equivalent to GP17. The interaction of the epididymal secretory protein with sperm suggests a possible role in the maturation process.

Antitumor activity of paramylon on sarcoma-180 in mice.

The antitumor effect of reserve polysaccharide, paramylon, from Euglena gracilis on the transplantable sarcoma-180 was examined in mice. This polysaccharide had an effect similar to that of lentinan. Paramylon, in a dose of 1 mug/g body weight, injected intraperitoneally 24 hr after tumor implantation had an inhibitory effect on the tumor growth, although without causing complete regression of the tumor. Alkaline-treated paramylon had a similar effect but at a smaller concentration than the native one. The inhibitory activity was not lost when the paramylon preparation was treated with pronase, DNase, or RNase. The antitumor effect might be a lymphocyte-mediated process. In tumors that were regressing after treatment, there was extensive outpouring of lymphoid cells with plasma cells and macrophages. A test conducted using paramylon ruled out the possibility of an interferon-mediated inhibiotry effect on tumor growth.